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:
Oats are crossbred to introduce traits of nutritional value, such as high fiber, or economical value, such as high yield. Each trait is influenced by many different genes or DNA sequences. Briefly, from each gene or from each DNA sequence, an expressed sequence or a messenger RNA (mRNA) is produced and this sequence is then used as a template to synthesize a protein. Each protein has a specific function to synthesize a specific product, such as the soluble fiber beta glucan. Any change in protein may cause a change in its function, so if a change causes an increase in a protein function that will result in an increase in a product. Each change, also known as a SNP, can be used as a marker or a reference for our genetic studies. Our overall objective is to identify these changes in oat DNA sequences and use this information for oat breeding. Sub-objectives 1 and 2 have been accomplished. We sequenced expressed sequences from 20 diverse oat lines representing North American oat lines and compared them to each other. Through comparison we identified DNA changes or SNPs in DNA sequence of 20 oat lines. Additionally, we screened six oat populations and 32 oat lines to validate these changes. We achieved the goal of 3,000 oat SNP markers for Sub-objective 3. Using a new strategy develop by the USDA ARS Aberdeen molecular genetics laboratory, these SNP markers were physically anchored to the appropriate oat chromosomes. This data will produce a more precise “road map” of the oat genome and this work will provide breeders a specific tool for desired traits in barley. Currently, as a part of Sub-objective 4, we developed SNP sequences for a high-throughput SNP assay, which will be used in genetic studies and marker assisted breeding of key oat traits. For Sub-objective 5, we identified sequences for genes controlling soluble and insoluble oat fiber. Specifically we sequenced all three alleles of CslF6 gene, which is responsible for production of beta glucan. We also sequenced the CslC9 gene, which is responsible for production of xyloglucan or insoluble fiber. We are currently sequencing CslF6 and CslC9 genes from an oat mutation population, which will be followed by chemical tests measuring the levels of beta glucan and xyloglucan in these mutant oat lines. Our goal is to validate the hypothesis that oat lines with a mutation or a change in CslF6 will have different level of beta glucan content, which is Sub-objective 6. Results of this project relate to Objective 1 of the parent project, “Develop, evaluate, and apply molecular tools, including molecular genotyping and transposon tagging, to small grains genetics and germplasm enhancement research”.