Presentation Type: Oral Presentation
Title: In-Depth Investigation of Soybean Seed-Filling Proteome and Comparison with a Parallel Study of Rapeseed
Author(s): Ganesh Agrawal^*  Martin Hajduch  Katherine Graham  Jay Thelen                       
Affiliation: University of Missouri, Biochemistry Department
Abstract:

To better understand the metabolic processes of seed filling in soybean, two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semi-continuous multi-dimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 non-redundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to parallel study of rapeseed to further understand the regulation of intermediary metabolism in protein- versus oil-rich seeds. Comparative analyses revealed: (i) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean and (ii) approximately 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests the mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways.

Presentation Type: Oral Presentation
Title: Rhg1 and Soybean Cyst Nematode - "What gene controls resistance?"
Author(s): Andrew Bent^*  Adam Heuberger  Sara Melito                         
Affiliation: University of Wisconsin - Madison
Abstract:

Soybean cyst nematode causes the greatest yield loss of any pathogen of U.S. soybeans. Almost all SCN-resistant U.S. soybean varieties have resistance that is controlled primarily by the Rhg1 locus derived from PI88788, and there is substantial curiosity about the molecular basis of this resistance. The Rhg1 locus was molecularly characterized and patented almost a decade ago, but the gene or genes at Rhg1 that contribute to SCN resistance remain unidentified. A candidate gene mapping to Rhg1 that encodes an LRR-kinase protein has been studied, but attempts to demonstrate SCN resistance function for this gene have not succeeded. This may be because this gene does not confer resistance, or because the assays used have had one or more weak points that prevent detection of function. Note, for example, that this resistance is partially dominant (“active” function may derive from resistant or susceptible parent), and that resistance is partial/quantitative, and that other loci make minor contributions to this SCN resistance. Our lab has recently joined the effort to identify the Rhg1 gene. We will describe our work to adapt, repeat and/or improve transgenic root production, SCN resistance assays that use these roots, gene annotation at the Rhg1 locus, fine-structure genetic mapping of resistance, and testing of candidate genes for SCN-resistance function.

Presentation Type: Oral Presentation
Title: Dissection of quantitative resistance in soybean against Phytophthora sojae using genetical genomics
Author(s): Brett Tyler^*  Lecong Zhou  Santiago Mideros  Lei Bao  Sucheta Tripathy  Konstantinos Krampis  Yongcai Mao  Bing Liu  Felipe Arredondo  Dominic Tucker   Regina Hanlon  Steven St. Martin  Ina Hoeschele  Anne Dorrance  Saghai Maroof 
Affiliation: Virginia Polytechnic Institute and State University
Abstract:

We have used a series of large-scale Affymetrix® microarray experiments to characterize mechanism of quantitative resistance in soybean against P. sojae. The first experiment was the transcriptional profiling of eight soybean genotypes with different quantitative resistance levels. Linear mixed model and contrast analysis with the 8 genotypes has revealed that a large number of genes were perturbed by the infection process, but only a small subset (756-1155 genes) were identified as associated with quantitative resistance. Gene expression changes common and specific to the most resistant cultivars were identified. The second experiment was a more detailed time-course and spatial profiling of 4 selected cultivars. In the third experiment, a population of 300 recombinant inbred lines (RILs) derived from a cross between Glycine max V71-370 and G. sojae PI407162 was used in order to combined gene expression profiling with QTL analysis. For genotyping of RILs we developed an algorithm for mining single feature polymorphisms (SFPs) from the expression data, crating a high density genetic map with an average markers separation of less than 2 cM. Genetical genomics and structural equation modeling are being used infer genetics networks associated with quantitative resistance.

Presentation Type: Poster
Title: Understanding Iron Deficiency Chlorosis in Soybean through Microarrays and the Soybean Genome Sequence
Author(s): Jamie O'Rourke^*  Rex Nelson  David Grant  Jeremy Schmutz  Carroll Vance  Steven Cannon  Michelle Graham  Randy Shoemaker               
Affiliation: Iowa State University
Abstract:

Soybean producers report up to a 23% yield loss due to Iron Deficiency Chlorosis (IDC) in the upper Midwest. The use of microarrays has allowed researchers to identify specific candidate genes involved in IDC. Near isogenic lines developed for their differential iron response, were grown hydroponically in iron sufficient and iron limited conditions. Transcriptional profiles of the plants were analyzed and compared using the Affymetrix® soybean chip. A comparison of Clark plants (efficient) (PI548553), grown under both conditions, identified 835 candidate genes putatively involved in soybeans' iron stress response. An identical comparison of IsoClark plants (inefficient) (PI547430) identified 200 candidate genes. The microarrays also identified 211 single feature polymorphisms (SFPs) distributed between the NILs. These SFPs represent a potential source of genetic variation involved in the differential iron stress response. Many of the SFPs are located in genes annotated as transcriptional regulators. Candidate gene sequences, SFPs, and markers known to lie within iron QTLs were aligned against the 7X build of the soybean genome. A sliding window analysis of the microarray data and the 7X genome, coupled with an iterative simulation of the data, showed the candidate genes exhibit clustering in the genome. The clusters represent regions of transcriptional importance to soybeans iron deficiency response. To our knowledge, this is the first time specific genes have been identified by microarray analysis and mapped to a region of the soybean genome corresponding to the trait of interest.

Presentation Type: Poster
Title: Functional genomics in soybean: assigning defense-related functionalities to soybean sequences
Author(s): Fu Daqi^*  Said Ghabrial  Aardra Kachroo                         
Affiliation: University of Kentucky
Abstract:

We recently reported the development of a bean pod mottle virus (BPMV)-based vector for employing virus-induced gene silencing in soybean. The availability of this vector and its successful employment for assigning functionalities to soybean sequences has great implications for functional genomics in this crop. Silencing the soybean stearoyl desaturase (SACPD) gene(s) reduced endogenous oleic acid levels, which in turn induced cell death, PR, and R gene expression. These phenotypes are very similar to those in the Arabidopsis SACPD mutant, ssi2. The SACPD-silenced soybean plants also showed enhanced resistance to multiple pathogens including Pseudomonas syringae and Phytophthora sojae. In addition to the SACPDs, several other components of soybean defense signaling pathways have been identified, silencing of which breaks down R-mediated resistance to viral and bacterial pathogens. Functional characterization of defense signaling components would aid in the engineering of novel, enduring, and broad-spectrum disease resistance in soybean.

Presentation Type: Oral Presentation
Title: Quantitative proteomic analysis of bean plants infected with virulent and avirulent strains of an obligate rust fungus
Author(s): Bret Cooper^*                             
Affiliation: USDA-ARS
Abstract:

The economically important dry bean plant Phaseolus vulgaris is a host for the rust fungus Uromyces appendiculatus. Resistance genes have been defined and have been used to protect the bean crop, but the proteins governing resistance are not well-resolved, especially compared to model plant-pathogen systems. To characterize the nature of resistance, we have used high-throughput tandem mass spectrometry to detect and analyze more than 3,000 proteins from infected bean leaves. By statistically comparing the amounts of proteins detected in a single plant variety that is either susceptible to infection or resistant, depending on the fungal strains introduced, we have distinguished resistance from susceptibility at a proteomic level. Several other plant proteomic responses, some which may favor the pathogen, also change during the course of infection. These results provide a basic foundation for understanding the proteomics of disease responses for a major crop plant.

Presentation Type: Poster
Title: Accumulation of Ricinoleic Acid in Seeds of Transgenic Soybean
Author(s): SooYoung Park^*  Mark Smith  Tom Clemente                         
Affiliation:
Abstract:

Castor bean (Ricinus communis) oil is highly versatile industrial oil used in an array of applications including plastics, foams, surfactants and biofuel. However, supply of castor oil is limited due to a number of negative characteristics, including presence of the highly toxic molecule, ricin, a by-product of castor bean meal, which limits its potential for domestic production. An avenue to circumvent the need for domestic production of castor bean is to produce the ricinoleic acid, which composes approximately 90% of the fatty acid profile of castor oil, in heterologous system, such as soybean. To this end, we introduced into soybean the caster oleate 12 hydroxylase gene under the control of the seed-specific phaseolin promoter. Fatty acid analysis of seeds carrying this transgene either displayed an mid-oleic acid phenotype (30% to 70% 18:1) without production of ricinoleic acid, or an elevated oleic acid phenotype (25% to 30% 18:1) with a concomitant production of ricinoleic acid ranging from trace levels up to 12%. In 2007, a field trial was conducted with one of the ricinoelic acid producing event designated 521-24. Yield estimates from the harvest under dryland conditions were 45bu/ac, with estimates of total protein and oil of 45.5% and 20%, respectively, based on NIR analysis. Ricinoleic acid levels from the harvest averaged 12.1%.

Presentation Type: Poster
Title: Evaluating Natural Variability of Soybean Proteins by Proteomic tools
Author(s): Savithiry Natarajan^*                             
Affiliation: USDA-ARS-PSI
Abstract:

Soybean is a rich and inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, yield and quality to meet the increasing global demand. Therefore, to ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations of different classes of proteins, storage, allergen and anti-nutritional protein profiles between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation.

Presentation Type: Poster
Title: Effect of Vacuum Infiltration and Dip-Wounding on Transformation of Soybean
Author(s): Xinlu Chen^*  Xiujuan Su  Henry T. Nguyen  Zhanyuan Zhang                       
Affiliation: University of Missouri
Abstract:

Effect of Vacuum Infiltration and Dip-Wounding on Transformation of Soybean
Xinlu Chen*, Xiujuan Su*, Henry T. Nguyen, and Zhanyuan Zhang*.
*Plant Transformation Core Facility, Division of Plant Sciences, National Center for Soybean Biotechnology, University of Missouri, Columbia, MO 65211 (zhangzh@missouri.edu).

Agrobacterium-mediated transformation of soybean (Glycine max) plays a unique role in studying gene functions and improving traits of this crop. However, transformation of most of soybean genotypes by Agrobacterium is still inefficient, despite recent progress. Because high infection of explants by Agrobacterium is a prerequisite for high frequency transformation, we evaluated the effect of two inoculation conditions on Agrobacterium infection. These two conditions included vacuum-infiltration and “dip-wounding” of soybean seedling node explants with Agrobacterium. The standard binary vector pZY102 was used which carries the bar gene as a selectable marker and the GUS reporter gene. The soybean genotype “Williams 82” was used throughout experiments. The infection status was determined at the end of shoot induction stage (via GUS staining) by the number of GUS-positive buds/shoots and sectors on per explant basis. Preliminary data showed that about 7.3% and 4% GUS-positive cloning buds and shoots developed on per explant inoculated using 5-min and 30-second vacuum-infiltration, respectively. These frequencies were higher than the standard procedure (2%). The 5-min vacuum-infiltration was also applied to twenty-four other soybean genotypes including Magellan, Bert and Maverick but all showed a reduced stable transformation at an average of 1% frequency, presumably due to the tissue damage by vacuum-infiltration. By contrast, the “dip-wounding”, i.e., dipping dissecting blade into Agrobacterium suspension medium before wounding cotyledonary nodal area was found to improve the transformation substantially from 0.0-2% to 2-5% (depending on vectors used) even when the seed vigor was poor causing significantly reduced transformation. This “dip-wounding” is now routinely used in our transformation protocol.

Presentation Type: Oral Presentation
Title: Soybean WRKY-type transcription factor genes GmWRKY13, GmWRKY21, and GmWRKY54 confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants
Author(s): Yun Zhou   Ai-Guo Tian  Hong-Feng Zou  Zong-Min Xie  Gang Lei  Jian Huan  Chun-Mei Wang  Hui-Wen Wang  Jin-Song Zhang  Shou-Yi Chen^*            
Affiliation: Institute of Genetics & Developmental Biology, CAS
Abstract:

WRKY-type transcription factors have multiple roles in plant defense response and developmental processes. Their roles in abiotic stress response remain obscure. In this study, we identified 64 GmWRKY genes from soybean and found that these genes were differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in yeast assay system and five had such ability. In DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with conserved WRKYGQK sequence in their DNA binding domain could bind to the W-box (TTGAC). However, the GmWRKY6 and GmWRKY21, with altered sequence WRKYGKK, lost the ability to bind W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54 were further investigated using transgenic approach. The GmWRKY21-transgenic Arabidopsis plants were more tolerant to cold stress whereas the GmWRKY54 conferred salt and drought tolerance possibly through regulation of DREB2A and STZ/Zat10. Transgenic plants overexpressing the GmWRKY13 showed more sensitivity to salt and mannitol stresses but reduced sensitivity to ABA when compared with wild type plants. In addition, the GmWRKY13-transgenic plants had more lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance and the GmWRKY13 may function in both lateral root development and abiotic stress response.

Presentation Type: Oral Presentation
Title: Developing low phytate crops
Author(s): Jinrui Shi^*  Hongyu Wang  Kathleen Schellin  Li Bailin  Stoop Johan  Ranch Jerry  Glassman Kimberly                 
Affiliation: Pioneer Hi-Bred International
Abstract:

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. Although low-phytate mutants provided a solution for managing the problems, breeding for commercial products was unsuccessful due to undesired agronomic characteristics associated with the mutants. We are taking transgenic approaches to modify phytate biosynthetic pathway. Silencing Ins(3)P synthase gene produced low-phytate seeds, but the germination was impaired. The low-phyate transgenic plants generated by suppressing various myo-inositol phosphate kinases either accumulate InsP intermediates or do not have a high level of Pi. The accumulation of InsPs may cause regulatory concerns, while the low level of Pi diminishes the commercial value of low-phytate products. Silencing maize lpa1 gene expression in an embryo-specific manner produced low-phytate and high-Pi transgenic seeds that germinate normally and have no significant seed dry weight reduction. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytate. Suppressing the homologous soybean MRP gene also generated low-phytate seed, suggesting that the strategy might be feasible for many crops.

Presentation Type: Oral Presentation
Title: Metabolic engineering of flavonoid biosynthesis revealed distinct functions of flavonoids in legume-rhizobia interactions
Author(s): Juan Zhang  Senthil Subramanian  Gary Stacey  Oliver Yu^*                       
Affiliation: Danforth Center
Abstract:

Flavonoid compounds have been implicated to play multiple roles in many plant developmental processes. However, the exact functions and the mode of action of these compounds are not clear in almost all these processes. Previously, flavonoids were shown be essential for legume-rhizobia symbiosis in both determinate (soybean) and indeterminate nodulating plants (M. truncatula). In this report, we used plant metabolic engineering to generate a series of transgenic M. truncatula roots that have different flavonoid profiles and studied nodule developments on these roots. We were able to supplement these roots with different flavonoid compounds to physiologically and biochemically complement the altered metabolic profile and study the role of individual flavonoid compounds during nodulation. More importantly, we were able to use an engineered strain of rhizobia that does not require flavonoids to nodulate the roots to further investigate the critical mechanism of the flavonoid compounds. Together, we demonstrated clearly that different flavonoid compounds function differently during M. truncatula nodule development. Our results show that flavones function as nod gene inducers primarily inside the roots whereas flavonol-related compounds functions as auxin transport inhibitors. Both groups of compounds are essential for normal nodulation. Our data provided supports for a new model for the critical roles flavonoids play during nodulation.

Presentation Type: Poster
Title: Molecular Genetic Analysis of Seed Protein Control at Linkage Group I in Soybean Near-Isogenic Lines
Author(s): Yung-Tsi Bolon*^*  Bindu Joseph* co-first author  Steven Cannon  Brian Diers  Andrew Farmer  Michelle Graham  Greg May  Gary Muehlbauer  James Specht  Nathan Weeks   Randy Shoemaker  Carroll Vance       
Affiliation: USDA-ARS, Plant Science Research Unit, St. Paul, MN
Abstract:

The molecular mechanisms that influence soybean seed composition are not well understood. Because the profitability of the soybean crop is affected by seed protein and oil content, insight into the genetic controls involved in these traits is important for future soybean improvement. Here, we examine the major soybean protein quantitative trait locus at Linkage Group I for candidate genes and pathways involved in seed protein and oil traits. Fine mapping and sequencing allowed us to define the introgressed QTL region at LG I which is estimated to span approximately 9 Mbp. In parallel, we used Affymetrix® soybean genome arrays to compare gene expression in a pair of near-isogenic lines that contrast in seed protein and oil. Gene expression profiles were obtained from multiple stages of the developing soybean seed. Analysis of differential gene expression and single feature polymorphisms, along with quantitative RT-PCR and sequencing confirmation highlighted a handful of candidate genes located within the protein QTL at LG I. High-throughput Solexa sequencing of the same samples supported our Affymetrix® data and revealed additional interesting phenomenon at LG I. Presumably, the variations we detected are related to the difference in protein and oil content between these two lines. These results demonstrate the power of gene expression analysis to contrast near-isogenic lines and illustrate how two complementary gene profiling technologies may aid in the annotation of genes in the soybean genome. Further investigation may provide new insight into the genes and pathways involved in protein and oil accumulation in the soybean seed.

Presentation Type: Poster
Title: Production of High Omega-3 Fatty Acid Soybean Oil as a Feedstock for Aquaculture
Author(s): Fareha Razvi^*  Pat Tenopir  Robert Weber  Steve Weirer  Tim O'Keefe  Michael Cremer  Bridgett Owen  George Graef  Jennica Lowell  Neil Sims   Tom Clemente         
Affiliation: Center for Plant Science Innovation, University of Nebraska-Lincoln
Abstract:

The identification and development of sustainable sources of protein and lipid will be required to maintain a plentiful and cost-effective supply of feed for the rapidly expanding aquaculture industry. Currently aquaculture feed relies upon both fishmeal and fish oil to meet the dietary needs of farm raised fish. As a means to address replacement of fish oil as a lipid source for the aquaculture industry, we report here on the development of novel soybeans that have potential to serve as a sustainable lipid source for farm raised fish. In soybean accumulation of high levels of stearidonic acid, a relatively rare omega-3 fatty acid, can be accomplished by co-expression of a delta 6 and delta 15 desaturase genes. To examine the capacity of a stearidonic acid producing soybean to synthesize the very long polyunsaturated omega-3 fatty acid eicosapentaenoic acid (EPA), a fatty acid elongase gene from zebra fish and a mammalian delta 5 destaurase gene were pyramided with the other desaturase genes. Soybean seed carrying this four gene stack displayed EPA accumulation up to 8% of the seed storage lipids. Studies are currently ongoing to evaluate the agronomics of this soybean germplasm and utility of both the stearidonic acid and EPA soybean oil as a dietary source of lipids for aquaculture.

Presentation Type: Poster
Title: Development of soybean oil low in palmitic acid and elevated in stearic and oleic acids
Author(s): Hyunwoo Park^*  George Graef  Mike Fromm  Tom Clemente                       
Affiliation: Department of Agronomy & Horticulture, University of Nebraska-Lincoln
Abstract:

Most commodity soybean oil has a fatty acid profile of app. 10% palmitic acid, 4% stearic acid, 17% oleic acid, 55% linoleic acid, and 10% linolenic acid. Oil high in oleic acid, and elevated in stearic acid, with a concomitant reduction in palmitic acid and polyunsaturated fatty acids offers improved functionality for both food and industrial applications. To produce soybean oil with such a fatty acid profile we stacked a transgene that carries a silencing element designed to simultaneously down-regulate in a seed-specific fashion a palmitoyl thioesterase (FatB) gene and a delta 12 desaturase gene, with a stearoyl ACP thioesterase seed-specific transgenic cassette, via sexual crossing. The parent that carries the silencing transgene was derived from a soybean event designated 335-13 which produces an oil high in oleic acid (>85%) and low in palmitic acid (<4%). This event, 335-13, was crossed to soybean plants that inherited the stearoyl ACP thioestersae gene from a set of transgenic events that displayed stearic acid levels ranging from 8% up to 15%. F1 seed from these crosses have generated fatty acid profiles with stearic acid ranging from 5% to 12%, and oleic acid levels ranging from 65% to 79%.

Presentation Type:
Title: Genomic sequence encoding 6-phytase gene from Glycine max
Author(s): Vinita Kumari^*                             
Affiliation: Indian Agricultural Research Institute
Abstract:

Phytases, the only recognized group of phosphatases, catalyze the stepwise removal of phosphates from seed phytins. In the present study a genomic sequence of phytase gene was amplified using long PCR. Cloning and characterization of the genomic sequence revealed a total length of 4220 bp containing 6 introns interrupting 7 exons. The transcribed sequence of the gene had an expected open reading frame of 1644 bp predicted to encode for 547 amino acids including an N-terminal signal peptide of 28 amino acids confirming its secretory status. Strong homology with the previously reported cDNA (GmPhy) as revealed in the Blast search indicated high degree sequence similarity to purple acid phosphatases including the conserved motifs and the signature metallophosphoesterase domains. mRNA expression for phytase gene as evaluated in the germinating cotyledons by RT-PCR revealed maximum transcript levels at around 11 days after germination. Single copy of the gene was detected during Southern analysis of the genomic DNA. Maximum enzymatic activity of the phytase was observed at the 11th day after germination which corresponded well with high transcript levels as seen around the same stage by semiquantitative RT-PCR.

