大竹蛏群体遗传多样性的研究
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摘要
大竹蛏（Solen grandis）是我国沿海潮间带常见的一种大型食用经济贝类，也是贝类养殖的潜在对象。本文应用扩增片段长度多态性（amplified fragment lengthpolymorphism, AFLP）分子标记和微卫星（simple sequence repeat,SSR）分子标记技术，对我国沿海大竹蛏的群体遗传多样性进行了分析，以期为大竹蛏的分子群体遗传积累资料。主要研究结果如下：
1、通过优化AFLP-PCR反应体系，筛选出7对AFLP引物，对5个地理群体的141只大竹蛏个体进行了扩增，共得到了403个位点，平均每组引物为57.5个位点，其片段大小在100～700bp之间。
2、对大竹蛏5个地理群体的遗传结构及遗传多样性的AFLP分析结果显示：丹东、烟台、莱州、南通4个群体的大竹蛏遗传结构相差不大，日照群体的多态位点比例高于其它4个群体，为79.16%，烟台群体相对较低为69.23%，结合Shannon’s信息指数（I）和Nei’s基因多样性指数（H）看，日照群体的遗传多样性水平较高，遗传变异较大，南通群体的遗传多样性水平较低。
3、通过对丹东、烟台、莱州、日照、南通5个群体之间的遗传相似系数和遗传距离进行分析的结果显示：丹东与南通群体之间的遗传距离最大为0.0910；烟台与日照群体间的遗传距离最小为0.0205。表明在5个大竹蛏群体中，日照与烟台群体之间的亲缘关系较近，丹东与南通群体间的亲缘关系较远。
4、应用分子变异分析（AMOVA）对5个群体的遗传变异来源进行分析的结果表明：大竹蛏5个群体在7对AFLP引物的检测下，其群体间遗传分化系数Φst=0.2250（p <0.001），群体间变异百分率为22.5%，而77.5%的变异则发生在群体内，说明群体的遗传变异主要来源于群体内个体间的遗传差异，但群体间也存在一定程度的基因渗透。
5、利用微卫星DNA技术对大竹蛏群体遗传多样性进行了研究。5对微卫星引物在5个大竹蛏群体中共扩增获得21个等位基因，每个微卫星位点所检测到的等位基因数为3～5个。5个群体的平均观测杂合度分别为0.2769～0.4267。多态信息含量为0.3592～0.3958。
6、Hardy-weinberg平衡及F检验数据显示大竹蛏群体的遗传结构发生了变化，表现为5个群体在某些位点都出现了偏离平衡的现象，遗传分化系数较小。Fst平均值为0.0751，提示大竹蛏群体处于中等分化水平，说明遗传变异主要来自于群体内个体间。
Solen grandis, an important economic shellfish, distribute along the coast of intertidalzone. Amplified fragment length polymorphism(AFLP) and microsatellite markers were usedto estimate population genetics of Solen grandis. The results of this study could provideinformation to the breed of the new species. The main results of this research are as following:
1. The AFLP system for Solen grandis was optimized including the time for DNA doubleenzymes digestion reaction, the dilute multiple for the per-amplification production anddifferent proportion in selective primers M+3/E+3. Then we selected seven primers to beused to the analysis of AFLP.141natural individuals from5populations of Solen grandiswere investigated using7primers. A total of403reproducible AFLP fragments, whichvaried most from100bp to700bp, were amplified among five populations,57.5fragmentsin every population averagely.
2. The genetic structure and genetic diversity for five geographical populations of Solengrandis shows: there’s little different from DD, YT, LZ, NT, The percentage loci ofpopulation from RZ are relatively higher the other four, was79.16%, YT got the lowestnumber was69.23%. But, both the Shannon’s index and Nei’s index showed that the orderof genetic diversity level was as follows: RZ were higher than others, and the lowest wasNT.
3. Comparing the genetic similarity coefficient and the genetic distance of5populationsfrom DD, YT, LZ, RZ and NT, the results showed that the genetic distance between DDand NT was the largest of0.091, and YT and RZ was the smallest of0.0205. The resultsindicated that, in the five natural populations, the relationship between RZ and YT wascloser, and the relationship between DD and NT was farther.
4. The results of Analysis of Molecular Variation (AMOVA) revealed that the diversity ofSolen grandis was divided into two parts,22.5%from inter-population and77.5%fromintra-population. Significant test showed that the genetic differentiation of Solen grandispopulation (Φst=0.225, P <0.001) within and among groups were significantly. So theresults indicated that the genetic variation within populations was the important factorreflecting on genetic differentiation.
5. Genetic diversity of Solen grandis populations was conducted using microsatellite DNA techniques.21alleles reproducible bands amplified with5primer combinations wereobtained from three populations and3-5were detected from each microsatellite locus.Average observed heterozygosity (Ho) of five populations was0.2769to0.4267, and theaverage value of polymorphism information content was0.3592to0.3958, respectively.
