海南沼虾遗传多样性的研究
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摘要
海南沼虾(Macrobrachium hainanense),隶属于甲壳纲,十足目,长臂虾科,沼虾属,是我国重要的淡水经济虾类。因其在河口附近繁殖且耐低温能力较弱,分布于浙江(瓯江以南)、广东、广西、福建、海南等地入海的河流。海南沼虾已应用于杂交育种研究,但有关其遗传多样性研究迄今仍为空白。本文采用SRAP分子标记技术对瓯江、闽江、珠江、万泉河、昌化江五个地理种群的海南沼虾遗传多样性进行了研究,并对珠江群体海南沼虾的ITS-1序列进行了分析。
①海南沼虾遗传多样性的研究
本文通过优化SRAP扩增的相关参数,建立了海南沼虾的SRAP反应体系。25μLSRAP扩增体系的最优参数如下：10×PCR反应缓冲液2.5μL,MgCl2(25mmoL／L)1.5μL,Taq DNA聚合酶(5U／μL)0.16μL,正反向引物(10μmol／L)各2.0μL,dNTP(2.5mmol／L)1.5μL,全基因组DNA(20ng／μL)1.0μL,ddH2O补足。
利用上述反应体系,从30对SRAP引物组合中筛选出15对扩增稳定且多态性较好的引物组合,并分析了瓯江、闽江、珠江、万泉河、昌化江五个地理种群海南沼虾的遗传多样性。共得到了255个清晰、稳定的位点,其中多态性位点为177个,多态性位点比例为69.4118%。五个地理种群多态性位点比例分别为47.45%、43.92%、53.33%、46.67%、43.92%；遗传杂合度分别为0.1799、0.1657、0.1839、0.1892、0.1763；Shannon信息指数分别为0.2746、0.2543、0.2876、0.2846、0.2658。两两群体间遗传分化指数(Gst)值在0.0887-0.2702之间,基因流值(Nm)在1.9135-5.1366之间,遗传距离值介于0.2472-0.3469之间,遗传相似系数为0.6531-0.7528。AMOVA分析结果表明：群体的遗传变异有17.64%是由群体间遗传变异引起的,且群体间遗传变异对总的遗传变异影响是显著的(Pst=0.176>0.15)。聚类分析结果表明：五个地理种群海南沼虾共分为两个类群,昌化江群体单独为一个类群；瓯江、珠江群体在遗传相似系数值为0.75时聚在一起；遗传相似系数值为0.738-0.742时,瓯江、珠江群体先后与闽江群体和万泉河群体聚在一起；当遗传相似系数值为0.67时,五个群体聚在一起。
②海南沼虾ITS-1序列分析
采用青虾(Macrobrachium nipponense)ITS-1扩增引物,对珠江群体海南沼虾基因组DNA进行扩增,扩增出大小为2000bp左右的单一亮带。用DNAStar软件分析海南沼虾ITS-1序列后得出：1)各海南沼虾个体间的平均遗传相似度为90.80357%,ITS-1片段长度在1630-1750bp之间；2)ITS-1序列中碱基A、G、C、G+C的含量百分比变化范围分别为：27%-30%(A),27%-31%(G),14%-16%(C),43%-46%(G+C)；3)ITS-1全序列中含有2个具有多态性的SSR位点,分别为位于258bp的(AG)n序列和1270bp的(GA)n序列。将海南沼虾ITS-1标准序列进行BLAST比对分析,结果表明：海南沼虾同青虾(M. nipponense)和刀额新对虾(Metapenaeus ensis)都有较高的同源性。
本文的研究填补了海南沼虾遗传多样性研究的空白,将为海南沼虾种质资源的有效保护和合理地开发利用提供理论依据；同时也为海南沼虾的遗传学研究提供了新的分子标记。
Macrobrachium hainanense, belonged to Macrobrachium, Palaemonidae, Decapoda, Crustacea, is one of the most important freshwater prawns for aquaculture in China. M. hainanense spawns near the estuary and its low-temperature tolerance is weak. As a result, it only exists in the rivers connected to the sea, which includes O'jiang river and other rivers in the south of O'jiang river. M. hainanense has been used for cross-breeding, but no work was reported on its genetic diversity. In this paper, genetic diversity of M. hainanense of five geographic populations, including O'jiang River(OJ), Minjiang River(MJ), Pearl River(PR), Wanquanhe River(WQ) and Changhuajiang River(CH), were investigated by SRAP markers and ITS-1 sequence of Pearl River population was analysed.
