【作者】 刘亚令；

【导师】 黄宏文；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 猕猴桃隶属于猕猴桃科(Actinidiaceae)猕猴桃属(Actinidia Lindl.)，为多年生木质藤本植物，雌雄异株，是一种重要的果树资源。在我国野生资源丰富，是半特有属。随着生态环境的破坏和猕猴桃产业化的发展，为了解决全世界猕猴桃产业支柱建立在单一的雌性品种上的遗传风险，需了解其自然资源现状，提出有效的保护和利用措施，通过育种手段，获得高产、优质的品种是实现猕猴桃产业健康发展的必经之路。另一方面，猕猴桃种间杂交和种内多倍化现象的普遍存在，使物种间界限不明显，了解种间的遗传结构和基因渐渗现象，可以为猕猴桃属植物的系统进化和分类关系提供重要的理论依据。本文结合以上几个方面，以中华猕猴桃(Actinidia chinensis)和美味猕猴桃(A．deliciosa)为主，同时涉及阔叶猕猴桃(A．latifolia)、毛花猕猴桃(A．eriantha)、绵毛猕猴桃(A．fulvicoma var．lanata)、金花猕猴桃(A．chrysantha)和京梨猕猴桃(A．callosa var．henryi)等7个分类群，利用SSR标记从DNA水平上对10个同域分布的不同物种自然居群进行了遗传多样性、居群遗传结构的分析以及种间杂交渐渗的探讨。主要结果如下：(1)利用9对SSR引物，在猕猴桃属7个物种共747个样本上共检测到253个等位基因，平均每位点等位基因数为28.11个，扩增等位基因数最多的位点为UDK96-009(48个)。在同域分布物种间的共有等位基因上，中华猕猴桃和美味猕猴桃的共有等位基因的比率最高，达60％以上；而湖北莽山(HM)毛花猕猴桃和金花猕猴桃物种间共有等位基因比率最低为18.5％。共享等位基因表型频率在物种间存在着差异，而且与各同域分布物种样本的交错程度或间距存在关联。(2)居群内遗传多样性分析表明：各物种居群内等位基因平均数A为4.45～14.44；多态位点百分率P=100，PIC值为0.644～0.870，表明猕猴桃属植物自然居群具有极高的遗传多样性。各物种居群间的分化系数依次为：美味猕猴桃(0.080)＜中华猕猴桃(0.086)＜毛花猕猴桃(0.116)＜金花猕猴桃(0.118)＜阔叶猕猴桃(0.119)。两两物种间的遗传分化系数平均范围在0.008～0.108，同域分布物种间的遗传分化低于物种内异域居群间的遗传分化，其中同域分布的中华猕猴桃和美味猕猴桃两物种间的基因流均为最高，其余物种间的基因流也都大于1。利用AMOVA分析结果同样表明：大部分遗传变异来源于居群内(79.45％)，居群间遗传分化系数为0.1698。(3)居群UPGMA聚类揭示中华猕猴桃和美味猕猴桃以地理区域优先聚类，其它物种多以物种分类群优先聚类，个体聚类结果显示多数个体聚在各自居群组内，但各地理居群并不按地理距离的远近聚类，这与Mantel相关性检测所揭示的居群间遗传距离与地理距离没有显著性相关的结果一致。中华／美味猕猴桃复合体近缘种间存在明显的共祖多态性和杂交渐渗现象，近缘种植株分布的交错程度或是否存在亚居群结构是影响杂交渐渗的因素之一。阔叶猕猴桃和毛花猕猴桃也存在着非对称性基因渐渗，表明亲缘关系较远的物种间杂交渐渗事件稀少，但存在个别同塑事件。(4)采用空间自相关分析方法对河南西峡和陕西商南同域分布的中华猕猴桃和美味猕猴桃自然居群SSR遗传变异的空间结构进行了分析，选用的9对SSR引物分别从两居群中共扩增出104个等位基因，选择其表型频率在20％～80％的SSR等位基因，运用等样本频率方法分别对中华猕猴桃和美味猕猴桃及其中华／美味猕猴桃复合体居群进行了空间自相关系数Moran’s I值计算。结果表明：中华猕猴桃和美味猕猴桃的遗传变异在两居群内均存在着一定程度的空间结构，尽管近半数或半数以上的等位基因在居群内表现为随机分布的空间模式，但也有相当比例的等位基因在居群内(29.6％～48.0％)和复合体居群内(51.0％，44.7％)呈现渐变、衰退、双向衰退或侵扰模式。而且其居群内遗传变异的空间分布规律都基本一致：相距在100 m以内，特别是30 m范围内的个体间的等位基因表现出显著性的正相关，随着地理距离的增大逐渐显示出负相关。本文通过SSR分子标记，运用不同的统计软件对结果进行分析，从不同的方面揭示了猕猴桃属植物中存在较高的遗传多样性和物种间的基因渐渗，进一步分析表明猕猴桃的遗传多样性和居群遗传结构不仅受其广域分布、远交、晚期分化等生活史特性的影响，同时还与猕猴桃的染色体基数高(x=29)、倍性复杂和种间杂交等因素密切相关。本研究结果有助于进一步探讨猕猴桃属植物的遗传变异、种群扩散及其地理系统发育进化等方面的规律，并为制定相应的保育策略和措施提供了基础数据和科学依据。更多还原

【Abstract】 Kiwifruit, belonging to the Actinidia Lindl. of family Actinidiaceae, is an important wild fruit resource in the world. It is a woody and dioecious liane, and most species of this genus are native to China. Although the wild resource of kiwifruit is very abundant, only few cultivars are under commercial cultivation, which pose great risk to the current global kiwifruit industry. Understanding genetic variation and population structue of natural populations of Actinidia is crucial importance for both genetic resourece conservation and breeding strategy deployment, especially for Actinidia chinesis, A. deliciosa, A. latifolia, and including A. eriantha, A. chrysantha, A. fulvicoma var. lanata, A. callosa var. henryi. On the other hand, due to the easy interspecies hybridization and highly polyploidization in Actinidia, it is difficult to delimitate species boundaries for this genus. Knowledge of genetic structure and introgression among populations may shed lights on the evolution and specialization of Actinidia. In this study, we used a set of microsatellite (also named SSR) data to investigate genetic diversity, population structure and interspecies introgression for 10 sympatric natural populations within seven Actinidia species. These results were as follows:(1) A total of 253 alleles were detected with the nine screened primer pairs for a total of 747 individuals. The loci UDK-96-009 has the most number of alleles with A=48, and the mean number of alleles across all loci is A=28.11. As for each sympatric population, a large number of common alleles (more than 60.0%) were found in both A. chinesis and A. deliciosa, whereas the lowest number of common alleles (18.5%) was detected in A. eriantha and A. chrysantha in HM-population. The phenotypic frequencies of shared alleles were different among populations-species comparison, which was correlated with the degree of mixture and