BarPlex v1.0: a multiplex PCR-based enrichment of genome-wide short segments that enable genetic studies in barley

Abstract

Over the past 15 years sequencing methodologies have advanced greatly enabling high-throughput sequencing based genotyping of crop plants. In this study, we developed BarPlex v1.0, a robust and cost-efficient genotyping approach in barley (Hordeum vulgare L.). In this multiplex PCR-based amplification of five-hundred genome-wide segments, followed by high-throughput sequencing of barcoded PCR products, we obtained hundreds to thousands of polymorphic markers. Comparison of genotyping with BarPlex v1.0 to genotyping-by-sequencing (GBS) revealed a similar genetic diversity. The polymorphic markers revealed by BarPlex v1.0 were highly accurate, with an average sequencing depth >700x and a data missing rate <0.5%. By analyzing 1,068 genotypes of wild barley (brittle rachis; Hordeum vulgare ssp. spontaneum L.), Tibetan semi-wild barley (brittle rachis), landraces, cultivars, as well as an F2 population, this assay has been robust in studies of population diversity, variety pedigree, heterozygosity discrimination, linkage mapping, as well as genome-wide association study (GWAS). Notably, a diversity analysis in a population of Tibetan semi-wild barley suggested a close relationship with Chinese landraces, but a dramatic decrease in its genetic diversity, inferring that Tibet was not a center of domestication for the native wild barley.