①Studies on genetic diversity of M. hainanense
SRAP reaction system of M. hainanense was established by optimizing PCR reaction factors. The optimal parameters of 25μL reaction system:10×PCR buffer 2.5μL, MgCl2 (25 mmoL/L) 1.5μL, Taq polymerase (5U/μL) 0.16μL, forward and reverse primer (10μmol/L) 2.0μL each, dNTP (2.5mmol/L) 1.5μL, genomic DNA (20ng/μL) 1.0μL, with ddH2O complement to 25μL.
With the SRAP reaction system,15 pairs of stably and well amplified primer combination were screened from 30 pairs of primer combination. Genetic diversity of five geographic populations named OJ, MJ, PR, WQ, CH for M. hainanense was studied.177 polymorphism loci out of 255 clear and stab loci, the presence ratio of polymorphism locus was 69.4118%. The percentage of polymorphism locus, heterozygosity, Shannon's information index for each population were 47.45%,43.92%,53.33%,46.67%,43.92%; 0.1799,0.1657,0.1839,0.1892,0.1763; 0.2746,0.2543,0.2876,0.2846,0.2658. Gst, Nm, genetic distance and genetic similarity coefficients for any two populations ranged from 0.0887-0.2702,1.9135-5.1366,0.2472-0.3469,0.6531-0.7528. With the method of AMOVA,17.64% genetic variation of populations was caused significantly by genetic variation of inter-populations (Pst=0.176>0.15). The Cluster analysis showed that the five geographic populations of M. Hainanense were divided into two groups and CH population was an independent group; OJ and PR population clustered when coefficient was 0.75; OJ, PR population clustered with MJ and WQ population when coefficient was 0.738-0.742; When coefficient was 0.67, all of populations clustered.
②Studies on ITS-1 of M. hainanense
By using of ITS-1 primer for Macrobrachium nipponense, ITS-1 of M. hainanense was amplified. A band length about 2,000bp was detected. ITS-1 of M. hainanense was analyzed by DNAStar and results as follows:①The length of ITS-1 was ranged from 1,630bp to1,750bp;②The content range of base A, G, C, and G+C were 27%-30%, 27%-31%,14%-16%,43%-46% respectively;③here are two polymorphism loci of SSR. One locus of (AG)n was at 258bp, the other locus of (GA)n was at 1270bp. Standard ITS-1 of M. hainanense was analyzed by BLAST method and the result showed that there was high homology between M. hainanens and M. nipponense, M. ensis.
This paper not only filled the gap of studies on genetic diversity for M. hainanense but provided the guides for scientific development and utilization of M. hainanense. In addition, new molecular markers were developed for studies of M. hainanense genetics.
①海南沼虾遗传多样性的研究
本文通过优化SRAP扩增的相关参数,建立了海南沼虾的SRAP反应体系。25μLSRAP扩增体系的最优参数如下：10×PCR反应缓冲液2.5μL,MgCl2(25mmoL／L)1.5μL,Taq DNA聚合酶(5U／μL)0.16μL,正反向引物(10μmol／L)各2.0μL,dNTP(2.5mmol／L)1.5μL,全基因组DNA(20ng／μL)1.0μL,ddH2O补足。
利用上述反应体系,从30对SRAP引物组合中筛选出15对扩增稳定且多态性较好的引物组合,并分析了瓯江、闽江、珠江、万泉河、昌化江五个地理种群海南沼虾的遗传多样性。共得到了255个清晰、稳定的位点,其中多态性位点为177个,多态性位点比例为69.4118%。五个地理种群多态性位点比例分别为47.45%、43.92%、53.33%、46.67%、43.92%；遗传杂合度分别为0.1799、0.1657、0.1839、0.1892、0.1763；Shannon信息指数分别为0.2746、0.2543、0.2876、0.2846、0.2658。两两群体间遗传分化指数(Gst)值在0.0887-0.2702之间,基因流值(Nm)在1.9135-5.1366之间,遗传距离值介于0.2472-0.3469之间,遗传相似系数为0.6531-0.7528。AMOVA分析结果表明：群体的遗传变异有17.64%是由群体间遗传变异引起的,且群体间遗传变异对总的遗传变异影响是显著的(Pst=0.176>0.15)。聚类分析结果表明：五个地理种群海南沼虾共分为两个类群,昌化江群体单独为一个类群；瓯江、珠江群体在遗传相似系数值为0.75时聚在一起；遗传相似系数值为0.738-0.742时,瓯江、珠江群体先后与闽江群体和万泉河群体聚在一起；当遗传相似系数值为0.67时,五个群体聚在一起。
②海南沼虾ITS-1序列分析
采用青虾(Macrobrachium nipponense)ITS-1扩增引物,对珠江群体海南沼虾基因组DNA进行扩增,扩增出大小为2000bp左右的单一亮带。用DNAStar软件分析海南沼虾ITS-1序列后得出：1)各海南沼虾个体间的平均遗传相似度为90.80357%,ITS-1片段长度在1630-1750bp之间；2)ITS-1序列中碱基A、G、C、G+C的含量百分比变化范围分别为：27%-30%(A),27%-31%(G),14%-16%(C),43%-46%(G+C)；3)ITS-1全序列中含有2个具有多态性的SSR位点,分别为位于258bp的(AG)n序列和1270bp的(GA)n序列。将海南沼虾ITS-1标准序列进行BLAST比对分析,结果表明：海南沼虾同青虾(M. nipponense)和刀额新对虾(Metapenaeus ensis)都有较高的同源性。
