1a. Objectives (from AD-416)
The objectives of this work are to: i) sequence hundreds of thousands of expressed-tagged (EST) sequences from 20 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, iv) develop a high-throughput SNP array (Illumina®) for use in genetic studies and MAB oat key oat traits, v) identify sequences for genes controlling soluble and insoluble oat fiber, and vi) validate the function of each fiber gene discovered.
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
Portions of sequence were generated for genes expressed in oat lines representing the diversity important to North American oat breeders. The portions were assembled into representations of approximately 650,000 genes. This effort increased the number of known oat genes sequence 21-fold. The sequence resource has been used to develop over 700 new oat genetic markers to date. Communication and exchange of work has taken place via meetings, conference calls, and laboratory visits The project is funded through a TRUST agreement with General Mills Inc.