Location: Plant Germplasm Introduction and Testing
2013 Annual Report
2. DNA plates will be shipped to the Institute for Genomic Diversity at Cornell University for sequencing.
3. Preparation of libraries for Next-Generation Sequencing (Elshire et al., 2011). Figure 1 shows the genotyping-by-sequencing (GBS) adaptors. A basic schematic of the protocol to be used for performing GBS is shown in Figure 2. Sequencing will be performed using Genome Analyzer II (Illumina, Inc., San Diego, CA) or similar. Raw sequence data is filtered, the DNA sequence aligned using The Basic Local Alignment Search Tool (BLAST) and SNPs called.
Genomic DNA libraries were prepared using a single enzyme and appropriate bar-coded adaptors that were developed at Cornell and published recently. After PCR amplification, the libraries were sequenced to an average depth of +1 Gbp per USDA accession. Following curation of the raw data, we called over 10,000 biallelic SNP loci that are represented in at least 50% of individuals sampled. Using 21,000 SNP loci, a preliminary STRUCTURE run suggests that the pea accessions fall into two clusters or subpopulations (K=2). Further analysis includes the reporting of such diversity measures as the number of subpopulations, Fst values/heterozygosity, observed and effective numbers of alleles, genetic distance, and phylogenetic trees. SNP loci were filtered based on number of individuals sampled at that locus, number of alleles, single SNP blocks or haplotypes, sequencing read depths, and alleles represented by a certain minor allele frequency. The processed, filtered SNPs will be used further for genome-wide association studies (GWAS). A manuscript is in preparation to further report the findings of this SCA. This progress contributes to the Objective 2 of the parental project: “Conduct genetic characterizations and phenotypic evaluations of genetic resources of the preceding crops and related wild species for priority genetic and agronomic traits”.