微卫星位点筛选方法综述

微卫星标记因其丰富的多态性和共显性等特点,已得到了广泛的应用.应用微卫星标记首先需要获得微卫星位点的序列信息,用来设计引物.获得微卫星位点的方法有多种,本文综述了获得和富集微卫星位点的常用方法.最简便、最省时的方法是从公共数据库(如EMBL、Genbank、EST数据库等)或已发表的文献中查找到微卫星位点,但只限于已经有序列数据发布的物种.第二种方法是种间转移扩增,即从相近物种的数据库中查找微卫星位点,或使用已有数据发表的遗传距离相近物种的微卫星标记.第三种方法是从基因组DNA中筛选微卫星位点,其中用于富集微卫星的方法有引物法、磁珠杂交法、尼龙膜杂交法以及RAPD技术法.     

Multilocus self-recognition systems in fungi as a cause of trans-species polymorphism

Trans-species polymorphism, meaning the presence of alleles in different species that are more similar to each other than they are to alleles in the same species, has been found at loci associated with vegetative incompatibility in filamentous fungi. If individuals differ at one or more of these loci (termed het for heterokaryon), they cannot form stable heterokaryons after vegetative fusion. At the het-c locus in Neurospora crassa and related species there is clear evidence of trans-species polymorphism: three alleles have persisted for approximately 30 million years. We analyze a population genetic model of multilocus vegetative incompatibility and find the conditions under which trans-species polymorphism will occur. In the model, several unlinked loci determine the vegetative compatibility group (VCG) of an individual. Individuals of different VCGs fail to form productive heterokaryons, while those of the same VCG form viable heterokaryons. However, viable heterokaryon formation between individuals of the same VCG results in a loss in fitness, presumably via transfer of infectious agents by hyphal fusion or exploitation by aggressive genotypes. The result is a form of balancing selection on all loci affecting an individual's VCG. We analyze this model by making use of a Markov chain/strong selection, weak mutation (SSWM) approximation. We find that trans-species polymorphism of the type that has been found at the het-c locus is expected to occur only when the appearance of new incompatibility alleles is strongly constrained, because the rate of mutation to such alleles is very low, because the number of possible incompatibility alleles at each locus is restricted, or because the number of incompatibility loci is limited.     

Genetic diversity and population history of golden monkeys (Rhinopithecus roxellana)

Golden monkey (Rhinopithecus roxellana), namely the snub-nosed monkey, is a well-known endangered primate, which distributes only in the central part of mainland China. As an effort to understand the current genetic status as well as population history of this species, we collected a sample of 32 individuals from four different regions, which cover the major habitat of this species. Forty-four allozyme loci were surveyed in our study by allozyme electrophoresis, none of which was found to be polymorphic. The void of polymorphism compared with that of other nonhuman primates is surprising particularly considering that the current population size is many times larger than that of some other endangered species. Since many independent loci are surveyed in this study, the most plausible explanation for our observation is that the population has experienced a recent bottleneck. We used a coalescent approach to explore various scenarios of population bottleneck and concluded that the most recent bottleneck could have happened within the last 15,000 years. Moreover, the proposed simulation approach could be useful to researchers who need to analyze the non- or low-polymorphism data.     

High population differentiation and unusual haplotype structure in a shade-intolerant pioneer tree species, Zanthoxylum ailanthoides (Rutaceae) revealed by analysis of DNA polymorphism at four nuclear loci

Differences in demographic history, life-history traits, and breeding systems affect nucleotide variation patterns. It is expected that shade-intolerant pioneer tree species have different patterns of genetic polymorphism and population structure than climax species. We studied patterns of nucleotide polymorphism at four putative starch pathway loci (agpSA, agpSB, agpL, and GBSSI) in Zanthoxylum ailanthoides , a shade-intolerant pioneer tree species that occupies forest gaps in warm-temperate forests of East Asia. Genetic diversity was lower within each population than among populations, and differentiation among populations was high across the loci ( F _ST = 0.32–0.64), as expected from the insect-pollinated breeding system and the metapopulation structure of this pioneer species. Numbers of haplotypes were smaller than those expected from the observed numbers of segregating sites. Single haplotypes accounted for more than 47% of all the sampled genes at the respective loci. These variation patterns were incompatible with neutral predictions for populations of a finite island model. Complex population dynamics, such as bottleneck and/or admixture, in the history of this pioneer tree species might have resulted in the observed patterns of genetic variation and population structure, which are different from those of climax wind-pollinated tree species, such as conifers. In contrast to the other loci investigated in this study, agpL showed nearly no variation in Z. ailanthoides (one singleton only), but there was some extent of variation in a closely related species, Zanthoxylum schinifolium . This suggests possibly a recent selective sweep at or near the locus in Z. ailanthoides .     