the distances of individuals from different species in each sympatric complex;(2) High levels of genetic diversity were observed within Actinidia populations, with the mean alleles A=4.45～14.44, percentage of polymorphic loci P=100, and polymorphism information content (PIC) values ranged from 0.644 to 0.870. The genetic differentiation between populations within species demonstrated A. deliciosa (0.080)＜A. chinesis (0.086)＜A. eriantha (0.116)＜A. chrysantha (0.118)＜A. latifolia (0.119). The mean values of genetic differentiation between species range from 0.008 to 0.108. All sympatric populations of A. deliciosa and A. chinesis have higher gene flow (Nm) than that of other species. The analysis of AMOVA showed that the genetic variation majorly distributed within populations (79.45), and 16.98% of the genetic variation was partitioned between populations.(3). The UPGMA dendrogram showed that A. deliciosa was closely related to A. chinesis according to geographic distances while other species clusted according to taxonomic units, suggesting a very close relationship of these two species. Mantel test revealed no significant correlation between the genetic distance and geographic distance. The individual cluster with Bayesian and Newhybrid methods showed that A. chinensis/ A. deliciosa complex was probably highly influneced by both shared ancestral gene pool and hybridization introgression. However, hybridization introgression is rarely occurred between less related species, for example asymmetrical interspecific introgressive hybridization was detected between A. eriantha and A. latifolia, although they are highly genetic differentiated. A stable hybrid zone has been characterized by important members of backcross genotypes in A. eriantha and A. latifolia. As for other species, cluster analysis also showed genetic crossing with different foreign species although only a small number of individuals has been included, suggesting a confirmed single species complex plus potential ecological plastic cline.(4) The spatial patterns of genetic variation in two populations (Henna-xixia population and Shannxi-shangnan population) with species A. chinensis and A. deliciosa were investigated using SSR markers. A total of 104 alleles were generated by nine pairs of SSR primers from two populations, and the alleles with phenotype frequency from 20% to 80% were chosen following equal gene frequency correlograms to calculate Moran’sⅠspatial autocorrelation coefficient for the two species and their complex (A. chinensis/A. deliciosa). Over half of alleles with random distribution pattern were found within populations, while large amount of alleles (29.6 %～48.0 % within populations, 51.0%, 44.7% for HX and SS complex, respectively) as cline, depression, and double depression or intrusion patterns, which suggesting that there were moderate spatial structures of genetic variation within populations of two species. The distribution patterns of genetic variation were similar either within the two populations or the complex. These demonstrated that allelic variation for individuals within 100 m (especially 30 m) distance had significantly positive correlation, but negative correlation appeared with the increasing of distance.In summary, the microsatellite analysis revealed a high intraspecies genetic diversity and interspecies introgression in the Actinidia, which could be attributed the combined impacts of wide-range and sympatric distribution, outcrossing, late differentiation and other life history characteristics, as well as genome size of the genus, complex ploidy level and interspecies hybridization. These results have implications for the study of mechanism of maintaining genetic variation, population expansion and phylogeography of Actinidia species, and also provide baseline data for the conservation and management of genetic resource of Actinida, especially for the sampling strategies for ex situ conservation.更多还原