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:
This project relates to the parent project’s Objective 2: “Develop methods to facilitate accelerated breeding for adaptive traits and utilization of germplasm diversity in barley and oat.” 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 message 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 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 single nucleotide polymorphism (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, 2, 3 and 4 have been accomplished. We sequenced expressed sequence tags (ESTs) from 20 diverse oat lines representing North American oat lines and compared them to each other. We also sequenced DNA sequences or markers known as 'DArTs' (Diversity Arrays Technology) and compared them to each other. A new technological approach, genotype-by-sequencing (GBS), has been used to sequence oat genomic DNA. Through comparison of all of these sequences, we identified DNA changes or SNPs in DNA sequence of 20 oat lines. We genotyped six oat populations and 32 oat lines to validate these changes. We achieved our milestone of 3,072 oat-based SNP markers that have been used to generate a high-throughput SNP array (Illumina®) for use in genetic studies and marker-assisted breeding of key oat traits. This array was called 6K oat chip or Infinium iSelectHD oat custom BeadChip and it is currently available to oat researchers and oat breeders at Illumina, Inc. Catalog # WG-401-1001. Using a new strategy developed by the USDA ARS Aberdeen molecular genetics laboratory, these SNP markers were physically anchored to the appropriate oat chromosomes and these results were published in a scientific journal. This work produced a more precise “road map” of the oat genome and provided a specific tool for breeders to determine various traits in oat populations. Currently, as a part of Sub-objective 5, we identified sequences for genes controlling soluble and insoluble oat fiber. We sequenced all three alleles of the CslF6 gene, which is responsible for production of beta glucan or soluble fiber. We also sequenced the CslC9 gene, which is responsible for production of xyloglucan or insoluble fiber. We sequenced the CslA7 gene, which is responsible for production of glucomannan and the CslH1 gene, which is responsible for production of beta glucan. We sequenced the CslF6 and CslC9 genes from an oat mutation population, which was followed by chemical tests measuring the levels of beta glucan in these mutant oat lines. We discovered oat mutant lines with higher beta glucan levels than the original oat line. Our goal was to validate our hypothesis stating that oat lines with a mutation or a change in CslF6 would have different level of beta glucan content, which is our Sub-objective 6. Sub-objective 6 is currently underway as function of various CslF6 alleles are being evaluated by using CslF6 alleles from different oat genomes for transformation of Arabidopsis, a model plant system. These experiments will validate function of different CslF6 alleles in oats in regards to the production of beta glucan in oat grain.