Balancing selection maintains sex determining alleles in multiple‐locus complementary sex determination

Hymenopteran species in which sex is determined through a haplo‐diploid mechanism known as complementary sex determination (CSD) are vulnerable to a unique form of inbreeding depression. Diploids heterozygous at one or more CSD loci develop into females but diploids homozygous at all loci develop into diploid males, which are generally sterile or inviable. Species with multiple polymorphic CSD loci (ml‐CSD) may have lower rates of diploid male production than species with a single CSD locus (sl‐CSD), but it is not clear if polymorphism is consistently maintained at all loci. Here, we assess the rate of diploid male production in a population of Cotesia rubecula, a two‐locus CSD parasitoid wasp species, approximately 20 years after the population was introduced for biological control. We show that diploid male production dropped from 8–13% in 2005 and 2006 to 3–4% by 2015. We also show from experimental crosses that the population maintained polymorphism at both CSD loci in 2015. We use theory and simulations to show that balancing selection on all CSD alleles promotes polymorphism at several loci in ml‐CSD populations. Our study supports the hypothesis that ml‐CSD populations have lower diploid male production and are more likely to persist than comparable sl‐CSD populations.     

Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates

Major histocompatibility complex (MHC) antigen-presenting genes are the most variable loci in vertebrate genomes. Host-parasite co-evolution is assumed to maintain the excessive polymorphism in the MHC loci. However, the molecular mechanisms underlying the striking diversity in the MHC remain contentious. The extent to which recombination contributes to the diversity at MHC loci in natural populations is still controversial, and there have been only few comparative studies that make quantitative estimates of recombination rates. In this study, we performed a comparative analysis for 15 different ungulates species to estimate the population recombination rate, and to quantify levels of selection. As expected for all species, we observed signatures of strong positive selection, and identified individual residues experiencing selection that were congruent with those constituting the peptide-binding region of the human DRB gene. However, in addition for each species, we also observed recombination rates that were significantly different from zero on the basis of likelihood-permutation tests, and in other non-quantitative analyses. Patterns of synonymous and non-synonymous sequence diversity were consistent with differing demographic histories between species, but recent simulation studies by other authors suggest inference of selection and recombination is likely to be robust to such deviations from standard models. If high rates of recombination are common in MHC genes of other taxa, re-evaluation of many inference-based phylogenetic analyses of MHC loci, such as estimates of the divergence time of alleles and trans-specific polymorphism, may be required.     

Modeling gynodioecy: novel scenarios for maintaining polymorphism

Nuclear-cytoplasmic gynodioecy is a breeding system of plants in which females and hermaphrodites co-occur in populations, and gender is jointly determined by cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Persistent polymorphism at both CMS and nuclear-restorer loci is necessary to maintain this breeding system. Theoretical models have explained how nuclear-cytoplasmic gynodioecy can be stable for certain assumptions. However, recent advances in our understanding of the genetics, population biology, and molecular mechanisms of sex determination in nuclear-cytoplasmic gynodioecious species suggest the utility of new models with different underlying assumptions. In this article, we examine different negative pleiotropic fitness effects of nuclear restorers (costs of restoration) using genetic and population assumptions based on recent literature. Specifically, we model populations with two CMS types and separate nuclear restorer loci for each CMS type. Under these assumptions, both overdominance for fitness and frequency-dependent selection at nuclear-restorer loci can support nuclear-cytoplasmic gynodioecy. Costs of restoration can be either dependent or independent of the cytoplasmic background. Seed fitness costs are more vulnerable to fixation of CMS types than pollen costs. Survivorship costs are effective at maintaining polymorphism even when total reproductive effects are low. Overall, our models display differences in the stability of nuclear-cytoplasmic gynodioecy and predicted population sex ratios that should be informative to researchers studying gynodioecy in the wild.     

