黄河三角洲柽柳植物的居群结构和遗传分化
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摘要
作为黄河三角洲关键建群物种之一,柽柳(T. chinensis)在维持当地生态系统平衡中起着重要作用。为了解柽柳居群遗传结构和进化动力学,本研究采用ISSR分子标记技术,通过比较分析黄河三角洲分布在不同盐碱生境下的五个柽柳居群的遗传多态,从柽柳居群的遗传多样性水平、居群内和居群间的遗传分化指数以及土壤盐成分对其居群遗传结构形成和进化的影响等方面进行了深入研究,我们的研究为黄河三角洲柽柳自然居群的种质保护和永续利用提供了必要的理论依据。主要结果如下：
一、首先建立了适合于柽柳(T. chinensis)的ISSR-PCR反应体系和扩增程序。25μL的优化体系中包括：10×PCR Buffer 2.5μL、Mg2+2 mM、dNTP 0.2 mM、引物0.4pM、模板DNA10ng、TaqDNA聚合酶1U。优化的PCR扩增程序为：94℃预变性10min; 94℃变性1min,49-51℃退火1min,延伸2min,39个循环；72℃终延伸10min。
二、从100条ISSR引物中筛选出10条扩增条带清晰、多态性高、重复性好的引物。
三、掌握了黄河三角洲柽柳居群遗传多样性水平和居群结构发展现状
利用10个ISSR引物对5个柽柳居群共140个个体的扩增,共得到78条带,其中多态性条带为62条,多态位点比率(P)为79.5%,等位基因平均数(4)为1.775±0.420,有效等位基因平均数(AE)为1.398±0.347,Nei遗传多样性指数(H)和Shannon多样性指数(I)分别为0.239±0.187和0.363±0.264。这些结果表明,柽柳居群在物种水平保持中等水平的遗传多样性。而在居群水平上其遗传多样性相对较低,各居群多态位点比率(P)、等位基因平均数(A)、有效等位基因平均数(AE)、Nei遗传多样性指数(H)和Shannon多样性指数(I)的平均值分别为56.3%、1.563±0.497、1.322±0.367、0.190±0.198和0.285±0.283,其中BHZX和YDD居群的遗传多样性相对较高,HHK居群的遗传多样性最低。
此外,研究发现黄河三角洲柽柳居群的遗传变异主要发生在居群内部,偏低的居群间遗传分化指数(Gst=0.159)表明居群间的遗传变异仅占柽柳总遗传变异的15.9%,而居群内的遗传多样性则占柽柳全部遗传多态的84.1%。AMOVA分析进一步验证了上述结论,同样显示大多数(83.1%)的遗传分化存在于居群内。而居群间高达2.637的基因流(Nm)则说明来自柽柳居群间频繁的基因交流可能是导致居群间分化程度降低的一个原因。
通过对五个柽柳居群的UPGMA聚类分析,发现可以将其分为三支,其中HHKW和YQE聚为一支、BHZX和YDD聚成一支,而HHK单独聚成第三支。这种聚类方式基本上按所在居群的土壤盐浓度排列,而和五个居群的地理分布格局没有关联性。进一步的相关性分析和Mental检验表明目前黄河三角洲柽柳居群间的遗传距离与地理距离之间没有相关性(r=-0.127,p>0.05),但居群遗传多样性和土壤含盐量却呈现显著的负相关(r=-0.958,p=0.005),这个结果说明土壤盐浓度在黄河三角洲柽柳居群遗传结构形成和进化中扮演着重要角色。
As one of leading constructive species in Yellow River Delta, Tamarix chinensis plays an important role on maintaining wetland ecological balance. To better understand the population genetic structure and evolutionary dynamic of local T. chinensis, five populations of T. chinensis in Yellow River Delta distributing in different habitats with variable soil salinity were investigated using inter-simple sequence repeat (ISSR) markers. The genetic diversity, the genetic differentiation indexes within and among populations, and the correlation between the population genetic structure and soil salinity were further compared and analyzed. The main results are as follows.
