Unlike many crop seeds which are large in size, forage seeds are relatively small. Moreover, some forage seeds from the same genus, or even different genera, share similar morphologies in terms of colour, size, weight and so on. Alfalfa (Medicago sativa L.) and burr medic (Medicago polymorpha L.) also share similar morphology. However, alfalfa is a perennial, cross-pollinated herbaceous species, whereas burr medic is considered an annual, self-pollinating plant. Therefore, a universal and rapid method of discriminating burr medic seeds from alfalfa seeds or other similar forage seeds from different plant species is needed. Previous methods of discriminating similar-looking forage seeds have been shown to be unreliable or time consuming. Here, a universal method that combines polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis techniques was developed to discriminate alfalfa and burr medic. Based on the differences in the lengths of homologous genes caused by insertions and deletions between two species, a pair of primers was designed to discriminate the accessions of 15 alfalfa and 9 burr medic via visualizing the clearly different PCR amplicon sizes. In addition, the melting curve analysis was performed to investigate the specificity and reliability of the primer pair. The results showed two different melting peaks between the two species, further demonstrating this method can accurately and sensitively identify the two species. Thus, we believe that this method has the great potential to be extensively applied for the identification of contaminated seeds and breeding of forages.

中文翻译：

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一种快速识别苜蓿和毛刺药用种子的通用方法，重点是区分不同的牧草种类

与许多较大的农作物种子不同，草料种子相对较小。此外，一些来自同一属甚至不同属的饲料种子在颜色、大小、重量等方面具有相似的形态。紫花苜蓿 ( Medicago sativa L.) 和 burr medic ( Medicago polymorphaL.) 也有相似的形态。然而，苜蓿是一种多年生、异花授粉的草本植物，而毛刺草被认为是一种一年生、自花授粉的植物。因此，需要一种通用且快速的方法来区分来自不同植物物种的苜蓿种子或其他类似的草料种子。先前区分外观相似的草料种子的方法已被证明是不可靠或耗时的。在这里，开发了一种结合聚合酶链反应 (PCR) 和聚丙烯酰胺凝胶电泳技术的通用方法来区分苜蓿和毛刺药。基于两个物种之间插入和缺失引起的同源基因长度的差异，设计了一对引物，通过可视化明显不同的 PCR 扩增子大小来区分 15 个苜蓿和 9 个 burr medic 的种质。此外，进行熔解曲线分析以研究引物对的特异性和可靠性。结果显示这两个物种之间存在两个不同的熔解峰，进一步证明该方法可以准确、灵敏地鉴定这两个物种。因此，我们相信该方法具有广泛应用于污染种子鉴定和牧草育种的巨大潜力。进一步证明该方法可以准确灵敏地识别这两个物种。因此，我们相信该方法具有广泛应用于污染种子鉴定和牧草育种的巨大潜力。进一步证明该方法可以准确灵敏地识别这两个物种。因此，我们相信该方法具有广泛应用于污染种子鉴定和牧草育种的巨大潜力。