青岚湖五种淡水蚌的群体遗传多样性及三角帆蚌微卫星DNA的分离
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摘要
利用北美Epioblasma capsaeformis和中欧Margaritifera margaritifera L．两种淡水双壳类的20对微卫星引物对江西青岚湖五种淡水蚌群体的基因组DNA进行了PCR扩增，筛选出8对多态性较高的引物，并用这8个微卫星座位分别对三角帆蚌(Hyriopsis cumingii)、褶纹冠蚌(Cristaria plicata)、洞穴丽蚌(Lamprotula caveata)、射线裂脊蚌(Schistodesmus lampreyanus)和中国尖嵴蚌(Acuticosta chinensis)五种蚌类群体的等位基因频率、遗传杂合度、有效等位基因数、多态信息含量等进行了遗传多样性分析，根据标准遗传距离对这五种蚌进行了聚类分析。并进一步利用PCR法分离了三角帆蚌的微卫星标记。主要研究结果如下：
1、五种蚌类群体的平均等位基因数为2.8750～4.000，平均观测杂合度(Ho)为0.4417～0.6333，平均期望杂合度(He)为0.4430～0.5706，平均多态信息含量(PIC)为0.368～0.498，五种蚌类群体表现出较高的遗传多样性。
2、有多个座位在不同蚌类群体中偏离哈代—温伯格平衡。微卫星位点Ecap6和MarMa3116仅在射线裂脊蚌中扩增出单一条带，可以作为鉴定射线裂脊蚌的特异性微卫星标记。
3、五种蚌类群体间的遗传距离在0.6228与1.4724之间，三角帆蚌和褶纹冠蚌之间的遗传距离最大，射线裂脊蚌和洞穴丽蚌之间的遗传距离最小。
4、用PCR法从三角帆蚌基因组DNA中筛选微卫星切实可行，而且避免了同位素污染，一般实验室即可满足实验条件。
5、运用PCR法在三角帆蚌的小片段基因组文库中能快速筛选含微卫星序列的重组阳性克隆，对筛选得到的31个阳性克隆进行测序，结果获得33个微卫星序列，其中perfect(完美型)25个，占75.8％；imperfect(非完美型)5个，占15.1％；compound(混合型)3个，占9.1％。对含微卫星的重组阳性克隆的测序结果表明，(AT／TC)_n在三角帆蚌的基因组DNA中含量比较丰富。
Freshwater mussels play an important role in many aquatic ecosystems, but little is known about their biodiversity and conservation genetics. In order to provide genetic information of freshwater mussels in China, microsatellite markers were used to investigate genetic diversity of five species of freshwater mussels (Hyriopsis cumingii, Cristaria plicata, Lamprotula caveata, Schistodesmus Iampreyanus, Acuticosta chinensis) in Qinglan Lake, Jiangxi Province.
8 primers for polymorphic DNA microsatellite loci were sieved from 20 primers which were developed from oyster mussel (EpiobIasrna capsaeforrnis) (Bivalvia: Unionidae) in North America and freshwater pearl mussel (Margaritifera margaritifera L.) (Bivalvia: Margaritiferidae) in Central Europe. we further described the isolation and characterization of the first microsatellite markers for H.cumingii, which were obtained by screening 58 recombinant clones. The main results are as follows:
1. Allelic richness ranged from 2 to 6 alleles per locus and averaged from 2.8750 to 4.000 alleles for five mussel populations. Heterozygosity levels varied from 0.4417 to 0.6333 for average observed heterozygosity (Ho) and 0.4430 to 0.5706 for average expected heterozygosity (He), and average polymorphism information content (PIC) ranged from 0.368 to 0.498, which revealed that all the five populations showed higher genetic diversity.
2. Several loci deviated from Hardy Weinberg Equilibrium. Single band was amplified in S.lampreyanus by loci MarMa3116, which can be regared as the specific microsatellite markers distinguishing S.lampreyanus from other species.
3. Genetic distance (Ds) among five mussel populations ranged from 0.6228 to 1.4724, revealed that the maximal Ds was 1.4724 between H. cumingii and C. plicata, and the minimal Ds was 0.6228 between S. lampreyanus and L. caveata.
4. The method of PCR screening of microsatellite sequences from H. cumingii was feasible and avoided the pollution of isotopes, which can be carried out in normal labs.
