草鱼不同群体（雌核发育、ENU诱变）的微卫星遗传结构分析及生长差异研究
发布时间：2018-03-13 11:38
本文选题：ENU诱变草鱼 切入点：草鲂杂交后代 出处：《上海海洋大学》2017年硕士论文 论文类型：学位论文
【摘要】：草鱼(Cetpharyngodon idellus)为我国特有的淡水养殖品种,在养殖鱼类中占有重要的经济地位。由于草鱼繁殖周期长,亲鱼个体大等原因,给应用传统选育方法育出优良品种造成一定的难度,目前草鱼尚无国家审定的良种。雌核发育为快速建立纯系的有效途径,利用微卫星标记对雌核发育后代的遗传多样性进行评估,在生产性能优良的长江水系草鱼的基础上进行良种选育可能是一种可行的方法。基于本实验室建立6龄ENU诱变草鱼群体,挑选4对优良亲本建立家系,经过生长性状对比筛选出具有明显生长差异的两个家系进行mstn1、mstn2基因的SNP研究,以期筛选出ENU诱变草鱼群体生长改良研究提供候选分子标记,进一步确定ENU诱变草鱼群体分子标记辅助育种方案。具体研究如下:(1)以紫外线灭活的团头鲂(Megalobrama amblycephala)精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导出长江水系优良F2代草鱼减数雌核发育子代。在后代中不仅存在雌核发育后代,还存在草鲂杂交后代,雌核发育后代的体型与草鱼一致,而草鲂杂交后代的体型介于草鱼与团头鲂之间。Partec CyFlow倍性分析仪测定结果显示:普通草鱼与雌核发育草鱼的相对DNA含量分别为23.01和22.72,二者的DNA含量接近;而高体型子代的相对DNA含量为25.38,介于草鱼与团头鲂(DNA含量28.21)之间,属于草鲂杂交后代。选取17个微卫星标记对草鱼群体、雌核发育草鱼群体和草鲂杂交后代的遗传多样性进行了检测,共检测出59个等位基因,其中43.18个有效等位基因。草鱼对照群体、草鲂杂交后代和雌核发育草鱼群体的平均等位基因依次为3.57、2.86和2.79,平均有效等位基因依次为2.93、2.37和1.96,平均期望杂合度在依次为0.6502、0.5573和0.3775,多态信息含量(PIC)平均值依次为0.5738、0.4649和0.3791。与草鱼对照群体相比,雌核发育草鱼群体的遗传多样性显著下降,表明通过减数雌核发育方法可获得纯合性较高的草鱼个体。构建了草鱼后代不同群体的DNA指纹模式图,筛选到不同群体的9个特异微卫星标记,为草鱼优良群体的选育奠定基础。(2)为了获得雌核发育ENU诱变草鱼群体的相关遗传参数,实验采用Partec CyFlow倍性分析仪测定ENU诱变草鱼群体(Q群体)和雌核发育ENU诱变草鱼群体(E群体)相对DNA含量分别为24.02和23.80,二者的DNA含量接近,均为二倍体。选取28个微卫星标记对Q群体和E群体多样性进行了检测。结果表明,E群体和Q群体的平均等位基因分别为3.7143、5.1786,平均有效等位基因分别为2.1857、4.0028,平均期望纯合度分别为0.5122、0.2814,平均期望杂合度分别为0.4878、0.7186,多态信息含量(PIC)平均值分别为0.4282、0.6606。从个体在微卫星位点的纯合率分析,在E群体中,每个个体的纯合度均小于1.00,说明没有完全纯合的个体。从每个微卫星位点在群体的纯合率分析,除了微卫星位点5476,HLJC118和HLJC81外,其他位点的纯合度以不同的速率得到明显的提高。研究结果表明经过减数雌核发育方法,ENU诱变草鱼群体的各微卫星位点的纯合度以不同的速率得到提升,遗传多样性明显降低,此方法可以获得纯合度较高的雌核发育ENU诱变草鱼个体,为ENU诱变草鱼良种选育提供重要遗传数据资料。(3)对4个ENU诱变草鱼家系进行生长对比,经过175天养殖,从4个家系的生长对比发现,从体重增长量的角度看,家系1和家系4相对于其他2个家系有明显的优势,从体重增重率来看,家系4和家系2相对于其他2个家系存在明显优势,综合来看家系4的优势最明显,家系3明显弱于其他3个家系。家系4的8个性状明显大于其他3个家系;家系1的体高、头长略大于家系2,其他的6个性状明显大于家系2和家系3;家系2的8个性状明显大于家系3。采用偏相关分析各个形态性状与体重的相关程度得出:家系1中有全长、体长、尾柄长、体厚,相关系数依次为0.357、0.619、0.608、0.396;家系2中有全长、体长、头长、体厚,相关系数依次为0.348、0.360、0.687、-0.384;家系3中有全长、体长、尾柄长、尾柄高,相关系数依次为0.529、0.449、-0.351、0.384;家系4中有全长、体长、体厚,相关系数依次为0.629、0.543、0.590,经过因子分析可以明显区分家系4与其他3个家系,以上统计方法可总结出家系4和家系3具有明显的生长差异,运用双向测序法对MSTN1、MSTN2基因在家系3、4中进行SNP位点筛选,对于MSTN1,家系3在465 nt C/G、467 nt G/A,而家系4在465 nt C/G均发生错义突变;对于MSTN2,家系3和4在912 nt C/T均发生同义突变,而家系3在1027 nt G/A、家系4在366 nt A/G均产生错义突变,位于非编码区1390 nt A/T和1401 nt G/A在两个家系均发现SNP位点。以上这些结果表明MSTN1、MSTN2基因的不同SNP位点与ENU诱变草鱼的生长性状存在紧密联系。
[Abstract]:Grass carp (Cetpharyngodon idellus) is a freshwater aquaculture species endemic to China, occupies an important position in the economic fishes. Due to the grass carp long reproductive cycle broodstock individuals and other reasons, is difficult to breed fine varieties of application of traditional breeding method, at present there is no national certification grass carp seed. Gynogenesis is the effective way to the rapid establishment of pure lines, were evaluated using microsatellite markers on genetic diversity of gynogenetic offspring, breeding may be a feasible method based on the Yangtze River grass carp production of excellent performance. The laboratory was established at the age of 6 ENU by grass carp group based on the selection of 4 to establish excellent parent families the growth traits were selected for mstn1 with two families significantly differences in growth, SNP of the mstn2 gene, in order to find out the grass carp ENU mutation population growth improvement research For the candidate molecular markers, to further determine the grass carp group by ENU molecular marker assisted breeding program. The specific studies are as follows: (1) with UV inactivated bream (Megalobrama amblycephala) sperm activation grass carp eggs, cold shock