基于RAD-seq技术的异型花SSR信息分析

Analysis on SSR in Sinoswertia tetraptera Base on RAD-seq

用RAD-seq(restriction-site associated DNA sequencing)对异型花(Sinoswertia tetraptera(Maxiowicz)T.N.Ho,S.W.Liu&J.Q.Liu)进行简化基因组测序,并借此分析异型花的SSR(simple sequence repeats)信息。利用SR search软件甄别所得序列中的SSR,得到了双端各有至少100 bp的SSR位点5 844个,其中5 339个(91.38%)成功设计引物,而三核苷酸SSR位点最多(3 323个);在能成功设计引物的SSR位点中,重复序列长度包括17种(12~36 bp);重复序列的基序共277种,其中五核苷酸基序种类最多(106种);随机挑选10对SSR引物,用4个异型花居群的32个个体检测检测其可用性和多态性,经PCR和聚丙烯酰氨凝胶电泳检测,有4对(ST2、ST3、ST6和ST10)成功扩增并表现出多态性;经GENEPOP 4.4对4个位点分析,显示其等位基因数量均值为6,多态性较高且不连锁(P<0.01);4个位点在多数居群中偏离哈迪温伯格平衡(P<0.01)且存在较高的纯合子数量(观测杂合度均值0.023),该结果归因于异型花主要进行自花授粉,在自然界中很难形成进行自由交配的居群;此外,ST2和ST6可在椭圆叶花锚(Halenia elliptica D.Don)中成功扩增,具有潜在通用性。本研究将为日后基于异型花SSR标记的相关研究提供数据库支持。

We used the restriction-site associated DNA sequencing （RAD-seq） technology to analyze simple sequence repeats（SSR） information of Sinoswertia tetraptera（Maxiowicz） T. N. Ho, S. W. Liu ＆ J. Q. Liu. The 5844 SSR loci, with at least 100 bp at two ends, were identified using SR search software. The 5339 （91.38%） loci＇ s primers were designed successfully. Among which, amount of tri-nucleotide SSR loci is the most（3323） ; repeat sequence length type number is 17, while repeat motif type number is 227, and type number of penta-nucleotide motif is the most（106）. We employed 32 individuals from 4 natural populations of S. tetraptera to estimate usability and polymorphism of 10 pairs of SSR primers selected randomly from the 5 339 pairs of primers. According to the result of PCR and Polyacrylamide gel electrophoresis, four pairs of primers （ ST2, ST3, ST6 and STI0） amplified favorably and showed polymorphism. By the GENEPOP 4.4, the mean number of alleles of the four loci is 6 ; these loci do not link to each other（ P 〈 0.01 ） ; these loci deviate fromHWE（P 〈 0.01 ） in most populations and have many homozygotes （observed heterozygosity mean 0. 023 ）, which due to the cleistogamous pollination mode of S. tetraptera. ST2 and ST6 were successfully amplified in Halenia elliptica. Our study will offer a SSR dataset in the future based on SSR markers of S. tetraptera.