5. Thirty-three microsatellites of the H. cumingii were obtained. Among the thirty-three microsatellites, There were 25 perfect ones (75.8%), 5 imperfect ones (15.1%) and 3 compound ones (9.1%). The experiment showed that microsatellite sequences characterized by (AT)_n and (TC/AG)_n abundant in genomic DNA of H. cumingii. Primers can bedesigned according to the flanking sequences of microsatellites and used in genetic analysis.
1、五种蚌类群体的平均等位基因数为2.8750～4.000，平均观测杂合度(Ho)为0.4417～0.6333，平均期望杂合度(He)为0.4430～0.5706，平均多态信息含量(PIC)为0.368～0.498，五种蚌类群体表现出较高的遗传多样性。
2、有多个座位在不同蚌类群体中偏离哈代—温伯格平衡。微卫星位点Ecap6和MarMa3116仅在射线裂脊蚌中扩增出单一条带，可以作为鉴定射线裂脊蚌的特异性微卫星标记。
3、五种蚌类群体间的遗传距离在0.6228与1.4724之间，三角帆蚌和褶纹冠蚌之间的遗传距离最大，射线裂脊蚌和洞穴丽蚌之间的遗传距离最小。
4、用PCR法从三角帆蚌基因组DNA中筛选微卫星切实可行，而且避免了同位素污染，一般实验室即可满足实验条件。
5、运用PCR法在三角帆蚌的小片段基因组文库中能快速筛选含微卫星序列的重组阳性克隆，对筛选得到的31个阳性克隆进行测序，结果获得33个微卫星序列，其中perfect(完美型)25个，占75.8％；imperfect(非完美型)5个，占15.1％；compound(混合型)3个，占9.1％。对含微卫星的重组阳性克隆的测序结果表明，(AT／TC)_n在三角帆蚌的基因组DNA中含量比较丰富。
Freshwater mussels play an important role in many aquatic ecosystems, but little is known about their biodiversity and conservation genetics. In order to provide genetic information of freshwater mussels in China, microsatellite markers were used to investigate genetic diversity of five species of freshwater mussels (Hyriopsis cumingii, Cristaria plicata, Lamprotula caveata, Schistodesmus Iampreyanus, Acuticosta chinensis) in Qinglan Lake, Jiangxi Province.
8 primers for polymorphic DNA microsatellite loci were sieved from 20 primers which were developed from oyster mussel (EpiobIasrna capsaeforrnis) (Bivalvia: Unionidae) in North America and freshwater pearl mussel (Margaritifera margaritifera L.) (Bivalvia: Margaritiferidae) in Central Europe. we further described the isolation and characterization of the first microsatellite markers for H.cumingii, which were obtained by screening 58 recombinant clones. The main results are as follows:
1. Allelic richness ranged from 2 to 6 alleles per locus and averaged from 2.8750 to 4.000 alleles for five mussel populations. Heterozygosity levels varied from 0.4417 to 0.6333 for average observed heterozygosity (Ho) and 0.4430 to 0.5706 for average expected heterozygosity (He), and average polymorphism information content (PIC) ranged from 0.368 to 0.498, which revealed that all the five populations showed higher genetic diversity.
2. Several loci deviated from Hardy Weinberg Equilibrium. Single band was amplified in S.lampreyanus by loci MarMa3116, which can be regared as the specific microsatellite markers distinguishing S.lampreyanus from other species.
3. Genetic distance (Ds) among five mussel populations ranged from 0.6228 to 1.4724, revealed that the maximal Ds was 1.4724 between H. cumingii and C. plicata, and the minimal Ds was 0.6228 between S. lampreyanus and L. caveata.
4. The method of PCR screening of microsatellite sequences from H. cumingii was feasible and avoided the pollution of isotopes, which can be carried out in normal labs.
5. Thirty-three microsatellites of the H. cumingii were obtained. Among the thirty-three microsatellites, There were 25 perfect ones (75.8%), 5 imperfect ones (15.1%) and 3 compound ones (9.1%). The experiment showed that microsatellite sequences characterized by (AT)_n and (TC/AG)_n abundant in genomic DNA of H. cumingii. Primers can bedesigned according to the flanking sequences of microsatellites and used in genetic analysis.
引文
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