Presentation Type:
Title: Genomic sequence encoding 6-phytase gene from Glycine max
Author(s): Vinita Kumari^*  Monica Jolly  Nirupama Dubey  Archana Sachdev                       
Affiliation: Indian Agricultural Research Institute
Abstract:

Phytases, the only recognized group of phosphatases, catalyze the stepwise removal of phosphates from seed phytins. In the present study a genomic sequence of phytase gene was amplified using long PCR. Cloning and characterization of the genomic sequence revealed a total length of 4220 bp containing 6 introns interrupting 7 exons. The transcribed sequence of the gene had an expected open reading frame of 1644 bp predicted to encode for 547 amino acids including an N-terminal signal peptide of 28 amino acids confirming its secretory status. Strong homology with the previously reported cDNA (GmPhy) as revealed in the Blast search indicated high degree sequence similarity to purple acid phosphatases including the conserved motifs and the signature metallophosphoesterase domains. mRNA expression for phytase gene as evaluated in the germinating cotyledons by RT-PCR revealed maximum transcript levels at around 11 days after germination. Single copy of the gene was detected during Southern analysis of the genomic DNA. Maximum enzymatic activity of the phytase was observed at the 11th day after germination which corresponded well with high transcript levels as seen around the same stage by semiquantitative RT-PCR.

Presentation Type: Oral Presentation
Title: Dissecting the molecular response of soybean to Asian soybean rust
Author(s): Martijn van de Mortel^*  Kathy T. Schneider  Michelle A. Graham  Ed J. Braun  Ricardo V. Abdelnoor  Reid D. Frederick  Thomas J. Baum  Steven A. Whitham               
Affiliation: Iowa State University, Department of Plant Pathology, Ames IA
Abstract:

Asian soybean rust (ASR) is now a threat to soybean production in all major growing regions in the world. This potentially devastating disease is caused by the obligate fungal pathogen Phakopsora pachyrhizi and resistance in soybean germplasm is limited. Moreover, this resistance is generally observed as low compatibility to the fungus, which occurs at a much later time (approximately 3 days after infection), compared to more typical resistance displayed as a hypersensitive reaction within the first 24h after infection. We have started to decipher the interactions between host and pathogen at the molecular level and categorized genes responsive to P. pachyrhizi in the Rpp2, Rpp3, and Rpp4 genetic backgrounds and in susceptible genotypes. Microscopic and gene expression analyses conducted at time points spanning early infection to symptom development have allowed us to correlate P. pachyrhizi growth and development with the timing of molecular events in the host. We observe that P. pachyrhizi induces a strong non-specific defense response upon infection, after which the fungus grows virtually undetected until haustoria form. The onset and magnitude of a second phase of differential gene expression patterns appear to be major determinants of resistance. In addition, we observe different classes of regulatory proteins differentially expressed in the various Rpp backgrounds, indicating these three resistance genes may activate specific signal transduction pathways. We hypothesize that the early responses represent an ineffective basal defense response. Rpp2, Rpp3, or Rpp4 subsequently mediates an effective resistance response after P. pachyrhizi forms haustoria and secretes effector proteins.

Presentation Type: Poster
Title: Genomic sequence encoding 6-phytase gene from Glycine max
Author(s): Vinita Kumari^*  Monica Jolly  Nirupama Dubey  Archana Sachdev                       
Affiliation: Indian Agricultural Research Institute
Abstract:

Phytases, the only recognized group of phosphatases, catalyze the stepwise removal of phosphates from seed phytins. In the present study a genomic sequence of phytase gene was amplified using long PCR. Cloning and characterization of the genomic sequence revealed a total length of 4220 bp containing 6 introns interrupting 7 exons. The transcribed sequence of the gene had an expected open reading frame of 1644 bp predicted to encode for 547 amino acids including an N-terminal signal peptide of 28 amino acids confirming its secretory status. Strong homology with the previously reported cDNA (GmPhy) as revealed in the Blast search indicated high degree sequence similarity to purple acid phosphatases including the conserved motifs and the signature metallophosphoesterase domains. mRNA expression for phytase gene as evaluated in the germinating cotyledons by RT-PCR revealed maximum transcript levels at around 11 days after germination. Single copy of the gene was detected during Southern analysis of the genomic DNA. Maximum enzymatic activity of the phytase was observed at the 11th day after germination which corresponded well with high transcript levels as seen around the same stage by semiquantitative RT-PCR.

Presentation Type: Poster
Title: Association Mapping and Transition of Elite Alleles in Major Cultivar Family Pedigrees of Soybean in China
Author(s): Junyi Gai^*  Tuanjie Zhao  Jun Zhang  Zixiang Wen  Deyue Yu                     
Affiliation: National Center for Soybean Improvement, Nanjing Agricultural University
Abstract:

The genotyping data of 85 SSR markers on 190 cultivars released in China were analyzed for association mapping of agronomic and quality traits under TASSEL GLM program. Forty-five SSR markers with 136 loci (times) associated with 11 traits, among those, 22 loci (times) consistent with QTLs mapped by family-based linkage procedure. A large part of the associated loci was different from those detected with 60 SSR markers on representative samples of 393 landraces and 196 wild accessions. Of the 190 released cultivars, 163 from five family pedigrees with 58-161, Xudou 1, Qihuang 1, Nannong 493-1, Nannong 1138-2 as their respective pedigree ancestor were analyzed for transition of elite alleles in each family pedigree. Two best alleles of each of the 9, 3, 2, 2 major loci, which explaining 91%, 36%, 13%, 31% total phenotypic variation for yield, 100-seed weight, protein content, and oil content were traced respectively. Each pedigree ancestor had its own elite alleles transited to its progenies but might lose during transition. The family pedigrees tended to get all the elite alleles, but with different frequency distributions. The high yield cultivars had average yield 2.36 times and average elite alleles 4.17 times of those of low yield cultivars, with the highest of 7 elite alleles out of 9 loci and the average only 2.33. However, some cultivars had high yield but less elite alleles and vise versa, which should be studied further. The results have provided a way of breeding by genotypic design with molecular markers.

Presentation Type: Oral Presentation
Title: Defining the Signatures of Positive Selection in the Soybean Genome as Targets for Mining the USDA Germplasm Collection for Crop Improvement
Author(s): David Hyten^*  Qijian Song  James Specht  Randall Nelson  Thomas Carter  Perry Cregan                   
Affiliation: USDA-ARS, Beltsville, MD
Abstract:

The G. max Asian landraces which are the most immediate result of domestication was the founding population of North American elite cultivars. Selection by breeders for agronomically favorable loci over the past 60 years would change allele frequencies and extend linkage disequilibrium (LD) in the genomic region surrounding the selected loci. This signature of selection in the genome is known as a selective sweep. To find putative regions that have undergone a positive selective sweep, we genotyped using the Illumina GoldenGate assay, 3,080 single nucleotide polymorphisms (SNPs) in 96 diverse G. max Asian landraces and 96 diverse elite cultivars. Several regions of the genome were found to have had a significant allele frequency change in the elite cultivars. The LD in these regions was assessed to determine which regions show extended amounts of LD which would indicate low amounts of haplotype variation in the elite cultivars. These regions that show significant allele frequency changes and high amounts of LD may contain genetic variation in the G. max Asian landraces which has not been tested in an elite germplasm background and may contain agronomically beneficial loci. While this analysis with 3,080 SNPs found several regions that may have undergone a selective sweep, the current project to genotype the USDA soybean germplasm collection with 50,000 SNPs will allow for a more thorough analysis of the genome and more sensitive tests to be performed for detecting selective sweeps that occurred in the creation of the elite cultivars.

Presentation Type: Oral Presentation
Title: Comparative functional genomics of drought stress response and resistance pathways in plants
Author(s): Arjun Krishnan  Shital Dixit  Aarati Karaba  Madana Ambavaram  Utlwang Batlang  Amal Harb  Peter Wittich  Saghai Maroof  Andy Pereira^*             
Affiliation: Virginia Tech, Blacksburg, VA
Abstract:

Global climate change, competing human needs, and irregular rain supplies threaten stable crop production that uses 70% of available water resources worldwide, making efficient water use and drought resistance important crop traits for improvement. However, the response and resistance of crop plants to drought or soil water-deficit is complex and classical breeding for the trait is difficult. To dissect conserved pathways involved in drought stress we study the model dicot and monoct reference plants Arabidopsis and rice, based on the hypothesis that perception, response and resistance to dehydration/drought have evolved in land plants through some common conserved mechanisms. The analysis of Arabidopsis and rice responses at the vegetative and reproductive stages to soil water deficit, simulating field conditions, reveals common regulated pathways and genes that can be dissected by reverse genetics approaches. In a forward genetics approach we have used a gain-of-function strategy in Arabidopsis to identify genes that impart improved drought resistance. The function of two Arabidopsis transcription factors we identified will be described in Arabidopsis and rice, to demonstrate applications for providing abiotic stress resistance and water use efficiency. Likewise, we assessed the function of Arabidopsis genes involved in leaf cuticle structure/composition, by regulated overexpression in rice and tomato, to improve plant-water relationships. We use Arabidopsis gene interaction networks integrated with rice ortholog information to analyze comparative gene functions between the two plants, as a model to extend to other plants such as maize and soybean for which we are generating gene expression data for drought stress.

Presentation Type: Poster
Title: Circadian clock regulated gene expression in soybean seeds
Author(s): Karen Kaczorowski^*                             
Affiliation: USDA-ARS
Abstract:

The circadian clock controls a number of important developmental and biochemical processes in plants. To get a better understanding of how circadian regulation may be important to the development and composition of soybean seeds, we profiled gene expression in developing soybean seeds using the Affymetrix® Soybean Genome array, which contains probes to approximately 37,000 soybean genes to identify genes with cycling expression. We compare these patterns of gene expression to circadian expressed gene data from whole Arabidopsis seedlings. We will discuss the implications of circadian control of gene expression in seeds for the mechanism of grain filling and determination of seed composition, and towards the identification of promoters for expression of recombinant proteins in soybeans.

Presentation Type: Oral Presentation
Title: Accelerated Yield Technology: a Platform for Increasing the Rate of Genetic Gain
Author(s): Scott Sebastian ^*                             
Affiliation: Pioneer Hi-Bred International
Abstract:

This seminar discusses the philosophy and key features of a molecular breeding strategy known commercially as 'Accelerated Yield Technology' or 'AYT'. AYT leverages proprietary association genetics techniques to identify QTL hotspots associated with long term selection for grain yield. The specific effects of these QTL are then determined within the context of specific breeding populations. The net result is a target genotype that leverages prior information but is optimized for each breeding population. This target gentoype can be created and used in "real time" within the context of a breeding program for early identification of progeny that are most likely to transgressive segregants for yield.

Presentation Type: Oral Presentation
Title: Adjusting Soybean Seed Composition: Better than Beano
Author(s): Emily Dierking^*  Kristin Bilyeu                           
Affiliation: University of Missouri-Columbia
Abstract:

As the use of soy products in foods and feeds increases, it is ever more important to consider the nutritional value of each component of the seed. The carbohydrate component is largely composed of sucrose, raffinose, and stachyose, which represent ~10% of the meal. Both raffinose and stachyose are considered anti-nutritional units; sucrose is considered nutritionally useful. Monogastric animals, including humans, lack the alpha-galactosidase enzyme required for the metabolism of raffinose and stachyose. However, microbes in the gut are able to utilize these carbohydrates causing flatulence and diarrhea; in livestock these complications ultimately reduce market value.The objective of this project is to resolve the undesirable effects of raffinose and stachyose in soybean seeds by elevating the metabolizable energy, sucrose, at the expense of raffinose and stachyose. This change in seed composition has the potential to increase the available metabolizable energy for non-ruminant animals as well as reduce the adverse side effects caused by microbes in the gut. Raffinose is formed by a galactosyl transfer from galactinol to sucrose, mediated by the enzyme raffinose synthase. To date, candidate raffinose synthase genes have been identified and characterized in Williams 82 and a low raffinose line, PI 200508. The genotype of one candidate gene has been completely associated with the low raffinose and stachyose phenotype through the use of a segregating population. Perfect molecular marker assays have been developed and can be utilized for more efficient breeding.

Presentation Type: Oral Presentation
Title: CLE peptide mimicry in soybean cyst nematode pathogenesis
Author(s): Jianying Wang  Amy Replogle  Xiaohong Wang  Eric Davis  Melissa Mitchum^*                     
Affiliation: Division of Plant Sciences and Bond Life Sciences Center, University of Missouri
Abstract:

The soybean cyst nematode (SCN), Heterodera glycines, is a sedentary endoparasite of soybean roots that causes extensive annual yield losses. SCN secretes proteins originating in its esophageal gland cells into selected root tissues to form unique and essential feeding cells. Small secreted peptides sharing similarity to the CLAVATA3/ESR (CLE) signaling peptide family of plants have been identified from SCN. Plant CLE peptides play important roles in plant growth and development including maintenance of the stem cell pool in the root meristem. Recent studies provide evidence of a role for SCN CLEs as ligand mimics of plant CLE peptides to developmentally reprogram the fate of root cells for feeding cell formation. Moreover, our data suggest a role for host-specific control of nematode CLE peptide recognition that could explain the specific adaptation of SCN to parasitize a limited number of plant species. Our studies provide novel insight into how nematode signals are perceived by plants to transduce signaling pathways that ultimately give rise to feeding cells within host plant roots. In the long term, we hope to apply our knowledge of the role of nematode CLEs in plant parasitism to devise novel strategies to engineer nematode resistant crop plants.

Presentation Type: Oral Presentation
Title: Healthier Soybean Oils via Biotechnology and Breeding
Author(s): Steve Schnebly^*                             
Affiliation: Pioneer Hi-Bred International
Abstract:

Conventional soybean oil is the most abundant vegetable oil in the world and requires hydrogenation to increase its stability for many food uses. This results in formation of trans-fatty acids, which have known coronary health risks. By modifying the fatty acid profiles of oilseeds through biotechnology and breeding, researchers have developed healthier oils which do not contain trans-fats. This presentation will review the progress that has been made for products such as low linolenic soy, high oleic soy, and provide an insight on product concepts which are slated for commercialization in the near future.

Presentation Type: Oral Presentation
Title: Gene expression responses in photosynthetic tissues to herbicides and pathogens
Author(s): Steven Clough^*  Jin Zhu  Patrick Tranel  William Patzoldt  Osman Radwan  Min Li  Bernarda Calla                 
Affiliation: USDA-ARS
Abstract:

When plants are attacked by pathogens, the photosynthetic tissue is often dramatically affected. The chloroplasts within this tissue can participate in defense by being a source of many plant secondary metabolites that serve as defense signaling compounds, antioxidants, and phytoalexins. The chloroplast electron transfer system can serve as a potential rapid source of reactive oxygen species. Therefore, it would be advantageous for a plant to control these processes as a defensive strategy, and for a pathogen to target the manipulation of chloroplasts during plant-pathogen interactions as a strategy to favor pathogen propagation. Many commercial herbicides also target chloroplast-related functions, as these tend to be vital to plants. For example, atrazine and bentazon both target photosystem II by blocking electron transfer to plastoquinone B; and glyphosate blocks EPSPS, a key enzyme of the chorismate pathway that is the source of essential aromatic amino acids. In an effort to identify responses that are pathogen or herbicide specific, or that are shared, we are comparing gene expression responses in soybean to herbicide-induced interruptions of specific chloroplast operations to the multitude of events triggered by various pathogens. Microarray time-course experiments, focused within the first 24 hours post treatment, have been completed and clustering results will be presented. Herbicides being used in this study are atrazine, bentazon, and glyphosate. Pathogen-soybean interactions being used in this study are: compatible and incompatible to Pseudomonas syringae, susceptible and resistant to Verguliforme solani (aka Fusarium solani) phytotoxin, and susceptible and resistant to Sclerotinia sclerotiorum.

Presentation Type: Poster
Title: Characterization of several transcription factors involved in seed development in soybean
Author(s): Deyue Yu^*  Fang Huang  Yingjun Chi  Qingchang Meng  Junyi Gai                     
Affiliation: Nanjing Agricultural University, National Center for Soybean Improvement, Nanjing 210095, China
Abstract:

Soybean is one of the most important oil crops in the world. With content of 40% protein and 20% fat on an average, soybean-derived product has been considered as “healthy food” in many countries. However, the manipulation of seed composition (protein and its composition, fat and its composition, etc) has been difficult since liitle is known about the mechanism of seed development and seed composition in soybean. Here, as a part work to illustrate the molecular mechanism of soybean seed development, we performed the microarray analysis of mRNA from various organs, including flowers, pods, leaves, and roots by application of Affymetrix® DNA Chips (including 35，611 transcripts). Our results revealed that there are about 220 genes expressed predominantly in both flowers and seeds. Among these genes, about 10% are putative transcription factors. Some of these genes were picked for expression specificity confirmation by real-time PCR, suggesting a good correlation between microarray analysis and real-time PCR analysis. Several genes encoding putative transcription factors, such as MADS-box genes, LFY-like genes and NAC-like genes, were chosen for further analysis by RT-PCR in developing seeds from 15 DAF (days after flowering) to 50 DAF. It was interesting to note that most, if not all, of these genes were found expressed in developing soybean seeds, with different levels and different dynamics, suggesting a very complex modulation of seed development in soybean. Results from transgenic plants with some of these transcription factors will be presented and discussed.

Presentation Type: Poster
Title: Endogenous miRNAs involving in Gly m Bd 28K gene expression and regulation
Author(s): Li Wang  Long-Guo Jin  Li-Juan Qiu^*                         
Affiliation: Institute of Crop Science,CAAS
Abstract:

MicroRNAs (miRNAs) are endogenous small RNAs that can have large-scale regulatory effects on development and stress responses in plants. One of the major soybean allergens, Gly m Bd 28K, can cause 5±8% of children and 1±2% adults sensitive to soybean ingestion. In this study, we try to understand Gly m Bd 28 deficient mechanism in order to develop Gly m Bd 28K-deficient soybean variety for soybean-sensitive individuals. Based on the genome sequence of Gly m Bd 28K gene we cloned, 9 miRNAs were predicted, and 3 of them had been cloned. Each of 3 formed a family consisted of a total of 12 potential Gly m Bd 28K-miRNAs from Gly m Bd 28K gene. Through the near-perfect complementarily blast between plant miRNAs and their target genes, we found one target gene for each Gly m Bd 28K-miRNA family. According to the target gene function, we found three Gly m Bd 28K-miRNA families are related to signal transduction (MYB [Glycine. max]), stress responses (early-responsive to dehydration protein-related [Arabidopsis thaliana] ) and plant development (triose-phosphate isomerase [Arabidopsis thaliana] ) respectively. It had been found that the MYB response elements existed in Gly m Bd 28K promoter region that may be regulated by Gly m Bd 28K-miRNAs target gene.

Presentation Type: Poster
Title: Development and mapping of a co-dominant SCAR marker linked to salt tolerance gene in soybean
Author(s): Rong-Xia Guan  Lei Tian  Zhang-Xiong Liu  Ru-Zhen Chang  Li-Juan Qiu^*                     
Affiliation: Institute of Crop Science, CAAS
Abstract:

Salt is an important abiotic stress that greatly affects plant growth and crop production. Breeding and use of salt tolerant cultivars is an effective way to overcome the problem. The goals of the present study were to convert a previously developed random amplified polymorphic DNA (RAPD) marker into a sequenced characterized amplified region (SCAR) marker, to map the salt tolerance gene, and to test the efficiency of markers for use in marker assisted selection (MAS). By cloning, sequencing and comparing the RAPD product between salt tolerant and sensitive cultivars, a co-dominant SCAR marker QS080064 was developed, which amplified a 500bp and a 410bp fragment in salt tolerant and sensitive parents, respectively. A fragment of 370bp was amplified in both of the parents. When this marker and other two polymorphic SSR markers were used to screen a F2 population derived from “Wenfeng 7×Union”. The salt tolerance gene was mapped to soybean linkage group N linked to SSR marker Sat_285 (18.5 cM) and SCAR marker QS080064 (1.4 cM). When the co-dominant marker was used to screen 55 resistant and 90 sensitive soybean germplasm, the identity between phenotype and genotype was 85.1%. The results indicated that this SCAR marker could be useful for marker assisted selection in soybean breeding.