Population genetics of Caenorhabditis elegans: the paradox of low polymorphism in a widespread species

Caenorhabditis elegans has become one of the most widely used model research organisms, yet we have little information on evolutionary processes and recent evolutionary history of this widespread species. We examined patterns of variation at 20 microsatellite loci in a sample of 23 natural isolates of C. elegans from various parts of the world. One-half of the loci were monomorphic among all strains, and overall genetic variation at microsatellite loci was low, relative to most other species. Some population structure was detected, but there was no association between the genetic and geographic distances among different natural isolates. Thus, despite the nearly worldwide occurrence of C. elegans, little evidence was found for local adaptation in strains derived from different parts of the world. The low levels of genetic variation within and among populations suggest that recent colonization and population expansion might have occurred. However, the patterns of variation are not consistent with population expansion. A possible explanation for the observed patterns is the action of background selection to reduce polymorphism, coupled with ongoing gene flow among populations worldwide.     

Population genetics of speciation in two closely related wild tomatoes (Solanum section Lycopersicon)

We present a multilocus sequencing study to assess patterns of polymorphism and divergence in the closely related wild tomato species, Solanum peruvianum and S. chilense (Solanum section Lycopersicon, Solanaceae). The data set comprises seven mapped nuclear loci (approximately 9.3 kb of analyzed sequence across loci) and four local population samples per species that cover much of the species' range (between 80 and 88 sequenced alleles across both species). We employ the analytical framework of divergence population genetics (DPG) in evaluating the utility of the "isolation" model of speciation to explain observed patterns of polymorphism and divergence. Whereas the isolation model is not rejected by goodness-of-fit criteria established via coalescent simulations, patterns of intragenic linkage disequilibrium provide evidence for postdivergence gene flow at two of the seven loci. These results suggest that speciation occurred under residual gene flow, implying that natural selection is one of the evolutionary forces driving the divergence of these tomato species. This inference is fully consistent with their recent divergence, conservatively estimated to be 相似文献   

Genome scans of DNA variability in humans reveal evidence for selective sweeps outside of Africa

The last 50,000-150,000 years of human history have been characterized by rapid demographic expansions and the colonization of novel environments outside of sub-Saharan Africa. Mass migrations outside the ancestral species range likely entailed many new selection pressures, suggesting that genetic adaptation to local environmental conditions may have been more prevalent in colonizing populations outside of sub-Saharan Africa. Here we report a test of this hypothesis using genome-wide patterns of DNA polymorphism. We conducted a multilocus scan of microsatellite variability to identify regions of the human genome that may have been subject to continent-specific hitchhiking events. Using published polymorphism data for a total of 624 autosomal loci in multiple populations of humans, we used coalescent simulations to identify candidate loci for geographically restricted selective sweeps. We identified a total of 13 loci that appeared as outliers in replicated population comparisons involving different reference samples for Africa. A disproportionate number of these loci exhibited reduced levels of relative variability in non-African populations alone, suggesting that recent episodes of positive selection have been more prevalent outside of sub-Saharan Africa.     

Genetic variation within and among populations of Arabidopsis thaliana.

We investigated levels of nucleotide polymorphism within and among populations of the highly self-fertilizing Brassicaceous species, Arabidopsis thaliana. Four-cutter RFLP data were collected at one mitochondrial and three nuclear loci from 115 isolines representing 11 worldwide population collections, as well as from seven commonly used ecotypes. The collections include multiple populations from North America and Eurasia, as well as two pairs of collections from locally proximate sites, and thus allow a hierarchical geographic analysis of polymorphism. We found no variation at the mitochondrial locus Nad5 and very low levels of intrapopulation nucleotide diversity at Adh, Dhs1, and Gpa1. Interpopulation nucleotide diversity was also consistently low among the loci, averaging 0.0014. gst, a measure of population differentiation, was estimated to be 0.643. Interestingly, we found no association between geographical distance between populations and genetic distance. Most haplotypes have a worldwide distribution, suggesting a recent expansion of the species or long-distance gene flow. The low level of polymorphism found in this study is consistent with theoretical models of neutral mutations and background selection in highly self-fertilizing species.     

Inferring the effects of demography and selection on Drosophila melanogaster populations from a chromosome-wide scan of DNA variation

Identifying regions of the Drosophila melanogaster genome that have been recent targets of positive Darwinian selection will provide evidence for adaptations that have helped this species to colonize temperate habitats. We have begun a search for such genomic regions by analyzing multiple loci (about 250) dispersed across the X chromosome in a putatively ancestral population from East Africa and a derived European population. For both populations we found evidence for past changes in population size. We estimated that a major bottleneck associated with the colonization of Europe occurred about 3,500-16,000 years ago. We also found that while this bottleneck can account for most of the reduction in variation observed in the European sample, there is a deficit of polymorphism in some genomic regions that cannot be explained by demography alone.     

