【作者】 李木旺；

【导师】 黄勇平；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 动物学， 2006， 博士

【副题名】遗传多样性分析、QTL分析、基因定位与分子标记辅助育种

【摘要】 分子标记是开展家蚕功能基因组研究的基础,目前这种技术已经在家蚕中得到了较为广泛的应用。然而,作为第二代分子标记的微卫星标记（Simple sequence repeats,SSR）在此之前在家蚕中仅有为数不多的报道。本研究利用本研究组通过建基因组文库-杂交-测序-设计引物的方法获得的SSR分子标记,进行了家蚕品种资源的遗传多样性分析、遗传连锁图构建及茧质性状的QTL定位、定位镇江株浓核病毒不感染基因nsd-Z并将连锁的SSR标记应用于分子标记辅助育种研究。主要研究结果如下:1、用26个SSR标记研究了31个代表性家蚕品种资源的遗传关系,这些SSR标记在家蚕品种间表现出丰富的多态性,等位片段数量在2-17个,平均为7.2个。每个SSR标记的平均杂合度在0-0.60之间,最高值为0.96。平均多态性指数为0.66（0.12-0.89之间）。用Nei（1978）的方法分析了各品种间的遗传距离,UPGMA聚类结果与传统意义上的形态分类方法接近。所获得的SSR数据经主成分分析支持UPGMA方法的聚类结果。2、用生产上广泛应用的家蚕品种菁松和茧质较差的品种兰10作为亲本组配了BC₁分离群体,用SSR标记构建了分子连锁图并对全茧量、茧层量、茧层率、蛹体重进行了QTL分析。与全茧量、蛹体重和茧层量相关的各3个QTL位点分别位于第1和第23连锁群。与茧层率相关的2个QTL位点分别位于第18和第19连锁群。3、利用家蚕减数分裂过程中雌性染色体上的基因不发生交换的特点,以对浓核病毒高度感染的家蚕品种Js和不感染的品种L10分别组配了2种类型的回交群体,即F1为雌的BC1F群体和F1为雄的BC1M群体,分别用于nsd-Z的定位和连锁分析。用22个BC1F群体筛选到7个与nsd-Z连锁的SSR标记。用190个BC1M群体构建了一个长度为80.6cM的连锁图,nsd-Z基因被定位在30cM处,与之最近的标记Fl0316与该基因距离4.4cM。4、利用分子标记辅助选择技术在连续回交群体中选择携带nsd-Z的优良个体。连续6代回交后自交,经济性状已经达到实用品种水平,经添食病原证实分子标记辅助选择是高效的育种手段之一。更多还原

【Abstract】 Molecular markers are very important in functional genome researches and it has beensuccessfully used in silkworm （Bombyx mori L.）. Although SSR markers （or microsatellites）have been increasingly used and shown advantages over other markers, they have not beenwidely used in silkworm. In order to pursue the application of SSR markers in theeconomically important insect, Bombyx mori, firstly, robustic SSR markers were obtained bythe flow chart of large insert genome DNA library construction, Southern blotting, sequencingand primer designing, then these markers were used to study the genetic diversity amongsilkworm germplasms, construct SSR linkage map, locate the nonsusceptible to （Zhenjiangstrain） densonucleosis virus gene （nsd-Z） and breed new silkworm strains using markerassisted selection method. The results are shown as follows.1. The genetic relationship among 31 different types of silkworm strains was studiedusing 26 SSR markers. All of the markers were polymorphic and unambiguously separatedsilkworm strains from each other. A total of 188 alleles were verified with a mean value of7.2 alleles/loci （range 2-17）. The average heterozygosity value for each SSR locus rangedfrom 0 to 0.60, and the highest was 0.96. The average polymorphism index content （PIC）was 0.66 （range 0.12-0.89）. UPGMA cluster analysis of Nei’s genetic distance groupedsilkworm strains based on their origin. Principal components analysis （PCA） for SSR datasupports their UPGMA clustering.2. A backcross population （BC1） was derived from two parentals, Jingsong, a widelyused strains in silk production and Lan10, which possesses short silk length. The SSRtechnique was employed for mapping the QTLs. The QTLs for the whole cocoon weight,cocoon shell weight, ratio of cocoon shell, weight of pupae were analyzed. Three QTLs forwhole cocoon weight, pupa weight and cocoon shell weight were mapped on linkage group 1and 23, respectively, and two QTLs were mapped to a same locus. Two QTLs for cocoonshell ratio were mapped on linkage group 18 and 19, respectively.3. Due to a lack of crossing over in females, reciprocal backcrossed F₁ （BC₁） progenywere used for linkage analysis and mapping of the nsd-Z gene using silkworm strains Js andL10, which are classified as being highly susceptible and non-susceptible to DNV-Z,respectively. The nsd-Z gene was found to be linked to seven SSR markers, using BC1Fpopulation. Using a reciprocal BC1M cross, we constructed a linkage map of 80.6 cM withnsd-Z mapped at 30 cM, with the closest SSR marker at a distance of 4.4 cM.4. Marker-assisted selection of nonsusceptible to DNV-Z silkworm strains wereexercised in predicting DNV-resistant type in backcrossed generations. One silkworm strainnonsusceptible to DNV-Z had been bred using this method and its economic characters weresimilar to the commercial silkworm strains. These PCR-based markers will be useful insilkworm breeding programs screening for this trait in segregating populations.更多还原