Genotyping for Species Identification and Diversity Assessment Using Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq).

Genotyping-by-sequencing (GBS) is a powerful approach for studying the genetic diversity of legume species. By using restriction enzymes or other methods to generate a reduced representation of the genome for sequencing, GBS can provide genome-wide single nucleotide polymorphisms (SNP) for diversity analysis at high throughput and low cost. Here we describe a novel double-digest restriction site-associated DNA sequencing (ddRAD-seq) approach. We also describe the downstream bioinformatic analysis of the sequencing data, including alignment to a reference genome, de novo assembly, SNP calling, phylogenetic analysis, and structure analysis.