Phylogenetic relationships and introgression patterns between incipient parapatric species of Italian brown trout (Salmo trutta L. complex)

Genetic variation at 47 protein loci was investigated in 16 wild brown trout populations from the Pô basin and three major domesticated stocks used for stocking this area. Twenty-four loci were polymorphic and large frequency differences were found at 15 of them. The most significant allozyme variations were congruent with the mtDNA sequence polymorphism previously observed in the same samples. We confirmed the occurrence of two parapatric incipient species, Salmo marmoratus and S. trutta fario , previously identified by morphological traits. These two species were fixed or nearly fixed for alternate alleles at eight loci (Nei's standard genetic distance = 0.16–0.18), but introgression was detected between adjacent samples of the two forms. Divergence levels at both mtDNA and nuclear loci suggested that the differentiation between S. marmoratus and S. trutta fario started between 3 and 1 million years before present. Variation at protein loci and mtDNA supported the hypothesis that the third species found in this area, S. carpio (an endemic population of the lake Garda) was issued from a recent hybridization of the two first species. Finally, we showed that three of the major Italian fish-farm strains originated from the Atlantic side and displayed substantial genetic differences with the natural populations of the Pô basin. Most of these populations were contaminated by stocking with introgression rate ranging from 0 to 70% and measures of protection and restoration of the rich genetic diversity present in this area should be urgently applied.     

Recent radiation of endemic Caribbean Drosophila of the dunni subgroup inferred from multilocus DNA sequence variation

Studies of island endemism provide a unique opportunity to elucidate fundamental mechanisms of speciation. Here we examine intra- and interspecific DNA sequence variation at four unlinked genetic loci among populations of the Drosophila dunni subgroup to provide a detailed genealogical portrait of the process of speciation among these island endemic species. Our data indicate two major rounds of diversification that have shaped the D. dunni subgroup. The first occurred 1.6-2.6 million years ago and separated three major lineages, one in Puerto Rico and the Virgin Islands, a second in the northern Lesser Antilles and Barbados, and a third in St. Vincent and Grenada. A second round of diversification occurred in the last 96,000 years in the northern Lesser Antilles and Barbados. The four distinct species that resulted from this recent round of diversification maintain relatively high amounts of genetic variation, similar to that of a closely related mainland species, and share extensive ancestral polymorphism. These data suggest a minimal role for population bottlenecks linked to founder events in the history of the D. dunni subgroup. Further, the recent divergence of these island populations highlights the extremely rapid development of reproductive isolation and distinct patterns of abdominal pigmentation that has occurred in these species.     

Evidence of gene conversion associated with a selective sweep in Drosophila melanogaster

Since Drosophila melanogaster colonized Europe from tropical Africa 10 to 15 thousand years ago, it is expected that adaptation has played a major role in this species in recent times. A previously conducted multilocus scan of noncoding DNA sequences on the X chromosome in an ancestral and a derived population of D. melanogaster revealed that some loci have been affected by directional selection in the European population. We investigated if the pattern of DNA sequence polymorphism in a region surrounding one of these loci can be explained by a hitchhiking event. We found strong evidence that the studied region around the gene unc-119 was shaped by a recent selective sweep, including a valley of reduced heterozygosity of 83.4 kb, a skew in the frequency spectrum, and significant linkage disequilibrium on one side of the valley. This region, however, was interrupted by gene conversion events leading to a strong haplotype structure in the center of the valley of reduced variation.     

Genetic Population Structure and Gene Flow in the Atlantic Cod Gadus Morhua: A Comparison of Allozyme and Nuclear RFLP Loci