1. Optimization of ISSR-PCR reaction system and amplified procedure
We optimized the reaction system and amplified procedure for T. chinensis, which was 25μL amplification reactions system containing 10×PCR Buffer 2.5μL,2 mM Mg2+, dNTP 0.2 mM, 0.4 pM primers,10 ng template DNA,1 unit of Taq DNA polymerase. The PCR amplified procedures was as follows:after a pre-denaturing of 5 min at 94℃, then 39 cycles of 1 min at 94℃for denaturation,1 min at a primer-appropriate temperature for annealing,2 min at 72℃for extension, and a final step of 10 min elongation at 72℃.
2. Ten effective ISSR-PCR primers were selected from 100 ISSR primers.
3. Obtainment of the information about the genetic diversities and population structure of T. chinensis in the Yellow River Delta
Using Inter Simple Sequence Repeat (ISSR) markers, five populations of T. chinensis, which consisted of 140 individuals, were analyzed in this study. Seventy-eight polymerase chain reaction fragments were scored, out of which 62 were polymorphic. The mean percentage of polymorphic loci (P) was79.5%, the number of alleles per locus (A) was 1.775±0.420, the effective number of alleles per locus (AE) was 1.398±0.347, and the mean Nei's gene diversity (h) and the mean Shannon's information index (I)were 0.239±0.187and 0.363±0.264, respectively. These indexes indicated that a moderate level of genetic diversities existed in T. chinensis populations of the Yellow River Delta. At the population level, however, a lower level of genetic diversity (P=56.3%;A=1.563±0.497;AE=1.322±0.367;h=0.190±0.198; I=0.285±0.283) was revealed. Among the five populations, the highest genetic diversities were found in both BHZX and YDD populations, and the lowest genetic diversity was obtained in HHK population.
The genetic differentiation coefficient Gst in present study was 0.159, which demonstrated that 15.9% of genetic variability existed among the T. chinensis populations. The estimated average number of migrants among populations (Nm) deduced by Gst was 2.637, implying a high level of gene flow exchanged among populations, which might be one reason for the less genetic differentiation among populations. Analysis of molecular variance (AMOVA) also revealed that most of the genetic variance (83.1%) of T. chinensis occurred within populations.
Unweighted pair group method with arithmetic mean (UPGMA) and showed that the populations with similar soil salinity had a close relationship, rather than the populations with a closer geographical distance. The mantel test showed that there was no correlation between genetic distance and geographical distance (r=-0.127, p>0.05), but A significant negative correlation between genetic diversity and soil salinity of 5 populations (r=-0.958, p=0.005) was displayed. This result showed that soil salinity played an important role in shaping the population genetic structure of T. chinensis in the Yellow River Delta.
一、首先建立了适合于柽柳(T. chinensis)的ISSR-PCR反应体系和扩增程序。25μL的优化体系中包括：10×PCR Buffer 2.5μL、Mg2+2 mM、dNTP 0.2 mM、引物0.4pM、模板DNA10ng、TaqDNA聚合酶1U。优化的PCR扩增程序为：94℃预变性10min; 94℃变性1min,49-51℃退火1min,延伸2min,39个循环；72℃终延伸10min。
二、从100条ISSR引物中筛选出10条扩增条带清晰、多态性高、重复性好的引物。
三、掌握了黄河三角洲柽柳居群遗传多样性水平和居群结构发展现状
利用10个ISSR引物对5个柽柳居群共140个个体的扩增,共得到78条带,其中多态性条带为62条,多态位点比率(P)为79.5%,等位基因平均数(4)为1.775±0.420,有效等位基因平均数(AE)为1.398±0.347,Nei遗传多样性指数(H)和Shannon多样性指数(I)分别为0.239±0.187和0.363±0.264。这些结果表明,柽柳居群在物种水平保持中等水平的遗传多样性。而在居群水平上其遗传多样性相对较低,各居群多态位点比率(P)、等位基因平均数(A)、有效等位基因平均数(AE)、Nei遗传多样性指数(H)和Shannon多样性指数(I)的平均值分别为56.3%、1.563±0.497、1.322±0.367、0.190±0.198和0.285±0.283,其中BHZX和YDD居群的遗传多样性相对较高,HHK居群的遗传多样性最低。
此外,研究发现黄河三角洲柽柳居群的遗传变异主要发生在居群内部,偏低的居群间遗传分化指数(Gst=0.159)表明居群间的遗传变异仅占柽柳总遗传变异的15.9%,而居群内的遗传多样性则占柽柳全部遗传多态的84.1%。AMOVA分析进一步验证了上述结论,同样显示大多数(83.1%)的遗传分化存在于居群内。而居群间高达2.637的基因流(Nm)则说明来自柽柳居群间频繁的基因交流可能是导致居群间分化程度降低的一个原因。
