越橘EST-SSR分子标记的开发及其遗传多样性分析

Development of expressed sequence tag-derived simple sequence and genetic diversity analysis of blueberry

【目的】探讨越橘EST序列中SSR位点的分布规律,开发越橘EST-SSR引物,并分析其在越橘品种遗传多样性研究中的作用。【方法】以91份越橘种质为材料,利用越橘果实转录组（RNA-Seq）的测序结果,通过SSRIT在线搜索,利用Primer Premier 5.0软件设计30对引物,并筛选扩增效果理想的引物,用筛选出的引物分析91份种质的多态性,构建91份种质的系统发育树。【结果】34 464条越橘unigene序列中含有SSR位点的序列有829条,SSR位点有913个,其中二核苷酸、三核苷酸重复是主要的SSR类型,分别占全部SSR位点的13.69%和81.27%。不同核苷酸数目重复基元的重复次数差异很大;越橘EST-SSR位点集中在11~15bp;三核苷酸和六核苷酸重基元复种类最多,在所有重复基元中GAA/TTC发生频率最高。设计的30对引物中有17对引物在供试越橘种质中扩增出理想的PCR产物,17对引物均有多态性。聚类分析结果显示,在遗传相似系数为0.69时,可以将供试越橘种质分成4组。【结论】EST-SSR标记可以用于越橘品种的鉴定与遗传多样性分析。

【Objective】The objective of this study was to analyze the SSR distribution in blueberry transcriptome,and develop new EST-SSR markers for studying the genetic diversity of Vaccinium germplasms.【Method】Using 91 Vaccinium germplasms for materials,SSR motifs were selected by SSRIT online software based on RNA-Seq results of blueberry fruits.A total of 30 primer pairs were designed by the software Primer Primer 5.0and phylogenetic tree was constructed by optimal primers to analyze the polymorphism of 91 germplasm resources.【Result】A total of 913 SSRs were identified from 34 464 Vaccinium unigenes.The dinucleotide and trinucleotide repeats were the dominant types with the frequency of 13.69%and 81.27%,respectively.The difference among different number of repeats of different SSR types waslarge.The most abundant repeat length of EST-SSR motif was 11-15 bp.Tri-nucleotide and hexa-nucleotide repeats had the most abundant repeat types and GAA/TTC was the most common motif.Among the30EST-SSR primers,amplified distinct bands and expected products were obtained by 17 pairs primers.The 17 polymorphic EST-SSR amplicons were validated by resequencing PCR products.Cluster analysis showed that 91 Vaccinium germplasms can be distinguished into 4groups with the genetic similarity coefficient of 0.69.【Conclusion】The EST-SSR markers can be used in germplasm identification and genetic analysis in Vaccinium.