广西乐业野生春兰iPBS遗传多样性分析与指纹图谱构建

Genetic diversity analysis and fingerprinting construction of wild Cymbidium goeringii from Leye,Guangxi by iPBS

【目的】分析广西乐业县野生春兰种质资源的遗传多样性,构建其DNA指纹图谱,为野生春兰种质资源的分类和鉴定提供科学依据。【方法】采用iPBS分子标记分析收集于广西乐业县81份野生春兰种质资源和4个春兰栽培品种的多态性位点比率(PPL)、多态性信息含量(PIC)、观测等位基因数(N a)、有效等位基因数(N e)、Nei’s基因多样性(H e)和Shannon信息指数(I)。从83条iPBS引物中筛选出扩增条带清晰、多态性高、重复性好的引物,对供试85份春兰种质的基因组DNA进行PCR扩增,统计凝胶电泳特征性谱带;采用POPGEN 1.32计算各野生春兰种质的遗传多样性指数,并基于其遗传相似系数进行UPGMA聚类;利用引物扩增条带多态性位点构建春兰种质数字指纹图谱。【结果】筛选出的6条引物扩增获得105条清晰谱带,其中,多态性条带95条,多态性比例为90.48%;85份春兰种质的平均PIC、平均N a、平均N e、平均H e和平均I分别为0.8050、1.7923、1.2204、0.2765和0.4590。供试春兰种质间的遗传相似性系数变辐为0.690~0.981,在相似系数0.704处,可将85份春兰种质划分为4个类群,其中,79个广西乐业野生春兰种质分布在第Ⅰ类群,第Ⅱ类群包括4个栽培品种,第Ⅲ和第Ⅳ类群分别只有1份广西乐业野生春兰种质。数字指纹图谱构建结果表明,利用2对iPBS引物(2249和2076)组合扩增的多态性位点即可将85份春兰种质全部区分,由此构建的乐业野生春兰种质数字指纹图谱可为野生春兰种质的鉴定提供科学依据。【结论】81份广西野生春兰种质表现出丰富的遗传多样性,基因交流较频繁,遗传背景相似。iPBS分子标记可用于春兰种质的鉴别及遗传多态性分析。

【Objective】The study aimed to analyze genetic diversity of wild Cymbidium goeringii germplasm resources in Leye county,Guangxi and to construct DNA fingerprint of the plant,so as to provide a scientific basis for the classification and identification of wild C.goeringii germplasm resources.【Method】Percentage of polymorphic loci(PPL),polymorphism information content(PIC),observed number of alleles(N a),effective number of alleles(N e),the Nei’s gene diversity(H e)and Shannon’s information index(I)of 81 samples of 4 wild C.goeringii cultivars in Leye county,Guangxi were analyzed by iPBS markers.Primers which were clear,of high polymorphism and good reproducibility were screened from 83 iPBS primers for PCR amplification of 85 C.goeringii germplasms and gel electrophoresis characteristic bands were recorded.POPGEN 1.32 software was used to analyze each genetic diversity index.UPGMA cluster analysis was conducted based on their genetic similarity coefficient.Digital fingerprint of C.goeringii germplasm was constructed according to polymorphism sites of primer amplification band.【Result】Through amplification of 6 primer,105 clear bands were obtained,in which 95 bands were polymorphic,accounting for 90.48%.The average values of PIC,N a,N e,H e and I were 0.8050,1.7923,1.2204,0.2765 and 0.4590,respectively.The genetic similarity coefficient among the germplasms ranged from 0.690 to 0.981.When genetic similarity coefficient was 0.704,85 germplasms could be divided into 4 groups,of which 79 germplasms of Leye were mainly distributed in group I,4 cultivars were distributed in groupⅡ,while only one species from Leye was distributed in groupsⅢandⅣrespectively.The digital fingerprint map constructing showed that 85 C.goeringii accessions were fully distinguished using the combined polymorphic sites amplified with 2 pairs of iPBS primers(2249 and 2076),thus the digital fingerprint was constructed,which could provide a scientific basis for the identification of wild C.goeringii germplasms.【Conclusion】Great genetic diversity was found among 81 C.goeringii germplasms in Guangxi with frequent gene communication and similar genetic conditions.iPBS can be applied for C.goeringii germplasm identification and genetic polymorphism analysis.