suppression method of the second polar body emission induced by long river system generation grass carp F2 with excellent meiotic gynogenetic offspring gynogenetic offspring. Not only exist in the offspring, there is grass bream hybrids and gynogenetic progeny are consistent with grass carp, and between hybrid grass bream size between grass carp.Partec and bream CyFlow ploidy analyzer results show: Grass Carp and grass carp common gynogenetic relative DNA content were 23.01 and 22.72, close to the DNA content of the two; and relatively high content of DNA type offspring was 25.38, between grass carp and bream (DNA content 28.21), belonging to the grass bream hybrids. Select 17 micro Microsatellite markers on the grass carp group, grass carp group and gynogenetic grass bream hybrids genetic diversity of detection, detected a total of 59 alleles, of which 43.18 effective alleles. Grass carp control groups, hybrids and gynogenetic grass bream grass carp group's average alleles were 3.57,2.86 and 2.79, the average effective alleles were 2.93,2.37 and 1.96, the average expected heterozygosity were 0.6502,0.5573 and 0.3775, the polymorphism information content (PIC) the average values were 0.5738,0.4649 and 0.3791. control group compared with grass carp, grass carp group was activated by genetic diversity decreased significantly, showed that the meiotic gynogenesis method can be obtained homozygous high grass carp individuals. To construct the DNA fingerprint pattern of grass carp offspring of different groups, different groups of screened 9 specific microsatellite markers for the breeding of excellent foundation grass carp group The foundation. (2) in order to obtain the genetic parameters of gynogenesis induced by ENU of grass carp group, grass carp group experimental determination of ENU mutation by Partec CyFlow ploidy analyzer (Q group) and Mitogynogenesis group grass carp ENU mutagenesis (E) relative DNA content were 24.02 and 23.80, close to the DNA content of the two. All diploid. Selected 28 microsatellite markers in Q population and E population diversity were detected. The results showed that E group and Q group the average alleles were 3.7143,5.1786, the average effective allele was 2.1857,4.0028, the average expected homozygosity was 0.5122,0.2814, the average expected heterozygosity was 0.4878,0.7186. The polymorphism information content (PIC) were respectively 0.4282,0.6606. from individuals homozygous rate analysis in microsatellite loci, in the E group, each individual homozygosity was less than 1, indicating that there is no completely homozygous individuals. From each microsatellite locus in the homozygous rate analysis group, in addition to the 5476 microsatellite loci, HLJC118 and HLJC81, other sites of homozygosity were obviously increased at different rates. The results indicate that after meiotic gynogenesis method, ENU mutation grass carp group each microsatellite locus homozygosity at different rates improved genetic diversity decreased significantly, this method can obtain high homozygosity of gynogenetic ENU by grass carp individual, provide important data for ENU genetic mutation breeding. Grass carp (3) of the 4 ENU mutation in grass carp family growth comparison, after 175 days of culture, the growth of the contrast from 4 the family, from the perspective of weight increase, family 1 and 4 has obvious advantages compared with the other 2 families, the weight gain rate, family 4 and 2 compared with the other obvious advantages of 2 families, to comprehensive 鐪嬪绯，
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