Presentation Type: Poster
Title: Development of SNP markers in soybean enabling haplotype analysis of an rhg1 candidate gene conferring resistance to soybean cyst nematode
Author(s): Yinghui Li^*  Chen Zhang  Zhongshan Gao   Marinus Johannes Maria Smulders   Zulu Ma   Zhangxiong Liu   Haiyang Nan   Ruzhen Chang*   Lijuan Qiu ^*             
Affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, 100081 Beijing, P. R. China
Abstract:

One of the most destructive pests of soybean (Glycine max (L.) Merr.) is the soybean cyst nematode (SCN, Heterodera glycines Ichinohe). Markers based on the SCN resistance gene will permit efficient marker-assisted selection (MAS). We sequenced the rhg1 gene, a likely candidate gene, in six resistant and two susceptible soybean genotypes and identified single nucleotide polymorphisms among the sequences. A total of 37 SNPs (11 in the coding region and the others in noncoding regions) were found. An agarose gel-based allele-specific and codominant PCR method was established for genotyping 6 of the SNPs. A total of 70 genotypes had been genotyped with the 6 SNPs and appeared to be only 9 different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the 6 SNPs loci in 70 genotypes, consistent with intensive directional selection. Strong LD pattern was detected among 5 SNPs. Two SNPs formed one haplotype that was completely associated with the SCN resistance. This had not been achieved before, as the best marker up to now, the microsatellite marker BACR-Satt309, maps 0.4 cM proximal to the rhg1 locus, so that resistant-associated alleles are present in all resistant plants, but also in a number of susceptible genotypes. Our new allele-specific PCR markers will therefore significantly improve the efficiency of MAS for development of SCN resistant varieties.

Presentation Type: Oral Presentation
Title: Field testing of the transgenic soybean for WUE trait
Author(s): Marie Petracek^*  Bala Karunanandaa                           
Affiliation: Monsanto Company
Abstract:

From screening hundreds of genes in our transgenic soybean pipeline, we have tested a number of genes that we hypothesize confer increased tolerance to water stress. We have employed strategies including growth chamber and field physiology and as well as isotope discrimination studies to further understand the effect of misexpression of these genes on water use efficiency (WUE). Use of isotopic discrimination as a tool in the identification of transgenic events that are of capacity or conductance types will be discussed.

Presentation Type: Poster
Title: Inheritance of salt (NaCl) tolerance in wild soybean (Glycine soja) accession PI483463
Author(s): Jeong-Dong Lee^*  J. Grover Shannon  Tri D. Vuong  Henry T. Nguyen                       
Affiliation: Division of Plant Sciences, University of Missouri-Delta Cenetr
Abstract:

Excessive salt can reduce soybean [Glycine max (L.) Merr.] yield in grower fields. Salt tolerant soybean cultivars are effective in reducing salt damage. Finding new gene sources for salt tolerance is important for breeding programs to reduce losses from salt injury. The objectives of this study were to determine the inheritance of salt tolerance in Glycine soja accession PI483463 and to test the allelic relationship with the tolerance gene from S-100 that is common in many southern US cultivars. Tolerant PI483463 (T) was crossed to salt sensitive cultivar, Hutcheson (S), for the inheritance study. For the allelism test, PI483463 (T) was crossed with the tolerant genotype S-100 (T). Roots of parents, F1 plants, F2 populations, and F2:3 lines from both populations were partially immersed in a 100 mM salt solution to determine tolerance. The F2 population from R x S cross segregated in a ratio of 3 (T): 1 (S) and the F2:3 lines segregated in a ratio of 1 (T): 2 (Segregating): 1 (S). The F2 population and F2:3 lines derived from PI483463 (T) x S-100 (T) segregated 15 (T): 1 (S) indicating tolerance genes from the two sources were different. Also, PI483463 amplified at a different size than the two salt tolerance SSR makers, Sat_091 and Satt237 flanking the salt tolerance allele from S-100. These results show that wild soybean PI483463 has a single dominant gene for salt tolerance which is different than the gene in S-100. Mapping studies will determine the specific location of this new allele.

Presentation Type: Poster
Title: Metabolomics and transcriptomics as tools to improve soybean resistance to aphids
Author(s): Gustavo MacIntosh^*  Mariana Chiozza  Matthew Studham  Charles Kanobe                       
Affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University
Abstract:

Soybean aphids reduce plant growth and produce important yield losses of up to 30%. Current control of the soybean aphid relies on application of costly chemical insecticides that harm beneficial non-target organisms and deteriorate the environment. Although some resistant varieties have been identified, they are poorly characterized and not yet introduced in commercial varieties. Alternatively, dissection of the molecular basis of plant defense response to pests can identify genes that can be used as targets in transgenic strategies to develop new resistant plant varieties. “-omics” approaches have identified a vast number of new targets for crop improvement. However, the same high-throughput nature of these technologies presents a problem: how do we determine what genes or metabolites, among the hundreds identified, are relevant for particular trait improvements? We use a systems approach that combines transcriptional and metabolomics profiles of soybean plants challenged with soybean aphids to extract relevant information. We identified several signaling pathways involved in the defense response. We also found that aphids have a profound effect on plant metabolism, in particular amino acids and lipid profiles. We are currently testing the role in the defense response of some of the genes identified by these methods, through the production of transgenic plants, or using Virus-induced Gene Silencing. Metabolic and transcriptional changes indicated that green leaf volatiles (GLV) are important components of the defense response, and that hydroperoxide lyase (HPL) could be the limiting step in GLV production. Transgenic soybean plants overexpressing HPL showed improved aphid resistance.

Presentation Type: Poster
Title: The Soybean Breeder’s Toolbox: Updated Features and New Data
Author(s): David Grant^*  Rex T. Nelson  Steven B. Cannon  Randy C. Shoemaker                       
Affiliation: USDA-ARS-CICGR, Iowa State University
Abstract:

The Soybean Breeder’s Toolbox is constantly being upgraded and improved with new genetic and genomic data and tools. The genetic, physical and sequence map displays are based on viewers developed by the GMOD project, and have been extended by us to provide additional functionality. The Soybean Breeders Toolbox (SBT) contains much of the data originally collected in SoyBase. Genetic and physical maps are shown using the comparative map viewer CMap. This viewer allows side-by-side displays of multiple physical or genetic maps for comparing homoeologous regions of the genome or the genetic and physical maps. Markers and QTL on the genetic maps and BACs and FPC contigs on the physical map are linked to additional detailed textual data in the SBT. Controlled vocabularies for soybean growth, development and phenotypic traits are being developed and integrated with the Plant Ontology Consortium and Plant Trait Ontologies. Soybean ontologies are linked as appropriate to genes and QTL in the SBT, thus allowing the SBT to be searched by trait or developmental stage as well as by the more usual gene or QTL name. Along with recently reported QTL and the final FPC-based Williams 82 physical map the SNP-based markers developed by Hyten et al. (2008) have been added to the SBT. These new sequence-based genetic markers allow detailed integration of the genetic and sequence maps. The JGI soybean genomic sequence is viewed using GBrowse. Data tracks include the genomic sequence, sequenced genetic markers, soybean BACs, repetitive sequences, unigene sets, and potential gene haplotypes.

Presentation Type: Oral Presentation
Title: Applilcation of functional genomics to the study of soybean biology
Author(s): Stacey Gary^*                             
Affiliation: National Center for Soybean Biotechnology, University of Missouri, Columbia, MO 65211
Abstract:

The soybean genome sequence predicts the presence of > 50000 genes. The future challenge will be to define the functions of these genes or, at least, those that are agronomically important. Functional genomic tools can provide useful information to aid in discerning gene function. Over the past few years, the soybean community has developed a variety of functional genomic platforms. Our lab is utilizing these platforms to study basic soybean biology, especially with regard to soybean-microbe interactions. We conducted numerous transcriptomic studies using different array formats. However, these platforms suffer from a lack of sensitivity. To address this, we developed a library of PCR primers directed toward all of the identified soybean transcription factors (TF) and used this to accurately quantify TF gene expression in a variety of tissues and in response to a variety of environmental cues. Likewise, proteomics is being utilized to characterize specific soybean tissues. In one case, we just completed a proteome reference map for soybean root hair cells. This provides a useful survey of the metabolism of this unique, terminally differentiated soybean cell. Metabolomic studies and an examination of soybean small RNA populations are underway, as well as more specific studies of the soybean cell wall and a variety of other experiments. Collectively, these data point the way to our ultimate goal of a systems-level view of soybean physiology and development.

Presentation Type: Oral Presentation
Title: Molecular mapping of an aphid resistance locus using whole-genome analysis and sequence data
Author(s): Matthew Hudson^*  Stephanie Bellendir  Ki-Seung Kim  Karen Kaczorowski  Brian Diers                     
Affiliation: University of Illinois
Abstract:

A strategy using near-isogenic lines (NILs) and Affymetrix Soybean GeneChip microarrays was employed to identify genetic markers closely linked to the soybean aphid resistance gene Rag1. Genomic DNA hybridization to Affymetrix arrays allowed the identification of a large number of Single Feature Polymorphisms (SFPs) between two parent lines, Dowling (resistant) and Dwight (susceptible). The Rag1 locus was then mapped to an interval of less than 5cM using Affymetrix array hybridization of genomic DNA from two pre-existing NILs, which were developed by back-crossing the Rag1 gene from Dowling into the Dwight background. Conventional genetic mapping using PCR-based SNP markers developed from the SFPs in the Rag1 interval allowed further narrowing of the gene position to a region defined by a physical contig sequenced by the soybean genome project. A genome-enabled SNP-discovery strategy using automated primer design and melt-curve polymorphism detection was then developed. This high throughput, directed approach is capable of producing SNP markers at around 10kb intervals throughout a fully sequenced region. This method was used to further fine map the locus to a region of around 150kb.

Presentation Type: Poster
Title: Systems biology approach to understand seed composition: A convergence of metabolomics, transcriptomics and metabolic flux data to expand the metabolic and regulatory network of developing soybean seeds
Author(s): Ling Li^*  Wenxu Zhou  Yusuf Demirkale  Dan Nettleton  Mark Westgate  Basil Nikolau  Eve Wurtele                 
Affiliation: Department of Genetics, Cellular, and Developmental Biology, Iowa State University
Abstract:

As the propagule that ensures the dissemination of plants, seeds also support human activity as one of the major products of agriculture. Seed reserves are synthesized by the programmed expression of a metabolic network during seed development. Although the basic biochemical processes by which imported carbon and nitrogen is converted to the final products, protein, oil and carbohydrate. However, there is a great deal to be learned concerning the molecular- genetic mechanisms that regulate this complex metabolic network. We have taken advantage of microarray technology to identify the global gene expression profile that regulates the developmental and biochemical network, and ultimately determines structure and composition of the mature seed. We have coupled this analysis with metabolomics analyses and metabolic flux analysis to gain insights into the metabolic and regulatory program that determines soybean seed composition.

Presentation Type: Poster
Title: Genetic Diversity for Seed Mineral Composition in Teramnus labialis, a Wild Relative of Soybean
Author(s): Michael Grusak^*                             
Affiliation: USDA-ARS Children's Nutrition Research Center, Houston, TX
Abstract:

Teramnus labialis (L.) Spreng. is a wild relative of soybean whose seeds are collected and used as a food source by tribal populations in Asia. In order to assess the potential of this legume to provide dietary minerals for humans, fourteen diverse accessions were grown under controlled, nutrient-replete conditions and seeds were harvested for mineral analysis. The germplasm originated from Indonesia, Africa, the Caribbean, and South America. Seed concentrations of phosphorus (P), potassium (K), sodium (Na), iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn) were found to fall within the range of published values for several cultivated grain legumes (including soybean), while calcium (Ca) and magnesium (Mg) were higher in Teramnus labialis seeds. Mineral concentrations across the diverse accessions showed ranges of 1.3- to 2.3-fold for the macronutrient minerals (Ca, Mg, P, K) and 1.8- to 15.9-fold for the micronutrient minerals (Fe, Cu, Mn, Zn, and Na). The existing genetic diversity in this wild legume, especially for the essential minerals Ca and Mg, could be exploited to develop Teramnus labialis as a new cultivated legume for tropical regions of the world. Furthermore, this species could serve as a model for understanding the mechanisms controlling Ca, Mg or K transport from vegetative tissues to developing seeds. Interestingly, T. labialis is closely aligned with soybean, as both are members of the Glycineae subtribe, and thus the molecular resources developed for soybean are likely transferable to and could be used to study mineral transport and partitioning in this wild relative.

Presentation Type: Poster
Title: T-DNA AND TRANSPOSON MUTAGENESIS IN SOYBEAN
Author(s): Melanie Mathieu  Shaoxing Huang  Zhanyuan Zhang  David Somers  Tom Clemente  Kan Wang  Henry NGuyen  Gary Stacey^*               
Affiliation: University of Missouri
Abstract:

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes. Therefore, a major focus is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed that approximately 70% disrupted known genes, with the majority matching sequences in the database derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by transposon mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.

Presentation Type: Poster
Title: Integration of the soybean genome sequence with genetic and physical maps to produce chromosome-scale assemblies
Author(s): Steven Cannon^*  Will Nelson  David Hyten  Qijian Song  Jeremy Schmutz  Jane Grimwood  Nathan Weeks  Myron Peto  Jianxin Ma  Greg May   Gary Stacey  Dan Rokhsar  Scott Jackson  Perry Cregan  Randy Shoemaker 
Affiliation: USDA-ARS and Iowa State U
Abstract:

Assembly of the soybean whole genome shotgun sequence (WGS) requires two conceptually different tasks: first, assembly of sequence reads into large, mostly-contiguous “scaffolds”; and second, ordering, orienting, and validation of the scaffolds to produce chromosome “pseudomolecules.” In the second phase, we used a variety of strategies to take advantage of the rich resources developed by the soybean research community. We first made an approximate ordering and orienting (O&O) of most 8x scaffolds using a consensus genetic map with 5503 markers, then made refinements by constructing a special-purpose high-resolution map (using a Williams 82 x G. soja PI468916 population with 444 RILs), with markers selected from potentially problematic regions in the 7x draft WGS. Next, we used comparisons of soybean to itself and to Medicago to help identify additional O&O problems. Because the Glycine genome has undergone a whole-genome duplication, each region usually matches one to three corresponding ("homoeologous") regions, which can also be used to identify O&O problems. In addition, we evaluated the assembly using genomic landmarks such as telomeric and centromeric repeats and ribosomal DNA. Finally, we used the curated soybean (cv. Williams) physical map, and additional clone pairs not used in the primary assembly, to identify scaffold associations and potential misassemblies. Iterating with these approaches and resources, we are approaching a genome sequence in which more than 96% of the WGS sequence, and virtually all of the gene-containing sequence, has been ordered, oriented, and positionally validated.

Presentation Type: Poster
Title: Shedding light on soybean oil synthesis utilizing Arabidopsis double-DGAT knockouts
Author(s): Gary Ratliff^*                             
Affiliation: University of Kentucky
Abstract:

Oil synthesis in soybeans, and oilseeds in general, is poorly understood. Diacylglycerol acyltransferase (DGAT) enzymes have been shown to be involved in the production of triacylglycerol (TAG), the main component of plant oil. Arabidopsis contains 2 DGAT enzymes, DGAT1 and DGAT2. Although knocking out DGAT1 in Arabidopsis resulted in a 50% reduction of seed oil, the effects of knocking out DGAT2 in this model oilseed are unknown, leaving a significant gap in our limited understanding of oil synthesis. This issue is addressed under the hypothesis that at least part of the remaining 50% of the oil in these DGAT1 null mutants is produced by DGAT2. Sets of seeds mutated by T-DNA insertion into either the DGAT1 or DGAT2 genes were obtained from The Arabidopsis Information Resource (TAIR). The DGAT1 and DGAT2 null mutants have been grown to maturity and phenotyped. Select lines of DGAT1 null mutants have been crossed with DGAT2 null mutants. The F2 generation of these crosses is being screened for double knockouts, and these seeds analyzed for oil content and composition. If the double null mutants have little or no oil, we may conclude that DGAT1 and 2 are mainly responsible for oil production in Arabidopsis. If significant oil is produced, then other enzymes, such as PDAT, may be important factors in oil production in Arabidopsis. This very important study has never been done, and will provide useful information for future work regardless of where the results fall.

Presentation Type: Poster
Title: Molecular Mapping of the Optimum® GAT® Herbicide Tolerance Trait in Soybean
Author(s): Julian Chaky^*  Connie Martin  Matthew Campbell  Jeffrey Hegstad  Nikki Roach  Mark Vogt  Sara Fischer  Janel Albertsen  Daniel Sherman  Lori Wickham   Andrea Erickson  Daria Schmidt       
Affiliation: Pioneer Hi-Bred International, Inc
Abstract:

Soybeans [Glycine max (L.) Merr.] with the Optimum® GAT® trait have dual mode of action herbicide tolerance to glyphosate (N-phosphonomethyl glycine) and acetolactate synthase (ALS) inhibitor herbicides. The objective of this study was to identify molecular markers linked to the Optimum® GAT® event DP356043-5 and to place the gat transgene onto the soybean molecular genetic map. An F2 population of 552 individuals was constructed from a cross between the original event generated in a pureline event DP356043-5 (GG) and a Pioneer Hi-Bred commercial cultivar. The F2 population was screened for segregation of three hundred twenty three polymorphic SNP-based markers. Mapping analyses revealed that event DP356043-5 was located on linkage group N of the soybean molecular genetic map. The availability of linked molecular markers will facilitate the genetic analysis of event DP356043-5 in relation to surrounding genes, enabling rapid forward breeding in soybean variety development programs.

Presentation Type: Poster
Title: Rapid and efficient protocols for DNA extraction and molecular identification of the Soybean (Glycine max., L.) growing Turkey
Author(s): Hasibe Cingilli VURAL^*                             
Affiliation: university
Abstract:

Four DNA extraction methods were used to obtain DNA from herbarium specimens of soybean and fresh soybean seeds, and germinated soybean seeds in this study. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. Four different DNA extraction methods were compared for the isolation of DNA from the soybean homogenates, namely the CTAB extraction method, Plant Genomic DNA Purification Kit method, EZ1 Nucleic acid isolation method, and DNA extraction with phenol purification and liquid nitrogen. The main goal of study is to evaluate various methods of DNA isolation in terms of DNA yield and amplification quality. To preserve DNA well, it is necessary to dry plants as fast as possible. Extraction results depend on how the plant material is prepared, and the type of chemicals or DNA isolation protocols used. Obtaining high quality DNA depends on the isolation technique used. Several methods that are useful for dry plant tissue from herbarium specimens have been described (Wittzell, 1999; Ristaino et al., 2001; Taylor and Swann, 1994). Four extraction protocols were compared using fresh material and dry herbarium specimens and seeds. Plant Genomic DNA Purification Kit method; the concentration of DNA in the second elution extract was very low, thus repeated elution was not applied. DNA extraction with phenol purification and liquid nitrogen; This technique is not quite suitable for fresh plant leaves. It is usually used for herbarium samples of at least 0.2 g. CTAB extraction method; This protocol is quite suitable for DNA isolation from the fresh plant material or germinated seed plantlets. The total DNA was isolated from approximately 0.5-1 g of fresh leaves (conserved in CTAB) of the same collection of sample. EZ1 Nucleic acid isolation method; This tehnique is quite useful for high yield and quality of DNA isolation from dried soybean seeds. In this methods, no further purification was needed for molecular analysis.

Presentation Type: Poster
Title: Cloning and functional analysis of two diacylglycerol acyltransferases genes from soybean (Glycine max)
Author(s): Runzhi Li^*  Keshun Yu  Tomoko Hatanaka  David Hildebrand                       
Affiliation: University of Kentucky
Abstract:

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA dependent acylation of sn-1,2-diacylglycerol (DAG) to form triacylglycerol (TAG) in the terminal step of seed oil formation. To understand the features of the gene encoding soybean DGAT and its role in soybean seed oil synthesis and accumulation, two full-length cDNAs encoding type 1 diacylglycerol acyltransferases (namely GmDGAT1a and GmDGAT1b) were cloned respectively from soybean developing seeds by the rapid amplification of cDNA ends (RACE) method. Alignment of the coding sequence showed that they shared identities by 94% and 95% at protein and DNA sequence, respectively. The genomic architectures of GmDGAT1a and GmDGAT1b both contain 14 introns and 15 exons. There are some differences in length of several exons and introns between GmDGAT1a and GmDGAT1b genomic sequences.

Enzymatic assays using microsomes from the yeast transformed by the two cDNA clones demonstrated that GmDGAT1a and GmDGAT1b have similar DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-diacylglycerol were preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. Expression analysis by qRT-PCR revealed that both transcript abundances were much higher in developing seeds than in than tissues including leaves, stem, roots and flowers. Soybean DGAT1a was mainly expressed in early stages of developing seeds while DGAT1b was highly expressed in later stages, suggesting soybean DGAT1b plays more important roles in TAG accumulation in soybean seed oil. A TILLING approach is being employed to identify mutants of GmDGAT1b for further elucidation of GmDGAT1 functions in soybean seed oil formation and plant growth and development together with K. Meksem and S. Liu at SIU.

