Alfalfa is one of the most economically valuable forage crops in the world. However, molecular cytogenetic studies in alfalfa lag far behind other cash crops and have reached a bottleneck. Here, we developed a novel chromosome identification system by designing 21 oligo probes in specific regions of each chromosome, which can be used as a barcode to simultaneously distinguish all chromosomes in a cell.

 

Using this system, we revealed the chromosome karyotype features and evolutionary differences among ten cultivated alfalfa varieties. Interestingly, we also found two chromosomal variation types i.e., aneuploidy and large chromosomal segment deletions in the seeds of three alfalfa varieties. Variation frequency analysis showed that only 7/173 seeds in those three alfalfa varieties had chromosome aberrations, which indicated that the inheritance and meiosis of alfalfa had evolved to a relatively stable state. Remarkably, 4/7 variation seeds were chromosome 2 aberrations, suggesting that chromosome 2 appears to be more susceptible to natural chromosomal aberrations than other chromosomes during inheritance.