Genome sequences of the five <i>Sitopsis</i> species of <i>Aegilops</i> and the origin of polyploid wheat B-subgenome

Summary Bread wheat ( Triticum aestivum L., BBAADD) is a major staple food crop worldwide. The diploid progenitors of the A- and D-subgenomes have been unequivocally identified, that of B however remains ambiguous and controversial but is suspected to be related to species of Aegilops , section Sitopsis . Here, we report the assembly of chromosome-level genome sequences of all five Sitopsis species, namely Ae. bicornis, Ae. longissima, Ae. searsii, Ae. sharonensis , and Ae. speltoides , as well as partial assembly of Ae. mutica genome for phylogenetic analysis. Our results support that the donor of bread wheat B-subgenome is a distinct, probably extinct, diploid species that diverged from an ancestral progenitor of the B-lineage similar to Ae. mutica and Ae. speltoides . The five Sitopsis species have variable genome sizes (4.11-5.89 Gb) with high proportions of repetitive sequences (85.99-89.81%); nonetheless, they retain high collinearity with other wheat genomes. Differences in genome size are primarily due to independent post-speciation amplification of transposons rather than to inter-specific genetic introgression. We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding. These resources provide a new roadmap for evolutionary and genetic studies of the wheat group. Significance The origin of the B-subgenome of hexaploid bread wheat remains unknown. Here we report the assembly of chromosome-level genome sequences of all five Sitopsis species of the genus Aegilops , which are previously considered as possible direct progenitors or contributors to the B-subgenome. Our comparative genomic analyses reveal that the B-subgenome originated from an unknown, most likely extinct species phylogenetically distinct from Ae. speltoides , its extant closest relative. We also provide evidence that Ae. speltoides is neither the direct progenitor of the G-subgenome of tetraploid wheat Triticum timopheevii . The high-quality Sitopsis genomes provide novel avenues to identify new important genes for wheat breeding.