【作者】 韩智科；

【导师】 刘萍；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 本文以三疣梭子蟹(Portunus trituberculatus)近交家系为实验材料,采用相关分析和通径分析方法、微卫星DNA标记技术(Microsatellite DNA markers, SSR),对三疣梭子蟹不同形态性状对体重的影响进行了分析;应用微卫星DNA标记技术对三疣梭子蟹近交家系遗传多样性分析和近交程度进行了分析。主要结果如下:随机选取2006年建立的三疣梭子蟹05-G4-1家系F2、F3、F4三个世代各30尾,分别测量全甲宽(X1)、甲宽(X2)、甲长(X3)、体高(X4)、体重(Y)共5个形态学指标。采用相关分析和通径分析的方法,分别计算了家系以形态性状为自变量(x)对体质量(y)的相关系数、通径系数和决定系数,进而对各性状的影响大小进行剖分。结果表明,该家系三个世代三疣梭子蟹的各形态性状与体质量的相关性均达到极显著(P<0.01)或显著水平(P<0.05)。三个世代各性状的通径系数达到而来显著水平;决定系数分析结果与通径分析的变化趋势一致。经显著性分析,将偏回归系数显著的变量对体质量建立多元回归方程,三个世代的回归方程如下,F2:Y=-52.467+0.301X1+0.201X2+0.252X3+0.439X4;F3:Y=-82.567+0.177X1+0.170X2+0.325X3+0.225X4;F4:Y=-81.261+0.283X1-0.071X2+0.161X3+0.361X4。三个方程的回归关系均达到了极显著水平(P<0.01)。根据FIASCO方法构建了三疣梭子蟹微卫星富集文库,对含微卫星DNA序列设计了71对引物并对其进行了多态性筛选,筛选出21对微卫星多态性引物,用于三疣梭子蟹野生群体30个个体的遗传多样性检测和标记的评价。21个位点共获得了188个等位基因,平均每个位点扩增得到8.9个等位基因。不同引物获得的等位基因数差异较大,从3～13个不等,其中Pot8、Pot37、Pot48、Pot53、Pot54、Pot66六个位点分别获得了11、12、12、11、13、11个等位基因,而Pot46仅获3个等位21个微卫星位点的期望杂合度的范围为0.659～0.889,PIC值均高于0.5,表明它们都有很高的杂合度。均可用于三疣梭子蟹种群遗传结构分析,为三疣梭子蟹品种选育、种系评估提供更多的微卫星DNA信息。利用l6对微卫星引物对三疣梭子蟹(Portunus trituberculatus )野生群体和连续4代兄妹交家系05-A2-1和05-G4-1进行了遗传分析。结果表明在野生群体中16个微卫星位点均为多态位点,但在05-A2-1中有9个多态位点,05-G4-1中有11个多态位点。在野生群体中共检测到136个等位基因,05-A2-1、05-G4-1分别为52、47个。近交家系的平均基因纯合率均较高,其中05-A2-1为55.87%,05-G4-1达56.69%,而野生群体平均基因纯合率为38.37%。近交家系平均杂合度和平均多态信息含量均较野生群体低。在近交系05-A2-1和05-G4-1中,群体间遗传相似性指数最大,其遗传距离最小,说明二者之间的亲缘关系最近。近交系遗传多样性明显降低,已具有较高的遗传相似性。利用16个多态性微卫星位点分析了三疣梭子蟹家系F1到F4 4个选育家系的遗传结构与遗传多样性变化情况。结果显示,随着选育的进行,4个世代家系遗传多样性指标值逐渐下降,F1到F416个微卫星位点的平均多态信息含量从0.6753下降到0.4061,平均等位基因数从3.5下降到2.1333,平均观测杂合度从0.6435下降到0.4774,平均等位基因纯合率从0.5669到0.4024。遗传距离逐渐增加,相邻世代间的遗传相似性逐步升高。随着选育的进行,结果表明,经过连续4代的选育,选育群体的遗传基础逐步得到纯化,基因型逐渐趋向纯合、稳定,经进一步的选育可望获得较稳定的品系。更多还原

【Abstract】 This article used Portunus trituberculatus of inbred family for the experimental material.We make study on Body Weight by the use of correlation analysis and path analysis.We also studied on the genetic diversity and the degree of inbreeding of Portunus trituberculatus in different years using microsatellite DNA markers. The main results are as follows:We randomly selected 30 individuals from 05-G4-1 family of Portunus trituberculatus,and sampled for measuring five morphometric traits including full carapace width(X1)、carapace width(X2)、carapace length(X3)、body height(X4)and body weight(Y).By correlation and path analysis,the results show that the correlation coeficients of each morphological trait to body weight are all at significant(P<0.05)or extremely significant level(P<0.01).The path coefficients in three years all reach significant level(P<0.05),the diversification of determination coeficients analysis is consistent with that of path . The morphometric traits which reach level of significance(P<0.05) are used to establish the multiple regression equations,which are Y=-52.467+0.301X1+0.201X2+0.252X3+0.439X4;Y=-82.567+0.177X1+0.170X2 + 0.325X3 + 0.225X4 ; Y= - 81.261 + 0.283X1 - 0.071X2 + 0.161X3 +0.361X4。