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)
cDNA libraries from 20 oat genotypes representing the genetic variation within N. American germplasm will be constructed (N = 64). The libraries will be sequenced using 454 GS FLX sequencing and the resulting information will be used to construct contigs which will be aligned to identify in silico SNPs. In addition, redundant DArT clones will be sequenced and alignments will be used to identify addition silico SNPs. From these sequences, 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 109 N. American cultivars and breeding lines, six mapping populations, and 32 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. In addition to this work, additional cDNA libraries from developing seeds will be constructed and sequenced. This infromation will be used to identify genes controlling soluble and insoluble fiber in oat. Once the transcripts from these genes are dissovered full length sequences will be obtained using comparative genomics. The sequences function will be validated by transformation of Arabidopsis and/or soybean. Further validation of the genes will be done using a tilling approach.
3. Progress Report
Soluble and in soluble dietary fiber are important constituents of whole oat grain. Of these fibers, beta-glucan has been shown to significantly impact human health particularly the risk factors leading to coronary heart disease. Understanding the variation within specific “blueprints” or sequences of genes controlling the construction of beta glucan and other fibers will allow target selection of lines with drastically increase fiber levels. The USDA-ARS Aberdeen has recently partnered with General Mills Inc. to elucidate the genetic blueprints of fiber development in various oat lines and to study the effects of small changes in the blueprints of genes controlling fiber development. To date, the blueprints of four specific genes controlling beta glucan production have been resolved and key variations increasing beta glucan levels have been discovered. Additionally, blueprints of three other gene families have been elucidated. A mutation population consisting of 15,000 mutants has been developed. The aforementioned genetic blueprints are now being screened in the population to identify variations. Once lines containing variations have been identified, seed from the plants will be evaluated for changes in fiber levels. This work will allow target development of high beta glucan lines possible in oat. This work directly relates to objective 3 of the current ARS Aberdeen project plan (5366-21000-024-00D) “Develop improved barley and oat cultivars meeting the needs of conventional and specialty markets for both dryland and irrigated production systems”. Project monitoring is accomplished through site visits, phone conversations, e-mail, and written reports.