【作者】 刘彦群；

【导师】 鲁成； 向仲怀；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2003， 博士

【摘要】 柞蚕业是我国的一项传统产业，在现代国民经济中仍然具有资源配置、经济和生态的合理性，有着广阔的发展前景。柞蚕业起源于我国，数百年的发展与传播在各地孕育了丰富的品种资源，截止今日，许多地方仍生存着野生的柞蚕资源，形成了世界上最大的柞蚕种质资源库。柞蚕种质资源是柞蚕业乃至整个蚕丝业可持续发展的必不可少的物质基础。研究不同地理品种的遗传差异、分类以及系统进化，可为柞蚕种质资源鉴定、保存、利用以及品种培育和推广提供理论基础，意义十分重大。本论文利用随机扩增多态DNA(RAPD)标记技术、mtDNA序列分析，以及种群遗传学研究方法研究了柞蚕种质资源的遗传变异、系统分类、种内水平的微进化等，其中遗传变异研究包括品种内、品种间、地理群体间、放养柞蚕与野生柞蚕间的遗传差异检测，这项研究不论是在品种选育、种质资源的利用等实际问题上，还是在探讨柞蚕品种乃至种群的分化等理论问题上均有重要的意义。主要结果如下： 1 柞蚕品种的DNA多态性分析 利用RAPD标记技术，以3个家蚕品种(大造、C108、7532)为对照，对4个有代表性的柞蚕品种(河41、四青、青黄1号、杏黄)的DNA多态性进行了分析。14个随机引物对28个个体的基因组DNA进行扩增，共得到143条DNA条带，其中多态性条带占96.5％。柞蚕品种内个体间的多态性为45.8％～49.4％；家蚕品种内个体间的多态性仅为9.09％～19.7％。柞蚕品种内个体间的遗传距离为0.133～0.238；家蚕品种内个体间的遗传距离为0.008～0.081。柞蚕不同品种的个体间的遗传距离为0.215～0.382；家蚕的为0.206～0.356。聚类分析时，柞蚕的各个个体均按品种聚在一起。 利用种群遗传学软件PopGen和多态性比率分别对柞蚕品种间和品种内的遗传变异分布进行了分析。柞蚕的DNA多态性有近60％来源于品种内个体间，仅40％来源于品种间；家蚕的DNA多态性来源于品种间的占75％，仅有25％来源于品种内个体间。利用Nei基因多样性指数估算的柞蚕品种内的遗传分化系数为0.5251，即有52.51％的遗传变异来源于品种内，来源于品种间的遗传变异仅占总遗传变异的47.49％；而家蚕品种间的遗传分化系数为0.8188，即有81.88％的遗传变异来源于品种间，来源于品种内的遗传变异仅有18.12％。两种计算方法的结果均反映出柞蚕的遗传变异来源于品种内个体间的部分要大于来源于品种间的部分，表明与品种纯度较高的家蚕相比，目前的柞蚕品种仍是一个遗传多样性十分丰富的群体，纯度较低。 对柞蚕品种的纯度较低的原因进行了讨论，认为与其起源驯化过程、生存的生态环境、品种的选育方法等是相一致的。讨论了家养动物的遗传变异在品种间和品种内的分布与人工驯化时间的关系。对品种选育的纯度与DNA多态性需求的平衡进行了讨论，并分析了对柞蚕品种选育的启示，提出了柞蚕品种选育的下一步的研究目标：寻找提高品种纯度与保持品种内的遗传多样性的平衡点。 2柞蚕品种资源的系统分类 利用RAPD标记技术对68个柞蚕品种（系）间的遗传差异进行了分析，并依此构建了柞蚕品种 的系统分类层次。利用33个随机5！物对68个柞蚕品种和2个外群对照（天蚕、家蚕）共扩增出364 条清晰DNA条带，其中360条（98．90％）呈多态性；68个柞蚕品种共扩增出296条带，多态性带占 90．gW。扩增带数最少的“33”有104条带，最多的“育绿”有171条带，大多数品种在13o～160 之间。68个品种（系）中只有9个品种产生了特有带，占12．68％．68个柞蚕品种（系）间的遗传 距离为o．1二卜o．3H，主要集中在o．观o～o．300之间，占总品种数的丑o．97％；有1巴．帕叽的品种对的 遗传距离小于0．2（M；遇传距离大于0．300的品种对，仅占0．95％。这说明，总体上柞蚕品种资源之 间的遗传距岛较小，牲程度较高，亲缘关系较近。 根据聚类晰结果将68个柞蚕品种（系）分成6个类群。柞蚕品种资源的的聚类结果表现出3 个特点：地域性聚类明显、类群间的聚类水平低、选择的作用明显（柞蚕品种具有较强的可塑性）。 聚类图中，辽宁、内荚古、吉林。、朝鲜的品种在聚类时互相聚在一起。河南、贵州、山东的品种均 各自聚为相对集中的一类．所以，结合地域性和聚类水平，可将68 ＋hs蚕品种（系）进一步划分为 3个大的类群，即山东类群、辽宁类群（包括辽宁、吉林、内蒙古和朝鲜的品种）和河南类群（包 括河南和贵州的品种）。 通过不同数据分析方法、建树方法、外群选择、位点数的比较，在位点数达到364时，采用单 匹配相似系数计算遗传距离，并用NJ法重建了系统发生树，通过与柞蚕品种传括的记载及生物学特 性相比较，最终确定了品种系统发生的较优树。在本试验及前人研究的基础上，总结了RAPD进行 系统学分析时5个注意事项：不同的物种所适用的方法不一定相同；分析的位点数必须足够；外群 以选择2个物种作为复合外群较好，选择亲缘关系较近的1个物种作为第1外群。另选亲缘关系较 远一些的。l个物种作为第2外群；数据更多还原