Presentation Type: Poster
Title: A point mutation conferring Gy4 null phenotype is remarkably conserved in diverse soybean germplasm
Author(s): Won-Seok Kim  Heo Jae Ho  Randall Nelson  Krishnan Hari^*                       
Affiliation: University of Missouri, Columbia, MO 65211
Abstract:

Tofu, a cheese-like food made by curdling soymilk, is a major dietary staple of Asian countries. Consumption of tofu and other soy products is steadily increasing in North America due to its well known health benefits. Soybean A5, A4 and B3 peptide null lines 'Enrei' and 'Raiden' are commonly utilized in breeding programs to develop high-quality tofu cultivars. To expand the genetic diversity it is desirable to identify and utilize other A5, A4 and B3 null genotypes in the development of improved tofu cultivars that are adapted to North American conditions. In this study, we have screened diverse soybean accessions from the USDA Soybean Germplasm Collection to identify Gy4 mutants, the locus the controls A5, A4 and B3 peptide production. Analysis of total seed proteins from 485 soybean lines by SDS-PAGE enabled the identification of 38 accessions that lacked the A5, A4 and B3 peptides. These accessions showed marked differences in seed size and seed coat color and represented different maturity groups ranging from 0 to IX. To ascertain the molecular basis for the lack of A5, A4 and B3 peptides in the newly identified Gy4 mutants, nucleotide sequence of a portion of the Gy4 gene was determined from eight soybean accessions representing different maturity groups. All the analyzed Gy4 mutants revealed a single point mutation that changed the translation initiation codon ATG to ATA resulting in the A5, A4 and B3 null phenotype. The newly identified Gy4 mutants from our study will enable plant breeders to expand the genetic diversity of North American food-quality soybeans and also aid in the development of hypoallergenic soybeans.

Presentation Type: Oral Presentation
Title: Comparative evolutionary dynamics of soybean and common bean
Author(s): Jessica Schlueter^*  Scott Jackson                           
Affiliation: Purdue University
Abstract:

Soybean and common bean (Phaseolus vulgaris) share a common ancestor separated by approximately 15 million years of divergence. As such, the relationship between these two Phaseoloids offer an excellent system for studying the varying evolutionary dynamics of these two genomes. Soybean, relative to common bean, has undergone a more recent large scale duplication following divergence of these two species. This is reflected in the genome size variation with soybean being ~1.13 Mb and common bean ~625 Mb. Undergoing work has led to the development of a BAC-based physical map of common bean consisting of 1,183 clone contigs and 6,385 singletons with an ~9x coverage of the genome. In addition, 89,017 BAC-end sequences relating to this physical map have been sequenced. Based upon these resources as well as the emerging genome sequence of soybean, relationships between the soybean genome and the common bean physical map can be identified. Using the software SyMap, large syntenic blocks of common bean contigs can be anchored to the soybean genome scaffolds. Current FISH work is being undertake to verify these relationships. The organization of repetitive sequences in each genome are also being studied. Preliminary results suggest that while soybean seems to have gene rich regions with few repetitive sequences, common bean may have more dispersed repetitive sequences.

Presentation Type: Poster
Title: Developmental Regulation of Oil Deposition in Arabidopsis Seeds with Application to Soybean
Author(s): Hongyun Wang^*  Matthew Hudson  Yun Lin                         
Affiliation: Department of Crop Sciences, University of Illinois, Urbana, IL 61801
Abstract:

Arabidopsis transcriptional factors LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), ABSCISIC ACID INSENSITIVE3 (ABI3), and ABSCISIC ACID INSENSITIVE5 (ABI5) are known to regulate multiple aspects of seed development. In an attempt to understand the developmental control of storage product accumulation, we observed the expression time course of the five transcripts. By extending the expression periods of the two early genes LEC1 and LEC2 in transgenic seeds, we demonstrated that the subsequent timing of FUS3, ABI3, and ABI5 transcripts depends on LEC1 and LEC2. Because a delayed onset or reduced level of FUS3 mRNA coincide with reduction of seed oil content in the transgenic seeds, the role of FUS3 in oil deposition was further examined. Analysis of published seed transcriptome data indicated that FUS3 transcript increases together with nearly all the plastidial fatty acid biosynthetic transcripts during development. The ability of FUS3 to induce fatty acid biosynthetic gene expression was further confirmed in transgenic Arabidopsis seedlings expressing a dexamethasone-inducible FUS3 cDNA. By accommodating the current evidence, we propose a hierarchical architecture of the transcriptional network in Arabidopsis seeds in which the oil biosynthetic pathway is integrated through the master transcriptional factor FUS3. A soybean homolog (GmFUS3) for FUS3 has been identified. Transgenic soybean plants are being generated in which the promoter of lectin drives a GmFUS3 transgene. Based on our Arabidopsis model, the transgenic seeds are predicted to express GmFUS3 at an elevated level and therefore accumulate a higher percentage of oil.

Presentation Type: Poster
Title: Preliminary identification of additional ribosomal protein gene family members in Glycine max
Author(s): Ronald Frank^*  Daniel Schwent  Cyriac Kandoth  Fikret Ercal                       
Affiliation: Missouri S&T Biological Sciences
Abstract:

Ribosomal proteins are encoded by small gene families ranging from 2-7 members each that exhibit high conservation in sequence indicative of strong functional constraints. We used one previously identified gene sequence from seven different ribosomal protein families of Glycine max as query to collect EST sequences from related genes. Contigs were assembled from EST sequences and potential ORFs identified. Alignment of ORFs and subsequent analysis of synonymous versus nonsynonymous substitutions identified contigs that represent putative gene family members. Results are presented for five Glycine max ribosomal gene families in which we have identified 2-5 additional members for each family. We also used one sequence from Arabidopsis thaliana as query to identify a family of ribosomal protein genes in Glycine max for which no member had been previously identified. This serves to illustrate the potential this strategy has to identify all ribosomal protein genes in Glycine max as well as other conserved gene families.

Presentation Type: Poster
Title: Development of High-throughput SNP Genotyping Assay for the Asian Soybean Rust Resistance Gene from ‘Hyuuga’ Soybean
Author(s): Bo-Keun Ha^*  Maria J. Monteros  H. Roger Boerma                         
Affiliation: The University of Georgia
Abstract:

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean production in the USA. A novel rust resistance gene, Rpp(?)Hyuuga, from the Japanese cultivar ‘Hyuuga’ has been identified and mapped to linkage group C2. Our objective was to identify single nucleotide polymorphism (SNP) markers associated with this ASR resistance gene and to develop a high throughput SNP genotyping assay for use in marker assisted selection (MAS). To discover SNPs near the Rpp(?)Hyuuga locus, we used the whole genome shotgun assembly of Williams 82 soybean. A total of 20 primers were designed every 50 to 100kbp within the templates of Scaffold 60 from 1300kbp to 2300kbp surrounding the Satt079 locus. A total of 45 SNPs were identified in eight PCR fragments. We used high-resolution melting curve analysis with unlabeled probes to genotype these SNPs in 10 selected recombinant inbred lines (RILs) from the cross of Dillon  Hyuuga. The identification of recombination events combined with the ASR reaction data for these 10 RILs indicated the Rpp(?)Hyuuga locus is located between 1.5Mbp and 3.0Mbp in Scaffold 60. Also, a SNP (G/T) at the 1847497bp position in Scaffold 60 can differentiate the Rpp(?)Hyuuga resistant genotypes from 32 ancestral soybean genotypes that theoretically represent 95% of the alleles in the genetic base of North American soybean and the previously reported ASR resistance genes (Rpp1, Rpp2, and Rpp4). We developed a melting curve assay with SimpleProbe chemistry at this SNP for detection on a Roche LightCycler 480.

Presentation Type: Poster
Title: Examining differential gene expression of homologues of the isoflavonoid pathway in soybean (Glycine max)
Author(s): Julie Livingstone^*  Martina Stromvik                           
Affiliation: McGill University
Abstract:

Bioinformatics, coupled with targeted molecular experiments, can help us to identify gene copies and understand how these genes are regulated (Strömvik et al. 2004). In the present study we employed the isoflavonoid pathway as a model system to gain a better understanding of gene regulation in plants. We have predicted and verified known and novel gene homologues in the recently released draft soybean genome by using the over 410,000 (redundant) Expressed Sequence Tags (ESTs) available in public databases (Shoemaker et al. 2002; Vodkin et al. 2004). Both molecular and bioinformatics techniques are applied to study gene expression of genes in the isoflavonoid pathway with the aim of determining in which cells the different homologues are being expressed. Contiguous sequences (contigs) were assembled from EST data to represent the specific genes in the isoflavonoid pathway. Novel transcripts of 2-hydroxyisoflavanone dehydratase and isoflavone-7-O-glucosyltransferase have been discovered in this data. To resolve cell specific gene expression and to determine the relative expression of different RNA transcripts, we will section soybean pods with developing seeds using a cryotome and use laser capture microdissection (LCM) coupled with quantitative real-time PCR (QRT-PCR).

References
Shoemaker R et al (2002). A compilation of soybean ESTs: generation and analysis. Genome 45:329-338
Strömvik MV, Thibaud-Nissen F, Vodkin LO (2004) Mining Soybean Expressed Sequence Tag and Microarray Data. Recent Adv Phytochemistry 38:177-195
Vodkin LO et al (2004) Microarrys for global expression constructed with low redundacy set of 27,500 sequenced cDNAs representing an array of development and physiological conditions of the soybean plant. BMC Genomics 5:73

Presentation Type: Oral Presentation
Title: Genomic Approach to Improvement of Soybean Resistance to Defoliating Insects
Author(s): Shuquan Zhu  Wayne Parrott  John All  Roger Boerma^*                       
Affiliation: Center for Applied Genetic Technologies, University of Georgia
Abstract:

Breeding for soybean resistance to defoliating insects is hindered by the quantitative inheritance of the resistance, linkage drag on yield from the exotic resistance sources, and a lack of understanding of the resistance mechanisms. To improve the breeding efficiency, we mapped the QTLs for the resistance, and detected at least four QTLs located on LG-E, -G, -H, and -M. QTL-E (Pb) and QTL-M provide both antixenosis and antibiosis, and explain about 30% of the phenotypic variation. QTL-H and QTL-G confer only antixenosis or antibiosis, respectively, and explain about 10% of the phenotypic variation. We have transferred these QTLs from different plant introductions into a MG VII cultivar Benning through marker-assisted selection, and confirmed the effects of these QTLs. We have found QTL-M, and -H have no linkage drag on yield in Benning near-isogenic line. In addition, QTL-M, -H, and -G significantly enhance the effectiveness of an Bt Cry 1Ac transgene. Currently we have been investigating how QTL-E interacts with the other three QTLs and the Cry 1Ac gene. We have been fine mapping the major QTL-M with the goal of positional cloning. Accordingly, a BAC library has been made from genomic DNA of the resistant PI 229358. So far, QTL-M has been mapped to a 0.11-cM region of LG-M flanked by the SNP marker SNP116 and the SSR marker Sat_426. The 0.11cM interval of PI 229358 is equivalent to about 250kbp based on the ‘Williams 82’ DNA sequence assembly recently released by JGI. This makes it feasible to clone QTL-M, which should provide key insight into the molecular mechanism involved in soybean resistance to defoliating insects.

Presentation Type: Poster
Title: Function and Expression of a Small Auxin Down Regulated Gene Associated with Soybean Seed Mutations, Development and Germination
Author(s): Delkin O. Gonzalez^*  Anne Marie Boone  Sarah Jones  Lila O. Vodkin                       
Affiliation: University of Illinois
Abstract:

The phytohormone auxin down regulates the expression of a group of genes known as ADR (auxin down regulated). Regulation occurs at the level of cytoplasmic mRNA of different family members. Previous results using soybean cDNA microarray analysis revealed that a particular ADR gene was statistically significantly under-expressed in the seed coats of a mutant line presenting an affected structural integrity of the seed coat. This particular mutation is characterized by a net pattern that creates breaks in the seed coat. Similarly, we also found differential levels of expression of this particular gene during seed development in the seed coat and cotyledons. Also by using a new soybean oligo chip we determined that the expression levels of this gene during soybean germination and emergence is highly dynamic, showing over and under expression levels at different stages during this process. In order to learn more about the function and localization of the product of this particular ADR gene, the corresponding cDNA clone was identified isolated and confirmed by DNA sequence analysis. A PCR strategy was designed to clone this gene in frame with a HIS tag for inducible expression analysis and purification in E. coli and polyclonal antibodies to different antigenic regions are being produced in rabbit using a synthetic peptide synthesis approach. With these tools in hand we intend to determine the tissue specificity and functionality of this small ADR peptide as well as its association with soybean seed coat mutations, seed development and germination.

Presentation Type: Poster
Title: The important positive and negative regulators of feedback loops of circadian clock are conserved in soybean and exhibit similar diurnal expression pattern to Arabidopsis thaliana genes
Author(s): Dhiraj Thakare^*  Randy Dinkins  Saratha Kumudini                         
Affiliation: Department of Plant and Soil Sciences, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312
Abstract:

Flowering time is a characteristic of great agronomic importance. Relative to such model species as Arabidopsis and rice, little is known of the genetic mechanisms controlling flowering in soybean. Soybean breeders have identified a series of seven loci, known as the E genes that mediated by photoperiod, condition flowering in soybean. The dominant E1 allele is considered to have the most dramatic effect in delaying flowering under an inductive long day (LD) photoperiod. Earlier studies have shown that soybean plants receive the flowering stimulus as early as 7 days after planting. In this study E-gene near isogenic lines OT89-5 (e1e1) and OT93-26 (E1E1) were selected and tested under a 20h LD photoperiod. Starting from 7 days after planting, tissue samples were collected every 3hrs over a 72-h period. The sampled tissue was used to conduct differential diurnal transcription analysis on putative soybean orthologs of Arabidopsis clock associated genes known to function as positive and negative regulator of interlocked transcription-translation feedback loops. Plants of OT93-26 flowered 7-10 days later than OT89-5 under LD conditions. The putative clock genes tested exhibited circadian rhythmicity similar to that of Arabidopsis thaliana genes, indicating possible evolutionarily conserved molecular mechanism between these species. Slight shift in the diurnal cycle of some of the genes on day 8 was observed otherwise little differences were detected among the E-gene NILs tested. This suggests that the impact of the E1 allele on flowering time may not be due to regulation of the tested clock genes orthologs during this time period.

Presentation Type: Poster
Title: Global gene expression profiles of early soybean seed development using microarrays
Author(s): Sarah I. Jones^*  Delkin O. Gonzalez  Lila O. Vodkin                         
Affiliation: University of Illinois at Urbana-Champaign
Abstract:

Recently, we have examined global gene expression profiles of soybean seed cotyledons during the stages of major accumulation of oils, proteins, and starches, as well as the desiccating and mature stages. It was discovered that genes related to cell growth and maintenance processes, as well as energy processes like photosynthesis, decreased in expression levels as the cotyledons approached the mature, dry stage. Genes involved with seed maturation and some storage proteins had their highest expression levels at the stage of largest fresh weight. However, genes encoding transcription factors, DNA binding proteins, RNA polymerase, etc. showed higher expression levels in the desiccating and dry seeds than in most of the green stages, perhaps indicating the preparation of pathways that will be needed later in the early stages of imbibition. We extended this study to include the gene expression profiles of five stages of development of whole soybean seeds from a few days to about three weeks after fertilization, using amplified mRNA. This is a time when the seeds are undergoing rapid and dramatic increases in cell number and tissue differentiation, including the embryo and later the cotyledons. The microarrays used with the early seed stages were constructed with a low redundancy set of 38,000 soybean cDNAs from more than 80 different libraries of various soybean tissues. Selected genes are also being examined using custom oligo arrays containing up to 40 repeats of the same gene across the slide.

Presentation Type: Poster
Title: Identifying Candidate Mutant Genes in Glabrous Soybeans by Microarray Analysis
Author(s): Matt Hunt^*  Lila Vodkin                           
Affiliation: University of Illinois at Urbana-Champaign
Abstract:

Many of the transcription factors involved in the initiation and morphogenesis of trichomes in Arabidopsis have been identified. Trichome development, however, has not been as well characterized in agronomically important species such as soybean and cotton. Glabrous (trichomeless) varieties of soybean are available in the USDA germplasm collection. These varieties have the potential to be used to identify key developmental genes involved in trichome formation outside of Arabidopsis. In order to identify candidate genes contributing to the mutant glabrous phenotype in soybean, a microarray study was initiated to compare Clark standard (CS) and Clark glabrous (CG) soybean isolines. The comparison was carried out on older and younger leaf tissues with two complete biological replicates of the experiments and sixteen technical replicates. This comparison was carried out using the soybean ~27,000 unigene cDNA microarray library set derived from various soybean tissues, developmental stages, and stress conditions (Vodkin et al. 2004). The data was used to identify candidate genes that may be involved in the glabrous developmental phenotype. Four genes of interest were identified from the microarray study. Data from northern blot analysis showed much higher gene expression values for two of the candidates in CS compared with CG. Southern blot analysis also showed distinct RFLP patterns between CS and CG for both of the above genes. ESTs representing these sequences were used to isolate soybean BACs containing the corresponding genomic sequence from the Williams cultivar. Using the genome sequence information, PCR mapping was conducted for the two candidate genes in CS and CG varieties.

Presentation Type: Oral Presentation
Title: Targeted Sequencing and Analyses of the Rpp4 Asian Soybean Rust Resistance Locus in Soybean.
Author(s): Jenelle Meyer^*  Danielle Silva  Ricardo Abdelnoor  Randy Shoemaker  Michelle Graham                     
Affiliation: USDA-ARS-CICGRU, Iowa State University
Abstract:

Asian Soybean Rust (ASR) is a formidable threat to soybean production in many areas of the world and has recently made its way to the United States. Thus far, only five sources of resistance have been identified (Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5). Previous work by Silva et al (2008) mapped the Rpp4 locus to linkage group G of soybean. SSR marker Satt288, located within 2 cM of the Rpp4 locus, was used to screen the Iowa State University/USDA-ARS ‘Williams 82’ BAC library. Primers designed from BAC-end sequences and SSR Sat_143 were used to anchor and further expand the contig. Skim sequencing of selected BACs within the contig revealed a cluster of disease resistance genes that had not been previously identified in soybean. Shotgun sequencing of two identified BACs identified three R genes of the NBS-LRR class within the Rpp4 locus. TBLASTX (E<10E-4) searches against the recently released soybean genome (JGI; www.phytozome.net) identified only one Rpp4 candidate gene (RCG) homolog within the soybean genome. Additional BLASTN (E<10E-4) searches were used to identify a duplicated region syntenic to the Rpp4 locus. While gene content and order were conserved in this region, no RCG homologs were present. The rarity of the RCG genes in the genome may correlate to the rarity of resistance to ASR. SSR markers have been developed within the RCG cluster to pinpoint the resistance gene and to aid in future breeding efforts.

Presentation Type: Poster
Title: A member of the highly conserved FWL (tomato FW2.2-like) gene family is essential for soybean nodule organogenesis
Author(s): Marc Libault  Xue-Cheng Zhang  Manjula Govindarajulu  Yee Tsuey Ong  Laurent Brechenmacher^*  R. Howard Berg  Andrea Hurley-Sommer  Christopher G. Taylor  Gary Stacey             
Affiliation: University of Missouri
Abstract:

In a previous study, we identified a soybean homolog of the tomato FW2.2 gene that was strongly induced during soybean nodule development. Several studies demonstrated that tomato FW2.2 gene controls 30% of the variance in fruit weight by negatively regulating cell division.
To better understand the function of this gene family, we mined different plant genomes and cDNA databases allowing us to identify 103 FW2.2 genes in 14 plant species [FW2.2-like (FWL) genes]. Since no FWL gene was identified in metazoans, we conclude that the FWL gene family is plant-specific. We performed a comprehensive characterization of this gene family based on comparative analysis of protein and DNA sequences encoded by the 103 FWL genes. This analysis revealed 2 exon-encoded motifs that are strongly conserved based upon their respective sizes and ow rate of non-synonymous sites.
Similar to fruit development, nodule development on legume roots involves de novo organogenesis requiring cell division. Contrary to indeterminate nodules (e.g.., as formed on Medicago truncatula), the formation of determinate nodules (e.g., as formed on Lotus japonicus and Glycine max) is largely the result of cell expansion after the initial cell divisions. Among the FWL genes identified in G. max and L. japonicus, GmFWL1 and 3, as well as LjFWL3, were strongly induced during nodulation. In contrast, none of the M. truncatula FWL genes were induced during indeterminate nodule development. Given the similarity of the soybean and lotus genes to the tomato FW2.2 gene and the strong sequence conservation in this gene family, we hypothesize that GmFWL1 and 3 and LjFWL3 play a significant role in determinate nodule organogenesis through controlling cell division. Indeed, RNAi-induced silencing of GmFWL1 expression led to a significant reduction in nodule formation and global changes in nuclear structure.

