【作者】 张桂玲；

【导师】 黎中宝；

【作者基本信息】 集美大学 ， 水产养殖， 2009， 硕士

【摘要】 长毛明对虾作为我国重要的海洋经济虾类,近年来由于严重开发过度,产卵场环境恶化等因素的影响,其资源量急剧下降,在2005年出版的《中国物种红色名录》一书中已将其列为濒危[EN]物种,因此对于长毛明对虾的资源保护及复壮已迫在眉睫。本研究运用等位酶、AFLP及SSR技术,以我国东南沿海9个海域的长毛明对虾为实验样本,对其群体遗传多样性及分化进行分析研究,以期为长毛明对虾的资源保护、复壮、良种选育及其资源的可持续利用等多个方面提供必要的遗传学基础。研究结果如下:1.应用聚丙烯酰胺凝胶电泳技术,对我国东南沿海9个海区的长毛明对虾群体进行杂合性分析。测定了8个酶系统,共检测到15个酶位点32个等位基因。有7个多态位点,它们是位点Me-1、Me-2、Mdh-2、Aat-1、Sod-2、Est-1、Amy-1,在这些位点有2～5个等位基因。结果表明,在长毛明对虾9个群体的全部多态位点中,共有6个位点符合Hardy-Weinberg平衡(p>0.05),30个位点偏离Hardy-Weinberg平衡(p<0.05);除宁德群体外,其他8个群体中共有13个多态位点表现为杂合子缺失,结合各群体中每个多态位点的观察杂合度和期望杂合度,认为导致杂合子缺乏的主要原因可能是种群内交、自然选择、Wahland效应、哑等位基因等。另外,在本研究中共有23个位点表现为杂合子过剩(F<0),且在长毛明对虾9个群体中均有出现,从而认为长毛明对虾的种质资源较好。2.采用等位酶分析技术获的的我国东南沿海9个群体的遗传多样性指标为:平均每个位点的等位基因的有效数目(Ae)为1.1447～1.2094,平均为1.1664;多态位点百分数为13.33%～40.00%,平均为26.67%;观察杂合度(Ho)为0.1311～0.1511,平均为0.1380;期望杂合度(He)为0.0720～0.1023,平均为0.0866。群体间遗传距离(D)为0.0002～0.0049;群体间遗传相似度为0.9951～0.9998;群体间的遗传分化系数GST为0.0327;基因流为(Nm)11.7358。采用AFLP技术对我国东南沿海9个群体进行遗传多样性及分化分析,利用8对引物,在270个个体中共扩增出508个位点。结果显示,长毛明对虾9个群体的有效等位基因数(Ae)介于1.1956～1.3988之间,平均为1.2770;多态位点百分比(P)介于41.34%～63.58%之间,平均为50.88%;Shannon氏指数(I)为0.1841～0.3425,平均0.2486;Nei’s基因多样性指数(H)为0.1194～0.2305,平均为0.1643;群体间遗传距离(D)平均为0.0626,基因流(Nm)为1.8124;将以上长毛明对虾遗传多样性参数的均值与一些经济虾类与蟹类的以对应实验方法所获得的均值相比较,认为长毛明对虾群体的遗传多样性在甲壳类中处于较高水平,且群体之间发生了较小的分化。3.运用统计软件SPSS11.5分别对采用等位酶技术和AFLP技术获得的长毛明对虾9个群体的遗传距离与对应的地理距离进行相关性分析,结果如下:(等位酶标记部分)Pearson Correlation=-0.022 , Sig.(2-tailed)=0.896>0.05 ; (AFLP标记部分)Pearson Correlations=0.146,Sig.(2-tailed)=0.394>0.05。从而认为长毛明对虾9个群体的遗传距离与地理距离之间无显著相关性。4.采用生物素—磁珠吸附微卫星富集法,构建长毛明对虾的微卫星文库。从文库中挑取864个克隆,选取108个≥500bp的片段进行测序,获得72个微卫星序列。用引物设计软件Primer Premier5.0设计引物41对,到目前为止,已从中筛选出6对可用的微卫星引物。更多还原

