基于EST-SSR分子标记的蝴蝶兰种质资源遗传多样性分析和倍性预测OA

Phalaenopsis spp.has extremely high economic and ornamental value.The breeding of its new varieties generally adopts interspecific and intraspecific hybridization,resulting in complex genetic background and ploidy.In this study,expressed sequence tag-simple sequence repeat(EST-SSR)molecular marker technology was used to conduct genetic diversity analysis,hybrid identification,cluster analysis and ploidy prediction on 31 Phalaenopsis germplasm resources as well as the reciprocal F₁ hybrids of'Treasure Map'and'075'.The aim was to provide a basis for the utilization and innovation of Phalaenopsis germplasm resources,molecular marker-assisted breeding,ploidy identification and authenticity identification of hybrid progeny.Based on 19 reported SSR primer pairs combined with the M13 universal primer,capillary fluorescence electrophoresis was performed.Ten pairs of EST-SSR primers were screened for genetic diversity,genetic relationship analysis and ploidy prediction of 31 germplasm resources of Phalaenopsis.Seven pairs of EST-SSR primers with paternal characteristic bands were screened for authenticity,cluster analysis and ploidy prediction of 50 F1 hybrid progeny lines,and 50 F1 hybrid progeny lines were identified by flow cytometry combined with chromosome counting.The results showed that a total of 233 alleles were detected in 10 pairs of primers,the average number of alleles detected at each locus was 23.3,the total number of effective alleles was 137.65,the average number of effective alleles at each locus was 13.76,and the polymorphism information content(PIC)value of 10 pairs of primers varied in the range of 0.32～0.91,only primer Pap-3754 had moderate polymorphism,and the other 9 primers had high polymorphism.The cluster analysis of 31 Phalaenopsis germplasm resources showed that the 31 Phalaenopsis germplasm resources were divided into 3 categories at the genetic similarity coefficient of 0.23,among which the classⅠand class Ⅱwere native to Phalaenopsis orchid,and the 25 materials of classⅢwere Phalaenopsis horticultural species.The authenticity of the hybrid F1 progeny showed that all 50 hybrid F1 progeny lines were identified as true hybrids.Cluster analysis of 50 crosses showed that the 49 of them tested were closely related to the maternal parent,and only one line,L25,was closely related to the paternal parent.Flow cytometry and chromosome counting results showed that the hybrid offspring of Phalaenopsis were tetraploid,and compared with the molecular marker results,the accuracy of EST-SSR molecular marker was as high as 98%.The results indicated that the EST-SSR molecular marker could be used for the analysis of genetic diversity,ploidy prediction and early identification of hybrid offspring of Phalaenopsis orchid.This study is of positive significance for improving the breeding efficiency of new varieties and exploring the genetic background of Phalaenopsis.