6. Hardy-Weinberg equilibrium chi-square test and F-test data showed that the geneticstructure of the populations had been changed, including decreased heterozygous sites,increased loci deviated from the equilibrium, and decreased coefficient of geneticdifferentiation between five populations. Fst value (0.0751) suggested the Solen grandishad relatively weak genetic differentiation and illustrated that the main genetic variationexisted among individuals within population rather than between populations.
1、通过优化AFLP-PCR反应体系，筛选出7对AFLP引物，对5个地理群体的141只大竹蛏个体进行了扩增，共得到了403个位点，平均每组引物为57.5个位点，其片段大小在100～700bp之间。
2、对大竹蛏5个地理群体的遗传结构及遗传多样性的AFLP分析结果显示：丹东、烟台、莱州、南通4个群体的大竹蛏遗传结构相差不大，日照群体的多态位点比例高于其它4个群体，为79.16%，烟台群体相对较低为69.23%，结合Shannon’s信息指数（I）和Nei’s基因多样性指数（H）看，日照群体的遗传多样性水平较高，遗传变异较大，南通群体的遗传多样性水平较低。
3、通过对丹东、烟台、莱州、日照、南通5个群体之间的遗传相似系数和遗传距离进行分析的结果显示：丹东与南通群体之间的遗传距离最大为0.0910；烟台与日照群体间的遗传距离最小为0.0205。表明在5个大竹蛏群体中，日照与烟台群体之间的亲缘关系较近，丹东与南通群体间的亲缘关系较远。
4、应用分子变异分析（AMOVA）对5个群体的遗传变异来源进行分析的结果表明：大竹蛏5个群体在7对AFLP引物的检测下，其群体间遗传分化系数Φst=0.2250（p <0.001），群体间变异百分率为22.5%，而77.5%的变异则发生在群体内，说明群体的遗传变异主要来源于群体内个体间的遗传差异，但群体间也存在一定程度的基因渗透。
5、利用微卫星DNA技术对大竹蛏群体遗传多样性进行了研究。5对微卫星引物在5个大竹蛏群体中共扩增获得21个等位基因，每个微卫星位点所检测到的等位基因数为3～5个。5个群体的平均观测杂合度分别为0.2769～0.4267。多态信息含量为0.3592～0.3958。
6、Hardy-weinberg平衡及F检验数据显示大竹蛏群体的遗传结构发生了变化，表现为5个群体在某些位点都出现了偏离平衡的现象，遗传分化系数较小。Fst平均值为0.0751，提示大竹蛏群体处于中等分化水平，说明遗传变异主要来自于群体内个体间。
Solen grandis, an important economic shellfish, distribute along the coast of intertidalzone. Amplified fragment length polymorphism(AFLP) and microsatellite markers were usedto estimate population genetics of Solen grandis. The results of this study could provideinformation to the breed of the new species. The main results of this research are as following:
1. The AFLP system for Solen grandis was optimized including the time for DNA doubleenzymes digestion reaction, the dilute multiple for the per-amplification production anddifferent proportion in selective primers M+3/E+3. Then we selected seven primers to beused to the analysis of AFLP.141natural individuals from5populations of Solen grandiswere investigated using7primers. A total of403reproducible AFLP fragments, whichvaried most from100bp to700bp, were amplified among five populations,57.5fragmentsin every population averagely.
2. The genetic structure and genetic diversity for five geographical populations of Solengrandis shows: there’s little different from DD, YT, LZ, NT, The percentage loci ofpopulation from RZ are relatively higher the other four, was79.16%, YT got the lowestnumber was69.23%. But, both the Shannon’s index and Nei’s index showed that the orderof genetic diversity level was as follows: RZ were higher than others, and the lowest wasNT.
3. Comparing the genetic similarity coefficient and the genetic distance of5populationsfrom DD, YT, LZ, RZ and NT, the results showed that the genetic distance between DDand NT was the largest of0.091, and YT and RZ was the smallest of0.0205. The resultsindicated that, in the five natural populations, the relationship between RZ and YT wascloser, and the relationship between DD and NT was farther.
4. The results of Analysis of Molecular Variation (AMOVA) revealed that the diversity ofSolen grandis was divided into two parts,22.5%from inter-population and77.5%fromintra-population. Significant test showed that the genetic differentiation of Solen grandispopulation (Φst=0.225, P <0.001) within and among groups were significantly. So theresults indicated that the genetic variation within populations was the important factorreflecting on genetic differentiation.
5. Genetic diversity of Solen grandis populations was conducted using microsatellite DNA techniques.21alleles reproducible bands amplified with5primer combinations wereobtained from three populations and3-5were detected from each microsatellite locus.Average observed heterozygosity (Ho) of five populations was0.2769to0.4267, and theaverage value of polymorphism information content was0.3592to0.3958, respectively.
6. Hardy-Weinberg equilibrium chi-square test and F-test data showed that the geneticstructure of the populations had been changed, including decreased heterozygous sites,increased loci deviated from the equilibrium, and decreased coefficient of geneticdifferentiation between five populations. Fst value (0.0751) suggested the Solen grandishad relatively weak genetic differentiation and illustrated that the main genetic variationexisted among individuals within population rather than between populations.
引文
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