珍稀植物太行花的遗传多样性研究
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摘要
太行花(Taihanggia rupestris Yu et Li)是1974年发现,1980年经俞德浚和李朝銮鉴定的蔷薇科单种属植物,为中国特有。太行花是蔷薇科草本仙女木族最原始二倍体植物,染色体2n = l4,属古老的残遗种。太行花花的结构由两性花向单性花演变,这在仙女木族进化上占有特殊的位置,对阐明蔷薇科一些类群的起源和演化问题具有重要意义。但由于其分布区狭小,生境独特,且植株稀少,随着植被的破坏而导致生境的改变,分布范围日益缩减,濒临灭绝;被列为我国二级保护植物。
一般而言,只有在弄清物种的遗传结构和遗传多样性现状的前提下,才能够制定出切实可行的保护策略,否则,任何物种保护措施的实施,都不会取得实质性的成效。分子标记能直接从DNA水平反映物种水平上的遗传差异,是研究植物遗传多样性的有效工具。ISSR标记具有简单、快速、无种属特异性、节约成本的优点,被广泛应用于遗传多样性的研究。本试验应用ISSR标记对10个居群220个太行花个体的遗传多样性进行了研究。主要结果如下:
(1)筛选并优化了太行花DNA的提取方法。通过比较常规CTAB法、改良CTAB法和SDS法对太行花叶片总DNA的提取效果,发现常规CTAB法提取的DNA难以完全溶解,且有褐化现象;SDS法提取的DNA产率及纯度都很低;改良CTAB法提取的DNA产率高且稳定,无明显降解,杂质少,OD260/OD280值在1.8左右,并以改良CTAB法提取的DNA为模板,应用线粒体通用引物扩增出了特异性的高效产物。
(2)从测试的50条ISSR随机引物中选出了扩增效果稳定、条带丰富的12条引物进行220个样品的ISSR检测。12条引物共扩增出184条清晰可识别的条带,每条引物扩增出的条带数在11～19之间,平均为15.33;各条带分子量在130bp～2000bp之间。在扩增出的184条带中,共有148条带为多态条带,物种水平上多态位点比率为80.43%,居群上平均多态条带的百分比(PPB)为47.01%;物种和居群水平的平均Nei遗传多样性(h)分别为0.2479和0.1865,Shannon信息指数(I)分别为0.3785和0.2726;物种水平上等位基因数目为1.8043,每个位点平均有效等位基因数为1.4126。
(3)10个居群间的Nei遗传一致性的平均值为0.9223,其中QPG与XXT遗传一致性最高,达到0.9728,DPB和SSM遗传一致性最低,为0.8706;居群间的遗传距离在0.034～0.144之间,其中QPG与XXT居群遗传距离最小,DPB和SSM居群间的遗传距离最大。
(4)基于Nei遗传距离,利用UPGMA聚类法对10个太行花居群进行聚类分析,10个居群聚为两个大组。其中南部居群(DPB、YDS、LHP、XXT、QPG)聚为一组,北部5个居群,即HHS、JG、SSM、NY、DLG,聚为另一组。主成分分析(PCA)显示,南部居群主要集中在坐标轴的右下方,而北部群体主要集中在坐标轴的左上方,主成分分析支持UPGMA聚类的结果。
(5)利用GenAlEx6.1软件进行mantel检验,结果显示居群间的遗传距离和地理距离之间有显著的相关性(r = 0.611, P<0.001),说明地理因素对太行花的群体遗传结构有较大的影响。(6)利用POPGENE 1.32分析软件,计算不同居群之间的遗传分化水平。结果表明,10个居群总的基因多样性Ht为0.2465,居群内的基因多样性Hs为0.1865,居群间的遗传分化系数Gst=0.2435,居群间的基因流Nm=1.5537。同时,AMOVA分析显示73.51%的总基因变异发生在居群内,26.49%的变异发生在居群之间,组间的变异为9.24%,AMOVA分析验证了Nei基因多样性对群体结构的分析结果。
Taihangia Rupestris Yu et Li was firstly found in 1974, and then formally described in 1980 as the only species of the Taihanggia belonging to the family Rosaceae. T. Rupestris is an perennial herb endemic to China. T. Rupestris is the most primitive diploid species (2n = l4) in the tribe Dryadeae belong to the family Rosaceae according to its morphology, anatomy of carpel, chromosome counting, microscopic and submicroscopic structure of pollen, so it is an ancient relic species. T. Rupestris, with the characterization of evolvement from bisexual flower to unisexual flower, is at an important systematic position in the tribe Dryadeae, and study on it could throw a new light on evolution of the tribe Dryadeae. However, due to its small distribution area, unique habitats and rare plants, the destruction range of Taihangia Rupestris becomes smaller. It is endangered specie and listed as protected plants in China.
