【作者】 马玉敏；

【导师】 陈学森； 束怀瑞；

【作者基本信息】 山东农业大学 ， 果树学， 2009， 博士

【摘要】 板栗(Castanea mollissim Blume)起源于中国大陆,种质资源极为丰富。在长期的生态适应过程中,中国板栗在各地迥异的地理气候条件下形成了不同的生态群,分为长江流域、华北、西北、东南、西南生态群。其中中国野生板栗被认为是世界栽培板栗的原生起源种,曾在世界栽培板栗的驯化过程中起过决定性的作用,并且蕴藏着大量的优良珍稀类型。但是,近二十年来,由于国内外市场的巨大需求,板栗良种产业化进程加快,从野生和半野生种质向良种集约化栽培迈进,野生资源遭到严重破坏,致使一些珍贵的种质消失。本文利用分子系统学的原理和方法,结合传统的表型研究体系,采用荧光AFLP标记对中国野生板栗群体遗传结构和遗传变异进行分析,并应用分子标记和表型数据,分别探讨了中国野生板栗核心种质构建方法,研究结果将对这一珍贵资源的科学保护和有效利用以及丰富生物进化理论有重要意义。主要研究结果如下:1.对中国板栗秦岭山脉生态区、泰沂山脉生态区和燕山山脉生态区的3个种下居群118个野生株系的叶片、果实形态和坚果主要营养成分进行了调查研究,结果表明,3个居群的叶片大小、叶柄长度和粗度以及果实形状、大小等形态性状的变异系数均在10%以上,表现出丰富的遗传多样性,其中以叶面积及单粒重变异幅度和变异系数最大;而果形指数变异幅度和变异系数最小,是较稳定的植物学性状,3个居群的变异趋势基本一致;3个居群118个野生株系果肉中水分、总糖、淀粉、蛋白质、脂肪、灰分和维生素C等各种主要营养成分的含量在单株间差异显著,变异系数6.2% - 28.3%,其中以坚果蛋白质含量的变异幅度和变异系数最大,遗传多样性丰富,3个居群的变异趋势基本一致;2.对中国野生板栗的同一性状在秦岭山脉、泰沂山脉和燕山山脉的3个不同生态区下的遗传变异研究表明:同一性状在不同居群间大都存在显著或极显著差异,其中单叶面积和单粒重以秦岭山脉居群最小,分别为56.34cm2和2.95g,而维生素C含量最高(96.7mg/100g鲜果),分别是燕山山脉和泰沂山脉居群的2.29倍和2.22倍。相关分析结果显示,单粒重和单叶面积与降雨量和经度显著正相关,与海拔高度显著负相关,而维生素C含量与降雨量显著负相关,与海拔高度正相关,表明环境对板栗表型性状的遗传变异具有较大影响;3.以长江流域生态群、华北生态群、西北生态群、东南生态群和西南生态群的120个中国板栗株系为试材,利用荧光AFLP标记对中国野生板栗五个生态群的群体遗传结构进行了研究,结果表明:8对EcoR I /Mse I引物(其中Mse I引物为FAM荧光标记物)平均扩增多态位点数为180.88,平均多态位点百分比为83.31%。五个生态群的多态位点数和多态位点百分比比较表明:长江流域生态群(A = 150.38;P = 73.80%)>华北生态群(A = 147.75;P = 71.93%)>西北生态群(A = 137.88;P = 68.00%)>东南生态群(A = 90.50;P = 50.32%)>西南生态群(A = 86.00;P = 48.29%);中国板栗在种级水平Nei’s基因多样度(H = 0.206)和Shannon信息指数(I = 0.326)显著或极显著高于群体水平。在群体水平上,长江流域生态群的Nei’s基因多样度和Shannon信息指数(H = 0.191;I = 0.302)高于华北生态群(H = 0.185;I = 0.295)和西北生态群(H = 0.182;I = 0.289),但无显著性差异,显著高于东南(H = 0.142;I = 0.229)和西南生态群(H = 0.139;I = 0.218);对中国野生板栗5个生态群的群体遗传多样性和群体遗传结构分析认为,长江流域生态群的遗传多样性最为丰富,揭示了长江流域为中国板栗遗传多样性中心的可能性;4.本研究对中国野生板栗群体遗传结构的参数------遗传分化系数和基因流进行了分析。中国野生板栗5个生态群的遗传分化系数(Gst = 0.0952)显示,中国野生板栗的遗传变异主要存在于群体内,占总变异的90.48%,而群体间的遗传变异仅占总变异的9.52%。通过遗传分化系数计算得Gst基因流Nm = 4.752,说明中国野生板栗5个生态群存在适度的基因交流,这种遗传变异空间结构模式的形成可能是长距离基因流、自然气候、地理距离隔离等诸因素综合作用的结果;5.中国野生板栗5个生态群的Nei’s群体遗传一致度在0.8876 - 0.9665之间,遗传距离在0.0341 - 0.1193之间,说明群体间的相似程度较高,遗传距离较小。对5个生态群用UPGMA法进行聚类分析发现,西南和东南生态群首先聚在一起,之后华北和西北生态群聚在一起,最后长江流域生态群同西北和华北生态群聚在一起,此聚类图从直观上表明西南生态群和东南生态群相似性最高,遗传关系最近;6.以297个中国野生板栗株系的19个表型性状的遗传多样性数据为基础,探讨了采用表型性状构建中国野生板栗核心种质的方法,结果表明,采用马氏距离聚类优于欧氏距离,5种聚类方法比较,类平均法、离差平法和法和最长距离法优于最短距离法和中间距离法,偏离度取样策略优于随机和优先取样策略。对构建的核心种质采用主成分分析(PCA)对其进一步的确认,分布图显示核心种质遍布整个主成分图,并且远离中心的,具有特异性状的株系都入选为核心株系,确保了核心种质的代表性。本研究显示,当取样比例为20%时,采用马氏距离,利用离差平法和法进行多次聚类,结合偏离度取样策略构建的核心种质最有代表性,是采用形态学数据构建中国野生板栗核心种质的最佳的方法;7.采用荧光AFLP分子标记,以120个中国板栗野生株系为材料,研究了利用分子标记构建中国野生板栗核心种质的方法。同对照随机取样策略比较,位点优先取样策略能构建一个更有代表性的核心种质。采用主坐标对位点优先取样策略构建的核心种质的代表性进行确认,分布图显示核心种质遍布了整个坐标图,确保了核心种质的代表性。当选取25个株系时,根据SM、Jaccard或Nei & Li遗传距离进行多次聚类,采用位点优先法,是构建中国野生板栗核心种质较合适的方法。更多还原

【Abstract】 Chestnut (Castanea mollissima Blume) is an ancient deciduous fruit tree crop in the temperate zones, which originates in China. Because of diversity of ecological inhabits and differences and selections of adaptability between chestnut for ecological environments, and a long selection by natural factors, chestnut trees evolutes and forms different geo-ecological groups under different geo-ecological conditions: the Yangtze group, the Northern China Group, the Northwest Group, the Southwest Group and the Southeast group. The diversity of 3 wild chestnut intraspecific populations was analyzed using morphological traits, the genetic diversity and population genetic structure for five geo-ecological groups using principles of molecular systematics and fluorescent-AFLP marker techniques, and the methods of constructing core collection were studied by morphology traits and molecular