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. 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.

中文翻译：

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Oligo-FISH 条形码染色体识别系统为同源四倍体栽培苜蓿的天然染色体畸变倾向提供了新的见解


苜蓿是世界上最具经济价值的饲料作物之一。然而，苜蓿的分子细胞遗传学研究远远落后于其他经济作物，并已达到瓶颈。在这里，我们开发了一种新型染色体识别系统，通过在每条染色体的特定区域设计21个寡核苷酸探针，这些探针可以用作条形码来同时区分细胞中的所有染色体。利用该系统，我们揭示了10个栽培苜蓿品种的染色体核型特征和进化差异。有趣的是，我们还在三个苜蓿品种的种子中发现了两种染色体变异类型，即非整倍体和染色体大片段缺失。变异频率分析表明，这3个苜蓿品种中仅有7/173粒种子存在染色体畸变，表明苜蓿的遗传和减数分裂已进化到相对稳定的状态。值得注意的是，4/7 变异种子是 2 号染色体畸变，这表明 2 号染色体在遗传过程中似乎比其他染色体更容易受到自然染色体畸变的影响。 DNA序列变异分析表明，2号染色体同源拷贝之间存在和缺失变异（PAV）的差异大于其他7条染色体。我们认为同源拷贝之间如此大的 PAV 差异可能为 2 号染色体自然畸变倾向提供物理基础。我们的研究为苜蓿的分子细胞遗传学提供了一个有价值且有效的工具，并为同源多倍体遗传过程中自然染色体畸变的倾向提供了新的见解。