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 10 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. DNA sequence variation analysis showed that the difference of presence and absence variations (PAVs) among homologous copies of chromosome 2 was larger than that of the other seven chromosomes. We suggest that such large PAV divergence among homologous copies may provide the physical basis for natural chromosome 2 aberrations propensity. Our study provides a valuable and efficient tool for alfalfa’s molecular cytogenetics and sheds new insights into the propensity for natural chromosome aberrations during autopolyploid inheritance.

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