三疣梭子蟹两种体色相关分子标记研究
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摘要
三疣梭子蟹(Portunus trituberculatus)作为重要的海洋经济动物,其常见体色(头胸甲)为茶绿色。近年来,在我国东部沿海海域海捕三疣梭子蟹中开始出现体色为紫色的一类梭子蟹群体,除体色不同外,二者表型特征并无显著差异。为确定两种体色三疣梭子蟹之间的关系,本文对两种体色三疣梭子蟹不同个体的线粒体COI和16srRNA基因进行了比较分析以判定体色的差异是否由于种间或者种下的遗传差异引起的。以两种体色三疣梭子蟹个体基因组DNA为模板对线粒体两个保守序列基因(COI和16srRNA)分别进行了PCR扩增,而后,将扩增产物分别通过聚丙烯酰胺凝胶电泳(PAGE),进行单链构象多态性分析(SSCP)。结果发现,两个保守序列基因在两种体色三疣梭子蟹群体中均没有特异性电泳条带出现;DNA测序及序列比对分析发现,COI和16srRNA基因在两种体色三疣梭子蟹群体中的核苷酸序列相似度分别达到了99.87%和99.88%,分子水平研究得到的较高的相似度也说明了二者较近的亲缘关系。这表明紫色三疣梭子蟹并未发生亚种的分化,即紫色和茶绿色梭子蟹群体属于同一个种。鉴于在体色统计的调查研究中发现了浅紫色、灰色及虎斑纹等一系列不同体色的三疣梭子蟹类型,并且在养殖实验中有蜕壳后体色发生改变的现象,因此推测三疣梭子蟹体色遗传调控基础是微效多基因控制的数量性状遗传。
在确立两种体色三疣梭子蟹亲缘关系的基础之上,进一步对紫色和茶绿色三疣梭子蟹群体基因组DNA进行体色相关RAPD分子标记的筛选及其遗传多样性分析。首先通过正交实验设计对RAPD-PCR反应体系中的重要参数进行优化,并建立了一套适合三疣梭子蟹PCR扩增反应的体系;而后,通过混合分组分析法(BSA),从80个随机引物中共筛选了30个重复性好、多态性高的引物用于RAPD分析。结果表明:紫色和茶绿色群体中的多态性位点比例分别为61.29%和62.44%,可见紫色和茶绿色梭子蟹群体遗传背景相似,遗传多样性也较丰富;茶绿色梭子蟹的Nei氏基因多样性指数和Shannon信息指数均稍小于紫色梭子蟹的Nei氏基因多样性指数和Shannon信息指数,这表明紫色和茶绿色梭子蟹遗传多样性相似,但紫色梭子蟹较茶绿色梭子蟹遗传多样性更为丰富。而紫色和茶绿色三疣梭子蟹之间高度的Nei氏无偏差遗传相似性指数和较近的遗传距离,均反映了两者的亲缘关系十分相近。RAPD分析所用的30个随机引物在两个群体中扩增条带图谱基本一致,没有单一引物可作为体色相关分子标记用以严格区分两个群体,而有两个随机引物(SBS A09和SBS G16)分别产生的各一条可检测的体色相关的扩增DNA条带在两个群体内以不同概率的形式出现,微卫星分子标记初步分析结果也显示了引物MIH-SSR在紫色和茶绿色群体内产生一条不同比率的体色相关特异性条带。这一现象与三疣梭子蟹的体色属于数量性状且由微效多基因控制的观点相一致;同时,这也说明两种体色梭子蟹的亲缘关系非常亲近。
There have been some crab individuals with purple carapaces and appendages newly discovered in the wild captured blue swimming crab Portunus trituberculatus in recent years, while the normal carapace color of the blue swimming crab is tea-green. Given the morphological characters of purple crabs identical with that of the tea-green ones except the body color, it is expected that the purple crabs and the tea-green ones were from the same species. In order to identify the phylogenetic relationships between them at the molecular level, we amplified and compared two conserved mitochondrial gene fragments of purple and tea-green crabs through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the COI and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.87% and 99.88% nucleotide sequence identity of the COI and 16srRNA genes respectively. These indicated that there was no species or subspecies differentiation between purple and tea-green crabs, meaning that they belonged to the same species, Portunus trituberculatus. And some crabs with other carapace colors were found in their natural habitats. It was expected that the carapace color may be regulated by multiple genes located on the chromosomes.
