合浦珠母贝微卫星DNA标记分离与遗传多样性研究
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摘要
合浦珠母贝广泛分布于中国、日本、印度、澳大利亚等国的热带和亚热带海区,是生产海水珍珠的主要贝类,具有极高的经济价值。本研究利用FIASCO法构建了合浦珠母贝的基因组微卫星富集文库,并设计引物进行PCR筛选,获得9个多态位点。利用这9个多态位点,对中国的野生和养殖合浦珠母贝群体以及中国、日本、澳大利亚的合浦珠母贝群体的遗传多样性和遗传分化进行了分析,目的是为合浦珠母贝的种质资源保护和遗传选育提供基础资料。
1.用FIASCO法构建的合浦珠母贝基因组微卫星富集文库中共有近2000个单菌落,随机挑选1289个克隆用探针引物(CA)_(15)和载体引物进行第2次筛选,获得阳性候选克隆357个。测序结果表明,297个克隆(83.2%)含有明显的微卫星重复单元,通过序列比对分析,最终获得了280个具有特异微卫星序列的阳性克隆,共含有479个微卫星结构域。获得的微卫星序列长度范围是119-787bp,平均为408bp。其中两碱基(CA/GT)n重复单元出现的次数最多,占所有分离的微卫星数目的76%,此外还发现(AG)n、(AT)n、(GC)n、(CAA)n、(AAG)n、(ATT)n、(CCT)n、(ATTT)n、(GTTT)n、(CAGA)n、(GAGT)n、(CCGT)n、(GGGT)n、(CAAAA)n等多种重复类型,共占分离微卫星数目的24.0%,平均每种占1.7%。在获得的479个微卫星位点中,完美型微卫星370个,占77.3%;非完美型95个,占19.8%;复合型14个,占2.9%。用引物设计软件Primer premier 3.0设计引物219对,合成49对,筛选出31对有效扩增的引物,种群扩增获得9个多态位点,多态位点比例为29.03%。
2.对我国北海野生与养殖群体、大亚湾野生与养殖群体、三亚野生与养殖群体进行PCR扩增后发现:6个群体在9个多态位点共检测出44个等位基因,每个位点的等位基因数从2-9个不等。平均期望杂合度和平均观察杂合度分别介于0.590-0.649和0.393-0.516之间。平均多态信息含量P/C值在0.530-0.586之间。平均杂合子偏离度D值均为负,表明6个群体存在一定的杂合子缺失现象。Hardy-Weinberg平衡的卡方检测发现,在所检测的54个数据中(6个种群×9个位点),有40个偏离遗传平衡状态。两两群体之间的遗传分化指数介于0.002-0.012之间,并且大部分差异显著。AMOVA分析表明,94.59%的遗传变异存在于群体内,群体间的遗传变异仅为总变异的5.41%。研究结果表明,6个群体的遗传多样性水平都比较高,适合进行遗传选育。
3.中国、日本、澳大利亚合浦珠母贝群体遗传多样性的微卫星分析表明:北海群体、大亚湾群体、三亚群体、澳大利亚群体和日本群体的平均等位基因数分别是4.889、4.889、4.889、4.000和4.111;平均期望杂合度分别是0.592、0.605、0.649、0.575、0.564;平均观察杂合度分别是0.400、0.516、0.485、0.390、0.433。Shannon多样性指数分别是1.131、1.177、1.251、1.028、1.025;平均多态信息含量PIC值分别是0.530、0.545、0.586、0.498、0.483,表明日本和澳大利亚群体的遗传多样性低于中国群体。Hardy-Weinberg平衡的卡方检测发现,大多数位点偏离平衡状态(34个)。两两群体之间的F_(ST)值在0.010-0.075之间。除北海与大亚湾群体之间、北海与三亚群体之间差异不显著外,其余两两之间差异极显著。AMOVA分析表明,96.22%的遗传变异存在于群体内,群体问的遗传变异仅为总遗传变异的3.78%,但群体间遗传分化显著(F_(SC)=0.038,P＜0.001)。将5个群体分成中国、日本、澳大利亚3组后发现,组问的遗传变异仅为总变异的3.04%,但组间差异并不显著(F_(CT)=0.030,P=0.104)。两两群体间的遗传距离为0.062-0.269。UPGMA系统树显示,北海群体单独为一支,其他4个群体为一支。结果表明,5个群体之间的遗传分化水平较低,其遗传隔离与群体之间的地理间隔无关。
The pearl oyster Pinctada fucata is widely distributed in tropic and subtropic oceans and seas of China, Japan, Indian and Australia. It is an economically important species that is cultured for pearl production in these areas. In the present study, microsatellite-enhanced genomic library of the pearl oyster was constructed. 219 pairs of primers were designed using Primer premier 3.0. After screening, nine polymorphic loci were obtained. Genetic diversity and differentiation in the wild and cultured populations in China as well as within and among five populations from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) were studied using the nine polymorphic loci. The purpose is to provide some information for the protection ofgermplasm resources and genetic improvement of the animal.
