三疣梭子蟹论文三疣梭子蟹育种核心种质群体有效含量.doc

三疣梭子蟹论文：三疣梭子蟹育种核心种质群体有效含量【中文摘要】本文以三疣梭子蟹（Portunus trituberculatus）核心基础群快速生长品系即“黄选1号”为实验材料,根据Doyle and Talbot提出的计算有效群体含量的计算公式,对2008年三疣梭子蟹快速生长品系核心育种群有效群体含量进行估算。2009年选择三疣梭子蟹快速生长品系的子代蟹子经越冬后,按有效群体Ne=1,2,3,5,10,20,50梯度进行生产,比较不同有效群体含量对其子代的影响;选择22对三疣梭子蟹多态性好的微卫星标记,经过引物间的搭配、组合测试构建了多重PCR扩增技术体系。多重PCR的反应条件,包括退火温度,Mg2+含量、dNTPs的浓度等参数,最终建立了15个三重PCR组合,3个四重PCR组合,利用其中的两组三重组合,一组四重组合对三疣梭子蟹核心基础群快速生长品系2008年及2009年进行了亲子鉴定,主要结果如下:通过对2008年核心基础群快速生长品系有效群体进行估算,得出实际群体有效含量为213,实际近交系数为0.0023,其近交系数较小,近交衰退控制在较低的水平。但三疣梭子蟹保种数量和留种方式仍然困扰育种群体控制,因此,借鉴畜禽的保种模式,从留种方式、雌雄比例、保种数量等方面对三疣梭子蟹快速生长品系核心基础群进行保种研究,结果表明,采用随机留种,雌雄1:1比例交尾,雌雄数量各131只。此保种模式既能保证三疣梭子蟹快速生长品系核心基础群遗传多样性处于较高水平,使其具有较大的选育潜力,又能使保种成本维持在一个合理水平。此研究模式也为理论保种模式,需要在以后的实际群体选育过程中逐步完善。2009年,选择三疣梭子蟹快速生长品系的子代蟹子经越冬后,按有效群体Ne=1,2,5,10,20,50梯度进行生产,对其溞状幼体第4期、大眼幼体及幼蟹第2期各期的存活率进行计数,发现存活率不存在显著差异（P0.05）。有效群体大小的增加对子代的生长和存活没有显著影响,值得指出的是,虽然在短期内对子一代的表型方面并不存在显著影响,但参加繁殖的亲本个体较少时,其后代群体内个体的亲缘关系较近,导致子代群体遗传多样性较低,因而对核心育种群产生的长期影响不容忽视,这将在今后的研究中加以探讨。选择本实验室研发的22对三疣梭子蟹多态性好的微卫星标记,经过引物间的搭配、组合测试构建了多重PCR扩增技术体系。分别建立了47个二重PCR组合,并在47个二重引物的组合基础上建立15个三重PCR组合和3个四重PCR组合。本实验优化多重PCR的反应条件,包括退火温度,Mg2+含量、dNTPs的浓度等参数。最终建立了多重PCR技术体系,该技术的建立为三疣梭子蟹品种选育、种系评估提供快速、有效分子检测技术。选出优化好的2组三重组合,1组四重微卫星多重PCR组合用于三疣梭子蟹快速生长品系的亲子鉴定,采用人工分型和Cervus3.0模拟的方法对其鉴定率达到了100%,这样可以快速了解各个家系在快速生长品系中的存活情况,指导快速生长品系的选育。根据各个亲本对后代的贡献率的不同,以及父母本的个数,计算了近交系数增量的大小F=0.0058,有效群体含量Ne=88,近交系数增量较小,维持在较低的水平,构建的家系比较符合群体选育水平。总之,微卫星多重PCR基因扫描技术为加速三疣梭子蟹选育进程提供快速高效的检测工具。【英文摘要】This article based on the Portunus trituberculatus core group of fast-growing strain of “Huangxuan NO.1”as the experimental materials.According to the formulas of effective population size ,which brought forward by Doyle and Talbot,we estimated the effective population size（Ne）for rapid growth line fundamental stock in P.trituberculatus. In 2009, we selected the fast-growing strains of the offsprings through winter, according to the effective population Ne = 1,2,3,5,10,20,50 gradient production, compare the content of their offspring effective population.Selecting 22 pairs of polymorphic microsatellite markers on P. trituberculatus, building multiple PCR amplification technology system after matches between the primers and combinatorial testing. three four PCR combinations were also established. In this study, multiplex PCR reaction conditions were optimized, including annealing temperature, Mg2+ concentration, dNTPs concentration and other parameters.Finally,15 combinations of triple PCR were established, three four PCR combinations were also established.Using one of the two groups of triple combination, a quadruple combination of core infrastructure group P. trituberculatu.rapid growth of strains 2008 and 2009 were paternity test,the results are as follows:According to the formulas of effective population size ,which brought forward by Doyle and Talbot,we estimated the effective population size （Ne） for rapid growth line fundamental stock in P. trituberculatus.The real Ne was 213, which corresponded to a rate of inbreeding （F） of 0.23%.Results indicated thatF is relatively small,and inbreeding depression was in a low level, however, the cost of conservation was too large, it did not meet the conservation requirements. In light of breed conservation model in livestock and poultry, we investigated rapid growth line fundamental stock in P. trituberculatus on methods of selecting parents and ratio of dams and sires and the size of conservation population. We make the rules as follow: random selecting parents, the