脊尾白虾微卫星分离及其遗传多样性分析
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摘要
研究了近缘物种微卫星标记对脊尾白虾的通用性。利用11对中国对虾(Penaeus chinensis)的微卫星引物对脊尾白虾(Exopalaemon carinicauda)进行PCR扩增。经过PCR条件优化,有2对中国对虾引物能在脊尾白虾中重复稳定地扩增出目的片段,分别为引物RS1101和引物72A64,且均能表现出多态性,可用于脊尾白虾遗传多样性分析。
利用磁珠富集法开发了脊尾白虾微卫星标记并对其特征进行了分析。首先将脊尾白虾基因组DNA经MboI酶切,连上接头,构建基因组PCR文库,然后用生物素标记的简单重复序列(CA)16作探针与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,经一系列的洗涤过程,去除磁珠表面不含有微卫星的片段,最后将吸附在磁珠上的片段洗脱,PCR扩增,再进行克隆和测序,即可得到微卫星序列。本研究选取61个阳性克隆,经测序,共获得32个微卫星序列,其中：完全型微卫星27个(84.38%),复合型微卫星1个(3.13%),非完全型微卫星4个(12.5%)；所得微卫星中以CA/GT为重复单位的序列30个,占93.75%,重复次数在20次以上的微卫星27个,占84.38%,最高重复次数为39次。用引物设计软件Oligo6.0设计引物23对。
利用微卫星标记研究了脊尾白虾群体的遗传多样性。用所筛选到的10对微卫星引物对江苏连云港、浙江三门和福建连江3个脊尾白虾群体的132尾个体基因组DNA进行PCR扩增,分析其群体遗传多样性。根据3个群体在10个微卫星位点上的PCR扩增图谱,计算出各群体的遗传多样性参数。结果显示：10个微卫星位点分别扩增到等位基因数为6-12,共计89个,平均等位基因数为8.90,平均有效等位基因数为7.43,平均遗传杂合度为0.863,平均多态信息含量为0.843,所有微卫星位点均呈高度多态性(PIC>0.5)。江苏连云港、浙江三门和福建连江3个群体的平均遗传杂合度分别为0.827、0.845、0.805；平均多态信息含量分别为0.792、0.815、0.771,说明这3个群体遗传多样性水平较高。3个群体中,浙江三门群体与福建连江群体间的遗传相似系数最高(0.6594),遗传距离最小(0.4164),说明这2个群体亲缘关系最近；江苏连云港群体与福建连江群体间的遗传相似性系数最低(0.6374),遗传距离最大(0.4504),可推断这2个群体亲缘关系最远。根据群体间的遗传距离采用UPMGA法进行聚类分析,结果显示：浙江三门群体与福建连江群体首先聚为一支,然后再与江苏连云港群体聚为一支。聚类分析结果说明,江苏连云港群体与其他2个群体亲缘关系最远,浙江三门群体与福建连江群体亲缘关系最近。由此可预测,为获得最大的杂种优势,可选用江苏连云港和福建连江两地的脊尾白虾进行杂交育种。
The transferability of Penaeus chinensis SSR markers to Exopalaemon carinicauda was explored. The conservativeness of the side sequences of the microsatellite in species of close genetic relationship has already been discovered.11 polymohic microsatellite primers identified in Penaeus chinensis were employed to amplify in the genome of Exopalaemon carinicauda. The conditions of polymerase chain reaction(PCR) were optimized for the fidelity of DNA synthesis during PCR amplification. It was found that 9 loci failed to be amplified and 2 loci (RSI 101 and 72A64) amplified specific products successfully. The two loci were polymorphism.
