基于山茶转录组的SSR标记开发及亲缘关系分析

Development of SSR markers based on transcriptome of Camellia japonica and analysis of genetic relationship

【目的】开发基于山茶转录组的EST-SSR分子标记,并应用该标记进行山茶种质的亲缘关系分析。【方法】本研究以山茶叶片转录组测序所获得的50 518条Unigenes为背景数据,利用MISA软件搜索SSR标记,设计并筛选SSR多态性引物,对8份山茶种质进行了UPGMA聚类。【结果】共检索到13 197个SSR位点,SSR的发生频率和分布频率分别为19.52%、26.12%,平均分布距离为4.33 kb;在6种SSR重复类型中,以单、二、三核苷酸这3种重复类型为主,分别占48.420%、34.917%和15.473%;出现频率高的基序类型为A/T、AG/CT和AT/TA,占总SSR位点的79.61%;三核苷酸中以AAG/CTT、ACC/GGT、AAT/ATT类型居多;在设计出的10 974对引物中,随机选用其中90对引物进行多态筛选,73对引物扩增产物与预期大小一致,有效扩增率为81.11%,29对引物存在扩增多态性,占39.73%。扩增得到72个等位基因,有效等位基因数平均达到3.264;观察杂合度和期望杂合度的平均值分别为0.208和0.638,引物多态信息含量平均为0.496;8份山茶种质在相似系数为0.58时,可以分为4类:山茶原种为Ⅰ类,‘黑蛋石’、‘红叶黑魔法’和‘黑骑士’为Ⅱ类,‘黑魔法’和‘金华美女’分别为Ⅲ类和Ⅳ类。【结论】山茶转录组测序的Unigenes信息可作为SSR标记开发的有效来源,为山茶的遗传多样性研究、亲缘关系鉴定以及分子标记辅助育种等奠定了一定的理论基础。

[Objective]Develop the EST-SSR markers based on transcriptome sequencing and apply these markers to analyze the genetic relationships of Camellia germplasms.[Method]Using 50 518 Unigenes obtained from the transcriptome sequencing of C.‘Red Leaved Black Magic’leaves as background data,SSR markers were searched out,and polymorphic primers were then designed and screened to construct the UPGMA evolutionary tree for 8 Camellia germplasms.[Result]13 197 SSR sites were founded out.The occurrence frequency and the distribution frequency were estimated to be 19.52%and 26.12%,respectively,with the average distribution distance of 4.33 kb.Among the 6 types of SSR repeat motifs,mono-nucleotide,di-nucleotide and tri-nucleotide were the frequent dominant motifs,accounting for 48.420%、34.917%and15.473%,respectively.The types of motifs with high occurrence frequency included A/T,AG/CT and AT/TA,accounting for 79.61%of the total SSR loci;AAG/CTT,ACC/GGT,AAT/ATT were in the majority among the tri-nucleotides;10 974 pairs of primers were designed,and 73 pairs of primers were finally used to amplify effectively based on 90 pairs selected randomly,resulting in the rate of effective amplification of 81.11%;29 primers showed some amplification polymorphism,accounting for 39.73%.The average number of effective alleles was estimated to be 3.264 with a total of 72 alleles.The mean values of observed heterozygosity(Ho)and expected heterozygosity(He)were measured to 0.208 and 0.638 respectively,as well as the average polymorphic information content(PIC)of 0.496;genetic relationship and cluster analysis indicated that eight Camellia germplasms could be classified into four categories at a similarity coefficient of 0.58:wild C.japonica in groupⅠ;C.‘Black Opal’,C.‘Red Leaved Black Magic’and C.‘Night Rider’together in groupⅡ;C.japonica‘Black Magic’and C.japonica‘Jinhua Meinǖ’in groupⅢand groupⅣrespectively.[Conclusion]The result showed that Camellia transcriptome Unigenes can be used as an effective source for SSR markers exploiting,which lays a theoretical foundation for studies on genetic diversity,identification of genetic relationship and marker-assisted breeding of Camellia.