There are almost 2000 colonies in the enriched library. The second PCR screening was performed using the primers of (CA)_(15) probe and T vector, and 357 positive clones were obtained. Sequencing results showed that 297 clones contained microsatellite repeat unit. By alignment, 280 microsatellite sequences were obtained after excluding redundancy, including 479 microsatellite doman. The sequence length ranged from 119 to 787bp with an average of 408bp. The (CA/GT)_n repeat unit was the most abundant motifs with a percentage of 76%. Several other repeat types were also detected, such as (AG)_n, (AT)n, (GC)n, (CAA)n, (AAG)n, (ATT)n, (CCT)n, (ATTT)n, (GTTT)n, (CAGA)n, (GAGT)n, (CCGT)n, (GGGT)n, (CAAAA)n, accounting for 24%, 1.7% for each type. Among all the 479 microsatellites, 370 (77.3%) sequences were perfect type, 95 (19.8%) were imperfect type and 14 (2.9%) were compound type. 219 pairs of primers were designed using Primer premier 3.0 and 49 pairs were synthized. As a result, 31 pairs of primers were screened effective for PCR amplification and nine loci showed polymorphism as tested by a population, and the percentage of polymorphic loci accounted for 29.03%.
Genetic diversity in the wild and cultured populations from Beibu Bay, Daya Bay, Sanya Bay was studied using the nine polymorphic markers. A total number of 44 alleles were detected and allele numbers varied from 2 to 9 among loci. The average expected heterozygosity ranged from 0.590 to 0.649, while the average observed heterozygosity ranged from 0.393 to 0.516 among populations. PIC value ranged from 0.530 to 0.586. The negative mean value of D indicated an overall deficit of heterozygotes for the six populations. Forty out of 54 tests were significantly deviated from Hardy-Weinberg equilibrium. AMOVA indicated that 94.59% variation resided within population, while genetic vatiation among populations was only 5.41%. The results showed that genetic diversity was high in all of the six populations, and these populations were competent for selective breeding.
Genetic diversity within and among five populations of the pearl oyster from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using nine microsatellite DNA markers. The average numbers of alleles for the five populations were 4.889, 4.889, 4.889, 4.000 and 4.111. The average expected heterozygosities were 0.592, 0.605, 0.649, 0.575, 0.564 respectively. And the mean levels of obsevered heterozygosity were 0.400, 0.516, 0.485, 0.390 and 0.433, respectively. Shannon's information indexes were 1.131, 1.177, 1.251, 1.028, 1.025. The average polymorphism information contents were 0.530, 0.545, 0.586, 0.498 and 0.483, respectively. These results indicated that genetic diversity of Japanese and Australian populations was lower than the three populations of China. Chi-square tests showed that most of the cases (34) in five populations deviated from Hardy-Weinberg equilibrium. Pairwise F_(ST) ranged from 0.010 to 0.075. Genetic differentiation among the five populations was significant except the values between Beihai and Daya Bay, and between Beihai and Sanya. AMOVA showed that 96.22% variation resided within populations, genetic vatiation among populations was only 3.78%,but significant (F_(SC)=0.038, P<0.001). After dividing the five populations into three groups, the results showed that genetic differentiation among the groups was 3.04% but not significant (F_(CT)=0.030, P=0.104). Pairwise genetic distances among populations ranged from 0.062 to 0.269. An UPGMA tree demonstrated that the population of Beihai was clustered into one group, and the other four populations were clustered into another group. All the results showed that genetic differentiation among the five populations was low, and the genetic distance was not correlated with the geographic distance.
