三疣梭子蟹不同地理群体遗传多样性和杂交育种的初步研究
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摘要
本文利用生物学性状多元分析和线粒体DNA基因片段序列分析技术对分别采自海州湾、莱州湾、辽东湾、舟山的三疣梭子蟹(Portunus trituberculatus)4个群体的遗传结构进行了比较分析,同时对三疣梭子蟹群体间杂交子代的形态学性状进行了研究,主要结果如下:
对三疣梭子蟹4个野生群体线粒体16S rRNA和COⅠ基因片段进行了PCR扩增和产物测序,分别得到长度为524bp和658bp的片段。两段序列的碱基组成均显示较高的A+T比例(16S rRNA基因70.8%,COⅠ基因63%),这与果蝇、虾类、蟹类等无脊椎动物的16S rRNA和COⅠ基因片段研究结果相似。通过对三疣梭子蟹16S rRNA和COⅠ基因片段遗传特征的研究,发现种内地理群体间变异较低,在16个样本中,16S rRNA基因序列中共检测到1个变异位点,2种单倍型;COⅠ基因序列中共检测到4个变异位点,5种单倍型。另外,以中华绒螯蟹为外群探讨了梭子蟹科(Portunidea)几个属种的系统进化关系。用MEGA4.0软件中的NJ法构建了系统进化树,基于16S rRNA和COⅠ两种片段的聚类结果均显示梭子蟹属(Portunus)与美青蟹属(Callinectes)亲缘关系最近,先聚在一起,然后再与蟳属(Charybdis)聚在一起,最后才与外群中华绒螯蟹聚在一起,这一结果与传统分类基本一致。
采用不同地理群体内自繁和种群间杂交的方法,进行了自繁和杂交子一代生长性状的比较研究。结果表明,80日龄时的杂交子一代在各项指标都表现出一定的杂种优势(0.24%—13.50%),LS在5个性状中都高于ZL。100日龄时杂交子一代表现出的杂种优势有所下降,杂种优势率在-0.88%—4.13%之间,甲长表现出杂种劣势。120日龄时杂交子一代的杂种优势范围为-1.91%—4.37%,甲长、甲宽表现出杂种劣势。可见不同杂交组合、不同性状、不同时间表现出的杂种优势有显著差异。ANOVA分析表明,80日龄、100日龄、120日龄时,5个生长性状在群体之间存在极显著差异。实验结果表明,三疣梭子蟹4个杂交子一代在生长性状上存在一定程度的杂种优势,可见种内群体间杂交是培育三疣梭子蟹优良新品系的重要方法之一。
另外,测量了海州湾、莱州湾、辽东湾、舟山4个选育二代群体100日龄三疣梭子蟹的5个外部形态性状,用SPSS11.0统计软件对这4个群体进行聚类分析、判别分析和单因子方差分析。聚类分析结果表明,海州湾和舟山2个群体形态最为接近,辽东湾和莱州湾2个群体形态较为接近。对样本所属群体进行判别分析,海州湾、莱州湾、辽东湾、舟山4个群体的判别准确率分别为80%、76.7%、80%和42.9%,综合判别率为70.3%。建立了4个群体各自的判别函数公式。通过对4项形态比例参数进行单因素方差分析,计算差异系数,发现4个参数在群体间存在极显著差异。3种分析结果均认为,4个群体三疣梭子蟹在形态上产生了一定程度的差异,但所有的差异均未达到亚种水平。
Genetic variation of Portunus trituberculatus was analyzed using multianalysis and DNA Sequencing technique. And the cross breeding among wild populations of P.trituberculatus was studied. The main results were shown as follows:
Mitochondrial 16S rRNA gene and COⅠgene fragments were amplified and sequenced from 4 wild populations of Portunus trituberculatus. Respectively, 524bp and 658bp long partial gene fragments of the 16S rRNA gene and the COⅠgene were obtained. A high percentage of A+T base composition in the two sequences was observed, which was similar to the results of studies on drosophila, shrimp, crab and other invertebrates. Little sequence variation was observed in 16S rRNA gene and COⅠgene fragments within species. 2 haplotypes were identified from 16 samples with 1 variation site of 16S rRNA gene and 5 haplotypes were identified from 16 samples with 4 variation sites of COⅠgene. In addition, the phylogenetic relationship among several genera of Portunidea was discussed using Eriochier sinensis as the out-group. The molecular phylogenetic trees were constructed by NJ method using software MEGA4.0, and Portunus was clustered with Callinectes first, then they were clustered with Charybdis,and finally with Eriochier sinensis. The results are consistent with classical taxonomy.
