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

【Objective】Based on primer binding site amplification(iPBS) and simple sequence inter repeat DNA polymorphism analysis(ISSR) molecular markers,the germplasm molecular marker identification and genetic diversity analysis 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 molecular 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 group Ⅱ, groupⅠcontained all P. bellatulum germplasms, and group Ⅲ contained all P. concolor germplasms. And a total 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 germplasms, group Ⅱ included 6 germplasms with difficult phenotype identification and 13 P. wenshanense germplasms, and group Ⅲ included 17 P. concolor germplasms. Cluster analysis results for the iPBS,ISSR and iPBS+ISSR molecular markers 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. concolor,but far from P. bellatulum. 【Conclusion】Both iPBS and ISSR molecular markers can be used for germplasm identification and genetic diversity analysis among the relative species of iPaphiopedilum. 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.