Presentation Type: Poster
Title: EST sequencing of Phaseolus vulgaris genes responsive to Uromyces appendiculatus infection also identifies soybean genes responding to Phakopsora pachyrhizi infection
Author(s): Sandra Thibivilliers  Trupti Joshi  Kimberly B. Campbell  Brian Scheffler  Roger Boerma  Dong Xu  Bret Cooper  Henry T. Nguyen  Gary Stacey^*             
Affiliation: University of Missouri, Columbia, MO
Abstract:

No soybean (Glycine max) cultivars showing field resistance to Asian soybean rust (ASR), caused by the biotrophic fungus Phakopsora pachyrhizi, are currently available. Four resistance genes (R genes, Rpp1 to 4) were identified but their field effectiveness was lost mainly due to adaptation by the fungus. In a closely related crop, Phaseolus vulgaris (common bean) several R genes were identified that confer durable resistance to Uromyces appendiculatus (bean rust) infection. We examined whether the common bean-rust pathosystem could be used as a surrogate to better understand the soybean-rust pathosystem. A suppressive, subtracted bean cDNA library was made enriched for sequences specifically expressed in plants induced with either a virulent bean rust strain, race 41, or an avirluent rust strain, race 49. A total of 10,581 unisequences were generated from this library allowing the identification of 6,202 new bean ESTs, significantly adding to the available EST sequences for this plant. Regulation of selected bean and rust bean genes in response to bean rust infection was confirmed by qRT-PCR. Gene expression was similar for both race 41 and 49 during the first 48 hours of the infection process but varied significantly at the later time points (72-96 hours). Likely soybean orthologues of 26 bean genes were identified and their regulation during ASR infection was analyzed. The results showed that 75% of these soybean genes were differentially regulated by ASR infection.

Presentation Type: Poster
Title: Validation of QTLs for Soybean Cyst Nematode resistance in two inter-specific sobean populations
Author(s): Mayumi Acosta^*  Istvan Rajcan                           
Affiliation: University of Guelph
Abstract:

Soybean Cyst nematode (SCN) (Heterodera glycines Ichinohe) is the most damaging soybean pest world-wide. Glycine soja, the soybean’s wild ancestor, may provide sources of new minor and major resistance genes that could help in developing more diverse SCN resistant varieties. In a previous study, novel quantitative trait loci (QTLs) associated with SCN resistance were identified on LG I, K and O in a population derived from the cross between Glycine max NKS08-80 and Glycine soja PI 464925b. In order to validate these QTLs and study the linkage relationship between loci that confer resistance to SCN, two inter-specific soybean recombinant inbred line (RIL) populations (P2 and P4) were studied. The P2 population consisted of 121 F2:5 lines derived from the cross between NK S08-80 and PI 458536 and the P4 population consisted of 209 F2:3 lines derived from the cross between OAC Shire and PI464925B. Phenotyping results showed SCN resistance data of each population were normally distributed. Extreme genotypes were found in both populations indicating transgressive segregation. P2 was genotyped with 50 SSR markers. The QTL on LG O, which derived resistance from G. soja was confirmed by composite interval mapping. The QTL was flanked by Satt466 and Satt345 and explained 12.2% of the phenotypic variation. Three epistatic interactions were detected explaining 30% of the phenotypic variation. Currently, RILs from P4 are being genotyped with 52 SSR markers. The study confirms the stability of QTL on LG O in a different genetic background that could help to develop more diverse resistant soybean varieties.

Presentation Type: Poster
Title: The lipoxygenase gene family: a genomic fossil of shared polyploidy between Glycine max and Medicago truncatula
Author(s): Jin Hee Shin^*  Kyujung Van  Dong Hyun Kim  Kyung Do Kim  Suk-Ha Lee                     
Affiliation: Seoul National University
Abstract:

Soybean lipoxygenases (Lxs) play important roles in plant resistance and beany flavor. Lxs comprises a multi-gene family including GmLx1, GmLx2, GmLx3, and many of them were characterized. Here, we report the tetrad genome structure of soybean Lx regions produced by ancient and recent polyploidy. And, two Lx regions in Medicago truncatula showing synteny with soybean were analyzed. Differential evolutionary rates between soybean and Medicago were observed and the median Ks values of Mt-Mt, Gm-Mt, and Gm-Gm paralogs were 0.75, 0.62, and 0.46, respectively. Thus, comparison of Gm-Mt paralogs (Ks=0.62) and Gm-Mt orthologs (Ks=0.45) elucidates the ancient duplication of Lx regions in the common ancestor prior to Medicago-Glycine split. After speciation, no Lx regions generated by another polyploidy was identified in Medicago, instead, tandem duplication of Lx genes were detected. On the other hand, a lineage specific duplication was observed in soybean, resulting two pairs of Lx regions. Each pair of soybean regions was co-orthologous to one Lx region in Medicago. A total of 34 Lx genes, 15 MtLx and 19 GmLx genes, were divided into two groups by phylogenetic analysis. In conclusion, this study shows that Lx gene family was evolved from two distinct Lx genes in the most recent common ancestor by segmental duplication, speciation, and following tandem duplication.

Presentation Type: Poster
Title: Genomic fractionation and consolidation of soybean genome around Rxp locus
Author(s): Kyung Do Kim^*  Dong Hyun Kim  Kyujung Van  Suk-Ha Lee                       
Affiliation: Seoul National University
Abstract:

Soybean [Glycine max (L.) Merr.] is an ancient tetraploid whose genome has gone through both neopolyploidy and paleopolyploidy events. Many studies revealed the structure produced by neopolyploidy, but still few are reported regarding paleopolyploidy due to high frequency of gene loss after duplication events. Gene retention between homeologous soybean regions and their orthologous Medicago truncatula regions provides a glimpse of ancestral genome of legume family. Therefore, excluding the evidence of neopolyploidy is the key to search the hidden structure produced by paleopolyploidy in soybean. Based on the novel approach that we have developed, we identified the four chromosomal regions of soybean related to Rxp locus that are evidence of both duplication events. Alignment of these regions allowed identification of syntenic blocks and showed heavily degenerated pairs caused by paleopolyploidy as well as highly conserved pairs produced by neopolyploidy. These genomic fractionation and consolidation indicates that the soybean regions have undergone differential rearrangement in genic and intergenic regions. Also, the two regions of M. truncatula are found to be orthologous to the soybean regions. Differential synonymous substitution rates between the soybean and M. truncatula regions are observed and colinearity among those varies as well, explaining the evolutionary origin of soybean and M. truncatula.

Presentation Type: Poster
Title: Molecular mapping of soybean aphid resistance genes in PI 567541B
Author(s): Guorong Zhang^*  Dechun Wang                           
Affiliation: Michigan State University
Abstract:

Soybean aphid (Aphis glycines Matsumura) is an important pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. PI 567541B is a newly discovered aphid resistance germplasm with early maturity characteristics. The objectives of this study were to map and validate the aphid resistance genes in PI 567541B using molecular markers. A mapping population of 228 recombinant inbred lines (RILs) derived from PI 567541B was investigated for the aphid resistance in both field and greenhouse trials. Two quantitative trait loci (QTLs) controlling the aphid resistance were found using the composite interval mapping method. PI 567541B conferred resistant alleles at both loci. An additive x additive interaction between these two QTLs was identified using the multiple interval mapping method. The two QTLs were localized on linkage group (LG) F and M, respectively. These two QTLs explained most of the phenotypic variation in both field and greenhouse trials. The QTL on LG M explained a large portion of the phenotypic variation. The QTL on LG F had much less effect than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an independent RIL population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B, but had various genetic backgrounds. Hence, the two new QTLs identified and validated in this study could be useful in improving soybean aphid resistance by marker-assisted selection.

Presentation Type: Poster
Title: Characterization of a soybean (Glycine max) polyubiquitin promoter in transgenic soybean
Author(s): Carlos M. Hernandez-Garcia^*  Robert A. Bouchard  Cheri A. Nemes  Joseph M. Chiera  John J. Finer                     
Affiliation: Department of Horticulture and Crop Science, OARDC/The Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA.
Abstract:

The success of plant genetic transformation relies greatly on the strength and specificity of the promoter used to drive genes of interest. We have previously reported high levels of transient GFP expression in lima bean cotyledonary tissue using a polyubiquitin promoter (Gmubi) from soybean (Glycine max). In this study we analyzed the gfp gene expression mediated by the Gmubi promoter in stably-transformed soybean tissues. Strong GFP expression was observed in proliferative transgenic embryogenic tissues during selection, which was retained during culture proliferation. GFP expression was generally consistent within transgenic events but variable across events. In whole plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, floral petals, pollen, pods and developing seeds in plants at both vegetative and reproductive growth stages. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. In epidermal cells, GFP was primarily localized in the nuclei and cytoplasm of guard cells. Introduction of the intron-less version of the Gmubi promoter displayed the same GFP expression pattern in almost all the tissues and organs of seedlings and adult plants albeit at lower intensities. Southern blot analysis of transgenic tissues confirmed integration of the gfp gene and transmission was followed up to the T3 generation. In conclusion, the Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.

Presentation Type: Poster
Title: Identification of Candidate Genes for the Rpp4 Asian Soybean Rust Resistance Locus in Soybean.
Author(s): Jenelle Meyer^*  Danielle Silva  Ricardo Abdelnoor  Randy Shoemaker  Michelle Graham                     
Affiliation: USDA-ARS CICGRU Iowa State University
Abstract:

Asian Soybean Rust (ASR) is caused by the fungus Phakospora pachyrhizi and is a threat to soybean production in many areas of the world including the United States. Thus far, only five sources of resistance have been identified (Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5). Previous work by Silva et al (2008) mapped the Rpp4 locus to linkage group G of soybean. SSR marker Satt288, located within 2 cM of the Rpp4 locus, was used to screen the Iowa State University/USDA-ARS ‘Williams 82’ BAC library. Primers designed from BAC-end sequences and SSR Sat_143 were used to anchor and further expand the contig. BAC-end sequences and skim sequencing of selected BACs within the contig revealed a cluster of R genes. Shotgun sequencing of two identified BACs identified three R genes of the NBS-LRR class within the Rpp4 locus. TBLASTX (E<10E-4) searches against the recently released soybean genome (JGI; www.phytozome.net) identified only one Rpp4 candidate gene (RCG) homolog within the soybean genome. A duplicated genomic region syntenic to the Rpp4 locus was identified by additional BLASTN (E<10E-4) searches. Although gene content and order were conserved, no RCG homologs were present in the duplicated region region. The rarity of the RCG genes in the genome may correlate to the rarity of resistance to ASR. SSR markers have been developed within the RCG cluster to pinpoint the resistance gene and to aid in future breeding efforts. These new markers may also be used to test the RCGs as possible candidates for resistance to other pathogens.

Presentation Type: Poster
Title: Genetic Evolution of Common Bean (Phaseolus vulgaris)
Author(s): Mirayda Torres-Torres^*  Scott Jackson                           
Affiliation: Purdue University
Abstract:

Phaseolus vulgaris (common bean) is one of the most important legumes worldwide. Common bean provides low cost protein (~20 to 25 percent protein) and is a good source of fiber, minerals, and vitamins. Common bean has an interesting domestication process in that there are two putative centers of domestication (South American and Center America). The domestication process has produced seven different races. Several studies have supported single or multiple domestications events in common bean. Our interest is to identify genes involved in the response to domestication, evaluate the evolution of these genes as the result of single or multiple domestication events, and map the DNA repeats on the chromosomes of common bean. Since there is little genomic sequence for common bean, we are using gene sequences from Glycine max (soybean), a closely related species, in order to design primers to amplify these genes from common bean. We will use a Bottom-Up approach in order to identify candidate genes involve in selection. Our idea is to be able to sequences several candidate genes in a diverse common bean population to evaluate for selection as well apply correlation analysis. It will allow us to identify genes in selection and see if these genes are a result of multiple or single domestication events. In the case of DNA repeats, we will clone common bean DNA repeats families found through informatic analyses. We will localize the DNA repeats in common bean chromosomes by performing fluorescence in situ hybridization. These analyses will permit us to study common bean genome evolution.

Presentation Type: Poster
Title: Legume Genomics as part of Plant Genomics at the National Center for Biotechnology Information
Author(s): Brian Smith-White^*  Anjana Raina Vatsan  Vyachevslav Chetvernin  Cliff Clausen  Wonhee Jang  John Lopez  Peter Meric  Sergey Resenchuk  Kirill Rotmistrovsky  Deanna Church   Greg Schuler  Tatiana Tatusova       
Affiliation: NCBI
Abstract:

Plant genomics is a simple expansion of the scope of genomics at the National Center for Biotechnology Information (NCBI). In addition to the tools for storage of and analysis of nucleotide sequence such as, respectively, GenBank and BLAST, genomics at NCBI includes databases that enable 1) monitoring the progress of genome sequencing projects (Genome Projects), 2) datamining of probes (Entrez Probe), 3) datamining of primer sequences (UniSTS), and 4) viewing of genome units (MapViewer). These resources have been populated with data from Glycine max, Medicago sativa, Medicago truncatula, Phaseolus vulgaris, Lotus corniculatus, and Vigna radiata. The use of these resources will be described.

Presentation Type: Oral Presentation
Title: The soybean tissue culture and genetic engineering center
Author(s): John Finer^*  Pete Lafayette  Wayne Parrott  Harold Trick  Jack Widholm  Lila Vodkin                   
Affiliation: The Ohio State University
Abstract:

The USB funded "Soybean Tissue Culture and Genetic Engineering Center" consists of five laboratories at four universities, and has been working to improve soybean genetic engineering, for both applied and basic research. To share our research tools with the soybean scientific community, procedures are posted on a web site at http://www.cropsoil.uga.edu/soy-engineering/ The Center has generated and evaluated many different transgenic soybeans events, with traits such as virus and insect resistance and altered amino acid, phytic acid, oil, carotenoid and isoflavone profiles in seeds. Since the Center’s transformation system focuses on somatic embryos, the technology is particularly useful to help research seed-specific traits. In addition, Center personnel have worked to isolate and characterize new soybean promoters and designed new vectors for gene stacking and for enhancing or reducing gene expression. Although the cultivar “Jack” is the best-suited for gene introduction work, transgenes have successfully been introduced into Williams 82 as well as other cultivars. Low copy transgene integration approaches have been combined with rapid transgenic plant production techniques to yield reliable, consistent and efficient methodologies for recovery of transgenic events. Efforts have recently been initiated to generate new levels of resistance to soybean root knot and cyst nematodes using nematode-derived genes and RNAi approaches. Twenty RNAi constructs were generated and divided among center personnel for coordinated introduction into soybean cultivars, previously screened for susceptibility. Preliminary results suggest that RNAi approaches appear to be useful for generating new sources of nematode resistance in soybean.

Presentation Type: Poster
Title: Isolation and preliminary characterization of 9 different polyubiquitin promoters from soybean
Author(s): John Finer^*  Xianfeng Chen  James Roberts  Thomas Laudeman  Michael Timko  Paul Rushton                   
Affiliation: The Ohio State University
Abstract:

The polyubiquitin gene family in soybean contains 3 moderately well characterized genes (Ubi1, 2, 3) as well as other family members that have received little to no attention. The promoters regulating these genes have likewise not been well characterized but show promise as strong constitutive promoters, based on gene expression data. We have now performed extensive characterization of one of these Gmubi promoters (Ubi3, see Hernandez-Garcia et al, this meeting) in transgenic plants, and have isolated and performed partial characterization of an additional 8 Gmubi promoters. Promoters from polyubiquitin genes containing tetrameric repeats of 76 amino acids were selected. All of the promoters (~1.4 kb) were PCR-amplified from genomic DNA of cultivar “Jack”, using primers based on the soybean genome sequence. Gmubi 1-7 contained introns of 532-725 bp in their 5 prime untranslated regions (UTRs), with splice acceptor sites that were directly adjacent to the translational start site. Gmubi 8 and 9 did not appear to contain introns within the 5 prime UTR. Phylogenetic analysis of the promoter regions revealed pairwise similarities in sequences (Gmubi 1 and 3, 2 and 7, 4 and 5, 8 and 9), which has been observed with other tetraploid crops. A possible cis-regulatory element with the consensus sequence of CACG(T/C)G(G/T)CACA was identified in all of the 9 promoters and is currently being evaluated using a minimal promoter gain-of-function system. The sequence of the element suggests that it may be an ACGT-containing ABA-responsive element or a G-box. All of the promoters direct moderate to high levels of gene expression in lima bean cotyledon transient expression assays.

Presentation Type: Oral Presentation
Title: The landscape of transposable elements in the soybean genome
Author(s): Jianchang Du^*  Zhixi Tian  Scott Jackson  Steven Cannon  Randy Shoemaker  Jianxin Ma                   
Affiliation: Agronomy, Purdue University, West Lafayette, IN 47907
Abstract:

The draft sequencing of the soybean genome has provided a unique opportunity to study structural and evolutionary dynamics of transposable elements in this economically important legume crop species. Using a combination of structure-based analysis and homology-based comparison, we have identified 6456 intact LTR-retrotransposons and 9238 solo-LTRs, which are classified into 486 distinct families with the copy numbers ranging from 1 to 1253. Of these families, 60% are gypsy-like elements, and the rest are copia-like elements. These elements, together with numerous truncated fragments or remnants, make up ~35% of the soybean genome. Based on the chromosomal distribution of these elements and their association with soybean centromere satellite repeats, three centromere-enriched families have been identified. Our data suggest that the majority (78%) of the intact LTR-retrotransposons were amplified in the past 3 million years, and 43% of them have undergone bursts in the past 1 million years. On the other hand, rapid removal of retrotransposon DNA by unequal recombination and illegitimate recombination has played a major role in counteracting the expansion of soybean genome caused by rapid proliferation of LTR-retrotransposons. Currently, we are identifying DNA transposons, such as Mutator and CACTA, and Helitron elements, and have obtained a preliminary collection. These transposable element datasets will facilitate the annotation of soybean genes and lay a foundation for further study of the organization, structure and evolution of the soybean genome.

Presentation Type: Poster
Title: Micrarray analysis of soybean treated with Fusarium virguliforme filtrate suggests a role of genes related to cell-wall modification and detoxification during resistance.
Author(s): Osman Radwan^*  Min Li  Steven Clough                         
Affiliation: University of Illinois, Dept Crop Science, IL
Abstract:

Among the four economically most important diseases of soybean [Glycine max (L.) Merrill.] worldwide is the disease called Sudden Death Syndrome (SDS) caused by Fusarium virguliforme (FV), formally known as F. solani f.sp glycines. This soil-borne fungus colonizes soybean roots causing root rot, and also releases a phytotoxin that is translocated to leaf tissues and causes interveinal chlorosis and necrosis leading to the symptoms of scorching and possible defoliation. Here, we report on a microarray study investigating the early response of soybean plants to the FV phytotoxin. Three varieties, with different levels of resistance were used: PI567.374 (highly resistant), Williams 82 (intermediate) and Essex (highly susceptible). Cross comparison of the gene expression profiles from different varieties allowed identification of some SDS defense genes that appear to be induced specifically in PI567.374, and another set of genes were induced commonly between the two more resistant varieties in comparison to susceptible Essex. Further functional annotations based on sequence homology suggested that some of the induced genes might encode proteins involved in cell-wall modification, detoxification, defense response, primary metabolism and membrane transport. Quantitative real-time reverse-transcribed PCR confirmed the differential transcript accumulation of a subset of these genes. Additional analyses are currently being conducted in whole plants challenged with natural root infection.

Presentation Type: Poster
Title: Molecular markers/QTL underlying disease/pest resistance/tolerance in soybean
Author(s): Wenbin Li^*  Yingpeng Han  Weili Teng                         
Affiliation: Northeast Agricultural University,China
Abstract:

Marker-assisted selection (MAS) could facilitate the development of disease/pest resistant cultivars of soybean. MAS is more efficient than selection based on the phenotype for a trait with low heritability. Gene introgression can readily be followed using molecular markers that are not influenced by the environmental conditions. Seven RIL populations were developed in this study. A complete randomized design was adapted with three locations and three years experiment. Five QTL associated with the tolerance to Phytophosora root rot, three QTL relevant to MSV resistance, two QTL associated with white mould, four QTL associated with SCN, three QTL relevant to soybean seed borer were detected, which would be beneficial for MAS to develop disease/pest resistant varieties of soybean.