Portunus trituberculatus is an economically valued crab species. However, rapid expansion in agro-industry of it in recent years in China yielded also some problems such as frequent diseases outbreak, breed/strain degeneration, and natural resource depletion. They have limited the sustainable development. In this research,matings and crosses within and among three different geographical populations(Zhoushan:ZS; Laizhou Bay:LZ; and Haizhou Bay:HZ)of the species were screened for new breed and strains.Seven populations of first filial generations have been obtained,of which three were of matings and 4 crossings.The characteristic indices of the first filial generations were measured on Days 80,100and 120.Results show that the heterosis was significant in the whole carapace width,carapace width,carapace length,body height,and body weight;for instance, the difference in heterosis of Day 80 one was 0.24%—13.50%. The heterosis in LS was greater than in ZL in all dimensions. On Day 100, the heterosis varied -0.88%—4.13% with hybrid weak ness in carapace length. On Day 120, the heterosis varied -1.91%—4.37% with hybrid weakness in carapace length and carapace width. Analysis with ANOVA program shows that some traits were significantly different among seven populations of the first filial generations. Hybridization was proved important in this study for increasing the growth performance of trituberculatus.
Five morphological characters of populations of four selected Portunus trituberculatus from Haizhou Bay, Laizhou Bay, Liaodong Bay and Zhoushan were analysed by clustering,discriminant methods and One-Way ANOVA .Crabs from Haizhou Bay and Zhoushan were pooled into one group while crabs from Liaodong Bay and Laizhou Bay were pooled into another group by cluster analysis. In discriminant analysis,The accuracy rate of discriminant on four farmed Portunus trituberculatus from Haizhou Bay, Laizhou Bay, Liaodong Bay and Zhoushan were 80%,76.7%,80% and 42.9%.Total accuracy rate of discriminant was 70.3%. The discriminant functions of the populations from four populations were established,respectively. One-Way ANOVA was conducted on 4 proportional parameters. The results showed that the four proporational parameters were significantly different among the four populations. In this study all results supported that there were some morphological differences among the four populations, but variations of all the morphologieal traits were not characterized subspecies.
对三疣梭子蟹4个野生群体线粒体16S rRNA和COⅠ基因片段进行了PCR扩增和产物测序,分别得到长度为524bp和658bp的片段。两段序列的碱基组成均显示较高的A+T比例(16S rRNA基因70.8%,COⅠ基因63%),这与果蝇、虾类、蟹类等无脊椎动物的16S rRNA和COⅠ基因片段研究结果相似。通过对三疣梭子蟹16S rRNA和COⅠ基因片段遗传特征的研究,发现种内地理群体间变异较低,在16个样本中,16S rRNA基因序列中共检测到1个变异位点,2种单倍型;COⅠ基因序列中共检测到4个变异位点,5种单倍型。另外,以中华绒螯蟹为外群探讨了梭子蟹科(Portunidea)几个属种的系统进化关系。用MEGA4.0软件中的NJ法构建了系统进化树,基于16S rRNA和COⅠ两种片段的聚类结果均显示梭子蟹属(Portunus)与美青蟹属(Callinectes)亲缘关系最近,先聚在一起,然后再与蟳属(Charybdis)聚在一起,最后才与外群中华绒螯蟹聚在一起,这一结果与传统分类基本一致。
采用不同地理群体内自繁和种群间杂交的方法,进行了自繁和杂交子一代生长性状的比较研究。结果表明,80日龄时的杂交子一代在各项指标都表现出一定的杂种优势(0.24%—13.50%),LS在5个性状中都高于ZL。100日龄时杂交子一代表现出的杂种优势有所下降,杂种优势率在-0.88%—4.13%之间,甲长表现出杂种劣势。120日龄时杂交子一代的杂种优势范围为-1.91%—4.37%,甲长、甲宽表现出杂种劣势。可见不同杂交组合、不同性状、不同时间表现出的杂种优势有显著差异。ANOVA分析表明,80日龄、100日龄、120日龄时,5个生长性状在群体之间存在极显著差异。实验结果表明,三疣梭子蟹4个杂交子一代在生长性状上存在一定程度的杂种优势,可见种内群体间杂交是培育三疣梭子蟹优良新品系的重要方法之一。
另外,测量了海州湾、莱州湾、辽东湾、舟山4个选育二代群体100日龄三疣梭子蟹的5个外部形态性状,用SPSS11.0统计软件对这4个群体进行聚类分析、判别分析和单因子方差分析。聚类分析结果表明,海州湾和舟山2个群体形态最为接近,辽东湾和莱州湾2个群体形态较为接近。对样本所属群体进行判别分析,海州湾、莱州湾、辽东湾、舟山4个群体的判别准确率分别为80%、76.7%、80%和42.9%,综合判别率为70.3%。建立了4个群体各自的判别函数公式。通过对4项形态比例参数进行单因素方差分析,计算差异系数,发现4个参数在群体间存在极显著差异。3种分析结果均认为,4个群体三疣梭子蟹在形态上产生了一定程度的差异,但所有的差异均未达到亚种水平。
Genetic variation of Portunus trituberculatus was analyzed using multianalysis and DNA Sequencing technique. And the cross breeding among wild populations of P.trituberculatus was studied. The main results were shown as follows:
