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基于SSR标记的白栎天然居群遗传多样性分析
Genetic diversity analysis among natural populations of Quercus fabri based on SSR markers
【目的】利用SSR分子标记,对白栎天然居群进行遗传多样性分析,为其种质资源的合理开发与保护提供指导。【方法】采用筛选出的SSR引物对3个群体进行遗传多样性与遗传结构研究,并计算遗传参数;基于个体间的遗传距离进行主成分分析,运用GenAlEx 6.5进行遗传距离与地理距离的相关性Mantel检验,软件Arlequin 用于分子方差分析,聚类分析采用MEGA 7中的UPGMA法;同时,使用Structure软件中的贝叶斯聚类算法探究群体遗传结构。【结果】6对SSR引物共检测到75个等位基因,平均有效等位基因数、观察杂合度和期望杂合度分别为7.16、0.70和 0.80,Shannon信息指数为2.05。AMOVA表明,群体内部的分子变异率高达97.41%,而群体之间的遗传变异仅为2.59%。黄山(YM)与天目山(TM)群体间的基因流为12.908,显著高于其他群体之间的交流水平,而二者间有着最近的遗传距离为0.272。贝叶斯法与UPGMA法聚类结果一致,表明来自YM与TM的个体亲缘关系更近。【结论】白栎具有较高的遗传多样性水平,且不同居群间的水平相当。YM和TM居群的基因交流最为密切,遗传分化不明显。白栎绝大部分遗传变异来源于群体内部,对于该物种的保护应减少不合理的人为开发行为,以原地保护为主,同时开展种质资源的收集与遗传改良工作。
【Objective】 This study used simple-sequence repeat (SSR) markers to analyze the genetic diversity on Quercus fabri Hance from natural populations. The study provides important advice concerning the reasonable exploitation and utilization of this resource. 【Method】 The analysis of the genetic diversity and genetic structure of Q. fabri was performed using screened SSR molecular markers. The genetic parameters were calculated and the principal coordinate analysis was applied based on the genetic distance among individuals. The analysis of molecular variance was conducted using Arlequin software. GenAlEx 6.5 was used to test the correlation between the genetic distance and geographic distance. MEGA 7 was used for the UPGMA phylogenetic analysis, and Structure was employed to perform the Bayesian clustering analysis to explore the genetic structure. 【Result】 A total of 75 alleles were detected from six markers. The average number of effective alleles, observed heterozygosity, and expected heterozygosity were 7.16, 0.70 and 0.80, respectively. The Shannon’s information index was 2.05. AMOVA demonstrated that the genetic variation within populations (97.41%) accounted for the main percentage, with a negligible variation among populations (2.59%). The gene flow between Huangshan Mountain (YM) and Tianmushan Mountain (TM) was 12.908, which was much higher than that among other populations, and the genetic distance between the two populations was the closest (0.272). The results of the Bayesian clustering analysis were in accordance with the result of the UPGMA phylogenetic analysis, in that samples from the YM and TM populations had closer phylogenetic relationships. 【Conclusion】 There was a high and coincident level of genetic diversity among natural populations of Q. fabri. The genetic communication between YM and TM was the most frequent, and the genetic variation between these two spots was minor. For Q. fabri, the heritable variation within populations accounted for the majority of genetic variation. When collecting germplasm resources for the genetic improvement of this species, we should avoid unreasonable artificial exploitation. In addition, insitu conservation should be taken into consideration.
Quercus fabri Hance / simple-sequence repeats (SSR) / genetic diversity / genetic structure
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熊仕发, 吴立文, 陈益存, 等. 不同种源白栎果实形态特征和营养成分含量变异分析[J]. 林业科学研究, 2020,33(2):93-102.
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The nonrecombinant, uniparentally inherited nature of organelle genomes makes them useful tools for evolutionary studies. However, in plants, detecting useful polymorphism at the population level is often difficult because of the low level of substitutions in the chloroplast genome, and because of the slow substitution rates and intramolecular recombination of mtDNA. Chloroplast microsatellites represent potentially useful markers to circumvent this problem and, to date, studies have demonstrated high levels of intraspecific variability. Here, we discuss the use of these markers in ecological and evolutionary studies of plants, as well as highlighting some of the potential problems associated with such use.