In general, we must know the genetic structure and genetic diversity of species before we make the conservation strategy. If not, any protection measures can not achieve the expected results. Molecular markers are useful tools in the Genetic Diversity study. Inter-simple sequence repeat (ISSR) is a molecular marker targeting simple sequence repeats (SSR), by which the length variation of DNA fragment between adjacent microsatellites is detected in the entire genome. Freedom from the necessity of obtaining flanking sequence information, high level of polymorphism and simple technology, ISSR has been widely used for Genetic Diversity studies. Inter-simple sequence repeat (ISSR) markers were employed to estimate the genetic diversity and genetic differentiation of ten populations. The main results are as follows:
(1)We filtered and optimized the DNA extraction method of Taihangia Rupestris. The total DNA was isolated from the leaf of Taihanggia rupestris YU et LI by the conventional CTAB method,improved CTAB method and SDS method. The results showed the extracts by conventional CTAB method were brown and could not dissolve completely, and those by SDS method had a low quality and yield. As for the improved CTAB method, there was a high production rate of the extracts,that was pure with little degradation,and A260/ A280 was 1.8 or so. With these leaf DNA serving as template, PCR at two loci of mitochondrial genomes was effective and special highly.
(2) We selected 12 ISSR primers from 50 ISSR primers for the Amplification of 220 samples. Amplification of the ISSR fragments in 220 individuals sampled with 12 primers generated 184 clearly identifiable bands, with a mean of 15.33 bands per primer. In all, there were 148 polymorphic bands, the percentage of polymorphic band (PPB) was 47.01 % on average and 80.43% at the species level. The Nei’s gene diversity (h) was estimated to be 0.1865 on average at the population level, 0.2479 at the species level, while Shannon indices (I) were 0.2726 and 0.3785 respectively. The number of alleles was estimated to be 1.8043 at the species level and the average effective number of alleles per locus was estimated to be 1.4126.
(3)Pairwise Nei’s unbiased measures of genetic identity of the ten populations was high as 0.9233 on average, QPG and XXT are the closest with 0.9728, whereas DPB and SSM are the most distant populations with a identity coefficient of 0.8706. Genetic distance was detected to be 0.034～0.144 between populations, QPG and XXT are the Least, whereas DPB and SSM are the largest.
(4)The UPGMA analysis clustered 10 populations into two groups. GroupⅠcontained populations DPB, YDS, LHP, QPG and XXT. GroupⅡincluded populations HHS、JG、SSM、NY、DLG. Principal coordinate analysis (PCA) shows: Samples from the southern population were founded in the lower and right side of the diagram, samples from the northern populations in the upper and left side, Principal component analysis supports the results of UPGMA clustering.
(5)Mantel test was performed using GenAlEx 6.1.The mantel test showed that there was significant positve correlation between geographical distance and genetic distance (r = 0.611, P<0.001), indicating the role of geographic isolation in shaping the present population genetic structure of Taihanggia Rupestris.