【Abstract】 Molecular systematics on germplasm resources of oak silkworm, Antheraea pernyi , including genetic diversity, cultivar taxonomy and microevolution within species, were investigated using Random Ampified Polymorphic DNA (RAPD), mtDNA sequence analysis and population genetic analysis. The genetic diversity among individuals within cultivar, cultivars, populations, and between oak silkworm and wild oak silkworm were detected. The results were as follows:1 Genetic diversity in cultivar of oak silkwormRAPD analysis was used to assess genetic variability in four cultivars of oak silkworm cultivars (He41, Siqing, Qinghuangl, Xinghuang) . Fourteen arbitrary 10-mer primers amplified 143 DNA fragments, 138 (96.5%) of which were polymorphic. The DNA polymorphism of different individuals within cultivar reached 45.8%-49.4%. The genetic distance value of different individuals within cultivar were 0.133-0.238; of different individuals among different cultivars were 0.215-0.382. The cluster analyses which were performed with UPGMA and NJ methods showed that all individuals were grouped according to its cultivar.The genetic variation distribution within and between cultivars were analyzed using polymorphism ratio and PopGen software. 60% DNA polymorphism was derived from different individuals within cultivar, and 40% from between cultivars.The total gene diversity (Nei’s index) was 0.2737, with a distribution within (52.51%) and between cultivars (47.49%); The results snowed that the present oak ouoworm cultivai was still a mixed population wirn lower purity.We discussed the possible reasons to lower purity of the cultivars, and thought that the results were probably cased by the shorter domestication history, reared-in-the-wild and mixed seed selection, et al. The genetic variation distribution within and between cultivars was to some extent related to the domestication history. We put forward a new viewpoint that the purity of seed selection keeps the balance with the DNA polymorphism demand. The research aim at oak silkworm seed selection was to determine how to keep the balance between improving cultivar purity and keeping the genetic diversity within cultivar.2 Genetic relationships of gerniplasm resources of A.pernyiThe genetic relationships of 68 A. pernyi cultivars were analyzed by random amplified polymorphic DNA (RAPD). A total of 296 DNA fragments were amplified by 33 primers selected among A. pernyi cultivars, 269 of which (90.88%) were polymorphic. Only nine (12.68%) of A. pernyi cultivars used had one or more cultivar-specific markers. The value of pairwise genetic distance were from 0.120 to 0.324, and most of them were in the range of 0.200 to 0.300. There were 99.05% of the cultivar pairs with parewise genetic distance less than 0.300, which had great genetic similarity. The percentage of 0.95% of cultivar pairs had better genetic differece, with pairwise genetic distance exceeding 0.300. Generally, there was great genetic similarity among A. pernyi cultivars.A dendrogram was constructed by Neighbor-Joining method. The A. pernyi cultvars used were classified into 6 genetic groups. The cultivars in a group were closely related to their geography distribution , and clustered into groups in a lower level and that the effect of selection on A. pernyi cultivar was obvious. We thought that these results were probably caused by the shorter radiation and differentiation history, the shorter artificial selection time and widely cultivar exchange among different producer area. It was reflected that A. pernyi, as a domesticated and reared-in-the-wild, was subjected to both artificial and natural selection. By combining the geography distribution clustering characteristics and cluster level, the 68 A. pernyi cultivars were divided into 3 larger groups: Shandong group, Henan group which included Henan cultivars and Guizhou cultivars , Liaoning group which included Liaoning cultivars, Jilin cultivars, Neimenggu cultivars and Chaoxian cultivars.Compared with different data analysis methods, outgroup更多还原