Chromosome-level reference genome assemblies and annotation of four diploid Aegilops

Background Aegilops species represent an important reservoir of genetic diversity for agronomic and end-use quality traits as well as resistance to biotic and abiotic stresses for wheat improvement. However, the limited availability of high-quality reference genomes has impeded the effective use of these wild relatives in breeding programs. Results Here, we present chromosome-level reference genome assemblies of four diploid Aegilops species: Ae. comosa , Ae. markgrafii , Ae. umbellulata and Ae. uniaristata . Using a hybrid strategy combining Pacific Bioscience (PacBio) HiFi sequencing, BioNano optical mapping and Hi-C scaffolding, we generated contiguous assemblies ranging from 3.58 to 4.48 Gb characterized by BUSCO completeness score ranging from 97.9 to 98.4%. RNASeq and IsoSeq data from multiple tissues were integrated to facilitate gene annotation. The annotation yielded a total of 47,246 to 51,203 protein-coding genes across the four species, while also characterizing the repeat element composition of these diploid Aegilops . Collinearity analyses identified conserved colinear blocks and revealed multiple intra- and inter-chromosomal rearrangements. Orthogroups shared among the Aegilops and Triticum species were used to perform a phylogenetic analysis that clarified evolutionary relationships among the genomes of this Aegilops/Triticum complex, where the diploid genomes of these four species clustered as a sister monophyletic clade within the D-lineage. Conclusions This study produced reference-quality genome assemblies of four diploid Aegilops species, completing the set of high-quality assemblies now available for all 11 diploid Aegilops species and establishing foundational genomic resources for comparative genomics, gene discovery, and the acceleration of wheat breeding strategies. Additionally, these genomic resources offer a critical framework for extending genomic characterization of tetraploid and hexaploid Aegilops species.