ratio of dams and sires is 1:1, and the number of dams and sires is 131, respectively. In this situation, genetic diversity of rapid growth line fundamental stock is preferably rich,which satisfies selective breeding, at the same time the cost on breed conservation is economical. This is only a theoretical model of breed conservation, and it needs the process of gradual improvement of breeding populations in the future. In 2009, we selected the fast-growing strains of portunus offspring crab through winter, press the effective population Ne = 1,2,5,10,20,50 gradient production, its Daphnia IV and megalopacrab survival rate of the measurement period, the survival rate is not significantly different （P 0.05）.Increase the effective population size of the growth and survival of F1 no significant effect, it is worth noting that although in the short term this effect （F1） aspects of the phenotype does not exist, but because of some small group of individuals to participate in reproduction, and itsgenerations of individuals within the group closer genetic relationship, the lower genetic diversity, and thus long-term impact on the （F2 and beyond） can not be ignored, which will be explored in future research.Selecting 22 pairs of polymorphic microsatellite markers on P.trituberculatus, building multiple PCR amplification technology system after matches between the primers and combinatorial testing. 47 combinations of double PCR were established, 15 combinations of triple PCR were established based on the 47 double PCR combinations, three four PCR combinations were also established. In this study, multiplex PCR reaction conditions were optimized, including annealing temperature, Mg2+ concentration, dNTPs concentration and other parameters. Multiple PCR technology system was established eventually, the establishment of this technology provides a fast, effective molecular detection technology for the portunus breeding, germ-line assessment.Selecting polymorphic PCR products with few null allele sites, the final combination of select 2 tripl notyping and Cervus3.0 artificial simulation method for its identification rate of 100%, so it can quickly understand the various families of the survial in“Huangxuan No. 1”, guiding the breeding of“Huangxuan No. 1”. According to each parents contribution of the different generations, as well as the number of parents, the calculated size of inbreeding coefficient incrementF= 0.0058, effective population size Ne = 88, small increments of inbreeding coefficient, remained low level, building the family level more in line with group selection. In summary, microsatellite multiplex PCR gene scanning technology supplies a fast and efficient tool for accelerating the breeding process of P. trituberculatu.【关键词】三疣梭子蟹 有效群体 微卫星多重PCR 亲缘鉴定【英文关键词】Portunus trituberculatus phenotypic correlation microsatellite DNA markers unique allele Parentage determinatio【目录】三疣梭子蟹育种核心种质群体有效含量摘要4-6ABSTRACT6-7第一章 引言11-221 三疣梭子蟹生物学11-122 分子遗传标记概述12-152.1 限制性片段长度多态性（Restrictive Fragment Length Polymorphism，RFLP）12-132.2 随机扩增多态性DNA（Ramdomly Amplified Polymorphism DNA，132.3 扩增片段长度多态性（Amplified Fragment Length Polymorphism，13-142.4 微卫星（Microsatellites）142.5 线粒体DNA（mtDNA）142.6 单核苷酸多态性（Single Nucleotide Polymorphism，SNP）14-153 三疣梭子蟹有效群体的研究进展15-173.1 在啮齿类动物方面的研究163.2 在水产动物方面的研究16-174 微卫星多重PCR 技术的发展及应用17-214.1 SSR 分析17-194.2 微卫星多重PCR19-204.3 分子标记与亲子鉴定20-215 研究目的及意义21-22第二章 三疣梭子蟹快速生长品系核心种质有效群体含量22-281 材料与方法22-241.1 材料来源221.2 实验