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采用SSR-PCR扩增技术,将实验室利用高通量测序与磁珠富集法结合开发得到的1 210对黍稷SSR引物进行筛选,筛选出在会宁大黄糜、陇糜8号、太原55号和会宁野糜子4个黍稷品种之间表现出条带清晰、多态性明显、重复性较好的引物;并通过提取DNA以及PCR过程中退火温度的简单优化来建立比较适宜的黍稷SSR分子标记反应体系。结果表明,在DNA提取过程中将ddH2O的量减少到100μL,增加65℃的水浴时间并增加4%β-巯基乙醇和DNA浓度等可以获得较高质量的DNA。利用优化好的反应体系从1 210对SSR引物中筛选出116对多态性较好的引物,占总数的9.59%,其中,群体一的2个亲本所特有的引物有36对,群体二的2个亲本中特有的引物有97对,2个群体共有的多态性引物有19对;对引物碱基结构分析发现,选用的1 210对引物中有1 173个双碱基重复SSR和36个三碱基重复SSR;筛选到的多态性引物中双碱基重复SSR有102个。三碱基重复SSR有14个。筛选出的多态性SSR标记能用于黍稷的遗传多样性和群体遗传结构研究。
SSR-PCR amplification was used to screen 1 210 pairs of barley SSR primers developed by high-throughput sequencing and magnetic bead enrichment in the laboratory. The results showed that SSR-PCR was used to screen the SSR primers of Huitian Dahuang Mi, Longmi 8, Taiyuan 55 and Ningbian Millet four broomcorn varieties showed a clear band, obvious polymorphism, better repeatability of primers; and DNA extraction and PCR process by the simple optimization of the annealing temperature to establish a more appropriate Millet SSR molecular marker reaction system. The results showed that high quality DNA could be obtained by reducing the amount of ddH2O to 100 μL during DNA extraction, increasing the water bath time at 65 ° C and increasing the concentration of β-mercaptoethanol and DNA by 4%. 116 optimized primers were screened from 1 210 pairs of SSR primers, accounting for 9.59% of the total, using an optimized reaction system. Among them, 36 pairs of primers specific to the two parents in group one and two There were 97 pairs of primers unique to the two parents and 19 pairs of polymorphic primers shared by the two populations. Based on the analysis of the base structure of the primers, 1 173 pairs of SSRs and 36 A three-base repeat SSR; two polymorphic SSR primers selected 102. There are 14 triple-repeat SSRs. Polymorphic SSR markers screened can be used to study the genetic diversity and population genetic structure of bream.