【作者】 司马杨虎；

【导师】 向仲怀； 鲁成；

【作者基本信息】 西南农业大学 ， 特种经济动物， 2003， 博士

【摘要】 家蚕是重要的经济昆虫，大多数重要经济性状都呈数量性状遗传，家蚕茧质性状就属于其中之一。需要用数理统计学的方法把控制某一数量性状的多个基因作为一个整体进行研究。但这种传统的方法无法鉴别出单个数量性状基因或染色体片断，更难确定数量性状基因座位(Quantitative trait loci,简称QTL)在染色体上的位置及其与其它基因的关系，从而也就大大限制了我们对目的数量性状的转移利用效率。构建高质量高密度的分子连锁图谱，可绘制出影响数量性状的所有主基因的详细连锁图，从而确定其在染色体上的位置、单个效应及互作效应，以便于人们更加有效的对目的基因进行操纵和转移利用。 因此把数量遗传学和分子遗传学相互结合，将形态性状构建的遗传图谱发展为高密度的分子连锁图，从分子水平上操纵家蚕一些重要经济性状基因，对实现分子育种具有十分重要的意义。本论文的主要研究结果如下： 1．家蚕AFLP分子连锁图谱的构建 本研究采用了两个遗传差异比较大的现行生产用家蚕品种872和湘晖作为作图亲本材料，杂交得F1代，自交后得F2代，从F2代随机抽取91个个体作为作图群体。 1．1 66组AFLP引物，共扩增出1559个多态性位点，每组引物扩增出23.6条多态性带。经X~2检测，1559个多态性位点中有692个位点符合3：1，占总多态性位点的44.39％，将302个来自母本湘晖，390个来自父本872的标记各自分为21个和28个连锁群，湘晖的21个连锁群，命名为a亚群，872的28个连锁群，命名为b亚群。692个有效位点中的550个构成了a、b两亚群，占有效位点的79.48％。占总多态性位点的35.28％，还有142个位点不连锁，不能进入连锁图中。 1．2 在a亚群中有233个位点连锁，还有69个位点不连锁。a亚群中各连锁群上标记数目变化范围在4-43个，连锁群的长度变化在22.3cM-424.3cM，标记的平均图距变化在3.39cM-17.43cM，位点间的平均距离为8.81cM。平均图距小于10cM的连锁群有14个，大于10cM小于20cM的连锁群有7个。ZI个连锁群上位点平均数为＊．l个，连锁群的平均长度为89CM。1．3在 b亚群中有 317个位点连锁，非连锁的位点有 73个。b亚群中各连锁群上标记数目变化范围在3－35个，连锁群的长度变化在2．4 CM－366．SCM，标记的平均图距变化在0．8。M－26．96CM，位点间的平均距离为9．26测。平均图距小于10cM的连锁群有18个，大于10c回小于20。饲的连锁群有7个。28个连锁群上位点平均数为11．31个，连锁群的平均长度为95．6。M。1．4 a亚群中21个连锁群的总长度为1868．IcMb亚群中28个连锁群的总长为2677．50cM，a、b两亚群平均总长为 2272．scM，a、b亚群平均距离为 9．00cMa。本研究构建的是一张密度较高的 AFLP分子连锁图，为后一步的 QTL基因定位和分子标记辅助选择（MS）奠定基础。2．不同群体大小和不同标诏数的作图比较 作图群体的大小是影响作图精度和作图效率的一个重要因子。目前关于构建分子连锁图谱最小有效群体，并无统一标准，本研究分别以群体大小91、sl、71、61、51和45个个体为材料，分析了不同群体的作图效果获得以下结果：相同参数条件下，随着群体的减小，分群数、不连锁标记数都表现出逐步减少的趋势。 随着群体的缩小，必须逐步调低交换值才能达到分群均匀的目的。同一群体随着交换值的逐步减小，分群数、不连锁标记数和二联体数逐步增多，尤其小群体分群数和不连锁标记数的变化则极为急剧，进入连锁群的标记数及其百分比也随之减小，总图距和平均图距也相应变小。二联体数的增多会影响图谱的质量，进入连锁群的标记数及其百分比的变少与交换值的设定值变小有关，总图距的减小与交换值的设定值和进人连锁群的标记数有关。平均图距的变小是由于交换值的设定值变小所致，交换值设定值变小，势必会将本来连锁的一些图距相对较大的标记视为不连锁标记或被划为不同的连锁群。 相同群体不同标记数在相同的作图参数条件下，随着标记数的减少，分群数有减少的趋势，但二联体数则以较多的标记群体为最少，而进入连锁群的标记数和总图距有明显的降低趋势，但平均图距以300个标记最小，其次为692个标记类型，542个标记和392个标记的平均图距较接近。因此根据Mapmaker分析对参数的设定要求和本研究分群作图效果，家蚕 FZ代作图群体至少不小于引个个体为宜：结合队L定位对群体和标记的要求，本研究以引个个体692个标记的综合效果最好，因为它覆盖的全基因组长度最长，更能代表数量性状的整体分布情况。3．1家蚕茧质性状的频数分布特征和性别效应的预测3．1．l家蚕茧质性状的频数分布特征 家蚕全茧量、茧层率和蛹体重的频数分布表现为明显的双峰特征，茧层量的频数分布则表现为单峰特征，说明家蚕的全茧量、茧层率和蛹体重性状存在性别效应。为了符合QTL分析对数量性状的要求，对家蚕的茧质性状的性别效应进行了预测和调整。3．互．2家蚕茧质性状性别效应的预测 采用以下分析模型：YI户尸“S勺，首先用 MINQUEmao，1971）法无偏地估计各项随机效应方差，再用 Zhu（19更多还原