markers, respectively, in order to provide science evidence for conservation and utilization, to construct core collection of C. mollissima Bl. and breed the cultivated chestnut. The main results are as follows:1. The diversity of leaf, petiole and nut in 3 wild intraspecific populations (the Qinling Mountains ecotype, the Taiyi Mountains ecotype, and the Yanshan Mountains ecotype) of C. mollissima Bl. were investigated. The results indicated: There were significant differences among and within population. The morphological traits, such as leaf length, width, leaf shape index, length of petiole and nut shape, size, color, have great morphological diversity with variation coefficient (CV) of more than 10%. The range of nut weight varied from 1.69g to 3.89g in the Qinling Mountains population with CV 18.3%. The range and CV of leaf area and nut weight were the largest, and those of leaf shape index were the smallest; The content of main nutrition composition (water, total sugar, starch, protein, fat, ash and Vc) in ripe nuts determined in obtained 118 seedlings at the Qinling Mountains, the Taiyi Mountains and the Yanshan Mountains was analyzed and presented considerable genetic variations with CV of 6.2% - 28.3%, and the range and CV of the protein were the largest. There were the same trends in the 3 wild intraspecific populations;2. Compared with the diversity of the same trait in the different conditions, the result showed that, there was significant difference between site conditions with smallest CV in leaf area and nut weight and highest CV of Vitamin C contents in the Qinling Mountains; Correlation analysis revealed that mean weight of a single nut with annual rainfall and the longitude of each ecotype, in contrast, they were negatively correlated with the altitude. Vitamin C contents in the nuts from different ecotype were negatively regulated by annul rainfall. These relationships indicated that environmental conditions had impact on genetic diversity in Chinese wild chestnut.3. Population genetic structure was studied using fluorescent-AFLP markers on 120 Chinese Chesnut (Castanea mollisima Blume) accessions collected from the Yangtze River group, the Northern China group, the Northwest group, the Southeast group, and the Southwest group of fluorescent-AFLP markers. The purpose of this study was to determine the genetic structure and genotypic diversity amongst the different eco-geographical populations of Chinese chestnut. The results showed that the average number of polymorphic loci (A) was 180.88, the percentage of polymorphic loci (P) was 83.31% by 8 pairs of EcoR I /MseI (Mse I - a FAM fluorescent marked primer) primers in Chinese chestnut of five groups. Analysis for the average number of polymorphic loci (A) and the percentage of polymorphic loci (P) in five Chinese chesnut groups indicated that the Yangtze River group (A = 150.38; P = 73.80%)> the Northern China group (A = 147.75; P = 71.93%)> the Northwest group (A = 137.88; P = 68.00%)> the Southeast group (A = 90.50; P = 50.32%)> the Southwest China group (A = 86.00; P = 48.29%); Nei’s gene diversity (H = 0.206) and Shannon information index (I = 0.326) at species level was significant or high significant higher than at group level. At group level, Nei’s gene diversity and Shannon information index (H = 0.191; I = 0.302) in the Yangtze River group was higher than those in the Northern China group (H = 0.185; I = 0.295)and in the Northwest group(H = 0.182; I = 0.289), there were no significant