Chromosome identification of Medicago polymorpha by oligonucleotide-based FISH

Medicago polymorpha (2n = 2x = 14) is a valuable forage legume, but the identification of its somatic chromosomes has been challenging due to a lack of distinctive chromosome morphological features. With appropriate probes, oligonucleotide-based FISH is a highly effective method for chromosome identification. However, there are no available probes for M. polymorpha. In this study, we isolated five tandem repeats from the M. polymorpha genome, named Mp51, Mp139, Mp167, Mp179, and Mp497. Mp51 showed two pairs of signals located at the pericentromere. Mp139 exhibited four pairs of signals, located at the pericentromere and short arm of chromosomes. Mp167 and Mp179 showed seven pairs of signals, respectively, concentrated in the pericentromere. Mp497 exhibited three pairs of signals, distributed across the pericentromere and proximal position of the chromosomes. The combined FISH results of Mp51 and Mp139 oligo probes with 5S rDNA and 18S-26S rDNA probes demonstrated distinct signal patterns for each chromosome, enabling the precise identification of all chromosome pairs. Finally, the visual identification of M. polymorpha chromosomes was resolved. This will provide useful cytological information for studying the chromosomal structure and behavior of M. polymorpha.