摘要：

为建立莴苣品种DNA分子快速鉴定技术体系,选择8个表型差异大、不同类型的莴苣品种,利用聚丙烯酰胺凝胶电泳从245对引物中初筛出81对引物,对初筛引物添加荧光标记,另选择90个有代表性的莴苣品种,利用毛细管电泳复筛出22对核心引物,依据扩增片段大小为各引物选择了相应的参照样品,采集了233个莴苣品种的DNA指纹数据。结果表明,22对核心引物在233个莴苣品种中共检测到269个等位基因,每对引物检测到等位基因为4~22个,平均为12.23个,PIC值为0.37~0.89,平均为0.73。聚类分析结果表明,233个供试品种主要划分为茎用和叶用两大类群,叶用类群中结球莴苣聚成一类,半结球莴苣趋于聚成一类,散叶莴苣难以聚成一类。对核心引物未能区分开的6组15个品种进行田间种植对比鉴定,3对品种表型性状无明显差异被判定为相同品种,表型性状有明显差异的品种大部分性状表达状态相同或相近,为近似品种,表明分子指纹聚类结果与基于表型的分类结果基本一致。此外,8组24个同名品种均能从分子上进行区分。利用筛选出的22对核心引物构建了233个莴苣品种的指纹数据库,建立了通用于茎用莴苣和叶用莴苣的品种鉴定技术体系,可用于莴苣品种和种质资源快速鉴定、遗传多样性分析和DUS测试辅助筛选近似品种等工作。

关键词: 莴苣, 品种鉴定体系, SSR标记, DNA指纹图谱, DUS测试

Abstract:

In order to establish a rapid DNA molecule identification system of lettuce varieties,8 lettuce varieties of different types with significant morphological differences were chosen and used for preliminary primer-screening,and 81 pairs of primary primers were selected from 245 pairs of primers by polyacrylamide gel electrophoresis. Labeled with fluorescence at 5' end of upstream,the 81 pairs of primary primers were re-screened with 90 lettuce varieties by capillary electrophoresis,and 22 pairs of core primers were finally screened out. The corresponding reference samples for each of the core primers were also selected according to the size of amplified fragments and a SSR fingerprint database of 233 varieties was constructed based on these core primers. The results showed that 22 pairs of core primers detected a total of 269 alleles in 233 varieties. For each pair of primers,the detected alleles ranged from 4 to 22,with an average of 12.23,and the PIC value ranged from 0.37 to 0.89,with an average of 0.73. Cluster analysis showed that 233 varieties were divided into two major groups of stem lettuce and leaf lettuce. Among the leaf groups,crisphead lettuce cluster into a group,half-hitch lettuce tended to cluster into a group,and looseleaf lettuce was difficult to cluster into a group. 15 varieties in 6 groups with no difference in SSR markers were compared in a field trail. 3 pairs of varieties with no significant morphological differences were determined as the same and other varieties from different groups were determined as similar varieties because of a few morphological differences. Thus,the result of DNA clustering was basically in agreement with that of the field morphological classification. In addition,24 varieties in 8 groups with the same name can be distinguished by the molecular markers. In conclusion,a fingerprint database of 233 lettuce varieties was constructed based on the 22 pairs of core primers and a variety identification system for stem lettuce and leaf lettuce was established,which could be used for rapid identification of lettuce varieties and germplasm resources,genetic diversity analysis and similar variety selection in DUS testing.

Key words: Lettuce, Variety identification system, SSR markers, DNA fingerprint, DUS testing