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Quercus acutissima is native to eastern Asia. It has a wide distribution in China and China is an important component in understanding the ecology and genetic structure of this species. Q. acutissima attained high economic value for hardwood product and can be managed as an energy tree species. To investigate the genetic variation of Q. acutissima provenances, 12 microsatellite primer pairs were used to analyze 672 trees sampled from 28 provenances of Q. acutissima in China. All of the tested microsatellite loci proved to be effective for the studied Q. acutissima provenances. The results revealed that allele numbers varied from 5 to 13 per locus, with an average of 8 alleles per locus. The mean observed heterozygosity and expected heterozygosity were 0.4927 and 0.7023, respectively. The relatedness of the provenances was studied using the arithmetic mean algorithm based on Nei's genetic distance and principal coordinates analysis. Interestingly, both approaches revealed two main groups: one consisted of the eastern Chinese provenances, and the other comprised of the western Chinese provenances. An analysis of molecular variance indicated that most genetic variation was contained within populations (84 %). The two microsatellite markers developed in this study may be employed for genetic characterization of other oak species. Considering the management or breeding programs of Q. acutissima provenances in China, we should treat each main group as a single gene resource.
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The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) n and (GA/CT) n repeats. The proportion of positive clones yielded estimates of 3x10(5) such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) n motif was more abundant than the (CA/GT) n motif in these clones. The (GA/CT) n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11-20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.
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GenAlEx: Genetic Analysis in Excel is a cross-platform package for population genetic analyses that runs within Microsoft Excel. GenAlEx offers analysis of diploid codominant, haploid and binary genetic loci and DNA sequences. Both frequency-based (F-statistics, heterozygosity, HWE, population assignment, relatedness) and distance-based (AMOVA, PCoA, Mantel tests, multivariate spatial autocorrelation) analyses are provided. New features include calculation of new estimators of population structure: G'(ST), G ''(ST), Jost's Dest and F'(ST) through AMOVA, Shannon Information analysis, linkage disequilibrium analysis for biallelic data and novel heterogeneity tests for spatial autocorrelation analysis. Export to more than 30 other data formats is provided. Teaching tutorials and expanded step-by-step output options are included. The comprehensive guide has been fully revised.
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Three methods for estimating the average level of gene flow in natural population are discussed and compared. The three methods are FST , rare alleles, and maximum likelihood. All three methods yield estimates of the combination of parameters (the number of migrants [Nm] in a demic model or the neighborhood size [4piDsigma(2) ] in a continuum model) that determines the relative importance of gene flow and genetic drift. We review the theory underlying these methods and derive new analytic results for the expectation of FST in stepping-stone and continuum models when small sets of samples are taken. We also compare the effectiveness of the different methods using a variety of simulated data. We found that the FST and rare-alleles methods yield comparable estimates under a wide variety of conditions when the population being sampled is demographically stable. They are roughly equally sensitive to selection and to variation in population structure, and they approach their equilibrium values at approximately the same rate. We found that two different maximum-likelihood methods tend to yield biased estimates when relatively small numbers of locations are sampled but more accurate estimates when larger numbers are sampled. Our conclusion is that, although FST and rare-alleles methods are expected to be equally effective in analyzing ideal data, practical problems in estimating the frequencies of rare alleles in electrophoretic studies suggest that FST is likely to be more useful under realistic conditions.
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The identification of genetically homogeneous groups of individuals is a long standing issue in population genetics. A recent Bayesian algorithm implemented in the software STRUCTURE allows the identification of such groups. However, the ability of this algorithm to detect the true number of clusters (K) in a sample of individuals when patterns of dispersal among populations are not homogeneous has not been tested. The goal of this study is to carry out such tests, using various dispersal scenarios from data generated with an individual-based model. We found that in most cases the estimated 'log probability of data' does not provide a correct estimation of the number of clusters, K. However, using an ad hoc statistic DeltaK based on the rate of change in the log probability of data between successive K values, we found that STRUCTURE accurately detects the uppermost hierarchical level of structure for the scenarios we tested. As might be expected, the results are sensitive to the type of genetic marker used (AFLP vs. microsatellite), the number of loci scored, the number of populations sampled, and the number of individuals typed in each sample.
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In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.
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魏高明. 苏皖4 种同域分布栎树的遗传变异与基因渐渗[D]. 南京: 南京林业大学, 2015.