Presentation Type: Poster
Title: Isolation and Characteristic of a RAV-like Transcription Factor Ortholog Associated with the Control of Photosynthesis and Senescence in Soybean
Author(s): Wenbin Li^*  Lin Zhao  Giulan Luo                         
Affiliation: Soybean Research Institute(Key Lab of soybean Biology in Education Ministry),Northeast Agricultural University,China
Abstract:

A cDNA library enriched for mRNAs encoding ESTs that increased in abundance during short days was constructed by SSH from leaf tissues of a photoperiod sensitive soybean. The proteins predicted to be encoded by the mRNAs were inferred to be involved in diverse functions such as transcription, signal transduction, programmed cell death, protein, carbohydrate macro-molecule degradation and stress responses. A full-length mRNA encoded the transcription factor RAV was cloned by RACE containing 1,380bp with an open reading frame. The GmRAV protein included an AP2/ERF domain and a B3 domain. GmRAV mRNA abundance was increased in SDs following leaf treatments with GA and ABA versus decreased following BR treatment. Transgenic tobacco expressing GmRAV driven by the 35S promoter showed morphological and characteristic alterations such as slower plant growth rate, shorter plants, reduced root elongation, smaller, much tender and greener leaves, fewer root numbers and delayed flowering time in comparison with controls. Dwarfism of the transgenic tobacco may be related to the strong inhibitory effect of GmRAV on the brassinosteroid (BR) response or the weaker stimulatory effect of GmRAV on the GA stem elongation response. The organ analyses of soybean showed that GmRAV mRNA abundances were higher in SDs than in LDs.

Presentation Type: Poster
Title: Mid-oleic Quantitative Trait Loci in Two Soybean Crosses
Author(s): Nabil Belbachir^*                             
Affiliation: South Dakota State University
Abstract:

Abstract

Mid-oleic Quantitative Trait Loci in two soybean crosses


Oleic acid is a major monounsaturated fatty acid that plays a vital role in soybean
oil quality. Soybean oil with high oleic acid increases stability at high cooking
temperatures and reduces the need for hydrogenation, thus reducing trans-fats.
Molecular breeding can be a valuable tool to detect the genomic region of the soybean
genome controlling this trait and develop soybean varieties with increased levels of oleic
acid content. Although many Quantitative Trait Loci (QTL) have been reported in
soybean for oleic acid content, none or very few have been confirmed in other
populations from the same or different gene pools. Moreover, there is inconsistent
information on the confirmation of previously reported QTL. Previous studies on the
oleic acid content in soybean oil have determined the presence of six quantitative trait
loci which are clustered in four different linkage groups (A1~96cM, D2~85cM, G~22-
53cM, and L~30-71cM). The objective of this study was to confirm the association of the
QTL candidates with oleic acid content and to determine the individual contributions of
specific QTL for oleic acid in three types of breeding populations. The plant materials
used in the study were derived from 140 F3 lines, 60 BC1F3 lines, and 90 F5 lines with
two replications. All three populations were developed by crossing a South Dakota line, vi
SD01-076R, to a North Carolina germplasm line, N98-4445A, which contained increased
levels of oleic acid. Nine microsatellite markers related to the QTL genomic regions were
used. Single factor ANOVA (SF-ANOVA) was used to identify candidate QTL. Four
QTL (Satt200 on LG-A1 with R2= 3.5%, Satt235 on LG-G with R2= 9.4%, Satt303 on
LG-G with R2= 4%, and Satt398 on LG-L with R2=4.6%) were confirmed in the F3 lines.
In the BC1F3 lines, two major QTL (Sat113 on LG-L with R2= 27.3% and Satt235 on
LG-G with R2=23.8%) and one minor QTL (Satt200 on LG-A1 with R2= 9.9%) were
significantly associated to oleic acid content. For F6 lines, only one significant QTL
(Satt235 on LG-G with R2=4.1%) was found to be associated with oleic acid content.
Based on two-factor ANOVA, there was no significant interaction between the QTL
identified in the F3 and F6 lines. Satt235 was consistently associated with oleic acid
content across all populations. QTL associated with oleic acid are not stable across
populations. The unconfirmed QTL may have been false positive or specific for the
original mapping population. This study shows the necessity for confirmation of novel
QTL in many different populations and environments. The validation of original QTL
will help the efficiency of MAS application in plant breeding programs and facilitate
QTL physical map establishment. Future work will include F2:4 population QTL
confirmation to further validate the QTL reported and to develop a soybean line with
elevated oleic acid content.

Presentation Type: Poster
Title: SoyXpress: A soybean transcriptome database
Author(s): Kei Chin Cheng  Martina Stromvik^*                           
Affiliation: McGill University
Abstract:

In order to detect potential unintended effects in the transcriptome of transgenic soybean, we developed SoyXpress [1]- a comprehensive database integrating 380,000 Expressed Sequence Tags (ESTs) and 36,000 Affymetrix probe sets, linked to Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways and SwissProt keywords. All annotation have links back to GO, KEGG and SwissProt and the keywords have links to PubMed and TOXLINE, to access further literature on each gene. SoyXpress is implemented in MySQL and uses a perl cgi web interface (http://soyxpress.agrenv.mcgill.ca/). The current mircroaray data in SoyXpress measures gene expression in the first tri-foliate leaves of two transgenic and three conventional soybean cultivars developed for Canadian growing conditions. The results show that our method is efficient in measuring global gene expression and greatly facilitates the elucidation of the particular differentially expressed genes [2]. Future experiments incorporating additional transgenic and conventional cultivars, as well as additional tissues, will further our understanding of the range of gene expression in crops that are generally recognized as safe (GRAS).


1. Cheng, K.C. and Strömvik, M.V. (2008) "SoyXpress: a database for exploring the soybean transcriptome". Submitted.
2. Cheng, K.C., Beaulieu, J., Iquira, E., Belzile, F.J., Fortin, M.G. and Strömvik, M.V. (2008) “Effect of transgenes on global gene expression in soybean is within the natural range of variation of their conventional counterparts.” Journal of Agricultural and Food Chemistry.56: 3057-67

Presentation Type: Poster
Title: Variation in Soybean Rust Reaction Response in a Set of Resistant Germplasm Accessions
Author(s): David R. Walker^*  Dario Narvaez  James J. Marois  David L. Wright                       
Affiliation: USDA-ARS Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, IL 61801
Abstract:

Soybean resistance to soybean rust (SBR), caused by Phakopsora pachyrhizi, is often associated with the formation of reddish-brown (RB) lesions, reduced disease incidence and severity, and/or prolongation of the latent period between infection and sporulation (referred to as “slow rusting”). The appearance of SBR symptoms on different resistant germplasm accessions from a field in Quincy, FL varied considerably in 2007. Nine of the resistant Plant Introductions (PIs) and four susceptible cultivars were subsequently evaluated in the greenhouse to determine whether similar variation would also occur under more controlled conditions. A diverse range of reactions to SBR was also observed in the greenhouse. PI 606440A developed “classic” RB lesions that were 1 mm or greater in diameter, with occasional uredinia but little or no sporulation. Other accessions, such as PI 417089A and PI 567104B developed much smaller (<1 mm) RB or brown lesions, while lesions developed by PI 506947 were often purplish in appearance. Apparent variation in the spread of disease upwards through the canopy and in the amount of chlorosis associated with rust lesions was observed among both the cultivars and the resistant accessions. These results may reflect different mechanisms of soybean resistance to SBR, and possibly differential sensitivity to toxins produced by the pathogen.

Presentation Type: Poster
Title: Soybean Cultivar Variation in Response to Elevated Ozone Concentration
Author(s): Amy Betzelberger^*  Justin McGrath  Kelly Gillespie  Randall Nelson  Elizabeth A. Ainsworth                     
Affiliation: Department of Plant Biology, University of Illinois, Urbana-Champaign, Urbana, IL 61801
Abstract:

Crop losses to ozone damage are conservatively estimated to cost $1 to $3 billion in the U.S. These costs will rise as surface-level ozone increases over this century. A critical step in maximizing soybean yield in a future of rising tropospheric ozone is identifying variation in cultivar responses, which depends upon rapid physiological and molecular screens for ozone tolerance and sensitivity. The aim of this study was to test the response of 10 soybean cultivars to growth at elevated ozone concentrations under field conditions at the Soybean Free Air Concentration Enrichment (SoyFACE) facility (www.soyface.uiuc.edu). Throughout the season, we compared photosynthesis, leaf area index, chlorophyll fluorescence, and the antioxidant capacity of 10 soybean cultivars grown at ambient ozone (41 ppb) and elevated ozone (83 ppb). All cultivars showed a significant decrease in seed yield, ranging from an 11.2% loss in Loda to a 36.4% loss in IA-3010. Maximum leaf area index and the antioxidant capacity of soybean cultivars were not consistently affected by growth at elevated ozone. However, light-saturated photosynthesis was significantly reduced (p<0.05) by 11.3% on average for all cultivars across the growing season. Stomatal conductance to water vapor was also significantly reduced (p<0.001) by 15% in elevated ozone. Both photosynthesis and the quantum yield of photosystem II were significantly correlated with seed yield in the cultivars, suggesting that these measures may be good physiological markers for yield response to elevated ozone. Future work will investigate variation in the molecular response of soybean cultivars to elevated ozone.

Presentation Type: Poster
Title: Title Test
Author(s): Michael Gonzales^*                             
Affiliation: SOYMAP
Abstract:

ABSTRACT TEST

Presentation Type: Poster
Title: Characterization and mapping of soybean aphid resistance and maturity traits from PI 230977
Author(s): C Carter^*  L Hesler  K Tilmon  R Scott                       
Affiliation: South Dakota State University
Abstract:

The soybean aphid (Aphis glycines Matsumura) has become a major pest of soybean in the United States. The primary source of host plant resistance to date has been the Rag1 gene from Dowling. However, some aphid biotypes have already begun to overcome this resistance. Hesler et al (J. Econ. Entomol. 100:1464-1469) have demonstrated that the soybean PI 230977 offers aphid resistance comparable to that of Dowling, with both antibiosis and antixenosis components. PI 230977 alleles differed from those of Dowling at 8 SSR loci spanning a 23 cM region flanking the Rag1 gene on MLG M, suggesting that PI 230977 provides an alternative source of aphid resistance. PI 230977 was crossed to two SD breeding lines and aphid resistance was analyzed in segregating F2 and F3 families in both greenhouse assays and caged field trials. Aphid resistance in F2-3 populations segregated as a quantitative or recessive trait, in contrast to the single dominant gene inheritance of Rag1 resistance. We are currently analyzing SSR markers dispersed throughout the genome in the F2 and F3 progeny of SD93-828 X PI230977. Analysis of aphid resistance and SSR markers in the F2 population indicated a tentative association with the CEW 9-1 and 1-3 insect resistance QTLs on MLG D1b; there was no association of aphid resistance with any markers on MLG M. Maturity and various morphological traits were associated with SSRs on MLGs L (days to flower, Satt229; branching, Satt229), K (branching, Satt055), and C2 (determinate/indeterminate, Satt376).

Presentation Type: Oral Presentation
Title: Discovery and genetic engineering of transcription factors towards drought tolerance through dissecting root characteristics in soybean
Author(s): Henry Nguyen^*  Babu Valliyodan  Son Tran  Dong Xu  Robert Sharp  Gary Stacey                   
Affiliation: National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, MO 65211
Abstract:

Drought is the major abiotic stress factor limiting crop productivity worldwide and in the US. It is reported that the average yield losses are more than 40% in soybean due to drought stress. Efforts to understand the physiological mechanisms and the genetic dissection of drought responses in legumes, especially in soybean, are still in the early stages. Root system plays vital role in plant adaptation and productivity under water-limited environments. Under mild or severe water deficit conditions, root can continue to grow while shoot growth is reduced or inhibited. In the signal transduction cascades from perception of stress signals to stress-responsive gene expression, various transcription factors (TFs) function not only as molecular switches for gene expression but also as interacting partners in the network. Focus on these networks is essential to understand the transcriptional regulatory mechanisms of root growth and plasticity under water deficits. Our overall goal is to understand functions of these regulatory switches to dissect their specific role in plant development and stress tolerance. Molecular tailoring of novel TFs has the potential to overcome a number of limitations in creating drought-tolerant transgenic soybean plants with better yield. To dissect the expression pattern of different TFs and signaling genes under water deficit conditions we have utilized microarray and in-house developed unique library of transcription factor genes in soybean. We have identified several drought related and root related transcription and signaling factors, and characterization and engineering of selected candidates through translational genomics pipeline are in progress.

Presentation Type: Poster
Title: Elevated carbon dioxide and elevated ozone concentrations alter patterns of soybean leaf growth
Author(s): Justin McGrath^*  Elizaberth Ainsworth                           
Affiliation: Department of Plant Biology, University of Illinois, Urbana-Champaign
Abstract:

Leaves are critical for harvesting light energy and taking up carbon dioxide. Changes in leaf expansion or development can integrate across the plant canopy and growing season to impact yield and plant-atmosphere fluxes. Although the effects of elevated carbon dioxide and ozone concentrations on leaf expansion have been studied individually, few studies examine leaf expansion in more realistic simulations of future conditions, with simultaneously elevated carbon dioxide and ozone. The purpose of this study was to examine leaf expansion in soybean exposed to elevated carbon dioxide and ozone in a field setting to determine how growth parameters such as final leaf area, leaf number, and growth rate were altered by climate change. Elevated carbon dioxide and elevated ozone increased and decreased leaf area index (LAI), respectively. Changes in LAI were due to changes in leaf number and individual leaf area. Leaves were larger upon unfolding in elevated carbon dioxide and smaller in elevated ozone, which compounded over time to alter final leaf area, even though relative growth rates were similar. Furthermore, carbohydrate concentrations were altered in old but not young leaves, indicating that changes in leaf size result from whole-plant, not leaf-level, responses. Changes in leaf area were related to cell parameters; leaves in elevated carbon dioxide had more cells and leaves in elevated ozone had smaller cells. Plant responses in the combined elevated carbon dioxide and ozone treatment were similar to the elevated carbon dioxide treatment, suggesting that rising carbon dioxide will protect against rising ozone concentrations.

Presentation Type: Poster
Title: Establishment of a protein reference map of soybean root hair cell by 2D gel electrophoresis and MudPIT
Author(s): Laurent Brechenmacher^*  Joohyun Lee  Sherri Sachdev  Zhao Song  Tran Hong Nha Nguyen  Joshi Trupti  Nathan Oehrle  Marc Libault  Brian Mooney  Dong Xu   Bret Cooper  Gary Stacey       
Affiliation: National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri-Columbia, Columbia, MO 65211
Abstract:

Root hairs are single tubular cells formed from the differentiation of epidermal cells, called trichoblasts, on primary and secondary roots. They are involved in water and nutrient uptake, anchorage of the plant into the soil and represent the colonization site of leguminous roots by rhizobia, soil bacteria that establish a nitrogen fixing symbiosis. Root hairs develop by polar cell expansion or tip growth, a unique mode of plant growth shared only with pollen tubes. We decided to comprehensively examine soybean root hair proteins by 2D gel electrophoresis and MudPIT analyses. Soybean was selected for this study due to its agronomic importance and its root size, which permits isolation of root hairs in sufficient amount for proteomic studies. Through this effort, we established the first soybean root hair proteome reference map by identifying more than 1000 proteins. Only 207 proteins were identified by both approaches. The proteins identified are mainly involved in basic cell metabolism (primary metabolism, protein synthesis and processing), but also in functions more specific to the root hair including nutrient uptake or vesicle trafficking.

Presentation Type: Poster
Title: Shedding light on soybean oil synthesis utilizing Arabidopsis double-DGAT knockouts
Author(s): Gary Ratliff^*  David Hildebrand                           
Affiliation: University of Kentucky
Abstract:

Presentation Type: Poster
Title: Soybean genotypes differ in relative amount of viral antigen after Bean pod mottle virus inoculation
Author(s): José L. Aponte-Rivera^*  George L. Graef  Julian M. Chaky  Loren Giesler  Amy Ziems  James E. Specht                   
Affiliation: Dep. of Agronomy, Univ. of Nebraska, Lincoln, NE 68583-0915
Abstract:

There is no documented resistance to BPMV, and yield losses of 3% to 52% have been reported. Most studies report no relationship between severity of foliar and seed mottling symptoms and presence of BPMV. Consequently, evaluation of soybean genotypes for tolerance to BPMV based on visual symptoms is not successful. The objective of this study was to evaluate response of three soybean genotypes to two inoculum amounts by measuring relative amount of viral antigen (RVA) in leaf tissue. Two soybean lines that were asymptomatic and one that showed severe leaf and seed mottling symptoms, based on three years of evaluation of inoculated plots, were used. Three experiments were conducted, each using a CRD with 5 replications. Unifoliolate leaves were inoculated at Vc using 1X or 2X inoculum levels. Leaf tissue was harvested at 24h, 2, 4, and 6 weeks after inoculation. For the 24h sampling time, the unifoliolate leaves were harvested. Later sampling treatments included at least the first trifoliolate leaf and the most recently developed leaf on the main stem. RVA was determined using a semi-quantitative ELISA method. Genotypes differed significantly for RVA over treatments and sampling times, with some significant interactions. However, at four weeks and later, RVA did not differ among nodes within a genotype. Genotype differences in RVA suggest suppression of virus replication, and that RVA might be used as an indicator of tolerance to BPMV in screening or genetic studies.

Presentation Type: Poster
Title: Molecular Genetics and Soybean Genomics Laboratory Research Program
Author(s): Theresa Musket^*  Nguyen Laboratory Members  Henry T. Nguyen                         
Affiliation: Division of Plant Sciences, University of Missouri
Abstract:

Research interests in the Nguyen laboratory focus on the molecular genetics of stress tolerance and the application of genomics and genetic engineering technologies to soybean improvement. There are two major research emphases in the laboratory: soybean genome mapping and molecular breeding; and functional genomics for drought tolerance and improvement of soybean seed composition. In the mapping and molecular breeding area, research involves: quantitative trait loci (QTL) discovery for breeder desired traits including soybean cyst nematode (SCN), pathogen resistance, seed composition, and abiotic stress tolerance; genome structure and organization through genetic and physical mapping and single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker development; and a molecular breeding program for marker assisted selection (MAS) for SCN and oleic acid content. The functional genomics area includes research in: physiology and genetic variation of soybean root growth; transcript, protein, and metabolite profiling of soybean tissues under drought stress; characterization of stress specific and tissue specific candidate genes for genetic and metabolic engineering for enhancing stress tolerance and yield in soybean; engineering value-added traits to improve soybean seed composition; studying the phytosterol pathway to increase sterol content; development of soybeans with superior oil traits; and the identification of bioactive compounds benefiting human health and functional foods. The Nguyen lab is also actively engaged in the soybean genome sequencing and annotation program of DOE-JGI.

Presentation Type: Poster
Title: Construction of proteome and metabolome maps of soybean to improve yield and value-added traits
Author(s): Babu Valliyodan^*  Laurent Brechenmacher  Jianlin Cheng  Dong Xu  Gary Stacey  Henry Nguyen                   
Affiliation: National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, MO 65211
Abstract:

Although the genetic blueprint of soybean is represented by the genome, its phenotype is a product of that blueprint manifested as the production of proteins and metabolites influencing growth characteristics, stress responses, seed composition and yield. Profiling soybean gene products will lay the foundation for a systems biology approach to key processes such as seed development, which will lead to the genetic improvement of yield and seed composition. Also, these approaches help in the search of novel bioactive compounds leading to the production of new generation soybean products for human health and nutrition. It is well known that environmental cues influence developmental phenotypes in plants. Different biotic stresses such as fungal diseases and abiotic stresses such as drought and flooding also elicit phenotypic responses from the genome. Thus, by studying the gene products, a direct correlation between response and specific peptides/metabolites can be made. This will lead to crop improvement either through breeding or transgenic efforts. Major objectives of this study are: a) to identify key soybean seed, leaf, and root proteins involved in development and biotic and abiotic stress responses; b) to establish a comprehensive set of chemical standards for soybean metabolites moving toward construction of a metabolome map with a focus on seed and drought effects on seed development and c) to compile a database linking proteomic and metabolite information and associate this information to value-added soybean traits and markers for assisted breeding. We are utilizing GC/MS, LC/MS and NMR approaches to identify key molecules for further characterization. This project is funded by the United Soybean Board.

