1a. Objectives (from AD-416):
The expected objective is an association mapping panel of 384 pea accessions genotyped for 20,000-plus SNPs based on genotyping-by-sequencing of the highly phenotyped pea core germplasm collection. The pea core population has been evaluated extensively for a variety of agronomic and morphological traits – seed weight, disease and pest resistances, stem and root traits, protein and macronutrient seed concentrations (Jermyn and Slinkard 1977; Kraft et al., 1998; Tedford and Inglis 1999; McPhee and Muehlbauer 1999, 2001; Malvick and Percich 1999; McPhee et al., 1999; Grunwald et al., 2003; Grusak et al., 2004, McPhee 2005; Coyne et al., 2005a). This high density genotyped panel can then be used for genome-wide association studies (GWAS) as demonstrated in a spring barley germplasm collection (Pasam et al., 2012) and for genomic selection (Heffner et a., 2010). This germplasm panel will be a valuable community resource (Mackey et al 2012). This pea core has been expanded from the refined pea core (Coyne et al 2005b) with the addition of Pisum subspecies, RIL mapping population parents, and snap pea lines.
1b. Approach (from AD-416):
1. The pea core population (384 accessions-adjusted with entries from Jim Myers) will be grown in the greenhouse. Leaf samples will be collected and DNA extracted into four 96-cell format plates using the Qiagen DNeasy kit. DNA concentrations will be determined using a fluorometer. Yields: 100 µl of 300-500 ng/µl DNA. 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.
3. Progress Report:
We have completed the genotyping of the USDA pea core collection (~384 accessions of cultivated pea) using libraries prepared and sequenced by high throughput next-generation sequencing (Illumina GAII). Principle findings are described in brief below. 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”.