蝴蝶兰属与钻喙兰属通用SSR引物的开发与应用

Development and Application of Universal SSR Molecular Marker Primers in Phalaenopsis and Rhynchostylis

利用蝴蝶兰属28个种的38份原生种资源和钻喙兰属3个种的9份原生种资源的简化基因组测序数据,开发筛选出多态性高、通用性强的19对蝴蝶兰属内种间通用、12对钻喙兰属内种间通用和12对两属间通用的SSR引物。采用荧光标记毛细管电泳进行等位变异检测,12对属间通用引物在两个属的47份种质中共检测到134个等位基因,每对引物扩增出等位基因6~17个,平均观测等位基因数(N_(a))为11.17,平均有效等位基因数(N_(e))为6.36,期望杂合度(H_(e))为0.66~0.88,观测杂合度(H_(o))为0.07~0.38。所有引物位点均呈高多态性,Shannon指数介于1.30~2.43,均值为1.88,说明引物的多态性极高,适用于蝴蝶兰属和钻喙兰属种质资源的鉴定。47份种质资源聚类结果和群体结构分析能较明确地揭示蝴蝶兰属和钻喙兰属种质的亲缘关系,与植物学分类结果基本一致。在蝴蝶兰‘Red Rose’与钻喙兰的跨属杂交F1代群体检测中,有23对通用引物具多态性,扩增出75个多态性条带,杂种之间的遗传相似系数介于0.58~1.00,母本与属间杂种的平均遗传相似性系数(0.62)高于父本(0.54),表明该跨属杂交F1代偏母系遗传,与表型观测结果吻合。

Based on simplified genome sequencing data of 38 germplasm resources from 28 native species of Phalaenopsis and nine germplasm resources from three native species of Rhynchostylis,19 pairs of universal SSR primers in Phalaenopsis,12 pairs of universal SSR primers in Rhynchostylis,and 12 pairs of universal SSR primers between the two genera were developed and selected for their high polymorphism and universality.Fluorescent-labeled capillary electrophoresis was used for allele variation detection.Among the 47 germplasm resources,a total of 134 alleles were detected by the 12 pairs of universal primers between genera,with 6–17 alleles amplified per pair of primers.The mean observed number of alleles(N_(a))was 11.17,the mean effective number of alleles(N_(e))was 6.36,and the expected Heterozygosity(H_(e))ranged from 0.66 to 0.88,while the observed heterozygosity(H_(o))ranged from 0.07 to 0.38.All primer sites showed high polymorphism,with Shannon's index ranging from 1.30 to 2.43 and a mean of 1.88,indicating that the primers have high polymorphism and were suitable for identifying germplasm resources of both two genera.Cluster analysis and population structure analysis of the 47 germplasm resources could clearly reveal the genetic relationships between Phalaenopsis and Rhynchostylis species,which is consistent with the results of botanical classification.In the detection of F1 hybrid populations between Phalaenopsis Jiuhbao Red Rose and Rhynchostylis retusa 23 pairs showed polymorphism,and a total of 75 polymorphic bands were amplified.The genetic similarity coefficient between hybrids ranged from 0.58 to 1.00,with an average genetic similarity coefficient between maternal parents and intergeneric hybrids(0.62)higher than that between paternal parents(0.54),indicating that the F1 generation of intergeneric hybrids showed maternal inheritance,which is consistent with the phenotypic observation results.