1.用FIASCO法构建的合浦珠母贝基因组微卫星富集文库中共有近2000个单菌落,随机挑选1289个克隆用探针引物(CA)_(15)和载体引物进行第2次筛选,获得阳性候选克隆357个。测序结果表明,297个克隆(83.2%)含有明显的微卫星重复单元,通过序列比对分析,最终获得了280个具有特异微卫星序列的阳性克隆,共含有479个微卫星结构域。获得的微卫星序列长度范围是119-787bp,平均为408bp。其中两碱基(CA/GT)n重复单元出现的次数最多,占所有分离的微卫星数目的76%,此外还发现(AG)n、(AT)n、(GC)n、(CAA)n、(AAG)n、(ATT)n、(CCT)n、(ATTT)n、(GTTT)n、(CAGA)n、(GAGT)n、(CCGT)n、(GGGT)n、(CAAAA)n等多种重复类型,共占分离微卫星数目的24.0%,平均每种占1.7%。在获得的479个微卫星位点中,完美型微卫星370个,占77.3%;非完美型95个,占19.8%;复合型14个,占2.9%。用引物设计软件Primer premier 3.0设计引物219对,合成49对,筛选出31对有效扩增的引物,种群扩增获得9个多态位点,多态位点比例为29.03%。
2.对我国北海野生与养殖群体、大亚湾野生与养殖群体、三亚野生与养殖群体进行PCR扩增后发现:6个群体在9个多态位点共检测出44个等位基因,每个位点的等位基因数从2-9个不等。平均期望杂合度和平均观察杂合度分别介于0.590-0.649和0.393-0.516之间。平均多态信息含量P/C值在0.530-0.586之间。平均杂合子偏离度D值均为负,表明6个群体存在一定的杂合子缺失现象。Hardy-Weinberg平衡的卡方检测发现,在所检测的54个数据中(6个种群×9个位点),有40个偏离遗传平衡状态。两两群体之间的遗传分化指数介于0.002-0.012之间,并且大部分差异显著。AMOVA分析表明,94.59%的遗传变异存在于群体内,群体间的遗传变异仅为总变异的5.41%。研究结果表明,6个群体的遗传多样性水平都比较高,适合进行遗传选育。
3.中国、日本、澳大利亚合浦珠母贝群体遗传多样性的微卫星分析表明:北海群体、大亚湾群体、三亚群体、澳大利亚群体和日本群体的平均等位基因数分别是4.889、4.889、4.889、4.000和4.111;平均期望杂合度分别是0.592、0.605、0.649、0.575、0.564;平均观察杂合度分别是0.400、0.516、0.485、0.390、0.433。Shannon多样性指数分别是1.131、1.177、1.251、1.028、1.025;平均多态信息含量PIC值分别是0.530、0.545、0.586、0.498、0.483,表明日本和澳大利亚群体的遗传多样性低于中国群体。Hardy-Weinberg平衡的卡方检测发现,大多数位点偏离平衡状态(34个)。两两群体之间的F_(ST)值在0.010-0.075之间。除北海与大亚湾群体之间、北海与三亚群体之间差异不显著外,其余两两之间差异极显著。AMOVA分析表明,96.22%的遗传变异存在于群体内,群体问的遗传变异仅为总遗传变异的3.78%,但群体间遗传分化显著(F_(SC)=0.038,P＜0.001)。将5个群体分成中国、日本、澳大利亚3组后发现,组问的遗传变异仅为总变异的3.04%,但组间差异并不显著(F_(CT)=0.030,P=0.104)。两两群体间的遗传距离为0.062-0.269。UPGMA系统树显示,北海群体单独为一支,其他4个群体为一支。结果表明,5个群体之间的遗传分化水平较低,其遗传隔离与群体之间的地理间隔无关。
The pearl oyster Pinctada fucata is widely distributed in tropic and subtropic oceans and seas of China, Japan, Indian and Australia. It is an economically important species that is cultured for pearl production in these areas. In the present study, microsatellite-enhanced genomic library of the pearl oyster was constructed. 219 pairs of primers were designed using Primer premier 3.0. After screening, nine polymorphic loci were obtained. Genetic diversity and differentiation in the wild and cultured populations in China as well as within and among five populations from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) were studied using the nine polymorphic loci. The purpose is to provide some information for the protection ofgermplasm resources and genetic improvement of the animal.
