Comparative oligo-FISH validates genome assemblies and delivers a precise karyotype for Lens Mill. species.

Chromosome structural rearrangements play a significant role in karyotype evolution and speciation. These rearrangements pose challenges for precise karyotyping and the assembly of a genus pan-genome for crops and their wild relatives. Lens culinaris, an important cool-season legume primarily grown in India and Canada, is a cultivated species with six wild relatives. All seven Lens species exhibit distinct chromosomal arrangements, which affect introgression and complicate the development of an accurate karyotype for the genus. Using gene synteny analysis between the cultivated Lens species and six wild relatives, we developed cross-species oligo-FISH (Fluorescent in situ hybridization) probes to in situ confirm genome assemblies and synteny. Chromosome spreads were used for oligo-FISH experiments, in which the DNA was denatured and a set of red and green oligo probes was hybridized to the metaphase chromosomes. The combination of both oligo sets/probes resulted in a distinct pattern for each Lens spp. chromosome, allowing the inference of a more robust karyotype for six Lens species. The karyotyping of Lens spp. confirmed the proper assignment of chromosomes in the genome assemblies and validated the rearrangements detected in the synteny analysis. The results attest to a higher sequence-level similarity among the closest related species despite the occurrence of several chromosomal structural changes among them. Oligo-FISH probes can be used in conjunction with plant genome assembly projects, supporting the delivery of a precise representation of the physical chromosomes of a species.