Below is a copy of the letter that is sent to users with their seed request. The letter explains how the mutants were generated and what users need to do to genotype and propagate the mutant lines. If you have any additional questions, please email maizetilling@purdue.edu.

     TILLING mutant seed stocks are currently housed at Purdue University. For each mutant, the TILLING Search Page and MaizeGDB have a link to a web-based order stock form. There is no charge for the seed, but we do ask the requestor to pay for the shipment (via 2-day Federal Express).

     In order to be sure that the mutant allele is still present in the line, we need to have least 250 g of bulked M3 seed from 8 or more successful pollinations. There are some lines that fell short of this target and have been designated "Contact maizetilling" because there is a slight chance that the mutant allele may be under-represented.

November 2, 2007

     This letter provides information about our TILLING inbred lines and how to obtain seed stocks. We have several EMS mutant populations and the details of each are described below. We are in the process of transferring the Maize TILLING seed stocks to the Maize Co-Op and having links for those stocks listed on the Maize GDB website so that you can just "click and order".

     If you are a TILLING user and wish to obtain seed for the mutations that were discovered in TILLING, please use the web-based ordering form that is accessible by clicking the "order seed stocks" on the TILLING results page (the link to this page was provided in the "Results email").

     If you are requesting seed for mutations that are now publically available or if you have any questions, please contact maizetilling@purdue.edu and we will let you know where the seed stocks are currently located and provide you with information for ordering the seed stocks from the Maize Co-Op.

     The EMS-mutagenized lines for TILLING were created in B73 and W22. Three of the W22 populations (03IN, 03HI, 04IN) harbor kernel markers from chromosome 9S (colorless, shrunken, bronze and waxy). The W22 male (mutagenized pollen) had the following genotype: c1, Sh1, Bz1/c1, Sh1, Bz1. The W22 female's genotype was C1, sh1, bz1, wx/C1, sh1, bz1, wx. Any W22 seed that you receive will be segregating purple, bronze and yellow kernels as well as shrunken and/or waxy kernels.

     One of our W22 populations, 05IN-W22TB, was generated by Tom Brutnell's laboratory as part of an EMS-screen for mutations that affect Ac transposition. These mutant lines are homozygous for Ac-im and contain a Ds element at the r locus. The presence of Ac-im in these lines should not pose any issues with analyzing your EMS-induced point mutation. The kernels that you receive will have various color patterns including fully colored, spotted and completely colorless. For more information about Ac-im, please refer to this paper from the Brutnell Laboratory: Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of Dissociation elements in maize. Genetics. 2005 Dec;171(4):1999-2012.

     The inbred background that the mutant is from is denoted by several identifiers. Mutants that are in B73 will have NS, NW, or a grid number-location (ex. 047-A6) identifier in the "stock" name. In addition, the "obtain stock" name will have B73 listed in it. Mutants from the W22 background will have P, PW or a 4-5-digit number (ex. 2367) in the "stock" name, and W22 will be listed in the "obtain stock" name. Mutants from the Brutnell Laboratory's Ac-im containing population will have "TB" as an identifier.

     The tissue used for DNA isolation was harvested from the M1 plant that is heterozygous for a given mutation. Each M1 plant, representing a unique mutant line, was selfed-giving rise to the M2 seed. For each line, we planted out 40 M2 seed and then randomly intermated them (also called "chain-sibbing"). We then bulked or pooled the ears from the crosses (8-12 successful crosses) resulting in the M3 seed. This seed (M3 seed) is what will be distributed to our users. Because we have taken the mutant lines out two generations, any other mutations also present in the line should be segregating randomly. In some cases, we are still bulking up the M3 line and will send M2 seed instead.

     You will receive at 10-40 K for each mutant and you need genotype each seedling to determine if it is heterozygous, homozygous wild type or homozygous recessive for the mutation of interest. In addition, you will need to bulk up the mutant line (either by selfing and/or back-crossing) in order to have sufficient seed for all your experimental needs. This additional round of crossing should further segregate your mutation from other mutations present in the line. In the TILLING results email you received, there was a link to the PARSESNP report that indicates restriction sites gained or lost in the mutant. Please note that these restriction sites are based on the genomic sequence you entered into CODDLE. If the sequence you submitted was not from B73 or W22, these restriction sites may not be present in the mutant. Also, we have observed several sequence polymorphisms between B73 and W22 and in some cases, the sequence submitted to us by the user. We have notified users when there are many differences between the two inbreds, however it is a good idea to confirm the presence (or absence) of a given restriction site in both inbreds if you plan to use RFLP analysis to genotype your mutant plants. If you have questions about the B73 and/or W22 sequence for you target, please email maizetilling@purdue.edu and we will send you the sequences.

     We are in the process of curating the phenotype data for our mutant lines. Any feedback that you could provide (i.e. field notes and/or photographs) with respect to mutant phenotypes etc. would be greatly appreciated.

     The chart below lists the seed we are sending you. Please do not hesitate to contact us if you have any questions or need any additional information.

Sincerely,

Rita Monde