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Natural introgression can cause negative effects where rare species experience genetic assimilation and invade by their abundant congeners. Quercus austrocochinchinensis and Q. kerrii (subgenus Cyclobalanopsis) are a pair of closely related species in the Indo-China area. Morphological intermediates of the two species have been reported in this region. In this study, we used AFLP, SSR and two key leaf morphological diagnostic traits to study the two Q. austrocochinchinensis populations, two pure Q. kerrii and two putative hybrid populations in China. Rates of individual admixture were examined using the Bayesian clustering programs STRUCTURE and NewHybrids, with no a priori species assignment. In total, we obtained 151 SSR alleles and 781 polymorphic loci of AFLP markers. Population differentiation inferred by SSR and AFLP was incoherent with recognized species boundaries. Bayesian admixture analyses and principal coordinate analysis identified more hybrids and backcrossed individuals than morphological intermediates in the populations. SSR inferred a wide genetic assimilation in Q. austrocochinchinensis, except for subpopulation D2 in the core area of Xi-Shuang-Ban-Na Nature Reserve (XSBN). However, AFLP recognized more Q. austrocochinchinensis purebreds than SSR. Analysis using NewHybrids on AFLP data indicated that these hybridized individuals were few F2 and predominantly backcrosses with both parental species. All these evidences indicate the formation of a hybrid swarm at XSBN where the two species co-exist. Both AFLP and SSR recognized that the core protected area of XSBN (D2) has a high percentage of Q. austrocochinchinensis purebreds and a unique germplasm. The Hainan population and the other subpopulations of XSBN of the species might have lost their genetic integrity. Our results revealed a clear genetic differentiation in the populations and subpopulations of Q. austrocochinchinensis and ongoing introgression between Q. austrocochinchinensis and Q. kerrii at the disturbed contact areas. Combining the results from genetic and morphological analyses, the conservation of subpopulation D2 should be prioritized. Conservation and restoration of the integrity of tropical ravine rainforest is an important long-term goal for the successful conservation of Q. austrocochinchinensis. The fine-scale landscape might play an essential role in shaping the spatial patterns of hybridization. Further studies are needed to evaluate these patterns and dynamics.
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Sympatric hybridizing oak species provide a model system for studying local adaptation. Disjunct populations of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill at the northern edge of their distribution may harbor important reservoirs of adaptive genetic variation. Genic (expressed sequence tag- simple sequence repeat = EST-SSR) and non-genic nuclear microsatellite (nuclear SSR = nSSR) markers were used to estimate neutral and potentially adaptive genetic variation in these two supposedly interfertile oak species showing different adaptations to drought. Eleven populations of putative Q. rubra and Q. ellipsoidalis located in the Western Upper Peninsula of Michigan were characterized using seven EST-SSRs and eight nSSRs. Bayesian cluster analysis revealed two distinct groups corresponding to each species with evidence of low levels of potential introgression. A comparison of the genetic structure of adult trees and seedlings revealed no evidence for selection against hybrids. Overall, similar levels of genetic variation and differentiation between populations and species were found at both EST-SSRs and nSSRs indicating that most EST-SSRs chosen reflect neutral variation. Two loci, 3A05 (nSSR) and GOT021 (EST-SSR, putative histidine kinase 4-like), were identified as putative outlier loci between species showing largely reduced variation in Q. ellipsoidalis. Future analyses of an increased number of EST-SSRs located in functional genes will allow the identification of genes involved in the reproductive isolation between both species.
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秦英英, 韩海荣, 康峰峰, 等. 基于SSR标记的山西省辽东栎自然居群遗传多样性分析[J]. 北京林业大学学报, 2012,34(2):61-65.
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Genetic diversity of five Quercus variabilis natural populations in four provinces of China was studied with microsatellite (SSR) markers. A relatively high level of genetic diversity was detected in Q. variabilis species with 16 polymorphic microsatellite loci. Average number of alleles (A) and effective number of alleles (N(e)) were 8.4375 and 5.9512 respectively. The mean expected heterozygosity (H(e)) was 0.8059 and Nei diversity index (h) was 0.8041. Higher diversity was found with the populations from the central range of the species in contrast to those from peripheral areas and human activities might decrease the genetic diversity of populations. The majority of genetic variation occurred within populations, which could be concluded from the low coefficient of genetic differentiation (F(st)=0.0455). In addition, significant correlation was found between geographical distance and genetic distance. All these results present a basis to the conservation and utilization of genetic diversity of Quercus variabilis.
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