Constructed under the FIASCO method Portunus microsatellite enriched library, Microsatellite-containing DNA sequence of 71pairs of primers and polymorphism screening was carried out ,selected 21 pairs of microsatellite polymorphic primers for Portunus swimming crab wild population genetic diversity of 30 individuals testing and evaluation of tags.A total of 21 sites were 188 alleles amplified per locus on average 8.9 alleles.Different primers were quite different alleles,ranging from 3 to 13,which Pot8, Pot37, Pot48, Pot53, Pot54, Pot66 six sites were obtained 11,12,12,11,13,11 alleles,but Pot46 only received 3 alleles.Allele size distribution in the 131~312bp,basically in line with the design theory primer product length.21microsatellite loci of expected heterozugosity in the range of 0.659~0.889,PIC values higher than 0.5,indicating that they have a high heterozygosity.Can be used for population genetic structure of Portunus analysis,trituberculatus breeding, germ-line assessment of microsatellite DNA for more information.We carried out the genetic analysis on wild population,inbred 05-A2-1 and 05-G4-1 of Portunus trituberculatus by the use of 16 microsatellite primers. The results showed that there were 16 microsatellite locis which were polymorphic in the wild population,but there were only 9 polymorphic locis in the 05-A2-1 and 11 polymorphic locis in the 05-G4-1. In the wild population,136 alleles were detected and there were 52 alleles in the 05-A2-1,47 alleles in the 05-G4-1. The inbred homozygous were high on average,of which 05-A2-1 was 55.87%,05-G4-1 was 56.69% and the average rate is 38.37% in the wild population. The inbred’s average heterozygosity and polymorphic information content is lower than the wild population. Between inbred 05-A2-1 and 05-G4-1,the population genetic similarity index is the largest and the genetic distance is smallest,indicating the closest relationship between them. The inbred genetic diversity decreased significantly and already has a high genetic purity.Sixteen microsatellite markers were used to analyze the genetic structure and genetic diversity of the selected populations of inbred Portunus rituberculatus from F1 to F4. The results showed that the genetic diversity of the breeding population was decreasing . The the average polymorphism information content (PIC) of the 16 microsatellite markers decreased from 0. 6753 to 0.4061, the average allele number from 3.5 to 2.1333, the average heterozygosity from 0.6435 to 0.4774 and the average rate of allele homozygous from 0.5669 to 0.4024. Increasing genetic distance, adjacent genetic similarity between generations gradually increased. With the breeding, the results show that after 4 consecutive generations of breeding.The genetic basis of breeding gradually purified homozygous and genotype gradually moving towards stable.We are expected to get more stable strains with further breeding.更多还原