Simple sequence repeat (SSR) markers are among more reliable markers widely used in population genetics and traditionally need intensive labor to develop. High-throughput sequencing (HTS) has enabled researches in screening whole DNA in order to develop dozens of reliable SSR markers to pave route for future studies. Annual medics (Medicago spp.) are specifically beneficial in agriculture, genetics, and pastureland management plans. In order to facilitate study of annual medics using SSRs and to test their effectiveness, HTS screening of whole DNA resulted in developing of hundreds of primer pairs published in this paper. Subsequent testing of candidate primers on Iranian M. orbicularis germplasm was narrowed down to six more variable primer pairs that generated 28 polymorphic bands with the allele size of 200–310 bp. Eleven populations were examined using our SSR markers. The average polymorphic information content was very high (> 0.90) for the primer combinations. Genetic diversity parameters were measured for all populations. A high level of polymorphism of 75% within populations was observed. We hypothesized that the self-pollinating reproductive system of M. orbicularis has led to the reduction of gene flow between populations and ultimately the low diversity among populations (24%). Different genotypes observed by structure analysis as well as regression and correlation analyses showed that genetical similarity was congruent with the geographical proximity. Our results suggest that our HTS-generated SSR markers can be used for the further study of other annual medics.

简单序列重复（SSR）标记是群体遗传学中广泛使用的更可靠标记之一，传统上需要大量劳动力来开发。高通量测序 (HTS) 使研究能够筛选整个 DNA，从而开发出数十种可靠的 SSR 标记，为未来的研究铺平道路。年度医疗人员（Medicago spp.）对农业、遗传学和牧场管理计划特别有益。为了促进年度医学人员使用 SSR 进行研究并测试其有效性，对整个 DNA 进行 HTS 筛选，开发了数百对引物对，并在本文中发表。随后对伊朗轮匝酵母种质上的候选引物进行测试，将 范围缩小到六个可变引物对，产生 28 个等位基因大小为 200-310 bp 的多态性条带。使用我们的 SSR 标记对 11 个群体进行了检查。引物组合的平均多态性信息含量非常高（> 0.90）。测量了所有种群的遗传多样性参数。观察到群体内 75% 的高水平多态性。我们假设轮状小 蠹的自花授粉生殖系统导致种群之间的基因流动减少，并最终导致种群之间的多样性较低（24％）。通过结构分析以及回归和相关分析观察到的不同基因型表明，遗传相似性与地理邻近性一致。我们的结果表明，我们的 HTS 生成的 SSR 标记可用于其他年度医生的进一步研究。