Chromosome-specific painting provides insights into the karyotype evolutionary direction and trajectory in the genus <i>Medicago</i>

Divergence in basic chromosome numbers among closely related species is widespread in plants, yet a fundamental question regarding the evolutionary direction of karyotype-whether descending (from higher to lower numbers) or ascending (from lower to higher)-remains contentious. Alfalfa (<i>Medicago sativa</i> L.), a key forage crop, displays two basic chromosome numbers (<i>x</i> = 8 and <i>x</i> = 7) within the genus, and whether this divergence arose through descending evolution from 8 to 7 or the reverse remains unclear. Here, we developed a set of chromosome-specific painting markers capable of tracing chromosomal evolutionary trajectories among <i>Medicago</i> species. Comparative cytological analysis of seven accessions (<i>x</i> = 8) from the <i>M. sativa</i> L. complex revealed conserved chromosomal synteny in both diploid and autotetraploid species, with no detectable interchromosomal rearrangements. In <i>Medicago polymorpha</i> (<i>x</i> = 7), we discovered that the divergence in basic chromosome numbers (<i>x</i> = 7 vs. <i>x</i> = 8) resulted from large-scale fission-fusion events involving chromosomes 3, 5, and 6, rather than the simple fusion of chromosomes 3 and 7 as previously published genomic hypotheses. Further supporting evidence from rDNA remodeling and phylogenetic analysis indicates a descending evolutionary pathway, with the ancestral <i>x</i> = 8 transitioning to <i>x</i> = 7 approximately Mid-Miocene (~12 million years ago). Our results offer new insights into <i>Medicago</i> speciation and evolutionary origins, and instantiate a strategy for studying karyotypic evolutionary direction in other plant taxa with similar chromosomal dynamics.