1a. Objectives (from AD-416)
The objectives of this work are to: i) sequence hundreds of thousands of expressed-tagged (EST) sequences from 32 diverse genotypes representing the N. American oat germplasm, ii) sequence the remaining DArT clones from previous work, iii) develop approximately 1,536 to 3,072 oat-based SNP markers from the aforementioned sequences, and iv) develop a high-throughput SNP array (Illumina®) for use in genetic studies and MAB oat key oat traits.
1b. Approach (from AD-416)
Normalized etiolated seedling and immature floret cDNA libraries from 32 oat genotypes representing the genetic variation within N. American germplasm will be constructed.(N = 64). The libraries will be evaluated for sequence length and relationship to know cereal ESTs using standard cloning and sequencing methods. Once high quality libraries have been confirmed, they will be sequenced using 454 GS FLX sequencing. The sequence information will be used to construct contigs which will be aligned to identify in silico SNPs. In addition to the 454 sequencing effort, redundant DArT clones will be sequenced and alignments will be used to identify addition silico SNPs. Once a solid database of in silico SNPs has been assembled, sequences containing the best 1536 in silico SNPs will be sent to Illumina® for development of a pilot Oat Oligo Pool Assay (OOPA) panel via the Illiumina® Assay Design Tool. Pilot OOPA SNPs will be validated across 128 N. American cultivars and breeding lines, two mapping populations, and 76 aneuploid-hybrid stocks. To achieve the goal of at least 1,536 to 3,072 SNP markers for oat, a second pilot OOPA panel will be developed and validated as previously described. Documents Trust with General Mills, Le Sueur, MN. Log 37966.
3. Progress Report
The current genetic sequence information for cultivated oat is too small to develop a useful genetic marker resource. Such markers would greatly aid development of new oat varieties. In order to overcome this problem ARS Aberdeen has partnered with General Mills Inc. via a Trust agreement to develop the needed sequences and markers. Twenty-four oat lines, which represent the genetic variation useful to North American Oat breeders, were carefully selected. Short fragments for expressed genes, known as RNA, were extracted from each of the 24 oat lines. Using these fragments, line-specific libraries of DNA sequences were developed. The libraries are currently being sequenced. Once completed, thousands of oat gene sequences will be used to develop new oat markers. Frequent site visits were made along with conference calls and email correspondence