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以三年桐品种对年桐发育中的种子为材料,构建c DNA文库,获得3202个原始EST序列,经过去除冗余和低质量序列后,获得1047条非冗余单一序列,利用MISA软件从其中的173个非冗余EST序列中搜索得到212个SSR位点。其中,二核苷酸重复类型为主导(68.39%),其次是三核苷酸重复类型(25.94%),其优势基元为AG/CT(43.87%)、AT/AT(19.34%)和AGC/GCT(5.66%)。针对可设计引物的68个序列进行EST-SSR引物设计,结合PCR扩增和数据分析,鉴定出14对多态性标记,用该14对引物对169份三年桐种质资源进行遗传多样性检测,共得到41个等位基因(Na),平均每对引物扩增出2.93个等位基因,期望杂合度(He)变幅为0.08~0.63,平均值为0.33,多态性信息含量(PIC)变幅为0.08~0.56,平均值为0.3,表明14对EST-SSR标记多态性较高,能较好地反映三年桐种质的遗传多样性。三年桐种质资源按群体进行非加权配对算术平均法(UPGMA)聚类分析,群体间的相似度变幅为0.9604~0.9986,遗传距离变幅为0.0022~0.0404,表明三年桐群体间的遗传多样性相对较低,亲缘关系较近。对169份三年桐种质资源个体进行聚类分析,结果显示不同地理种源的遗传关系缺乏明显的地理结构。
A total of 3,202 EST sequences were obtained from the seeds of three year old Triticum aestivum L. grown in Tongtong, and 1047 non-redundant single sequences were obtained after removing the redundant and low quality sequences. Using MISA software, Of 173 non-redundant EST sequences, 212 SSR loci were found. Among them, the dinucleotide repeat types were predominant (68.39%), followed by trinucleotide repeat types (25.94%). The dominant motifs were AG / CT (43.87%), AT / AT /GCT(5.66%). Sixty-eight polymorphic markers were identified by PCR amplification and data analysis of 68 sequences of designed primers, and 169 of them were used for genetic diversity analysis A total of 41 alleles (Na) were obtained, with an average of 2.93 alleles per primer pair. The expected heterozygosity (He) ranged from 0.08 to 0.63 with an average of 0.33. The polymorphism information content PIC) ranged from 0.08 to 0.56 with an average of 0.3, indicating that 14 EST-SSR markers were highly polymorphic and could well reflect the genetic diversity of Triticeae. Three-year Tung germplasm resources were clustered by unweighted pairwise arithmetic average method (UPGMA). The variation range of similarity between groups was 0.9604 ~ 0.9986, and the genetic distance range was 0.0022 ~ 0.0404, indicating that among the three groups, Relatively low genetic diversity, close genetic relationship. Cluster analysis of 169 Triticeae germplasm resources showed that the genetic relationship of different geographic provenances lacked a clear geographical structure.