The SSR markers were developed and characterization in Exopalaemon carinicauda. Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA. In this experiment, we isolated microsatellite from Exopalaemon carinicauda genome with this method. Exopalaemon carinicauda genomic DNA was extracted and digested with restriction enzyme MboI. Fragments were ligated with artificial linkers(20bp). They were used to create a "whole genome PCR library". This genomic DNA was hybridized with a biotin-labeled microsatellite probe (CA)16. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. The selected DNAs are then amplified using primers designed complementary to the linkers, cloned into the pGEM-T vector and sequenced. In this experiment, we selected 61 positive clones. From these positive clones, we isolated 32 microsatellite DNA sequences and all most (about 93.75%) of them were with corn repeats CA/GT, among which 84.38% microsatellites were perfect repeats,12.5% imperfect repeats and 3.13% compound repeats; 84.38% microsatellites repeated more than 20 times and the biggest repead number was 39. This allowed us to designed 23 pairs of primer with the software Oligo 6.0.
Genetic diversity among three populations of Exopalaemon carinicauda distributing in Jiangsu, Zhejiang and Fujian province were studied by using microsatellite analysis technology.10 pairs of microsatellite primers of Exopalaemon carinicauda which were selected and proved to amplify successfully on Exopalaemon carinicauda. Amplified PCR products were imagined by Biolight. Average observed heterozygosity and average expected heterozygosity were calculated by Popgene3.2 software. The results showed that 6 to 12 alleles per pair in the three populations were amplified. The total number of alleles is 89 and the average number of the alleles per pair was 8.90; the average number of the effective alleles per pair was 7.43; the average heterozygosity was 0.863; the average polymorphism information contents(PIC) was 0.843 and all the 10 loci presented high polymorphic(PIC>0.5). The average heterozygosity of the three Exopalaemon carinicauda populations in Jiangsu, Zhejiang and Fujian province were 0.827,0.845,0.805; the average PIC were 0.792,0.815,0.771. The results indicated that the genetic diversity of the three populations was relatively abundance, and the genetic variability was high. The highest genetic similarity index(0.6594) and the lowest genetic distance(0.4164) were found between Zhejiang population and Fujian population, which indicated a closer phylogenetic relationship between these two populations. On the other hand, the lowest genetic similarity index(0.6374) and the highest genetic distance(0.4504) existed between Jiangsu population and Fujian population, which suggested a farthest phylogenetic relationship between these two populations. Phylogenetic relationship of the three populations was analyzed by using UPGMA methods based on their genetic distances and the results showed that Zhejiang population and Fujian population were clustered in the first group, then the Jiangsu population was in the second group.From the results we can predict that in order to get the maximum heterosis, we can choose the two populations of Exopalaemon carinicauda from Jiangsu and Fujian for hybrid breeding.
利用磁珠富集法开发了脊尾白虾微卫星标记并对其特征进行了分析。首先将脊尾白虾基因组DNA经MboI酶切,连上接头,构建基因组PCR文库,然后用生物素标记的简单重复序列(CA)16作探针与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,经一系列的洗涤过程,去除磁珠表面不含有微卫星的片段,最后将吸附在磁珠上的片段洗脱,PCR扩增,再进行克隆和测序,即可得到微卫星序列。本研究选取61个阳性克隆,经测序,共获得32个微卫星序列,其中：完全型微卫星27个(84.38%),复合型微卫星1个(3.13%),非完全型微卫星4个(12.5%)；所得微卫星中以CA/GT为重复单位的序列30个,占93.75%,重复次数在20次以上的微卫星27个,占84.38%,最高重复次数为39次。用引物设计软件Oligo6.0设计引物23对。
利用微卫星标记研究了脊尾白虾群体的遗传多样性。用所筛选到的10对微卫星引物对江苏连云港、浙江三门和福建连江3个脊尾白虾群体的132尾个体基因组DNA进行PCR扩增,分析其群体遗传多样性。根据3个群体在10个微卫星位点上的PCR扩增图谱,计算出各群体的遗传多样性参数。结果显示：10个微卫星位点分别扩增到等位基因数为6-12,共计89个,平均等位基因数为8.90,平均有效等位基因数为7.43,平均遗传杂合度为0.863,平均多态信息含量为0.843,所有微卫星位点均呈高度多态性(PIC>0.5)。江苏连云港、浙江三门和福建连江3个群体的平均遗传杂合度分别为0.827、0.845、0.805；平均多态信息含量分别为0.792、0.815、0.771,说明这3个群体遗传多样性水平较高。3个群体中,浙江三门群体与福建连江群体间的遗传相似系数最高(0.6594),遗传距离最小(0.4164),说明这2个群体亲缘关系最近；江苏连云港群体与福建连江群体间的遗传相似性系数最低(0.6374),遗传距离最大(0.4504),可推断这2个群体亲缘关系最远。根据群体间的遗传距离采用UPMGA法进行聚类分析,结果显示：浙江三门群体与福建连江群体首先聚为一支,然后再与江苏连云港群体聚为一支。聚类分析结果说明,江苏连云港群体与其他2个群体亲缘关系最远,浙江三门群体与福建连江群体亲缘关系最近。由此可预测,为获得最大的杂种优势,可选用江苏连云港和福建连江两地的脊尾白虾进行杂交育种。