Presentation Type: Poster
Title: Progress in Development of a Reference Genetic Map and Integration of Soybean Physical Maps
Author(s): Xiaolei Wu^*  Rajesh PN  Tri D. Vuong  Vinh T. Nguyen  Theresa A. Musket  Chengwei Ren  Gary Stacey  J. Grover Shannon  David A. Sleper  Henry T. Nguyen            
Affiliation: Division of Plant Sciences, University of Missouri
Abstract:

Soybean BAC-based physical maps provide a useful platform for effective and high-throughput gene and QTL cloning, EST mapping, marker development, genome sequencing, and comparative genomics research. Soybean physical maps for “Forrest” and “Williams 82” representing southern and northern U.S. soybean germplasm base have been constructed with different fingerprinting methods. These physical maps are complementary for coverage of gaps. More than 2,000 genetic markers and 5,000 gene-based STS markers have been anchored onto the Williams 82 physical map but only a limited number of markers have been anchored to the Forrest physical map. We are using a Forrest x Williams 82 mapping population containing 1,025 F8 RILs to construct a reference genetic map. Our approach involves selection of a core set of this mapping population using SSR markers followed by high-resolution mapping using SNP markers. In addition to the public SNP markers, we have generated extensive polymorphic marker resources (SSR, SFP, Indels, and SNP) that will be used for the genetic mapping in this population. Moreover, we have developed telomere-specific markers and candidate gene-associated markers that will be placed on this reference genetic map. Our goal is to construct a high resolution genetic map that will allow us to integrate and utilize the physical maps to target QTL regions of interest.

Presentation Type: Poster
Title: Inheritance and Mapping of Soybean Aphid Resistance from Soybean Accession PI71506
Author(s): Andrew Van Nurden^*  Roy Scott  Catherine Carter  Louis Hesler  Kelly TIlmon                     
Affiliation: South Dakota State University
Abstract:

Soybean aphids (Aphis glycines Matsumura) continually establish populations of economic importance in soybean [Glycine max (L.) Merr.] production areas. Insecticide application costs prompt the development of resistant varieties. The soybean germplasm accession PI71506 has been shown to convey antixenotic host plant resistance. PI71506 alleles differed from those of Dowling at 7 of 8 SSR loci spanning a 23 cM region encompassing the Rag1 aphid resistance gene. The objective of this study is to determine the inheritance of resistance from PI71506 and map its location in the genome. We made crosses between SD1111R and PI71506 to create F2 populations and subsequent F3 families for resistance screening. Individual plants were screened in no-choice green house tests utilizing small sticky cages. Pooled F2 data in chi square tests revealed a goodness of fit of P>0.25 for a single dominant gene segregation of 3:1. We are currently screening the parents and F2 populations with polymorphic SSR markers for QTL analysis and genetic mapping of the F2 populations. F3 families are also being screened in choice tests during the 2008 growing season under an aphid tent. Data and DNA samples will be collected from F3 families for continued QTL and mapping analysis.

Presentation Type: Poster
Title: Overexpression of AP2 transcription factor improves drought tolerance in soybean
Author(s): Satish Guttikonda^*  Babu Valliyodan  Henry Nguyen                         
Affiliation: Division of Plant Sciences, University of Missouri, Columbia, MO, 65211
Abstract:

Drought is one of the major limiting factors for plant growth and crop productivity. Plants respond and adapt to these stresses through various biochemical and physiological processes. Several genes induced by drought stress include osmolytes and transcription factors. Presently most of the abiotic stress related genes were constitutively expressed and the overexpression of the transgene causes additional building blocks and energy which may affect the normal growth of transgenic plants. The major objective of this experiment is to develop drought tolerant transgenic soybean plants by comparing constitutive and abscisic acid inducible promoters. We have engineered APETELA2 transcription factor from Arabidopsis to soybean. Several transgenic lines were generated and molecular analyses were performed to confirm transgene integration. Drought experiments conducted with homozygous lines indicate that the overexpression transcription factor with constitutive and abscisic acid inducible promoters maintain high water content and show leaf dehydration tolerance without any major pleotropic effects when compared to wild type under prolonged gradual water stress. The experiment results demonstrate the feasibility of engineering soybean for increased drought tolerance by expressing stress responsive genes.

Presentation Type: Poster
Title: GS52 apyrase plays a critical role during soybean nodulation
Author(s): Manjula Govindarajulu^*  Sung-Yong Kim  Marc Libault  R. Howard Berg  Kiwamu Tanaka  Gary Stacey  Christopher G. Taylor                 
Affiliation: Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132 USA
Abstract:

Apyrases are nonenergy-coupled NTPases that hydrolyze nucleoside tri- (NTP) and di-phosphates (NDP) to nucleotide mono-phosphates (NMP) and orthophosphate. GS52, a soybean ecto-apyrase, was previously shown to be induced very early in response to inoculation with the symbiotic bacterium Bradyrhizobium japonicum. Over-expression of the GS52 ecto-apyrase in Lotus japonicus increased the level of rhizobial infection and enhanced nodulation. These data suggest a critical role for the GS52 ecto-apyrase during nodulation. To test this hypothesis, we utilized RNA interference (RNAi) to silence GS52 expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Transcript levels of GS52 were significantly reduced in silenced GS52 roots and exhibited reduced numbers of mature nodules. Development of the nodule primordium and subsequent nodule maturation were significantly suppressed in the GS52 silenced roots. Transmission electron micrographs of GS52 silenced root nodules showed early senescence and infected cortical cells were devoid of symbiosome containing bacteroids. Application of exogenous adenosine di-phosphate (ADP) to silenced GS52 roots restored full nodule development. Restored nodules contained bacteroids, thus indicating that extracellular ADP is important during nodulation. These results clearly suggest that GS52 ecto-apyrase catalytic activity is critical for the early rhizobium infection process, initiation of nodule primordium development and subsequent nodule organogenesis in soybean.

Presentation Type: Poster
Title: Genetic Mapping of Soybean Seed Protein QTLs
Author(s): Piyaporn Phansak^*  Watcharin Soonsuwon^*  James E. Specht  George L. Graef  Perry B. Cregan  David L. Hyten                   
Affiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68583-0915
Abstract:

A quantitative trait locus (QTL) is the chromosomal location of a gene governing that trait. QTL identification is the first step towards using marker-assisted selection (MAS) to introgress desirable QTL alleles into elite high-yield cultivars. Hundreds of high protein PIs exist in the USDA germplasm collection, and are obviously a source of high protein alleles, but few PIs have been characterized as to their allelic status at known (or unknown) protein QTLs. Although 76 protein QTLs are listed in SoyBase, many are likely repeat discoveries of the same QTL(s), given the typical +/- 10cM confidence intervals associated with QTL positions. The additive effect for these 76 QTLs was never greater than the respective 1.2% and 0.85% estimates obtained for the Linkage Group (LG)-I and LG-E QTLs. All remaining QTLs had a smaller apparent additive effect (or even smaller true effect, given the Beavis Effect). Discovery of QTLs different from the LG-I and LG-E QTLs, but still expressing a large additive effect, would be of great interest. We have mated 17 high protein (48% or more) germplasm accessions to high-yield public cultivars with normal protein (42% or less) to generate 17 populations. The 240 F2 plants in each population will produce F2.3 seed progenies in 2008 that will be phenotyped for seed protein content. Using a decile-based selective genotyping protocol, a few SSR markers known to be tightly linked to the LG-I and LG-E QTLs, and a panel of 1536 SNP markers, we intend to identify all protein QTLs of high additive effect in each population.

Presentation Type: Poster
Title: Phosphoproteome of Soybean Root Hairs Colonized by Bradyrhizobium japonicum
Author(s): Tran Hong Nha Nguyen^*  Laurent Brechenmacher  Gary Stacey                         
Affiliation: National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 Vietnam Education Foundation Fellow
Abstract:

The interaction between soybean (Glycine max) and the bacterium Bradyrhizobium japonicum (USDA110) leads to the establishment of a nitrogen fixing symbiosis. This symbiosis results in the formation of nodule in which the bacteria reduce atmospheric nitrogen to ammonia which constitutes a source of nitrogen that can be used by the plant. During the first stages of G. max- B. japonicum interaction, (iso)flavonoids secreted by soybean activate the synthesis of the B. japonicum lipo-chitin Nod factor. This Nod factor triggers early events of the infection process including root hair curling, infection thread development and formation of a nodule primordium [1]. The molecular mechanisms of these events are not completely understood.
In this study, we investigated protein phosphorylation in isolated root hair cells after inoculation with B. japonicum. Root hair proteins were extracted, separated by 2 dimension gel electrophoresis and stained by Pro-Q Diamond. At 24 hours after infection, 450 to 600 putative phosphoproteins were detected, of which 18 were found significantly regulated by B. japonicum infection. We are currently analyzed by mass spectrometry those phosphoproteins that respond to B. japonicum inoculation from 15 min to 3 hours after inoculation.
1. Stacey G., et al (2006). Curr. Opin. Plant Biol. 9: p. 110.

Presentation Type: Poster
Title: Association between SDS and SCN resistance in four populations with PI88788 SCN resistance
Author(s): Timothy Pruski^*  Jacob Delheimer  Brian Diers                         
Affiliation: Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801
Abstract:

Sudden death syndrome of soybean in North America is caused by the soil borne fungus, Fusarium virguliforme. The fungus attacks, invades, and inhabits the roots of susceptible soybean plants causing what can be a substantial root rot. Though the Fusarium is confined to the roots, the fungus can produce a toxin that when transported to the leaves will cause severe leaf chlorosis and necrosis that greatly diminishes the photosynthetic capability of the plant. Breeding for resistance to the pathogen is one strategy employed to help combat this problem. QTL for resistance to the leaf scorch caused by sudden death syndrome have been mapped in a number of populations and linkage groups. One of the more commonly identified and largest effect SDS resistance QTL is located near the top of linkage group G near the SCN resistance gene, rhg1. In this study, four populations segregating for PI88788 derived SCN resistance display field resistance to leaf scorch caused by sudden death syndrome. The resistance to the leaf scorch caused by SDS significantly co-segregates with the SSR marker Satt309 which in turn is closely linked to rhg1. Greenhouse tests in the absence of SCN with one of these populations failed to significantly associate SDS leaf scorch resistance with the SSR marker Satt309. This suggests that the SDS resistance linked to Satt309 in field tests is the result of the SCN resistance gene rhg1, not from a separate SDS resistance QTL.

Presentation Type: Poster
Title: Functional Versus Total Genetic Diversity in Soybean Association Mapping Panels
Author(s): Chris Grainger^*  Istvan Rajcan  Elizabeth Lee                         
Affiliation: University of Guelph
Abstract:

Genetic diversity is a key component in association mapping studies which aim to identify causative allelic polymorphisms for traits of interest. Many association panels consist of a wide array of diverse germplasm sources in order to capture maximum genetic diversity; however it is functional genetic diversity which is most beneficial to improving or creating useful cultivars. The purpose of this study is to examine the requirement for a genetically diverse germplasm panel (defined by molecular markers) versus a functionally diverse germplasm panel (defined by pedigree) of soybean [Glycine max (L.) Merr.] lines in identifying genomic regions associated with increased yield. Preliminary work has consisted of genotyping a number of soybean lines with SSR markers to ascertain the genetic diversity of the breeding lines. Future work will focus on both SSR and SNP genotyping for further genetic diversity and association analysis

Presentation Type: Poster
Title: Development of a New Herbicide Tolerance Trait to Improve Weed Control in Glyphosate Tolerant Soybean
Author(s): Cory Cui^*  Jill Bryan  Greg Gilles  Nathan Vanopdorp  Terry Wright  Thomas Greene  Steve Thompson                 
Affiliation: Dow AgroSciences
Abstract:

A synthetic gene (AAD12) has been developed by Dow AgroSciences from Delftia acidovorans. In planta this gene produces an enzyme that deactivates several herbicides having an aryloxyalkanoate moiety, including Phenoxy auxins (e.g., 2,4-D, MCPA) and Pyridyloxy auxins (e.g., fluroxypyr, triclopyr). The gene was introduced into soybean (Glycine max L.). In this presentation, we describe transformed soybean plant event characterization at the molecular level from generation to generation, and plant performance in the greenhouse and field. We have selected several high quality, simple events that are expressing the target protein at the levels that effectively protect the soybean events from 2,4-D applications. Protection or tolerance is observed from the early seedling stage to the reproductive stage at application rates exceeding 4 times typical herbicide use rates. Agronomic performance of individual events is comparable to iso-lines. Based on our data, it is clear that this technology enables broadcasting of mixtures of 2,4-D and glyphosate from pre-emergence to the end of the glyphosate application window, improves and enhances the performance of glyphosate & glufosinate cropping systems, improves the control of “hard to kill” broadleaf weeds, reduces selection pressure for glyphosate resistance and sustains the glyphosate cropping system.

Presentation Type: Poster
Title: A CYTOGENETIC APPROACH FOR SOYBEAN CHROMOSOME IDENTIFICATION
Author(s): Christian S. Hans^*  Scott Jackson                           
Affiliation: Purdue University, Department of Horticulture
Abstract:

The twenty chromosomes of soybean are small and lack distinguishing characteristics. Due to this difficulty, soybean has lagged behind organisms such as maize and wheat for cytogenetic studies. With the current sequencing progress of the soybean genome, it has become paramount to develop tools that can be used identify chromosomes unambiguously. Utilizing fluorescent in situ hybridization (FISH) to align each linkage group, the BACs were labeled with biotin and digoxigenin to use two-color detection on DAPI counterstained chromosomes. The aim of this research was to have a set BAC clones that have been mapped to opposite ends of each linkage group, which would parlay into a clone on each arm of each chromosome. The outcome has identified only a few BACs that have single FISH loci, but has identified many BACs that have duplicate signals on arms of other chromosomes (indicative of paleopolyploidy). BACs containing repetitive sequences, both dispersed and localized, were also found . The end result is a set of clones that can be used to unambiguously identify specific chromosomes that correspond to molecular linkage groups. These tools not only give a baseline for further structural study but also generate insight into how the soybean genome is organized and how it has evolved post duplication.

Presentation Type: Poster
Title: Quantitative Trait Loci Analysis and Transcription Factor Profiling for Resistance to Soybean Cyst Nematode (Heterodera glycines Ichinohe)
Author(s): T. D. Vuong^*  J. Wilcox  Md. S. Islam  S. L. Brown  X. Wu  B. Valliyodan  D. A. Sleper  J.G, Shannon  H.T. Nguyen             
Affiliation: University of Missouri-Columbia
Abstract:

Soybean cyst nematode (SCN) is the most destructive pest of soybean in the United States. Host plant resistance is an effective approach to control SCN. The continuation of growing the same resistant cultivar(s) could result in SCN population shifts and loss of SCN resistance. Among soybean plant introductions (PI) evaluated for SCN resistance, a few PIs have been reported to be highly resistant to multi-HG types of SCN. The objectives of our research were: to confirm previously reported quantitative trait loci (QTL); to identify novel QTL; and to profile soybean root-related transcription factors (TFs) to identify molecular switches regulating SCN resistance in the resistant PIs. Two mapping populations of F2:3 lines derived from the crosses of Magellan (S) and PI 438489B (Pop1) and PI 567516C (Pop 2) (R) were phenotyped for six HG types (PA1, -2,-3, -5, -14, and TN19) and genotyped with simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers. Genetic linkage maps were constructed using the JoinMap program. Following the normality test of the female index (FI, %), permutation tests and composite interval mapping (CIM) were performed to identify and map QTL using the MapQTL program. Repeated greenhouse assays were initiated to collect root samples inoculated with J2 juvenile nematodes for TFs profiling. Results of QTL analysis showed that in addition to previously reported QTL, three novel QTL associated with resistance to multi-HG types of SCN were mapped on LGs C1 (Pop 1), -G and -O (Pop 2). These results are in agreement with the previous report of broad-spectrum resistance of PI 438489B and PI 567516C. Studies using F7 recombinant inbred lines (RILs) developed from these populations to confirm the novelty of these QTL and soybean root-related TFs profiling are in progress.

Presentation Type: Poster
Title: Genomic Analysis of a Seed Protein QTL Region on Soybean LG I
Author(s): Bindu Joseph^*  Yung-Tsi Bolon  Brian Diers  Steven Cannon  Michelle Graham  Andrew Farmer  Greg May  James Specht  Nathan Weeks  Nevin Young   Perry Cregan  Carroll Vance  Randy Shoemaker     
Affiliation: Dept. of Agronomy, Iowa State Univ., Ames, IA-50011, USA
Abstract:

A seed protein QTL mapped to an interval between Satt239 and Satt496 on LG I (Chung et al, 2003) increases the seed protein content up to 2 percent when homozygous for the high protein allele. A genomic analysis of the LG I QTL region was undertaken to understand the genetic basis of seed protein variation in soybean. Physical mapping of the QTL region was carried out and a minimal tiling path (MTP) of eleven BACs was identified. Eight BACs from the MTP were sequenced. Alignment of the BAC sequences onto the preliminary sequence of the soybean genome followed by SSR analysis defined the LG I QTL region to be approximately 8.5 M bases, spanning between the SSR markers Sat_174 and Satt354. Identification of candidate genes within this region and verification of the candidacy of these genes is underway. The homoeologous region of the LG I QTL region was determined to be on LG O, which is also reported to contain a weak seed protein QTL. Approximately 50 percent of the predicted genes in the LG I QTL region had duplicated copies on the corresponding LG O region. Expression profiling of the conserved genes in the LG I and the LG O regions utilizing soybean EST database as well as seed derived transcripts from a pair of near-isogenic lines contrasting in seed protein and oil is in progress.

Presentation Type: Poster
Title: Temporal Analysis of Protein Phosphorylation Changes in the Soybean Seed Proteome after Pod Detachment
Author(s): Norma Houston^*  Ganesh Agrawal  Jay Thelen                         
Affiliation: University of Missouri – Columbia, Department of Biochemistry
Abstract:

Partitioning of photosynthate among carbohydrates, oils, and proteins within the developing seed is of agronomic interest. To better understand the role of protein phosphorylation in response to photosynthate supply, global quantitative phosphoproteomics was performed on developing seed from detached soybean pods. Soybean pods, 28 days after flowering, were detached from the stem in biological triplicate and seeds were harvested at 0, 6, 24, and 48 hours after pod excision. Whole seed proteins for each harvested time point were isolated under denaturing conditions, and fractionated by high-resolution two-dimensional gel electrophoresis. Phosphoproteins were detected in gel using Pro-Q Diamond stain followed by laser imaging. Gels were then stained with colloidal Coomassie to visualize total protein. Spot detection and quantitation was performed using ImageMaster software. Differential expression (p-value <0.05) was established for 253 phosphoproteins and 458 non-phosphoproteins based on statistical significance of protein expression changes between the control (0 hours) and at least two experimental (6, 24, 48 hours) samples. To date, 40 phosphoproteins and 101 non-phosphoproteins have been identified by LC/MS-MS. Among the differential proteins, a total of seven glycolytic enzymes were identified, five of which are phosphoproteins. One such glycolytic phosphoprotein, triose-phosphate isomerase, showed a six-fold increase six hours after pod excision and decreased to twice the control level by 24 hours. Collectively, these data suggest regulation of photosynthate assimilation and partitioning in seed may be more complex than previously thought.

Presentation Type: Poster
Title: Effects of genetic and environmental factors on accumulation of the important bioactive compounds in soybean seeds.
Author(s): Anatoliy Lygin^*  Ibrahim Moussa  Rendall Nelson  Jack Widholm  Vera Lozovaya                     
Affiliation: UIUC
Abstract:

We studied the effects of temperature and soil moisture conditions on concentrations, composition and ratios of isoflavones, saponins, spingolipids and sterols in the seeds of French soybean cvs. Imari and Queen and U.S. cvs. Jack and Loda. These compounds potentially can be beneficial for human and animal health and improve soybean profitability.
We found that concentrations of these compounds significantly vary among the seeds of different cvs. and are affected by environmental conditions. Total phytosterol contents were higher in the French cvs. than in the U.S. cvs. under all treatments with sitosterol being the major sterol in all genotypes. We also found an increased accumulation of sitosterol and total phytosterols under stress conditions of high temperature and drought. Plant sterols in the human diet are beneficial since they have a cholesterol-lowering effect due to both lowered cholesterol synthesis and intestinal absorption of cholesterol, and the cholesterol-lowering effect of plant stigmasterol. Identification of genotypes with enhanced accumulation of phytosterols in seeds could allow breeders and genetic engineers to combine desirable traits to produce soybeans with enhanced health promoting properties. Data on the variation in seed concentration of the important bioactive compounds isoflavones, sphingolipids, and saponins in different genotypes and under different environmental conditions will be also presented.
This study was supported in part by funds from the CPBR, Agrofresh, Inc., Illinois Soybean Program Operating Board, the Illinois Soybean Disease Biotechnology Center, the Illinois Agricultural Experiment Station, and the USDA Agricultural Research Service.

