橄榄转录组SSR信息分析及分子标记开发与应用

Analysis of Canarium album Transcriptome SSR Information and Molecular Marker Development and Application

选取不同品种(系)橄榄成熟果进行转录组测序,结果获得296314条序列,应用MISA软件进行SSR位点分析,获得86084个SSR位点,分布在70686条序列中,SSR在所检测序列中出现频率为23.86%,其中54735条序列含有两个及以上的SSR位点,占比达68.4%;橄榄转录组中SSR序列以复合型核苷酸、单核苷酸、二核苷酸重复类型为主,三者占SSR总数的88.88%;单核苷酸、二核苷酸重复类型中优势基元分别为A/T、AG/CT/TC/GA。以6个不同橄榄品系(种)为研究对象,对随机挑选的99对SSR引物进行有效性与多态性筛选,最终开发了53个有效性SSR分子标记,12个多态性SSR分子标记。利用开发的12个多态性EST-SSR标记对59份橄榄种质进行多态性评价与群体结构分析,结果共检测到48个多态性位点,香农多样性指数平均0.876,多态信息含量平均0.426。利用混群模型分组、UPGMA聚类和PCA对59份橄榄种质进行群体结构分析,混群模型分析结果与UPGMA聚类结果、主成分分析结果有类群上的交叉,但类群数有所不同,混群模型将其划分为3个类群;UPGMA聚类分析在遗传相似系数为0.68处划分为2大种群,与PCA方法分类结果相同。研究结果认为开发的12对SSR标记遗传多样性丰富、有效,为橄榄种质资源鉴别提供可靠的基础工具。

The mature fruits of different varieties(lines)of Canarium album L.were selected for transcriptome sequencing,and 296314 sequences were obtained.MISA software was used to analyze SSR loci,and 86084 SSR loci were obtained,distributed in 70686 sequences.The frequency of SSR in the detected sequences was 23.86%,of which 54735 sequences contained two or more SSR loci,accounting for 68.4%.The SSR sequences in the Chinese olive transcriptome were mainly composed of compound nucleotide,single nucleotide and dinucleotide repeats,accounting for 88.88%of the total SSRs.The dominant motifs in mononucleotide and dinucleotide repeat types were A/T and AG/CT/TC/GA,respectively.The effectiveness and polymorphism of 99 pairs of SSR primers randomly selected from six different Chinese olive lines(species)were screened.Finally,53 effective SSR molecular markers and 12 polymorphic SSR molecular markers were developed.The polymorphism evaluation and population structure analysis of 59 Chinese olive germplasm were carried out by using 12 polymorphic EST-SSR markers.A total of 48 polymorphic loci were detected.The average Shannon diversity index was 0.876,and the average polymorphic information content was 0.426.The population structure of 59 Chinese olive germplasms was analyzed by three methods of mixed group model grouping,UPGMA clustering and PCA.The results of mixed group model analysis were crossed with those of UPGMA clustering and principal component analysis,but the number of groups was different.The mixed group model was divided into three groups.UPGMA cluster analysis was divided into two populations at the genetic similarity coefficient of 0.68,which was the same as the PCA method.The results of the study concluded that the 12 pairs of SSR markers developed were genetically diverse and valid,and provided a reliable basic tool for Chinese olive germplasm resource identification.