The transferability of Penaeus chinensis SSR markers to Exopalaemon carinicauda was explored. The conservativeness of the side sequences of the microsatellite in species of close genetic relationship has already been discovered.11 polymohic microsatellite primers identified in Penaeus chinensis were employed to amplify in the genome of Exopalaemon carinicauda. The conditions of polymerase chain reaction(PCR) were optimized for the fidelity of DNA synthesis during PCR amplification. It was found that 9 loci failed to be amplified and 2 loci (RSI 101 and 72A64) amplified specific products successfully. The two loci were polymorphism.
The SSR markers were developed and characterization in Exopalaemon carinicauda. Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA. In this experiment, we isolated microsatellite from Exopalaemon carinicauda genome with this method. Exopalaemon carinicauda genomic DNA was extracted and digested with restriction enzyme MboI. Fragments were ligated with artificial linkers(20bp). They were used to create a "whole genome PCR library". This genomic DNA was hybridized with a biotin-labeled microsatellite probe (CA)16. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. The selected DNAs are then amplified using primers designed complementary to the linkers, cloned into the pGEM-T vector and sequenced. In this experiment, we selected 61 positive clones. From these positive clones, we isolated 32 microsatellite DNA sequences and all most (about 93.75%) of them were with corn repeats CA/GT, among which 84.38% microsatellites were perfect repeats,12.5% imperfect repeats and 3.13% compound repeats; 84.38% microsatellites repeated more than 20 times and the biggest repead number was 39. This allowed us to designed 23 pairs of primer with the software Oligo 6.0.
Genetic diversity among three populations of Exopalaemon carinicauda distributing in Jiangsu, Zhejiang and Fujian province were studied by using microsatellite analysis technology.10 pairs of microsatellite primers of Exopalaemon carinicauda which were selected and proved to amplify successfully on Exopalaemon carinicauda. Amplified PCR products were imagined by Biolight. Average observed heterozygosity and average expected heterozygosity were calculated by Popgene3.2 software. The results showed that 6 to 12 alleles per pair in the three populations were amplified. The total number of alleles is 89 and the average number of the alleles per pair was 8.90; the average number of the effective alleles per pair was 7.43; the average heterozygosity was 0.863; the average polymorphism information contents(PIC) was 0.843 and all the 10 loci presented high polymorphic(PIC>0.5). The average heterozygosity of the three Exopalaemon carinicauda populations in Jiangsu, Zhejiang and Fujian province were 0.827,0.845,0.805; the average PIC were 0.792,0.815,0.771. The results indicated that the genetic diversity of the three populations was relatively abundance, and the genetic variability was high. The highest genetic similarity index(0.6594) and the lowest genetic distance(0.4164) were found between Zhejiang population and Fujian population, which indicated a closer phylogenetic relationship between these two populations. On the other hand, the lowest genetic similarity index(0.6374) and the highest genetic distance(0.4504) existed between Jiangsu population and Fujian population, which suggested a farthest phylogenetic relationship between these two populations. Phylogenetic relationship of the three populations was analyzed by using UPGMA methods based on their genetic distances and the results showed that Zhejiang population and Fujian population were clustered in the first group, then the Jiangsu population was in the second group.From the results we can predict that in order to get the maximum heterosis, we can choose the two populations of Exopalaemon carinicauda from Jiangsu and Fujian for hybrid breeding.
引文
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