Presentation Type: Poster
Title: Building bridges between the disparate worlds of soybean breeding and soybean genomics.
Author(s): Jason Gillman^*  Garcia Arturo  Bilyeu Kristin                         
Affiliation: USDA-ARS
Abstract:

With the recent release of the initial draft of the soybean genomic assembly (Glyma0, http://www.phytozome.net/soybean.php) a new realm in soybean research has opened up. Although great progress has been made in the assembly of the genomic information into a format accessible to the soybean community, it is currently limited to supercontigs. While possible to search small sequences using the BLAST algorithm, there is currently no information present on the site indicating to which linkage group a supercontig belongs. As a result, the genomic information is currently of little use in plant breeding efforts. In essence, the physical sequence information is disconnected from the existing genetic map. In order to remedy this problem, we utilized reiterative BLAST searches, using known genetic markers, to assign the current supercontig structure to linkage groups. The aim of this work is to correlate genomic supercontigs with known linkage group-specific genetic markers; in essence, connecting the genetic and physical maps. As an example of this approach, we will present the genomic location, supercontig regions and linkage groups corresponding to the 4 FAD3 (fatty acid desaturase 3) genes present in the soybean genome.

Presentation Type: Poster
Title: RAPD markers for rust resistance in Brazilian soybean cultivars
Author(s): Marcelo Marchi  Sandra Trevisoli  José Pinheiro  Romeu Khiil  Eberson Calvo  Antonio Di Mauro^*                   
Affiliation: UNESP-Brazil
Abstract:

RAPD markers for rust resistance in Brazilian soybean cultivars - Molecular markers linked to resistance genes for diseases are very important mainly when associated to assisted selection. Thus, the present work aimed to detect RAPD markers linked to one soybean rust resistance locus. Two populations from PI 459025 x COODETEC 208 cross were obtained. In the F2 and F3 generations, these populations were artificially inoculated and evaluated for the Phakopsora pachyrhizi reaction, by the lesion type classification (RB – resistant and TAN – susceptible). Using the phenotypic results, the Bulked Segregant Analysis was performed for two DNA bulks from homozygous resistant and homozygous susceptible plants, respectively. From the 600 random RAPD primers analyzed, three of them identified polymorphic fragments between contrasting bulks and parents. Chi-square test confirmed the monogenic with complete dominance segregation for the pathogen resistance and the 3:1 segregation of bands presence for the markers. The three markers are linked to the resistance locus in repulsion phase and located at 4.5, 6.2 and 10.2 cM from it, so they are promising in assisted selection for soybean rust resistance. We now are developing SCAR marker for this gene.

Presentation Type: Poster
Title: Identification of SNP markers in differentially expressed genes for Iron Deficiency Chlorosis.
Author(s): Greg Peiffer^*  Jamie O'Rourke  Marie Romero  Michelle Graham  Randy Shoemaker                     
Affiliation: Iowa State University
Abstract:

Within calcareous soils of the upper Midwest, soybeans have a production disadvantage when they are unable to invoke an iron (Fe) deficiency stress response. Affymetrix GeneChip data for soybean showed that there are over 200 differentially expressed genes between Clark and a Clark isoline, isolines developed for their response to Fe. Eight Fe QTLs have been mapped on the 20 soybean linkage groups. However most of the differentially expressed genes don’t align with the QTL. This leads us to believe that transcription factors that act upon these genes may reside in the QTLs and have specific binding activity that differs between Fe efficient and inefficient cultivars. To test this we identified the transcription factors found by a microarray study and predicted their full sequence by aligning the corresponding ESTs with the 7x genome scaffolds and by analysis with FGENESH (softberry.com) to determine their promoter regions. Two primer pairs were made for each candidate transcription factor, one pair spanning the promoter region and the other spanning at least one exon. Primer pairs were used to amplify the targeted regions across eight genotypes, each differing in their response to IDC. The amplified regions from each genotype were then aligned and potential SNPs were identified. These SNPs will then analyzed using Sequenom’s iPlex genotyping system. SNPs associated with efficiency or inefficiency may then be used by breeders in field studies.

Presentation Type: Poster
Title: Re-annotation of the physical map of Glycine max for polyploid-like regions by BAC end sequence driven whole genome shotgun read assembly
Author(s): Navinder Saini  Jeffry Shultz  David Lightfoot^*                         
Affiliation: Biotech Center, Jabalpur, India
Abstract:

The soybean genome is composed of approximately four thousand interspersed homeologous regions with 1, 2 or 4 copies per haploid genome. The genome has been sequenced by whole genome shotgun sequence (WGS). Here the aim was to use BAC end sequences (BES) derived from three minimum tile paths (MTP) to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches. Results show that when sequence divergence was 1-10%, the copy number of homeologous regions could be identified from sequence variation in WGS reads overlapping BES. Homeolog sequence variants (HSVs) were single nucleotide polymorphisms (SNPs; 89%) and single nucleotide indels (SNIs 10%). Larger indels were rare but present (1%). Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5-10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed. The use of HSVs like SNPs and SNIs to characterize BACs will improve contig building methods. The data show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable deconvolution and positioning of sequence scaffolds (see BES_scaffolds section of SoyGD).

Presentation Type: Poster
Title: Both separate and common loci underlie the resistance inherited from Glycine max cultivar ‘Hartwig’ to three soybean cyst nematode populations.
Author(s): Kazi Samreen  Jeffry Shultz  Jason Bond  Prakash Arelli  David Lightfoot^*                     
Affiliation: SIUC
Abstract:

Cultivars vary in their resistance to different HG Types (populations) of the soybean cyst nematode (SCN), Heterodera glycines. The loci for resistance to H. glycines HG Type 1.3- (race 14) and HG Type 1.2.5 (race 2) of the soybean cyst nematode may vary in their locations. The aims were to compare the inheritance of resistance to three nematode HG Types in a population segregating for resistance to SCN and to identify the underlying quantitative trait loci (QTL). ‘Hartwig’, a soybean cultivar resistant to most SCN HG Types was crossed with the susceptible cultivar ‘Flyer’. A total of 92 F5-derived recombinant inbred lines (RILs) and 144 molecular markers were used for map development. The rhg1 associated QTL found in earlier studies were confirmed and shown to underlie resistance to all three HG Types in RILs (Satt309; HG Type 0, P= 0.0001 R2 = 22%; HG Type 1.3.., Satt275, P = 0.001, R2 = 14%) and NILs (Satt309; HG Type 1.2.5, P= 0.001 R2 = 24%). A new QTL underlying resistance to HG Type 1.2.5-was detected on LG D2 (Satt574; P= 0.001, R2 = 11%) among 14 RILs resistant to the other HG types and was confirmed in a small NIL population consisting of 60 plants of ten genotypes (P= 0.04). This QTL is located in an interval previously associated with resistance to both SDS leaf scorch and SCN HG Type 1.3.6.7. The QTL detected will allow marker assisted selection for multigeneic resistance to complex nematode populations in combination with other traits.

Presentation Type: Poster
Title: Proteome Analysis of In Soybean Near Isogenic Lines (NILs) Polymorphic At The rhg1 Resistance Locus Challenged With Heterodera glycines, The Soybean Cyst Nematode (SCN)
Author(s): Ahmad Afzal  Navinder Saini  Aparna Natajaran  Javed Iqbal  David Lightfoot^*                     
Affiliation: SIUC
Abstract:

Yellow dwarf disease cause by Heterodera glycines I., the soybean cyst nematode (SCN), is the most devastating soybean disease. Host resistance requires the resistance allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cell formed at the nematode feeding site about 24-48 h after feeding commences. Little is known about how the host response to infection is mediated. This study aimed to identify proteins that increased in abundance due to SCN infection in both resistant and susceptible near isogeneic lines that contrasted in their rhg1 alleles (NILs) . Using 2D gel electrophoresis, proteins extracted from near isogenic lines of soybean were resolved. Gel image analysis showed more than 1,000 resolved spots in the pH 3–10 range. Differentially abundant proteins were trypsin digested and analyzed using quadruple TOF (Q-TOF) tandem mass spectrometry. Positive protein identifications for 24 of the 30 gel spots corresponded to 28 different proteins. The identified proteins were grouped according to function. Most proteins were primarily implicated in signal sensing and mechanisms underlying disease resistance. The proteins included novel players in the SCN defense cascade. Together with genetic data the evidence suggests the rhg1 locus contains a cluster of genes that control different parts of the response to SCN.

Presentation Type: Poster
Title: Identification of an active endogenous transposon from the W4 locus in soybean
Author(s): Madan Bhattacharyya^*  Min Xu  Hargeet Brar  Sehiza Grosic  Reid Palmer                     
Affiliation: Iowa State University
Abstract:

In soybean [Glycine max (L.) Merr.], W4 is one of the loci that control anthocyanin biosynthesis in flowers and hypocotyls. A putative transposable element was suggested to reside within or adjacent to this locus in the mutable T322 line resulting in the w4-m allele. We have shown that the W4 locus encodes the dihydroflavonol-4-reductase 2 (DFR2). The w4-m allele in T322 is caused by the insertion of a CACTA-like transposable element, Tgmw4m, in the second intron of DFR2. Terminal inverted repeats (TIR) of the element are similar to those of other soybean transposable elements, e.g. Tgm family and Tgm-express1. We investigated footprints of several germinal revertant plants with purple flowers generated from a single progenitor mutable T322 plant. Distinct footprints were observed among the revertant plants. This result confirmed that excision of the element is responsible for gaining function of DFR2 among the germinal revertants with purple flowers. The w4-dp (dilute purple flowers) and w4-p alleles (pale flowers) were isolated as germinal revertants from the mutable T322 line. Both alleles mapped to W4. Sequencing of the promoters revealed that following excision from DFR2 intron 2, Tgmw4m inserted into the promoter of DFR2 in these functional mutant alleles. Two putative cis-elements, CCAAT motif and E-box (CACGTG) positioned at -1080 and -1068 were interrupted due to Tgmw4m insertion in these two alleles resulting in altered cell-specific DFR2 expression patterns in these functional mutants. Tgmw4m contains a transposes with high identity to that of En/Spm-type transposons isolated from maize. We investigated the extent of transposition events among the progenies of two sister progenitor T322 mutable plants. Approximately 40% of the families descended from a mutable plant contain one or more revertant plants with only purple flowers. We will discuss the applicability of this highly active endogenous transposable element in functional analyses of soybean genes.

Presentation Type: Poster
Title: SEQUENCE COMPARISON OF HOMEOLOGOUS REGIONS OF SOYBEAN AND LEGUME SPECIES REVEALS RAPID GENOME EVOLUTION WITHIN SYNTENIC REGIONS IN LEGUME SPECIES
Author(s): Jer-Young Lin^*  Christian Hans   Scott Jackson                         
Affiliation: Department of Agronomy and Purdue Genetics Program, Purdue University, West Lafayette, IN 47907, USA
Abstract:

Soybean has undergone at least two rounds of whole genome duplication followed by diploidization resulting in mosaic of duplicated segments. In order to understand the impact of polyploidy on soybean genome structure, two homeologous regions totaling about 2Mb belonging to chromosome 15 and chromosome 8 were sequenced to reveal the level of variation between homeologous regions in soybean. The large-scale rearrangement of mosaic phenomenon (rearrangement breakpoints) derived from the diploidization process was found within this 1Mb window. High levels of micro-synteny were observed although duplications, inversions and indels between the two homeologous regions were
also found. Another tropical season legume species, Phaseolus vulgaris (common bean) ~19 MY diverged from soybean, was chosen and the corresponding orthologous region was defined and sequenced. Extensive gene colinearity phenomenon was observed among both homeologous regions in soybean and the corresponding region from Phaseolus. An even longer range comparison was made to another legume species, Medicago truncatula, which is ~50 MY diverged from either Phaseolus or soybean. Obvious synteny was observed for about half of the orthologous region (500 Kb) among tropical season legume species. Our results demonstrated the effects of genome duplication, genome shuffling and gene retention/loss at the molecular level on legume genome evolution.

Presentation Type: Poster
Title: Identification of an active endogenous transposon from the W4 locus in soybean
Author(s): Hargeet Brar  Madan Bhattacharyya^*                           
Affiliation: Iowa State University
Abstract:

Sudden death syndrome (SDS), caused by Fusarium virguliforme (Fv), is a serious fungal disease of soybean. Fv attacks only the roots. The pathogen has never been identified from the above ground parts of the diseased plants. Therefore, it has been hypothesized that foliar symptoms are caused by the translocation of a phytotoxin(s) released by the fungus to the roots. Cell-free Fv culture filtrates cause foliar SDS symptoms, known as leaf scorch. Treatment of cell-free Fv culture filtrates with an endolytic protease, Proteinase K, abolished the phytotoxic activity of cell-free Fv culture filtrates. This suggests that the phytotoxin(s) is proteinaceous. To identify the specific protein that causes leaf scorch, cell-free Fv culture filtrates were separated by gel filtration and then by native-polyacrylamide gels electrophoresis. A low molecular weight protein (~13.5 kDa) was shown to produce leaf scorch. Mice monoclonal antibodies were generated against the proteinaceous phytotoxin. Antibodies were used to screen an Fv cDNA library. A cDNA clone (Fv Tox1) encoding an unknown ~14 kDa protein was identified. The proprotein encoded by the cDNA contains a signal peptide which is cleaved from the mature protein identified from the cell-free Fv culture filtrates. The FvTox1 gene (both mature and proprotein forms) was cloned into Escherchia coli expression vector pRSET and expressed using IPTG. The E. coli expressed proteins are currently being tested for production of SDS symptoms in soybean seedlings.

Presentation Type: Poster
Title: Analysis of the activity of the soybean laccase encoded within the Rfs2/rhg1 locus for resistance to SDS and SCN
Author(s): Ahmad Afzal  Rubina Ahsan  Khalid Meksem  Javed Iqbal  David Lightfoot^*  Aziz Jamai                   
Affiliation: SIUC
Abstract:

Resistance to soybean (Glycine max (L) Merr.) SDS caused by Fusarium virguliforme was underlain by several QTL. Four QTL clustered on linkage group G (QRfs 1-4). QRfs2 was fine mapped and DNA sequences of BAC clones from soybean cultivars “Forrest” and “Asgrow 3244,” sequenced. The QRfs2 locus was clustered with, or pleiotropic to, the Rhg1 locus for resistance to soybean cyst nematode (SCN; Heterodera glycines). A group of five genes were found within the Rfs2/rhg1 locus. One encoded an unusual diphenol oxidase (laccase; EC 1.10.3.2). The aim of this study was to characterize this member of the soybean laccase gene-family. Sequence comparisons showed the protein coding regions were identical in resistant cultivar Forrest and susceptible cultivars Asgrow 3244 and Williams 83.. However, potentially significant differences were present in the 6 of the 7 introns and the promoter regions. Transcript abundance (TA) was examined in a set of recombinant inbred lines (RILs) and cultivars that varied for resistance to SDS using roots at 10 days post-inoculation. However, there was no significant correlation between TA and SDS resistance among genotypes contrasting for the Rfs2/rhg1 locus . Expression of the protein in yeast pYES2/NTB expression vector yielded the predicted 70.3 kDa protein. The variant nature of the laccase was shown by the weak activity with common substrates. Although some laccases are involved in cell wall fortification by increasing the lignin synthesis, our studies indicate that mere presence of the laccase at Rfs2/rhg1 locus does not make it a strong candidate gene for QRfs2.

Presentation Type: Poster
Title: Towards Isolation of an Arabidopsis Non-host resistance gene that confers immunity against the Soybean pathogen Phytophthora sojae
Author(s): Rishi Sumit  Devinder Sandhu  Min Xu  Madan Bhattacharyya^*                       
Affiliation: Iowa State University
Abstract:

Phytophthora sojae is one of the most destructive soybean pathogens that causes stem and root rot disease. Arabidopsis thaliana is immune to this pathogen because of nonhost resistance mechanism. Recently, the Arabidopsis pen1 mutant in the ecotype, Columbia with a mutation in a syntaxin protein showed enhanced penetration by the barley powdery mildew pathogen, Blumeria graminis f. sp. hordei. pen1 is also penetrated by the soybean pathogen Phytophthera sojae. The ecotype, Columbia is otherwise immune to P. soaje. We have remutagenised the Arabidopsis pen-1 with the chemical mutagen, ethyl mthane sufonate (EMS). More than 3,000 individual M2 families were harvested from EMS-treated M1 plants. From an extensive screening of the mutant families and their selfing generations led to identification of 30 putative mutants. One of the Phytophthora sojae susceptible (pss) mutants, pss1 was crossed to ecotype Niederzenz. The F2:3 families developed from individual F2 plants of this cross were leaf inoculated to identify homozygous and heterozygous resistant F3 families and homozygous susceptible families. The F2:3families segregated in a 1:2:1 :: Resistant Homozygous : Resistant Heterozygous : Susceptible Homozygous ratio suggesting single gene inheritance for the mutated gene. These data also suggested that pss1 functions independently of pen1. Homozygous resistant and susceptible families were individually bulked to identify linked molecular markers. Currently, we are in the process of mapping the pss1 gene.

Presentation Type: Poster
Title: Expression of the Pleurotus ostreatus (oyster mushroom) ∆9 desaturase in Glycine max, Nictotiana tobacum and Arabidopsis thaliana.
Author(s): Sreenivasa Rao TJV^*  Tara Burke  Rao Suryadevara  David Hildebrand                       
Affiliation: University of Kentucky
Abstract:

A Pleurotus ostreatus (P.ostreatus) ∆9 desaturase gene encoding an enzyme with activity to palmitoleic acid containing substrates was cloned and characterized. The P.ostreatus is an edible basidiomycete of high nutritional value for its high level of vitamins and proteins and unsaturated fatty acids. The P. ostreatus is produced industrially for applications such as the manufacture of paper pulp, cosmetics and in the pharmaceutical industry and it is also reported to have anti-cancer properties. Desaturases that convert palmitate to palmitoleate are useful for improving the health value of foods. We are interested in the utility of P. ostreatus desaturase in improving vegetable oils. Recently there has been a considerable interest in increasing the palmitoleic acid (16:1, Δ9) content in diets for health purposes. Palmitoleic acid is reported to lower the risk of cardiovascular and cerebrovascular diseases. Palmitoleic acid is also more desirable than the main mid-chain saturated fatty acid in many vegetable oils, palmitic acid (16:0). Very high palmitoleic acid oil might have industrial value. Yeast ole1 mutant transformants expressing the P. ostreatus Δ9 desaturase have shown high levels of palmitoleic acid (16:1). The P. ostreatus Δ9 desaturase gene was cloned under a seed-specific promoter and the same gene have also cloned separately under a constitutive promoter and placed into a plant transformation vector. We have also cloned the Δ9 fatty acid desaturase gene from Saccharomyces cerevisiae under a seed specific promoter and a constitutive promoter as a control for the P. ostreatus work. We have introduced the entire gene constructs as a whole plasmids or linearized DNA fragments into soybean, tobacco and Arabidopsis through Biolistic and Agrobacterium transformation methods and the effects on fatty acid composition will be presented.

Presentation Type: Poster
Title: Development of SNP markers for the soybean aphid resistance gene Rag1 and fine mapping this gene in soybean
Author(s): Ki-Seung Kim^*  Karen Kaczorowski  Matthew Hudson  Brian W. Diers                       
Affiliation: University of Illinois
Abstract:

The soybean aphid [Aphis glycines Matsumura] is an important soybean [Glycine max (L.) Merr] pest in North America. Although a single dominant gene Rag1 was mapped to soybean LG M in the cultivar ‘Dowling’, there was a large gap between markers and the gene. Therefore, the development of additional genetic markers more closely linked to Rag1 are needed to accurately position the gene which will improve the effectiveness of marker assisted selection (MAS) in developing soybean aphid resistant varieties. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the region of Rag1 and to position these SNPs using a large population of BC4F2 plants from a cross between Dowling and susceptible cultivar Dwight. 808 BC4F2 plants were screened with Satt463 and Satt540, which were believed to flank the gene. 111 plants showing recombination between these markers were harvested. Hybridization of genomic DNA onto Affymetrix soybean GeneChip microarrays and sequencings of Dowling and Dwight for the Rag1 region were done to discover SNPs. 42 SNPs, 5 small insertions/deletions (InDels), and 10 recombinations were identified in the region of Rag1. Based on aphid tests and SNP genotyping for each recombinant line, Rag1 was located in a 115kb region between two SNP markers, 4338 and 56B. These efforts have resulted in the identification of markers that are closely linked to Rag1 that can be used in MAS for aphid resistance and have narrowed the interval containing the gene. This information is being used in efforts to clone this gene.

Presentation Type: Poster
Title: Development of SNP markers for the soybean aphid resistance gene Rag1 and fine mapping this gene in soybean
Author(s): Ki-Seung Kim^*  Karen Kaczorowski Kaczorowski  Matthew Hudson Hudson   Brian W. Diers