He Run-Li, Han Yi-Li, Wang Fang, Liu Ji-Quan, Wang Li-Hua
College of Traditional Chinese Materia Medica, Shanxi University of Traditional Chinese Medicine, Taiyuan 030619, China.
Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture, Yunnan Provincial Key Laboratory of Agriculture Biotechnology, Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agriculture Sciences, Kunming 650223, China.
Zhongguo Zhong Yao Za Zhi. 2018 May;43(9):1838-1843. doi: 10.19540/j.cnki.cjcmm.20180307.002.
In order to enrich the library of SSR and provide more powerful tools for molecular marker-assisted breeding in Astragalus membranaceus var. mongholicus, simple sequence repeats (SSR) loci in its transcriptome were searched in 18 040 unigenes (>=1 kb) by using MISA. SSR loci information was analyzed and SSR primers were designed by Primer 3. Furthermore, 110 pairs of primers were randomly selected for the polymorphic analysis on 20 plants collected from different habitats. A total of 5 640 SSRs were found in the transcriptome of A. membranaceus var. mongholicus, distributed in 4 462 unigenes with the distribution frequency of 31.26%. SSR loci occurred every 6 514 bp. Mono-nucleotide repeat was the main type, accounted for as much as 36.72% of all SSRs, followed by tri-nucleotide(32.57%) and di-nucleotide(27.73%) repeat motif. Among all 75 repeat types, A/T(2 026) was the predominant one followed by AG/CT(1 179), AAG/CTT(477). For validating the availability of the SSR primers designed using Primer 3, 110 pairs of primers were randomly selected for PCR amplification. Among them, 97 pairs of primers (88.18%) produced clear and reproductive bands. Using 19 pairs of primers showed polymorphism, 20 plants were divded into two groups by UPGMA. There are numerous SSRs in A. membranaceus var. mongholicus transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity, molecular identification, and marker-assisted breeding study for this plant.
为丰富蒙古黄芪简单序列重复（SSR）文库，为其分子标记辅助育种提供更有力的工具，利用MISA在18040条单基因序列（≥1 kb）中搜索其转录组中的SSR位点。对SSR位点信息进行分析，并使用Primer 3设计SSR引物。此外，随机选择110对引物对从不同生境采集的20株植株进行多态性分析。在蒙古黄芪转录组中共发现5640个SSR，分布于4462条单基因序列中，分布频率为31.26%。SSR位点每6514 bp出现一次。单核苷酸重复是主要类型，占所有SSR的36.72%，其次是三核苷酸重复（32.57%）和二核苷酸重复（27.73%）。在所有75种重复类型中，A/T（2026）是最主要的，其次是AG/CT（1179）、AAG/CTT（477）。为验证使用Primer 3设计的SSR引物的有效性，随机选择110对引物进行PCR扩增。其中，97对引物（88.18%）产生清晰且可重复的条带。使用19对具有多态性的引物，通过非加权组平均法（UPGMA）将20株植株分为两组。蒙古黄芪转录组中有大量高频且类型多样的SSR，这将为该植物的遗传多样性、分子鉴定和标记辅助育种研究提供丰富的候选分子标记。