There are almost 2000 colonies in the enriched library. The second PCR screening was performed using the primers of (CA)_(15) probe and T vector, and 357 positive clones were obtained. Sequencing results showed that 297 clones contained microsatellite repeat unit. By alignment, 280 microsatellite sequences were obtained after excluding redundancy, including 479 microsatellite doman. The sequence length ranged from 119 to 787bp with an average of 408bp. The (CA/GT)_n repeat unit was the most abundant motifs with a percentage of 76%. Several other repeat types were also detected, such as (AG)_n, (AT)n, (GC)n, (CAA)n, (AAG)n, (ATT)n, (CCT)n, (ATTT)n, (GTTT)n, (CAGA)n, (GAGT)n, (CCGT)n, (GGGT)n, (CAAAA)n, accounting for 24%, 1.7% for each type. Among all the 479 microsatellites, 370 (77.3%) sequences were perfect type, 95 (19.8%) were imperfect type and 14 (2.9%) were compound type. 219 pairs of primers were designed using Primer premier 3.0 and 49 pairs were synthized. As a result, 31 pairs of primers were screened effective for PCR amplification and nine loci showed polymorphism as tested by a population, and the percentage of polymorphic loci accounted for 29.03%.
Genetic diversity in the wild and cultured populations from Beibu Bay, Daya Bay, Sanya Bay was studied using the nine polymorphic markers. A total number of 44 alleles were detected and allele numbers varied from 2 to 9 among loci. The average expected heterozygosity ranged from 0.590 to 0.649, while the average observed heterozygosity ranged from 0.393 to 0.516 among populations. PIC value ranged from 0.530 to 0.586. The negative mean value of D indicated an overall deficit of heterozygotes for the six populations. Forty out of 54 tests were significantly deviated from Hardy-Weinberg equilibrium. AMOVA indicated that 94.59% variation resided within population, while genetic vatiation among populations was only 5.41%. The results showed that genetic diversity was high in all of the six populations, and these populations were competent for selective breeding.
Genetic diversity within and among five populations of the pearl oyster from China (Beibu Bay, Daya Bay, Sanya Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using nine microsatellite DNA markers. The average numbers of alleles for the five populations were 4.889, 4.889, 4.889, 4.000 and 4.111. The average expected heterozygosities were 0.592, 0.605, 0.649, 0.575, 0.564 respectively. And the mean levels of obsevered heterozygosity were 0.400, 0.516, 0.485, 0.390 and 0.433, respectively. Shannon's information indexes were 1.131, 1.177, 1.251, 1.028, 1.025. The average polymorphism information contents were 0.530, 0.545, 0.586, 0.498 and 0.483, respectively. These results indicated that genetic diversity of Japanese and Australian populations was lower than the three populations of China. Chi-square tests showed that most of the cases (34) in five populations deviated from Hardy-Weinberg equilibrium. Pairwise F_(ST) ranged from 0.010 to 0.075. Genetic differentiation among the five populations was significant except the values between Beihai and Daya Bay, and between Beihai and Sanya. AMOVA showed that 96.22% variation resided within populations, genetic vatiation among populations was only 3.78%,but significant (F_(SC)=0.038, P<0.001). After dividing the five populations into three groups, the results showed that genetic differentiation among the groups was 3.04% but not significant (F_(CT)=0.030, P=0.104). Pairwise genetic distances among populations ranged from 0.062 to 0.269. An UPGMA tree demonstrated that the population of Beihai was clustered into one group, and the other four populations were clustered into another group. All the results showed that genetic differentiation among the five populations was low, and the genetic distance was not correlated with the geographic distance.
引文
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