Disentangling sources of gene tree discordance for Hordeum species via target-enriched sequencing assays

Hordeum is an economically and evolutionarily important genus within the Triticeae tribe of the family Poaceae, and contains 33 widely distributed and diverse species which cytologically represent four subgenomes (H, Xa, Xu and I). These wild species (except Hordeum spontaneum, which is the primary gene pool of barley) are secondary or tertiary gene-pool germplasms for barley and wheat improvement, and uncovering their complicated evolutionary relationships would benefit for future breeding programs. Here, we developed a complexity-reduced pipeline via capturing genome-wide distributed fragments via two novel target-enriched assays (HorCap v1.0 and BarPlex v1.0) in conjugation with high-throughput sequencing of the enrichments. Both assays were tested for genotyping 40 species from three genera (Hordeum, Triticum, and Aegilops) containing 82 samples 67 accessions. Either of both assays worked efficiently in genotyping, while integration of both assays can significantly improve the robustness and resolution of the Hordeum phylogenetic trees. Interestingly, the incomplete lineage sorting (ILS) was inferred for the first time as the major factor causing phylogenetic discordance among the four subgenomes, whereas in New World species (carrying I genome) post-speciation introgression events were revealed. Through revising the evolutionary relationships of the Hordeum species based on an ancestral state reconstruction for the diploids and parental donor inference for the polyploids, our results raised new queries about the Hordeum phylogeny. Moreover, both newly-developed assays are applicable in genotyping and phylogenetic analysis of Hordeum and other Triticeae wild species.