(6)Using POPGENE 1.32 to estimate the level of genetic differentiation among the different populations. The result shows that the total genetic diversity of ten populations(Ht) was estimated to be 0.2465 and genetic diversity within populations(Hs) was detected to be 0.1865. Genetic differentiation coefficient between Population(Gst) was 0.2435 and gene flow(Nm) among populations was estimated to be 1.5537.Analysis of molecular variance revealed that 73.51% of the total genetic variability occurred within populations, 26.49 % was found among populations, in which only 9.24% resided between regions, indicating a low level of among–population genetic differentiation. The results of the population structure analysis by AMOVA and Nei genetic diversity is the same.
一般而言,只有在弄清物种的遗传结构和遗传多样性现状的前提下,才能够制定出切实可行的保护策略,否则,任何物种保护措施的实施,都不会取得实质性的成效。分子标记能直接从DNA水平反映物种水平上的遗传差异,是研究植物遗传多样性的有效工具。ISSR标记具有简单、快速、无种属特异性、节约成本的优点,被广泛应用于遗传多样性的研究。本试验应用ISSR标记对10个居群220个太行花个体的遗传多样性进行了研究。主要结果如下:
(1)筛选并优化了太行花DNA的提取方法。通过比较常规CTAB法、改良CTAB法和SDS法对太行花叶片总DNA的提取效果,发现常规CTAB法提取的DNA难以完全溶解,且有褐化现象;SDS法提取的DNA产率及纯度都很低;改良CTAB法提取的DNA产率高且稳定,无明显降解,杂质少,OD260/OD280值在1.8左右,并以改良CTAB法提取的DNA为模板,应用线粒体通用引物扩增出了特异性的高效产物。
(2)从测试的50条ISSR随机引物中选出了扩增效果稳定、条带丰富的12条引物进行220个样品的ISSR检测。12条引物共扩增出184条清晰可识别的条带,每条引物扩增出的条带数在11～19之间,平均为15.33;各条带分子量在130bp～2000bp之间。在扩增出的184条带中,共有148条带为多态条带,物种水平上多态位点比率为80.43%,居群上平均多态条带的百分比(PPB)为47.01%;物种和居群水平的平均Nei遗传多样性(h)分别为0.2479和0.1865,Shannon信息指数(I)分别为0.3785和0.2726;物种水平上等位基因数目为1.8043,每个位点平均有效等位基因数为1.4126。
(3)10个居群间的Nei遗传一致性的平均值为0.9223,其中QPG与XXT遗传一致性最高,达到0.9728,DPB和SSM遗传一致性最低,为0.8706;居群间的遗传距离在0.034～0.144之间,其中QPG与XXT居群遗传距离最小,DPB和SSM居群间的遗传距离最大。
(4)基于Nei遗传距离,利用UPGMA聚类法对10个太行花居群进行聚类分析,10个居群聚为两个大组。其中南部居群(DPB、YDS、LHP、XXT、QPG)聚为一组,北部5个居群,即HHS、JG、SSM、NY、DLG,聚为另一组。主成分分析(PCA)显示,南部居群主要集中在坐标轴的右下方,而北部群体主要集中在坐标轴的左上方,主成分分析支持UPGMA聚类的结果。
(5)利用GenAlEx6.1软件进行mantel检验,结果显示居群间的遗传距离和地理距离之间有显著的相关性(r = 0.611, P<0.001),说明地理因素对太行花的群体遗传结构有较大的影响。(6)利用POPGENE 1.32分析软件,计算不同居群之间的遗传分化水平。结果表明,10个居群总的基因多样性Ht为0.2465,居群内的基因多样性Hs为0.1865,居群间的遗传分化系数Gst=0.2435,居群间的基因流Nm=1.5537。同时,AMOVA分析显示73.51%的总基因变异发生在居群内,26.49%的变异发生在居群之间,组间的变异为9.24%,AMOVA分析验证了Nei基因多样性对群体结构的分析结果。
Taihangia Rupestris Yu et Li was firstly found in 1974, and then formally described in 1980 as the only species of the Taihanggia belonging to the family Rosaceae. T. Rupestris is an perennial herb endemic to China. T. Rupestris is the most primitive diploid species (2n = l4) in the tribe Dryadeae belong to the family Rosaceae according to its morphology, anatomy of carpel, chromosome counting, microscopic and submicroscopic structure of pollen, so it is an ancient relic species. T. Rupestris, with the characterization of evolvement from bisexual flower to unisexual flower, is at an important systematic position in the tribe Dryadeae, and study on it could throw a new light on evolution of the tribe Dryadeae. However, due to its small distribution area, unique habitats and rare plants, the destruction range of Taihangia Rupestris becomes smaller. It is endangered specie and listed as protected plants in China.