本文的研究填补了海南沼虾遗传多样性研究的空白,将为海南沼虾种质资源的有效保护和合理地开发利用提供理论依据；同时也为海南沼虾的遗传学研究提供了新的分子标记。
Macrobrachium hainanense, belonged to Macrobrachium, Palaemonidae, Decapoda, Crustacea, is one of the most important freshwater prawns for aquaculture in China. M. hainanense spawns near the estuary and its low-temperature tolerance is weak. As a result, it only exists in the rivers connected to the sea, which includes O'jiang river and other rivers in the south of O'jiang river. M. hainanense has been used for cross-breeding, but no work was reported on its genetic diversity. In this paper, genetic diversity of M. hainanense of five geographic populations, including O'jiang River(OJ), Minjiang River(MJ), Pearl River(PR), Wanquanhe River(WQ) and Changhuajiang River(CH), were investigated by SRAP markers and ITS-1 sequence of Pearl River population was analysed.
①Studies on genetic diversity of M. hainanense
SRAP reaction system of M. hainanense was established by optimizing PCR reaction factors. The optimal parameters of 25μL reaction system:10×PCR buffer 2.5μL, MgCl2 (25 mmoL/L) 1.5μL, Taq polymerase (5U/μL) 0.16μL, forward and reverse primer (10μmol/L) 2.0μL each, dNTP (2.5mmol/L) 1.5μL, genomic DNA (20ng/μL) 1.0μL, with ddH2O complement to 25μL.
With the SRAP reaction system,15 pairs of stably and well amplified primer combination were screened from 30 pairs of primer combination. Genetic diversity of five geographic populations named OJ, MJ, PR, WQ, CH for M. hainanense was studied.177 polymorphism loci out of 255 clear and stab loci, the presence ratio of polymorphism locus was 69.4118%. The percentage of polymorphism locus, heterozygosity, Shannon's information index for each population were 47.45%,43.92%,53.33%,46.67%,43.92%; 0.1799,0.1657,0.1839,0.1892,0.1763; 0.2746,0.2543,0.2876,0.2846,0.2658. Gst, Nm, genetic distance and genetic similarity coefficients for any two populations ranged from 0.0887-0.2702,1.9135-5.1366,0.2472-0.3469,0.6531-0.7528. With the method of AMOVA,17.64% genetic variation of populations was caused significantly by genetic variation of inter-populations (Pst=0.176>0.15). The Cluster analysis showed that the five geographic populations of M. Hainanense were divided into two groups and CH population was an independent group; OJ and PR population clustered when coefficient was 0.75; OJ, PR population clustered with MJ and WQ population when coefficient was 0.738-0.742; When coefficient was 0.67, all of populations clustered.
②Studies on ITS-1 of M. hainanense
By using of ITS-1 primer for Macrobrachium nipponense, ITS-1 of M. hainanense was amplified. A band length about 2,000bp was detected. ITS-1 of M. hainanense was analyzed by DNAStar and results as follows:①The length of ITS-1 was ranged from 1,630bp to1,750bp;②The content range of base A, G, C, and G+C were 27%-30%, 27%-31%,14%-16%,43%-46% respectively;③here are two polymorphism loci of SSR. One locus of (AG)n was at 258bp, the other locus of (GA)n was at 1270bp. Standard ITS-1 of M. hainanense was analyzed by BLAST method and the result showed that there was high homology between M. hainanens and M. nipponense, M. ensis.
This paper not only filled the gap of studies on genetic diversity for M. hainanense but provided the guides for scientific development and utilization of M. hainanense. In addition, new molecular markers were developed for studies of M. hainanense genetics.
引文
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