【Abstract】 Domestic silkworm is an importantly economic insect, most economic traits of which show quantitative heredity, and cocoon quality is one of them. Hereditary of quantitative traits shows continuous variations, mainly due to the piling effect of polygenes, and polygenes controlling one quantitative trait should be studied as a unit through mathematics and statistics. However, the traditional methods limit the efficiency of utilizing desired quantitative traits greatly for being unable to identify single gene or chromosome segment controlling quantitative traits, and more difficult to make clear the locations of quantitative trait loci (QTL) on chromosomes and their relations with other genes. It is advisable to construct a high quality and density linkage map containing all the major genes which have great effect on the quantitative traits and hence to make clear their locations on chromosomes, single effect and mutual effect in order to monitor and to use the target genes more effectively.It is more and more difficult for geneticists to improve the present varieties through traditional theory and methods. Therefore it is very meaningful to realize molecular breeding through combining quantitative genetics with molecular genetics and developing genetic linkage map based on morphological characters into a molecular linkage map with a high density and thus to monitor the important economic characters of domestic silkworm on the basis of molecular level. What we have carried out in the dissertation is mainly to construct a molecular linkage map with a high density using amplified fragment length polymorphism (AFLP), locate and analyze the quantitative traits of domestic silkworm such as cocoon quality and pupal weight, evaluate the direct selection for the regular pattern of DNA polymorphism of different inbred lines and their hybrid offsprings through the directed differentiation selection for the cocoon quality of domestic silkworm, and to breed a new excellent race of domestic silkworm using in both spring and autumn on the basis of combing traditional breeding with the techniques of DNA molecular marker.1. Constructing a molecular linkage map with a high density using AFLPTwo presently used varieties 872 and Xianghui, which had broad genetic background, were used to construct a map as parents. F2 generation was self crossed from FI generation, which derived Irom the parent 872 and Xianghui, and 91 individuals were selected randomly from the F2 generation as a group to constructing a map.1.1 Total 1559 polymorphic bands were amplified using 66 AFLP primer combinations and 23.6 polymorphic bands were amplified by a pair of primer combination on average. Of all the 1559 polymorphic bands, there were 692 polymorphic bands conforming to the ratio of 3:1 after X2 detection, accounting 44.39% of all. The detected fragments ranged from 100bp to 1000bp. Of the 692 effective polymorphic loci, 550 loci were used to construct a and b subgroup, accounting 79.48% of the effective loci and 35.28% of all the polymorphic loci. The other 142 polymorphic loci didn’t link each other and thus couldn’t be used to construct the linkage map. The 302 polymorphic loci from the female parent Xianghui and the other 390 polymorphic loci from the male parent 872 were divided into 21 and 28 linkage groups respectively. 21 linkage groups from Xianghui were named as a subgroup and 28 linkage groups from 872 b subgroup.1.2 In a subgroup, 233 polymorphic loci linked each other while the other 69 polymorphic loci didn’t; The number of markers ranged from^ to 43 in the individual linkage group of a subgroup, the average map distance of which was from 2.96cM to 13.08cM. The average distance between 2 loci was 8.81cM while the length of linkage group was from 22.3cM to 424.3cM. Of the 21 linkage groups in a subgroup, 19 had an average distance less than 10cM while 2 had an average distance between 10cM to 20cM. The average length of 21 linkage groups was 89cM (Table.2 ) and on each linkage group there were 11.1 loci on average.更多还原