【作者】 沈程文；

【导师】 黄意欢； 黄建安；

【作者基本信息】 湖南农业大学 ， 茶学， 2007， 博士

【摘要】 茶树育种和资源保存的成功依赖于对基因库资源遗传变异的数量、分布及其进化关系充分地了解和掌握。利用传统的形态和农艺性状相结合考察茶树的遗传变异和进化关系比较困难,而分子标记则是研究茶树遗传多样性和遗传分化的有效工具。本研究采用形态鉴定与SRAP、AFLP、ISSR3种分子标记对25份广东茶树种质资源和5份对照品种的遗传多样性进行了系统评价和分类研究,主要研究结论如下:1.采用10个表型性状对30份茶树种质进行鉴定和分析,各性状的平均变异系数为32.15%。其中茸毛的变异系数最大,为42.41%,变异系数最小的是芽叶生育期,为18.52%。基于表型性状的聚类分析表明,30份种质被分为4组:第1组有17个品种,第2组有10个品种,第3组为云南大叶种和凌云白毛茶2个对照品种,第4组仅海南大叶种1个对照品种。2.首次将SRAP标记应用于茶树种质资源研究,用21对SRAP引物对30份种质基因组DNA的分析,共扩增出127条带,其中114条为多态性带,比例为88.67%。每对引物组合的谱带数和多态性带数分别为6.05个和5.43个。在遗传距离0.3880处,30份茶树种质分为A、B、C三类,其中83.33%归到A类中。在遗传距离0.3140处,又可将A群划分为Ⅰ、Ⅱ、Ⅲ三个亚群。其中第Ⅰ亚群包括13个品种,第Ⅱ亚群包括2个品种,第Ⅲ亚群包括10个品种。3.利用5对AFLP引物在30份茶树种质中共扩增出401条带,平均每对引物扩增80.2条带,多态性条带338条,占总带数的84.3%。30个茶树群体品种之间以清凉山茶和清远笔架茶遗传距离最小(0.1275),桂北大叶种和凤凰水仙之间的遗传距离最大(0.6075),平均遗传距离为0.3571。结果表明广东茶树群体品种遗传多样性十分丰富。在遗传距离为0.40处取一结合线,可以将30个茶树品种分为3大类。从聚类结果可以看出,广东群体品种亲缘关系的多元性和相对集中性。4.利用15个ISSR引物在30份茶树种质中共扩增出104条带,平均每对引物扩增6.93条带,多态性条带101条,占总带数的96.89%。30份茶树群体品种的平均遗传距离为0.5018,其中平远锅苫茶与所有材料的平均遗传距离为O.7600,远高于平均水平。在遗传距离0.6500处,可将30份茶树种质分为A、B、C三类:它们分别包括18、10和2个品种。其中18个品种归到A类中,占60.00%。在遗传距离0.5500处,又可将A群划分为Ⅰ、Ⅱ二个亚群。其中第Ⅰ亚群包括14个品种;第Ⅱ亚群包括4个品种。5.首次综合运用SRAP、AFLP和ISSR3种标记方法的遗传距离结果进行相关分析,结果表明,AFLP标记与ISSR两种标记分析的遗传距离的相关性达到极显著性水平(r=0.3089,P<0.01);SRAP分析与ISSR分析的相关性达到显著性水平(r=0.1577,P<0.05);而SRAP分析与AFLP分析间相关性不显著(t=-0.0975,P>0.05)。利用灰色关联分析方法对SRAP、AFLP、ISSR标记与表型性状的聚类结果关联程度进行了研究,表型性状聚类结果与SRAP标记关联度居第一,ISSR标记的居第二;AFLP标记的居第三。6.通过对30份茶树种质的SRAP、AFLP和ISSR标记的整合和聚类分析,在遗传距离0.40处可将它们分成6大类群:第一类群包括广州小叶白心、广州小叶青心和平远锅苫茶共3个种质。第二类群只有9个种质,分别是罗定小叶、兴宁官田茶、广宁大叶青心、普宁小叶、广宁青桂大叶、高州白心、乐昌苦茶以及桂北大叶种、江西宁洲种两个对照品种。第三类群只有11个种质,包括东源上莞茶、清凉山茶、丰顺马图茶、连南大叶、乐昌白毛茶、仁化白毛茶、乳源白毛茶、台山白云茶、清远笔架茶、五华天竺山茶、南昆山白毛茶。第四类群包括了4个种质,即阳春白毛茶、惠阳小叶、云南大叶种和海南大叶种。第五类群仅1个种质即凌云白毛茶。第六类群聚集了凤凰水仙和石古坪乌龙2个乌龙茶品种。其分类较各种标记的单独使用更为合理地揭示了种质问的遗传关系,这说明方法间的不可替代性和各种标记综合的互补优势。以上研究,从形态学、分子水平上揭示了广东茶树种质的遗传多样性,为广东茶树种质资源保存以及优良品种的选育积累了数据,对广东茶树品种保护和利用具有重要意义。更多还原

