Genetic Diversity Analysis with 454 Pyrosequencing and Genomic Reduction Confirmed the Eastern and Western Division in the Cultivated Barley Gene Pool

Next-generation DNA sequencing (NGS) technologies can survey sequence variation on a genome-wide scale, but their utility for crop genetic diversity analysis is poorly known. Many challenges remain in their applications, including sampling complex genomes, identifying single nucleotide polymorphisms (SNPs), and analyzing missing data. This study presented a practical application of the Roche 454 GS FLX Titanium technology in combination with genomic reduction and an advanced bioinformatics tool to analyze the genetic relationships of 16 diverse barley (Hordeum vulgare L.) landraces. A full 454 run generated roughly 1.7 million sequence reads with a total length of 612 Mbp. Application of the computational pipeline called DIAL (de novo identification of alleles) identified 2578 contigs and 3980 SNPs. Sanger sequencing of four barley samples confirmed 85 of the 100 selected contigs and 288 of the 620 putative SNPs and identified 735 new SNPs and 39 new indels. Several diversity analyses revealed the eastern and western division in the barley samples. The division is compatible with those inferred with 156 microsatellite alleles of the same 16 samples and consistent with our current knowledge about cultivated barley. These results help to illustrate the utility of NGS technologies for crop diversity studies. The NGS application also provides a new informative set of genomic resources for barley research.