differences; but they were significant higer than those in the Southeast group(H = 0.142; I = 0.229)and the Southwest group(H = 0.139; I = 0.218).The analysis of five population diversity and genetic structure from five geo-ecological groups showed that genetic diversity of wild chestnut population in the Yangtze Group was most abundant. This suggested that chestnut originated in the Yangtze geo-ecological group, then diffused to the northern China and the southern China by natural factors.4. The parameters--genetic differentiation coefficient and gene flow of population genetic structure in wild chestnut were analyzed in this study. Genetic differentiation coefficient (Gst = 0.0952) for five wild chestnut groups showed that wild chestnut genetic variation was mainly within the groups and accounted for 90.48% of total variations. The genetic variation between groups accounted for 9.52% of total variations. The gene flow Nm was 4.752 according to the genetic differentiation coefficient between groups (Gst = 0.0952). This indicated that there are partly gene exchanges among five wild chestnut groups. It is suggested that the main way of gene exchanges could be main factors of the long distance gene flow, natural climate, geographical barriers geography distance et al.5. Nei’s genetic identities in five wild chestnut groups were between 0.8876 - 0.9665. Genetic distances were between 0.0341 - 0.1193. It was suggested that there were higher similarity between various geo-ecological groups in wild chestnut and lower genetic distance. The similarity between the Southwest group and the Southeast group was highest (99.06%), which showed that genetic differentiation was lowest between both groups in wild chestnut. The results from UPGMA cluster analysis for five groups showed that the Southeast and Southwest wild chestnut in the southern China was clustered together firstly and the Northern China and Northwest wild chestnut in northern China, then the Yangtze Group and the Northern China and Northwest group were clustered together. This indicated further that the similarity between the Southeast group and the Southwest group wild chestnut was highest and genetic relationship was closest.6. The diversity of 7 leaf traits and 12 nut traits from 297 accessions of C. mollissima Bl. was used to study method for constructing C. mollissima Bl. core collection using morphology. The results showed that Mahalanobis distance was the much better than Euclidean distance; UPGMA, Ward’s method and Complete linkage was better than Single linkage and Median method, and deviation sampling was more suitable than random sampling and preferred sampling for constructing core collection. When 20% accessions were selected, Mahalanobis distance and Ward’s method using stepwise clustering combining with Deviation sampling can construct a most reprehensive core collection and was the most suitable method for constructing C. mollissima Bl. core collection.7. The method for constructing core collection of C. mollissima Bl. based on molecular markers data was proposed, according to fluorescent AFLP marker, using 120 accessions of C. mollissima Bl. Compared with the random sampling strategy, allele preferred sampling could construct more representative core collections. When 25 accessions of C. mollissima Bl. was selected, allele preferred sampling strategy combined with SM, Jaccard or Nei & Li genetic distances using stepwise clustering was the suitable method for constructing C. mollissima Bl. core collection.更多还原