思茅松转录组SSR分析及标记开发

doi:10.13560/j.cnki.biotech.bull.1985.2017.05.010

摘要/Abstract

摘要： 基于思茅松转录组测序数据进行其SSR标记开发,为丰富思茅松SSR数据库提供参考。采用MISA软件对思茅松59 636条Unigene进行SSR搜索,共获得3 745个SSR位点,分布频率为6.28%,平均11.36 kb出现一个SSR位点。SSR重复类型以三核苷酸基序为主,其次是二核苷酸基序,所占比例分别为49%和24%,其主要重复单元分别为AGC/CTG和AT/TA。随机挑选224个位点进行引物设计及合成,琼脂糖凝胶检测发现其中29个位点具有多态性且效果良好,但仅12个位点符合SSR重复类型的倍数大小且扩增效率高于85%。利用这12对荧光标记引物,对2个居群42份样本进行遗传多样性分析,共检测到35个等位基因,多态性信息量（PIC）为0.093 2-0.480 9,平均为0.306 9。4个位点为低度多态位点（0<PIC<0.25）,8个位点为中度多态位点（0.25<PIC<0.5）。这12个标记可用于思茅松遗传多样性分析、遗传图谱构建、基因定位及克隆等研究,旨为思茅松分子标记辅助育种及变异水平等研究提供技术支持。

关键词: 思茅松, 转录组, SSR, 引物开发

Abstract: In Simao pine（Pinus kesiya var. langbianensis）breeding programs,lack of co-dominant genetic markers constrains the development of molecular marker assisted breeding. The aim of this study is to develop SSR molecular markers using transcriptome sequencing data. Searching the SSR loci from 59636 unigenes of P. kesiya var. langbianensis with MISA software,total 3745 SSRs were obtained,accounting for 6.28% of the total unigenes,averagely one SSR per 11.36 kb. Trinucleotide and dinucleotide repeats were dominant types among SSRs with the ratio of 49% and 24%,and others were only 27%. AGC/CTG was the most trinucleotide motif and AT/TA was the most dinucleotide motif. The primers were designed and synthetized based on randomly selected 224 SSR loci,verification by agarose gel electrophoresis revealed that 29 loci showed clear polymorphism. While only 12 of the 29 loci satisfied the multiple fragments of SSR repeat motifs in capillary electrophoresis and their amplification efficiencies were higher than 85%. Genetic diversity of 42 Simao pine samples from Jingdong and Puwen populations was investigated with these 12 fluorescently labeled primers. A total of 35 alleles were detected,the polymorphism information content（PIC）of all loci ranged from 0.0932-0.4809（the mean was 0.3069）. Among these,4 loci were classified as lowly polymorphic ones（0 < PIC <0.25）and 8 loci as moderately polymorphic ones（0.25 < PIC <0.5）. Conclusively,these 12 SSRs may be used in future studies on genetic diversity,linkage mapping,gene location and cloning,providing the technical support for molecular marker assisted breeding and mutation in P. kesiya var. langbianensis.

Key words: Pinus kesiya var. Langbianensis, transcriptome, SSR, primer development

赵能, 原晓龙, 缪福俊, 王毅, 陈伟, 李江, 吴涛, 王娟. 思茅松转录组SSR分析及标记开发[J]. 生物技术通报, 2017, 33(5): 71-77.

ZHAO Neng, YUAN Xiao-long, MIAO Fu-jun, WANG Yi, CHEN Wei, LI Jiang, WU Tao, WANG Juan. Development of SSR Molecular Markers Based on Transcriptome Data of Pinus kesiya var. langbianensis[J]. Biotechnology Bulletin, 2017, 33(5): 71-77.

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