【Abstract】 Fenneropenaeus Penicillatus is an important marine commercial animal in China, but its resource is falling sharply recently because of serious over-exploitation,environmental degradation of spawning grounds and other factors.A book called China Species Red List, which was published in 2005, has classified it as an endangered[EN]species.Thus the protection of F. Penicillatus and rejuvenation are imminent. Nine F. Penicillatus stocks from nine different coastal areas were analyzed by allozyme ,AFLP ,SSR technologies in order to study the genetic diversity and differentiation of the nine F. Penicillatus stocks. The aim of our study was to provide necessary information for resource protection, rejuvenation, breeding and sustainable use of resources in many aspects. The results were as follows:1.Polyacrylamide gel electrophoresis technology was used to analyze the allozyme heterozygosity of nine F. Penicillatus stocks.Eight enzymes coded by 15 loci were clearly resolved in the nine stocks.Seven loci (Me-1,Me-2,Mdh-2,Aat-1,Sod-2,Est-1,Amy-1) were polymorphic among the nine stocks exclaimed.The results also showed that a total of 6 loci meet the Hardy-Weinberg balance (p>0.05) and 30 loci deviated from the Hardy-Weinberg balance (p<0.05) of all polymorphic loci.Except the Ningde stock,13 loci from other eight stocks showed heterozygote deficiencies.Combined with information of the observed heterozygosity and expected heterozygosity,heterozygote deficiencies may mainly due to inbreeding,natural selection,Wahland effects,dumb allele and so on. In addition,in this study a total of 23 loci showed heterozygote excess (F<0) in nine stocks,which showed that the germplasm resources of F. Penicillatus is good.2.The allozyme analysis showed that the effective number of alleles (Ae) ranged from 1.1447 to 1.2094,with an average of 1.1664;percentage of polymorphic loci ranged from 13.33% to 40.00%, with an average of 26.67%;observed heterozygosity (Ho) ranged from 0.1311 to 0.1511,with an average of 0.1380;expected heterozygosity (He) ranged from 0.0720 to 0.1023, with an average of 0.0866.Genetic distance (D) was from 0.0002 to 0.0049,genetic similarity was from 0.9951 to 0.9998. And the genetic differentiation index GST among the nine stocks was 0.0327 and gene flow (Nm) was 11.7358.The AFLP technology was used to analysis the genetic diversity and differentiation of the nine F. Penicillatus stocks. Of all the 270 individuals,508 loci were amplified by eight pairs of primers.The results showed that the effective number of alleles (Ae) ranged from 1.1956 to 1.3988, with an average of 1.2770;the percentage of polymorphic loci (P) ranged from 41.34 to 63.58, with an average of 50.88%;the shannon’s index (I) ranged from 0.1841 to 0.3425, with an average of 0.2486; Nei’s genetic diversity index (H) ranged from 0.1194 to 0.2305, with an average of 0.1643.The average genetic distance (D) was 0.0626, and the gene flow (Nm) was1.8124.Comparing the genetic diversity from our study with that of other economy shrimps and crabs,which was obtained by corresponding experimental methods,our study showed that the genetic diversity of F. Penicillatus was at a high level,and the genetic differentiation was low.3. Useing statistical software SPSS11.5 to analyze the correlation between genetic distance and corresponding geographical distance of the nine F. Penicillatus stocks using allozyme and AFLP technologies. The results are as follows: (allozyme marker part) Pearson Correlation =-0.022,Sig.(2-tailed) =0.896>0.05;(AFLP marker part)Pearson Correlations=0.146, Sig.(2-tailed)=0.394>0.05.So our study showed that there was no significant correlation between genetic distance and geographical distance between nine stocks.4. Useing magnetic-bead enrichment method to construct microsatellite library in F. penicillatus. In this experiment, 108 DNA fragments (more than 500bp) were chosen from 864 colonies. 72 microsatellite sequences were obtained by analyzing the 108 DNA fragments. 41 pairs of primers were designed by Primer Premier 5.0. Up until now, 6 pairs of primers were selected which could be used to amply special fragments.更多还原