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 economic value, such as high yield. Each trait is influenced by many different genes or DNA sequences. During gene expression, the DNA sequence is used as a template to produce a message RNA (mRNA) which is used, in turn, as a template to synthesize a protein. Each protein plays a specific role within the oat plant, such as in the synthesis of beta glucan, an important dietary fiber with beneficial effects on human health. Changes in DNA sequence may cause changes in protein level or function which may influence the levels of key traits. A difference among oat lines in DNA sequence known as a single nucleotide polymorphism (SNP) may also be used as a marker, or reference, in genetic studies. In this project, SNPs for oat genetic research and to aid in the development of new oat varieties were identified using comparison of both expressed-gene (mRNA) sequence and genomic DNA sequence. Sub-objectives 1, 2, 3 and 4 have been completed: RNA from 20 oat lines, selected to represent the genetic variation important to North American Oat Breeders, was extracted and used to construct line-specific libraries of DNA sequence. From these libraries, DNA sequence reads were assembled into representations of approximately 650,000 genes. Comparisons of gene sequences across oat lines were used to identify single nucleotide polymorphisms (SNP) as potential genetic markers in oat breeding. The DNA sequences of markers known as Diversity Arrays Technology (DArTs) were also compared across lines. Over 3,072 SNP markers were identified from these sequence sources. SNP sequences were used to develop a high-throughput genotyping assay, the Infinium iSelectHD oat custom BeadChip (Illumina®), for use in genetic studies and marker-assisted breeding for important oat traits. Whole-genome DNA of the same 20 oat lines was used in an additional approach to SNP identification called genotype-by-sequencing. In this approach fragments of genomic DNA were sequenced and aligned for comparison across oat lines. Over 200,000 DNA variations were identified through this process. SNPs were validated using 6 oat genetic mapping populations. Through comparison of these oat maps, over 3,000 SNPs have been assigned a position on the oat genetic map, physically anchored to the appropriate oat chromosome. Sub-objective 5 has been completed and sub-objective 6 has been partially completed: Four specific genes - CslA7, CslC9, CslF6 and CslH1 - controlling production of beta glucan were chosen for in-depth study. Comparison of these sequences resulted in the discovery of key variations increasing beta glucan levels. In addition, a population of 15,000 mutant oat plants was developed and screened for mutations within the 4 selected genes. Mutant lines were then characterized for changes in soluble fiber levels. Through this process, an oat line was identified with altered beta glucan levels due to a mutation in the CslF6 gene. CslF6 allele function is being further validated by transforming Arabidopsis, a model plant system, with CslF6 alleles from different oat genomes. This research has increased the number of sequenced oat genes by 21-fold and has created a sequence resource used to develop new oat genetic markers. A high-throughput genotyping assay was developed, the Infinium iSelectHD oat custom BeadChip, and is currently available through Illumina, Inc. Catalog# WG-401-1001. Thousands of additional SNPs have been identified using genotyping by sequencing technology and, overall, more than 3,000 SNPs have been placed on a composite oat genetic map. This map is currently being finalized for use in whole-genome association analyses aimed at discovering regions of the oat genome that control key traits.