基于iPBS和ISSR分子标记的兜兰属同色组种质鉴定及遗传多样性分析

Identification and genetic diversity of Paphiopedilum sect.Concoloria germplasm resources based on iPBS and ISSR molecular markers

【目的】基于引物结合位点扩增(iPBS)和简单序列重复间DNA多态性(ISSR)分子标记对兜兰属同色组种质进行鉴定及遗传多样性分析,筛选出可靠的种质鉴定方法,为兜兰属种质鉴定、遗传多样性分析及分子育种提供科学依据。【方法】采用iPBS和ISSR分子标记对47份兜兰属同色组种质进行种质鉴定,并分析其亲缘关系和遗传多样性,挖掘可用于鉴别3个近缘种的分子标记。【结果】利用7条iPBS引物共扩增出117个位点,其中多态性位点100个,多态性比率为85.47%;47份供试种质的遗传相似系数为0.73~0.97;在遗传相似系数0.76处可将47份供试种质划分为三大类群,其中6份难鉴定种质与已知的文山兜兰聚为第Ⅱ类群,第Ⅰ类群包含了所有巨瓣兜兰种质,第Ⅲ类群包含了所有的同色兜兰种质。7条ISSR引物共扩增出115个位点,其中多态性位点111个,多态性比率为96.52%;47份供试种质的遗传相似系数为0.68~0.94,在遗传相似系数为0.73处可将47份供试种质划分为三大类群。第Ⅰ类群包括11个巨瓣兜兰种质,第Ⅱ类群包括表型难鉴定的6份种质及13个文山兜兰种质,第Ⅲ类群包括17个同色兜兰种质。iPBS和ISSR单独聚类结果和二者综合聚类分析结果略有差异,但种质聚类鉴定结果具有较高的一致性,均将47份兜兰属同色组种质分为三大类群,且6份难鉴定个体均与文山兜兰聚为一类,巨瓣兜兰和同色兜兰各自聚为一类。3个聚类分析结果均表明,文山兜兰与同色兜兰的亲缘关系较近,与巨瓣兜兰的亲缘关系较远。【结论】iPBS和ISSR分子标记均可用于兜兰属近缘种间的种质鉴定及遗传多样性分析,二者结合分析的方法可在DNA水平上更准确有效地鉴定易混淆种质,多种标记联合数据的聚类分析结果,能更全面客观地反映种质间的遗传多态性及亲缘关系。

【Objective】Based on primer binding site amplification(iPBS)and simple sequence inter repeat DNA poly-morphism analysis(ISSR)molecular markers,the germplasm molecular marker identification and genetic diversity analy-sis of Paphiopedilum sect.Concoloria were conducted to screened out reliable germplasm identification method,and to provide scientific basis for Paphiopedilum germplasm identification,genetic relationship,genetic diversity analysis and molecular breeding.【Method】Used iPBS and ISSR techniques to mine molecular markers for germplasm identification of 47 Paphiopedilum sect.Concoloria germplasms,and their relationships and genetic diversity were analyzed,and molecu-lar markers which could indentify the three species were explored.【Result】A total of 117 loci were amplified by 7 primers of iPBS,of which 100 were polymorphic loci,the rate of polymorphic loci was 85.47%;the genetic similarity coefficient of 47 tested germplasms was 0.73-0.97.At a genetic similarity coefficient of 0.76,47 germplasms could be divided into three major groups,among them,six germplasms difficult to identify were grouped with known P.wenshanense to form groupⅡ,groupⅠcontained all P.bellatulum germplasms,and groupⅢcontained all P.concolor germplasms.And a to-tal of 115 loci were amplified by 7 ISSR primers,of which 111 were polymorphic loci,the rate of polymorphic loci was 96.52%.The genetic similarity coefficient of the 47 tested germplasms was 0.68~0.94.At a genetic similarity coefficient of 0.73,the 47 tested germplasms could be divided into three major groups.The groupⅠincluded 11 P.bellatulum germ-plasms,groupⅡincluded 6 germplasms with difficult phenotype identification and 13 P.wenshanense germplasms,and groupⅢincluded 17 P.concolor germplasms.Cluster analysis results for the iPBS,ISSR and iPBS+ISSR molecular mar�kers were slightly different,but germplasm cluster identification results were highly consistent.All the 47 germplasm were divided into three groups,and the six hard-to-identify individuals were clustered with P.wenshanense.P.bellatulum and P.concolor clustered into a group respectively.All the three cluster analysis showed that P.wenshanense was close to P.con-color,but far from P.bellatulum.【Conclusion】Both iPBS and ISSR molecular markers can be used for germplasm identi-fication and genetic diversity analysis among the relative species of Paphiopedilum.The combination of the two molecular markers allows for a more accurate and efficient identification of confounding germplasm at the DNA level.The results of clustering analysis of the combined data from multiple markers can more comprehensively and objectively reflect genetic polymorphisms and genetic relationships among germplasms.