Mitochondrial 16S rRNA gene and COⅠgene fragments were amplified and sequenced from 4 wild populations of Portunus trituberculatus. Respectively, 524bp and 658bp long partial gene fragments of the 16S rRNA gene and the COⅠgene were obtained. A high percentage of A+T base composition in the two sequences was observed, which was similar to the results of studies on drosophila, shrimp, crab and other invertebrates. Little sequence variation was observed in 16S rRNA gene and COⅠgene fragments within species. 2 haplotypes were identified from 16 samples with 1 variation site of 16S rRNA gene and 5 haplotypes were identified from 16 samples with 4 variation sites of COⅠgene. In addition, the phylogenetic relationship among several genera of Portunidea was discussed using Eriochier sinensis as the out-group. The molecular phylogenetic trees were constructed by NJ method using software MEGA4.0, and Portunus was clustered with Callinectes first, then they were clustered with Charybdis,and finally with Eriochier sinensis. The results are consistent with classical taxonomy.
Portunus trituberculatus is an economically valued crab species. However, rapid expansion in agro-industry of it in recent years in China yielded also some problems such as frequent diseases outbreak, breed/strain degeneration, and natural resource depletion. They have limited the sustainable development. In this research,matings and crosses within and among three different geographical populations(Zhoushan:ZS; Laizhou Bay:LZ; and Haizhou Bay:HZ)of the species were screened for new breed and strains.Seven populations of first filial generations have been obtained,of which three were of matings and 4 crossings.The characteristic indices of the first filial generations were measured on Days 80,100and 120.Results show that the heterosis was significant in the whole carapace width,carapace width,carapace length,body height,and body weight;for instance, the difference in heterosis of Day 80 one was 0.24%—13.50%. The heterosis in LS was greater than in ZL in all dimensions. On Day 100, the heterosis varied -0.88%—4.13% with hybrid weak ness in carapace length. On Day 120, the heterosis varied -1.91%—4.37% with hybrid weakness in carapace length and carapace width. Analysis with ANOVA program shows that some traits were significantly different among seven populations of the first filial generations. Hybridization was proved important in this study for increasing the growth performance of trituberculatus.
Five morphological characters of populations of four selected Portunus trituberculatus from Haizhou Bay, Laizhou Bay, Liaodong Bay and Zhoushan were analysed by clustering,discriminant methods and One-Way ANOVA .Crabs from Haizhou Bay and Zhoushan were pooled into one group while crabs from Liaodong Bay and Laizhou Bay were pooled into another group by cluster analysis. In discriminant analysis,The accuracy rate of discriminant on four farmed Portunus trituberculatus from Haizhou Bay, Laizhou Bay, Liaodong Bay and Zhoushan were 80%,76.7%,80% and 42.9%.Total accuracy rate of discriminant was 70.3%. The discriminant functions of the populations from four populations were established,respectively. One-Way ANOVA was conducted on 4 proportional parameters. The results showed that the four proporational parameters were significantly different among the four populations. In this study all results supported that there were some morphological differences among the four populations, but variations of all the morphologieal traits were not characterized subspecies.
引文
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