Then the phylogenetic relationships and genetic differences were determined between purple and tea-green crab individuals of P. trituberculatus by RAPD technique. 40 color-different crab individuals were evaluated using 80 RAPD primers. 30 primers were selected using bulk segregation analysis (BSA) and RAPD-PCR analysis. The results showed that the ratio of polymorphic loci was 61.29%, Nei's gene diversity was 0.3756, Shannon's information index was 0.5545 among purple crab individuals; and 62.44% the ratio of polymorphic loci, 0.3451 Nei's gene diversity, 0.5099 Shannon's information index among tea-green crab individuals. It was clear that the genetic diversity between purple and tea-green crabs was similar, which indicated close phylogenetic relationships between the purple and tea-green crabs. In addition, two primers (SBS A09 and SBS G16) got specific DNA bands by comparing forty individuals of color-different crab individuals through RAPD-PCR analysis and the following electrophoresis. One specific DNA band (750~1000 bp) was discovered using the primer SBS A09, with 22.22% and 75.00% polymorphism within purple and tea-green crab individuals respectively; and one DNA band (1000~2000 bp) from the primer SBS G16 showed 35.00% and 75.00% polymorphism within purple and tea-green crab individuals respectively. And the SSR primer MIH-SSR also got specific DNA bands within color-different crab individuals. It indicated that the body color transmision was affected by the heredity of some quantitative characters. These indicated close phylogenetic relationships between purple and tea-green crabs.
在确立两种体色三疣梭子蟹亲缘关系的基础之上,进一步对紫色和茶绿色三疣梭子蟹群体基因组DNA进行体色相关RAPD分子标记的筛选及其遗传多样性分析。首先通过正交实验设计对RAPD-PCR反应体系中的重要参数进行优化,并建立了一套适合三疣梭子蟹PCR扩增反应的体系;而后,通过混合分组分析法(BSA),从80个随机引物中共筛选了30个重复性好、多态性高的引物用于RAPD分析。结果表明:紫色和茶绿色群体中的多态性位点比例分别为61.29%和62.44%,可见紫色和茶绿色梭子蟹群体遗传背景相似,遗传多样性也较丰富;茶绿色梭子蟹的Nei氏基因多样性指数和Shannon信息指数均稍小于紫色梭子蟹的Nei氏基因多样性指数和Shannon信息指数,这表明紫色和茶绿色梭子蟹遗传多样性相似,但紫色梭子蟹较茶绿色梭子蟹遗传多样性更为丰富。而紫色和茶绿色三疣梭子蟹之间高度的Nei氏无偏差遗传相似性指数和较近的遗传距离,均反映了两者的亲缘关系十分相近。RAPD分析所用的30个随机引物在两个群体中扩增条带图谱基本一致,没有单一引物可作为体色相关分子标记用以严格区分两个群体,而有两个随机引物(SBS A09和SBS G16)分别产生的各一条可检测的体色相关的扩增DNA条带在两个群体内以不同概率的形式出现,微卫星分子标记初步分析结果也显示了引物MIH-SSR在紫色和茶绿色群体内产生一条不同比率的体色相关特异性条带。这一现象与三疣梭子蟹的体色属于数量性状且由微效多基因控制的观点相一致;同时,这也说明两种体色梭子蟹的亲缘关系非常亲近。
There have been some crab individuals with purple carapaces and appendages newly discovered in the wild captured blue swimming crab Portunus trituberculatus in recent years, while the normal carapace color of the blue swimming crab is tea-green. Given the morphological characters of purple crabs identical with that of the tea-green ones except the body color, it is expected that the purple crabs and the tea-green ones were from the same species. In order to identify the phylogenetic relationships between them at the molecular level, we amplified and compared two conserved mitochondrial gene fragments of purple and tea-green crabs through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the COI and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.87% and 99.88% nucleotide sequence identity of the COI and 16srRNA genes respectively. These indicated that there was no species or subspecies differentiation between purple and tea-green crabs, meaning that they belonged to the same species, Portunus trituberculatus. And some crabs with other carapace colors were found in their natural habitats. It was expected that the carapace color may be regulated by multiple genes located on the chromosomes.
Then the phylogenetic relationships and genetic differences were determined between purple and tea-green crab individuals of P. trituberculatus by RAPD technique. 40 color-different crab individuals were evaluated using 80 RAPD primers. 30 primers were selected using bulk segregation analysis (BSA) and RAPD-PCR analysis. The results showed that the ratio of polymorphic loci was 61.29%, Nei's gene diversity was 0.3756, Shannon's information index was 0.5545 among purple crab individuals; and 62.44% the ratio of polymorphic loci, 0.3451 Nei's gene diversity, 0.5099 Shannon's information index among tea-green crab individuals. It was clear that the genetic diversity between purple and tea-green crabs was similar, which indicated close phylogenetic relationships between the purple and tea-green crabs. In addition, two primers (SBS A09 and SBS G16) got specific DNA bands by comparing forty individuals of color-different crab individuals through RAPD-PCR analysis and the following electrophoresis. One specific DNA band (750~1000 bp) was discovered using the primer SBS A09, with 22.22% and 75.00% polymorphism within purple and tea-green crab individuals respectively; and one DNA band (1000~2000 bp) from the primer SBS G16 showed 35.00% and 75.00% polymorphism within purple and tea-green crab individuals respectively. And the SSR primer MIH-SSR also got specific DNA bands within color-different crab individuals. It indicated that the body color transmision was affected by the heredity of some quantitative characters. These indicated close phylogenetic relationships between purple and tea-green crabs.
引文
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