基于锦绣杜鹃花蕾转录组的SSR标记开发及应用

Development and Application of SSR Markers Based on Buds Transcriptomic Data of Rhododendron pulchrum Planch.

[目的]探究锦绣杜鹃(Rhododendron pulchrum Planch.)EST-SSR的类型、分布频率和分布特征,开发有效的SSR标记,并验证其在遗传多样性研究和跨物种转移中的应用潜力。[方法]采用RNA-seq技术对锦绣杜鹃‘紫鹤’品种的花蕾进行转录组测序,MISA软件对组装的unigenes内部的SSR位点进行检索,分析其类型、分布频率。利用Primer3.0软件设计引物,并对锦绣杜鹃群体和其近缘种映山红群体进行多样性检测,利用POPGENE-PC2.2软件计算等位基因数、有效等位基因数、多态性位点百分率、Shannon’s信息指数、Nei氏多样性指数、观察杂合度、期望杂合度等参数。[结果]锦绣杜鹃‘紫鹤’花蕾转录组共有49527个unigenes(43766249bp),从中筛选出16120个SSR位点(24.46%),发生频率为1·(2.7kb)^-1。微卫星的重复次数主要集中在524次之间,二核苷酸发生频率最高(9624个,59.70%),其次是单核苷酸(3738个,23.19%),频率最低的为五核苷酸(42个,0.26%)。频率最高的重复基序有A/T、AG/CT、AAG/CTT、AGG/CCT、ACC/GGT、AGC/GCT、AAAG/CTTT、AAGAG/CTCTT、AGAGGG/CCCTCT等。选用13个多态性SSR标记对锦绣杜鹃群体进行PCR扩增,共得到71个等位基因,平均每个位点3~9个;有效等位基因数在各SSR位点之间变化范围为1.684~5.930;观察杂合度HO和期望杂合度HE的变化范围分别为0.000~1.000和0.433~0.848,平均值分别为0.696±0.426和0.705±0.129;Shannon’s信息指数I和Nei氏多样性指数h的变化范围分别为0.736~1.961和0.406~0.831,平均值分别为1.376±0.339和0.683±0.131。此13个标记在映山红群体中的跨物种扩增成功率为100%,并反映出丰富的遗传多样性。2个群体间的遗传差异93.4%存在于群体内部。[结论]基于AG/CT重复基序开发的13个SSR标记具有高度的多态性,为后续杜鹃花属植物的遗传多样性研究、遗传图谱构建、基因定位、分子标记辅助育种等研究奠定了基础。大别山野生映山红资源具有较高的遗传多样性,且杂合度过剩,遗传变异主要发生在群体内部。

[Objective] The types,distribution frequencies and characteristics of expressed sequence tag-simple sequence repeats (EST-SSRs) were clarified through transcriptome analysis of buds of Rhododendron pulchrum Planch (“Zihe” variety).Moreover,the development of polymorphic SSR markers were performed,as well as their usage in genetic diversity analysis and potential for cross-amplification in related species.[Method] RNA-seq data from buds of R.pulchrum were searched with MISA software.The characteristics of SSR loci were analyzed and SSR primers were designed with Primer 3.0.The effective primer pairs were used in genetic diversity analysis of R.pulchrum and R.simsii populations.The genetic parameters of population were calculated with POPGENE-pc 2.2 software.[Result] Totally,49 527 unigenes (43 766 249 bp) were obtained,and 16 120 SSR loci 1.(2.7 kb)^-1 were searched,which accounted for 24.46% of the total unigenes.Repeat numbers of most SSR loci ranged between 5-24.Dinuclotide repeat was the most abundant type with a frequency of 59.70%( 9 624 ),followed by mono-nucleotide repeat (3 738,23.19%),and the least type was penta-nucleotide repeat (42,0.26%).Moreover,the typical motifs were A/T,AG/CT,AAG/CTT,AGG/CCT,ACC/GGT,AGC/GCT、AAAG/CTTT,AAGAG/CTCTT,and AGAGGG/CCCTCT.The availability and polymorphism of the thirteen SSR markers selected were clarified in R.pulchrum population.A total of 71 alleles was scored,and the amount of allele ( Na ) and effective number of allele (Ne ) per locus ranged from 3-9 and 1.684-5.930,respectively.The observed heterozygosity ( HO) and expected heterozygosity (H E) varied from 0.000 to 1.000 and from 0.433 to 0.848,with the mean values of 0.696±0.426 and 0.705±0.129,respectively.Shannon’s information index ( I ) and Nei’s gene diversity (h ) ranged from 0.736 to 1.961 and from 0.406 to 0.831,with the average values of 1.376±0.339 and 0.683±0.131,respectively.All these SSR markers could be successfully cross-amplified in the related species R.simsii,which also showed high genetic diversity.The genetic variation existed mainly among populations.[Conclusion] The 13 polymorphic SSR markers based on unigenes containing (AG/CT)n loci will benefit for following genetic diversity analysis,genetic map construction,gene mapping,and molecular marker assisted breeding of Rhododendron species.Natural R.simsii germplasm resources possess high genetic diversity.Moreover,excess heterozygosity is observed,and genetic variation is mainly maintained among populations.