【Abstract】 The tea[Camellia sinensis (L.) O. Kuntze] breeding and resources preservation depend on the fully understanding of the genetic variation amount, distribution and evolution of Gene Tank. It is difficult to study the tea’s inherited disorders and evolution by traditional measures such as form and agronomic characters analysis, while molecular marker is an effective method in studying the genetic diversity and genetic differentiation. In this study, genetic diversity in twenty-five local cultivars of the tea plants in Guangdong province and five contrastive cultivars from different regions were assessed using morphological, SRAP, AFLP and ISSR makers for the first time, and the genetic relationship among the accessions was revealed by cluster analysis at various levels. The main results are as follows:1. Analysis on the 10 morphological characters of the 30 tea cultivars showed that the average coefficient variation was 32.15%, the biggest was the fine-hair (42.41%) , and the smallest was growth period of tea leaves (18.52%) . The cluster analysis of morphological characters showed that the 30 tea cultivars could be divided into 4 groups. There were 17 in the first group, 10 in the second group, and the third group contained two contrastive cultivars: Yunnan-dayezhong, and Lingyun-baimaocha, there was only one group-Hainan-dayezhong in the forth group.2. In this study, genetic diversity in tea plant was assessed using SRAP makers for the first time at home and abroad, which showed that SRAP was an effective, cheap and dependable marker for polymorphism analysis in tea plant. A total of 127 amplified fragments and 114 polymorphic fragments were detected by applying 21 SRAP primer combinations. The polymorphic fragment percentage was 88.67%. The number of amplified fragments and polymorphic fragments per primer combination were 6.05 and 5.43 respectively. 30 tea accessions were divided into four groups of A, B and C at genetic distance of 0.388, 83.33% of which were classified into group A. Group A could also be divided into I, II and IIIsub-groups at genetic distance of 0.314. There were 13 in the I subgroup, 2 cultivars in the II subgroup and 10 in the III subgroup.3. The AFLP silver staining protocol was used for amplification reaction in the experiment, twenty-five local cultivars of tea plant in Guangdong province and five contrastive cultivars were tested and analyzed using 5 primer combinations, 401 bands were obtained, 338 bands (84.3%) of them were found to be polymorphic,80.2 bands were obtained by every one primer pair on the average. The amplification fragment ranged from 50bp to 580bp. Among the 30 cultivars, the greatest genetic distances was between Guibei-daye and Fenghuang-shuixian (0.6075) , the lowest genetic distance was between Qingliangshan-cha and Qingyuan-bijiacha (0.1275) . It was reported here that the local tea plant of Guangdong province was characterized by a high genetic diversity. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA) of SPSS 12.0. 30 cultivars could be divided into three groups according to the combine line 0.40 of the genetic distance. This result shows that local Guangdong tea plants’ consanguineous relationship is diversified and concentrative.4. The 15 ISSR primers generated 104 polymorphic bands in the 30 cultivars, 101 bands (96.89%) of which were found to be polymorphic,6.93 bands were obtained by every oneprimer pair on the average. Among the 30 cultivars the average genetic distances were 0.5018 ,the greatest genetic distances was between Pingyuan-guoducha and others (0.7600) . We find 30 cultivars can be divided into three groups according to the combine line 0.6500 of the genetic distance which containe 18、10 and 2 cultivars respectively. The 18 cultivars can be grouped to A type(60.00%), according to the combine line 0.5500 of the genetic distance. A type can be divided into I, II subgroups, the I subgroup containes 14 cultivars and the II subgroup containes 4.5. There was remarkable correlation between the genetic distance of AFLP and ISSR (r =0.3089, P<0.01) molecular markers, significant correlation between SRAP and ISSR (r =0.1577, P< 0.05) , while the result of SRAP and AFLP had no significant correlation (r =-0.0975, P > 0.05). Based on the gray relational and analytical method, a relational level among SRAP, AFLP and ISSR makers and morphological cluster dendrogram was studied. The following conclusions were drawn. The relational level of SRAP marker lied in the fist place, the relational level of ISSR marker was in the second, and the relational level of AFLP marker was in the third.6. By analyzing the integrated data from SRAP、AFLP and ISSR data, they were classified into 6 groups, according to the combine line 0.40 of the genetic distance. The first group included 3 cultivars, those were Guangzhou-xiaoyebaixin, Guangzhou-xiaoyeqingxin and Pingyuan-guoducha, the second group contained 9 cultivars, those were Luoding-xiaoye, Xingning-guantiancha, Guangning-dayeqingxin, Puning-xiaoye, Guangning-qingguidaye, Gaozhou-baixin, Lechang-kucha, Guibei-dayezhong and Jiangxi-ningzhouzhong, the third group included 4 cultivars, those were Qingliangshan-cha, Fengshun-matucha, Liannan-daye, Dongyuan-shangguancha, Lechang-baimaocha, Renhua-baimaocha, Ruyuan-baimaocha, Taishan-baiyuncha, Qingyuan-bijiacha, Wuhuatianzhushan-cha and Nankunshan-baimaocha, the forth group included 4 cultivars, those were Yangchun-baimaocha, Huiyang-xiaoye, Yunnan-dayezhong and Hainan-dayezhong, the fifth group was Lingyun-baimaocha, the sixth group included Fenghuang-shuixian and Shiguping-wulong. The results reflected the genetic relationship among tea accessions more rationally than using different markers separately. This told us that different methods could not be replaced simply and it was feasible and proper to integrate various data.In summary, the quantitative criteria for evaluating genetic diversity and sampling strategy for genetic resources conservation and utilization of local cultivars of Guangdong province tea plant had been provided, and the results could have been used to guide genetic resources collection, conservation and elite breed selection of tea plant. This study was significant for genetic diversity research and conservation of local cultivars of tea plant in Guangdong province.更多还原