In general, we must know the genetic structure and genetic diversity of species before we make the conservation strategy. If not, any protection measures can not achieve the expected results. Molecular markers are useful tools in the Genetic Diversity study. Inter-simple sequence repeat (ISSR) is a molecular marker targeting simple sequence repeats (SSR), by which the length variation of DNA fragment between adjacent microsatellites is detected in the entire genome. Freedom from the necessity of obtaining flanking sequence information, high level of polymorphism and simple technology, ISSR has been widely used for Genetic Diversity studies. Inter-simple sequence repeat (ISSR) markers were employed to estimate the genetic diversity and genetic differentiation of ten populations. The main results are as follows:
(1)We filtered and optimized the DNA extraction method of Taihangia Rupestris. The total DNA was isolated from the leaf of Taihanggia rupestris YU et LI by the conventional CTAB method,improved CTAB method and SDS method. The results showed the extracts by conventional CTAB method were brown and could not dissolve completely, and those by SDS method had a low quality and yield. As for the improved CTAB method, there was a high production rate of the extracts,that was pure with little degradation,and A260/ A280 was 1.8 or so. With these leaf DNA serving as template, PCR at two loci of mitochondrial genomes was effective and special highly.
(2) We selected 12 ISSR primers from 50 ISSR primers for the Amplification of 220 samples. Amplification of the ISSR fragments in 220 individuals sampled with 12 primers generated 184 clearly identifiable bands, with a mean of 15.33 bands per primer. In all, there were 148 polymorphic bands, the percentage of polymorphic band (PPB) was 47.01 % on average and 80.43% at the species level. The Nei’s gene diversity (h) was estimated to be 0.1865 on average at the population level, 0.2479 at the species level, while Shannon indices (I) were 0.2726 and 0.3785 respectively. The number of alleles was estimated to be 1.8043 at the species level and the average effective number of alleles per locus was estimated to be 1.4126.
(3)Pairwise Nei’s unbiased measures of genetic identity of the ten populations was high as 0.9233 on average, QPG and XXT are the closest with 0.9728, whereas DPB and SSM are the most distant populations with a identity coefficient of 0.8706. Genetic distance was detected to be 0.034～0.144 between populations, QPG and XXT are the Least, whereas DPB and SSM are the largest.
(4)The UPGMA analysis clustered 10 populations into two groups. GroupⅠcontained populations DPB, YDS, LHP, QPG and XXT. GroupⅡincluded populations HHS、JG、SSM、NY、DLG. Principal coordinate analysis (PCA) shows: Samples from the southern population were founded in the lower and right side of the diagram, samples from the northern populations in the upper and left side, Principal component analysis supports the results of UPGMA clustering.
(5)Mantel test was performed using GenAlEx 6.1.The mantel test showed that there was significant positve correlation between geographical distance and genetic distance (r = 0.611, P<0.001), indicating the role of geographic isolation in shaping the present population genetic structure of Taihanggia Rupestris.
(6)Using POPGENE 1.32 to estimate the level of genetic differentiation among the different populations. The result shows that the total genetic diversity of ten populations(Ht) was estimated to be 0.2465 and genetic diversity within populations(Hs) was detected to be 0.1865. Genetic differentiation coefficient between Population(Gst) was 0.2435 and gene flow(Nm) among populations was estimated to be 1.5537.Analysis of molecular variance revealed that 73.51% of the total genetic variability occurred within populations, 26.49 % was found among populations, in which only 9.24% resided between regions, indicating a low level of among–population genetic differentiation. The results of the population structure analysis by AMOVA and Nei genetic diversity is the same.
引文
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