通过对五个柽柳居群的UPGMA聚类分析,发现可以将其分为三支,其中HHKW和YQE聚为一支、BHZX和YDD聚成一支,而HHK单独聚成第三支。这种聚类方式基本上按所在居群的土壤盐浓度排列,而和五个居群的地理分布格局没有关联性。进一步的相关性分析和Mental检验表明目前黄河三角洲柽柳居群间的遗传距离与地理距离之间没有相关性(r=-0.127,p>0.05),但居群遗传多样性和土壤含盐量却呈现显著的负相关(r=-0.958,p=0.005),这个结果说明土壤盐浓度在黄河三角洲柽柳居群遗传结构形成和进化中扮演着重要角色。
As one of leading constructive species in Yellow River Delta, Tamarix chinensis plays an important role on maintaining wetland ecological balance. To better understand the population genetic structure and evolutionary dynamic of local T. chinensis, five populations of T. chinensis in Yellow River Delta distributing in different habitats with variable soil salinity were investigated using inter-simple sequence repeat (ISSR) markers. The genetic diversity, the genetic differentiation indexes within and among populations, and the correlation between the population genetic structure and soil salinity were further compared and analyzed. The main results are as follows.
1. Optimization of ISSR-PCR reaction system and amplified procedure
We optimized the reaction system and amplified procedure for T. chinensis, which was 25μL amplification reactions system containing 10×PCR Buffer 2.5μL,2 mM Mg2+, dNTP 0.2 mM, 0.4 pM primers,10 ng template DNA,1 unit of Taq DNA polymerase. The PCR amplified procedures was as follows:after a pre-denaturing of 5 min at 94℃, then 39 cycles of 1 min at 94℃for denaturation,1 min at a primer-appropriate temperature for annealing,2 min at 72℃for extension, and a final step of 10 min elongation at 72℃.
2. Ten effective ISSR-PCR primers were selected from 100 ISSR primers.
3. Obtainment of the information about the genetic diversities and population structure of T. chinensis in the Yellow River Delta
Using Inter Simple Sequence Repeat (ISSR) markers, five populations of T. chinensis, which consisted of 140 individuals, were analyzed in this study. Seventy-eight polymerase chain reaction fragments were scored, out of which 62 were polymorphic. The mean percentage of polymorphic loci (P) was79.5%, the number of alleles per locus (A) was 1.775±0.420, the effective number of alleles per locus (AE) was 1.398±0.347, and the mean Nei's gene diversity (h) and the mean Shannon's information index (I)were 0.239±0.187and 0.363±0.264, respectively. These indexes indicated that a moderate level of genetic diversities existed in T. chinensis populations of the Yellow River Delta. At the population level, however, a lower level of genetic diversity (P=56.3%;A=1.563±0.497;AE=1.322±0.367;h=0.190±0.198; I=0.285±0.283) was revealed. Among the five populations, the highest genetic diversities were found in both BHZX and YDD populations, and the lowest genetic diversity was obtained in HHK population.
The genetic differentiation coefficient Gst in present study was 0.159, which demonstrated that 15.9% of genetic variability existed among the T. chinensis populations. The estimated average number of migrants among populations (Nm) deduced by Gst was 2.637, implying a high level of gene flow exchanged among populations, which might be one reason for the less genetic differentiation among populations. Analysis of molecular variance (AMOVA) also revealed that most of the genetic variance (83.1%) of T. chinensis occurred within populations.
Unweighted pair group method with arithmetic mean (UPGMA) and showed that the populations with similar soil salinity had a close relationship, rather than the populations with a closer geographical distance. The mantel test showed that there was no correlation between genetic distance and geographical distance (r=-0.127, p>0.05), but A significant negative correlation between genetic diversity and soil salinity of 5 populations (r=-0.958, p=0.005) was displayed. This result showed that soil salinity played an important role in shaping the population genetic structure of T. chinensis in the Yellow River Delta.
引文
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