High levels of gene flow have been implicated in producing uniform patterns of allozyme variation among populations of many marine fish species. We have examined whether gene flow is responsible for the limited population structure in the Atlantic cod, Gadus morhua L., by comparing the previously published patterns of variation at 10 allozyme loci to 17 nuclear restriction fragment length polymorphism (RFLP) loci scored by 11 anonymous cDNA clones. Unlike the allozyme loci, highly significant differences were observed among all populations at the DNA markers in a pattern consistent with an isolation-by-distance model of population structure. The magnitude of allele frequency variation at the nuclear RFLP loci significantly exceeded that observed at the protein loci (χ(2) = 24.6, d.f. = 5, P < 0.001). Estimates of gene flow from the private alleles method were similar for the allozymes and nuclear RFLPs. From the infinite island model, however, estimates of gene flow from the DNA markers were fivefold lower than indicated by the proteins. The discrepancy between gene flow estimates, combined with the observation of a large excess of rare RFLP alleles, suggests that the Atlantic cod has undergone a recent expansion in population size and that populations are significantly displaced from equilibrium. Because gene flow is a process that affects all loci equally, the heterogeneity observed among populations at the DNA level eliminates gene flow as the explanation for the homogeneous allozyme patterns. Our results suggest that a recent origin of cod populations has acted to constrain the extent of population differentiation observed at weakly polymorphic loci and implicate a role for selection in affecting the distribution of protein variation among natural populations in this species.     

Estimating parameters of speciation models based on refined summaries of the joint site-frequency spectrum

Understanding the processes and conditions under which populations diverge to give rise to distinct species is a central question in evolutionary biology. Since recently diverged populations have high levels of shared polymorphisms, it is challenging to distinguish between recent divergence with no (or very low) inter-population gene flow and older splitting events with subsequent gene flow. Recently published methods to infer speciation parameters under the isolation-migration framework are based on summarizing polymorphism data at multiple loci in two species using the joint site-frequency spectrum (JSFS). We have developed two improvements of these methods based on a more extensive use of the JSFS classes of polymorphisms for species with high intra-locus recombination rates. First, using a likelihood based method, we demonstrate that taking into account low-frequency polymorphisms shared between species significantly improves the joint estimation of the divergence time and gene flow between species. Second, we introduce a local linear regression algorithm that considerably reduces the computational time and allows for the estimation of unequal rates of gene flow between species. We also investigate which summary statistics from the JSFS allow the greatest estimation accuracy for divergence time and migration rates for low (around 10) and high (around 100) numbers of loci. Focusing on cases with low numbers of loci and high intra-locus recombination rates we show that our methods for the estimation of divergence time and migration rates are more precise than existing approaches.     

Class I Mhc genes of cichlid fishes: identification, expression, and polymorphism

 Cichlid fishes of the East African Rift Valley lakes constitute an important model of adaptive radiation. Explosive speciation in the Great Lakes, in some cases as recently as 12 400 years ago, generated large species flocks that have been the focus of evolutionary studies for some time. The studies have, however, been hampered by the paucity of biochemical markers for phylogenetic reconstruction. Here, we describe a set of markers which should help to alleviate this problem. They are the class I genes of the major histocompatibility complex. We provide evidence for the existence of at least 17 class I loci in cichlid fishes, and for extensive polymorphism of three of these loci. Since the polymorphism has a trans-species character, it will be possible to use it in investigating the founding events of the individual species. The sequences of the cichlid class I fishes support the monophyly of actinopterygian fish on the one hand, and of tetrapods on the other. Received: 4 November 1996 / Revised: 19 December 1996     

Perspective: gene divergence, population divergence, and the variance in coalescence time in phylogeographic studies

Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (theta) to T and the effect of uncertainty in theta on estimates of population divergence (tau) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on tau decrease when the range over which ancestral population size theta is assumed to be distributed decreases and when tau increases; they generally exclude zero when tau/(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding tau = 0 is roughly that l tau/(4Ne) > 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time.     

Development of microsatellite markers for the endangered Neotropical tree species Tibouchina papyrus (Melastomataceae)

We isolated and characterized 12 microsatellite loci for Tibouchina papyrus (Melastomataceae), an endangered species with narrow and disjunct range, endemics to a few localities in "cerrado rupestre" from Central Brazil. These microsatellites were obtained by sequencing of a genomic shotgun library for primer design. Leaves from 96 individuals collected in the three known local populations were genotyped using the 12 primers designed to analyze the polymorphisms at each locus. The number of alleles per locus ranged from one to six; two loci were monomorphic. Among the polymorphic loci, expected heterozygosities ranged from 0.161 to 0.714. Combined paternity exclusion probability was 0.957 and combined genetic identity (0.051) was high for studies on parentage. Tibouchina papyrus is a rare and endemic tree species of outcrop quartzite and sandstone soils, with highly isolated populations, which may have lead to the low degree of polymorphism that we detected. Also, motifs of most loci are larger than dinucleotide, which typically display lower levels of polymorphism.