款冬转录组SSR标记开发及其多态性研究

Development and polymorphism analysis of SSR molecular markers in Tussilago farfara transcriptome

目的分析款冬转录组数据的简单重复序列标记(SSR)位点信息并设计特异引物,以期为款冬分子标记辅助育种提供有力工具。方法对款冬转录组测序数据库中长度1 kb以上的18 938条unigene,利用MISA软件搜索SSR位点信息,利用Primer3设计引物,并随机选择55对SSR引物分析18份不同来源款冬材料的多态性。结果共搜索到4688个SSR位点,分布于3844条unigene中,SSR出现频率为24.75%,平均7979bp含1个SSR位点。SSR位点中三核苷酸重复类型出现频率最高(37.12%),其次是单核苷酸重复(32.36%)和二核苷酸重复(28.20%)。共有60种重复基元,其中A/T(31.42%)、AG/CT(12.80%)、ATC/ATG(9.62%)是优势重复基元。随机选择55对引物进行多态性验证分析,其中42对有扩增产物,14对具稳定可重复的多态性;利用UPGMA作图,将18份样品分为3类。结论款冬转录组中SSR位点出现频率高,类型丰富,多态性高,为其遗传多样性分析、遗传图谱构建和分子标记辅助育种等研究提供了候选分子标记。

Objective The SSR loci information in the transcriptome of Tussilago farfara was analyzed and specific primers were designed,so as to provide powerful tools for molecular marker-assisted breeding in this plant.Methods SSR loci in 18938 unigenes with length of 1 kb or more obtained by transcriptome sequencing were searched by using MISA.SSR primers were designed by Primer3 and 55 pairs were randomly selected for the polymorphic analysis on 18 samples collected from different habitats.Results A total of 4688 SSRs were detected in the transcriptome of T.farfara,distributed in 3844 unigenes with the distribution frequency of 24.75%.SSR loci occurred every 7979 bp.Trinucleotide repeats appeared to be the most abundant SSRs with a frequency of 37.12%,followed by mononucleotide(32.36%)and dinucleotide(28.20%).Among all 60 repeat motifs,A/T(31.42%),AG/CT(12.80%),and ATC/ATG(9.62%)were the predominant repeat types.For validating the availability of the SSR primers,55 pairs of primers were randomly selected for polymorphism analysis.Among them,42 pairs(76.36%)produced clear and reproductive bands and 14 pairs showed polymorphism.Eighteen plants were divided into three groups by UPGMA.Conclusion The SSR markers in the transcriptome of T.farfara show high frequency,rich type,and high polymorphism,which will provide the abundant candidate markers for genetic diversity,genetic mapping construction and marker-assisted breeding study for this plant.