导　　师： 杨官品

学科专业： 071007

授予学位： 硕士

作　　者： ;

机构地区： 中国海洋大学

摘　　要： 鲢/(Hypophthalmichthys molitrix/)是中国最大的池塘养殖的四大家鱼之一。四大家鱼的养殖可追溯到1000多年前的唐朝,是中国大陆居民最重要的蛋白质来源之一。四大家鱼已经引入到世界20多个国家和地区,用于食用或者控制水质。为加强鲢遗传学研究,已经构建了鲢SSR-AFLP性别平均连锁图谱。为增加连锁图谱的标记密度,并且在一定程度上避开SSR标记开发的高成本和困难性,本研究尝试开发了基于InDel的限制性片段长度多态性标记/(InDel-RFLP/)并用于鲢图谱构建。 微卫星/(microsatellite DNA/)和碱基插入缺失/(insertion-deletion, InDel/)是真核生物基因组的常见变异。为避开微卫星标记开发的困难性,本研究基于随机测序的鲢基因组片段,尝试开发基于InDel的限制性片段长度多态性标记/(InDel-RFLP/)。共133个标记中,65个在一个野生鲢群体中表现多态性/(该野生鲢群体采自长江流域荆州段,31个个体/)。共检测到161个等位基因,每位点等位基因数从2到4不等,平均值2.5个。六十五个多态性标记中,有35个定位到鲢SSR-AFLP性别平均连锁图谱上。实验还发现:长约2kb的鲢基因组片段比长约1kb的片段更适用于InDel-RFLP标记的开发。设计特异引物,扩增基因组片段,然后用4种限制性内切酶/(BsuR I、Hha I、Taq I和Vsp I/)之一切成适宜聚丙烯酰胺凝胶电泳分离的小片段,电泳分离、银染分型。该标记系统避免了对InDel精确定位的困难,通过高分辨PAGE分型,可为鲢连锁图谱构建和性状定位提供丰富的标记位点。 1.遗传多样性分析 设计的133对InDel-RFLP引物中,65对在鲢野生群体中表现多态性。每位点等位基因数从2到4不等,平均2.5。观测杂合度在0.036-1.000之间变化,期望杂合度在0.070-0.701之间变化。5个位点/(ZP02、ZP26、ZP47、P06和P17/)显著偏离哈温平衡。ZP26与ZP81之间连锁不平衡。InDel-RFLP标记可用于评价鲢 Silver carp /(Hypophthalmichthys molitrix/) is one of the most intensively pond-cultured fish species in China. Other popular cultured pond fish species in China include bighead carp /(Aristichthys nobilis/), grass carp /(Ctenopharyngodon idella/) and black carp /(Mylopharyngodon piceus/). The cultivation of these four fish species dates back more than 1000 years to the Tang Dynasty. They are the main sources of protein for humans in China's mainland. They have been introduced into more than 20 countries and regions of the world either for human consumption or for either their ability to aid in water quality control. In order to initiate the genetic study of silver carp, a combined SSR and AFLP sex average linkage map has been constructed. However, the map should be densified with more sequence tagged markers. In order to detour the limitations faced in the development of SSR markers, our interest in this study was to develop InDels based restriction fragment length polymorphism /(InDel-RFLP/) markers for silver carp. Microsatellites /(or simple sequence repeats, SSRs/) and insertion-deletions /(InDels/) are the common variations across eukaryotic genomes. In order to detour the limitations faced in the development of SSR markers, 133 randomly sequenced genomic fragments were used to develop InDel based restriction fragment length polymorphism /(InDel-RFLP/) markers for silver carp /(Hypophthalmichthys molitrix/). Of them, 65 were polymorphic in a wild silver carp population containing 31 individuals collected from Jingzhou fragment /(the middle reach/) of Yangtze River. In total, 161 alleles were identified. The allele number per locus varied from 2 to 4 with an average of 2.5. Thirty five InDel-RFLPs were assigned to the sex average SSR-AFLP /(amplified fragment length polymorphism/) linkage map of silver carp. It was found that ca.2kb fragments were more useful to developing InDel-RFLP markers than ca.1kb fragments. The fragments were amplified from the genomic DNA with the PCR products digested with one o

关 键 词： 鲢 碱基插入缺失 限制性片段长度多态性 连锁图谱 遗传多样性

领　　域： [农业科学] [生物学]