微卫星标记及其在蚜虫种群生物学研究中的应用

微卫星（SSR）是以1～6个碱基为重复单位组成的简单串联重复序列,具有丰度高、多态性高、共显性遗传、选择中性和可重复性好等优点。微卫星标记技术可分为三种类型：基于杂交的SSR指纹,基于PCR的SSR指纹和单位点SSR的PCR扩增。前两者不需要知道基因组序列信息,属于多位点标记;后者则需要前期工作,属于单位点标记,即通常所指的微卫星标记。到目前为止,共在14种蚜中筛选到141个微卫星位点,并发表了相关的引物序列,这给今后的相关研究提供了丰富的共享资源。业已证明微卫星引物在邻近种之间有一定的通用性。微卫星标记严格遵守孟德尔遗传规律,已经被用来推断某些蚜虫有性生殖的情况。同种蚜虫中可能同时存在有性系和无性系,微卫星研究证明有性系比无性系有更高的遗传多样性,无性系通常表现出杂合过剩和连锁不平衡。蚜虫的迁飞规律适合用微卫星标记加以研究,已有的研究显示在具有高度迁飞特性的种类中,地理种群间的遗传分化程度低,基因频率的相似性高。存在广泛分布的相同的多位点基因型是迁飞性蚜虫的另一个重要特征。在我国,微卫星应用于蚜虫生物学研究的工作还较少,鉴于该种标记的优良特性和巨大的潜力,本文建议今后相关的研究应该首先考虑微卫星标记。     

Alteration of pathogenicity-linked life-history traits by resistance of its host Solanum tuberosum impacts sexual reproduction of the plant pathogenic oomycete Phytophthora infestans

Although sexual reproduction implies a cost, it represents an evolutionary advantage for the adaptation and survival of facultative sexual pathogens. Understanding the maintenance of sex in pathogens requires to analyse how host resistance will impact their sexual reproduction through the alteration of their life-history traits. We explored this experimentally using potato (Solanum tuberosum) and one of its pathogens, the heterothallic oomycete Phytophthora infestans. Sexual reproduction was highest on hosts favouring asexual multiplication of the pathogen, suggesting similar nutritional requirements for both sexual and asexual sporulation. Sexual reproduction was also highest on hosts decreasing the latent period, probably because of a trade-off between growth and reproduction. Distinguishing host effects on each pathogenic trait remains however uneasy, as most life-history traits linked to pathogenicity were not independent of each other. We argue that sexual reproduction of P. infestans is an adaptation to survive when the host is susceptible and rapidly destroyed.     

Microsatellite analysis revealed genetic diversity and population structure among Chinese cashmere goats

Most cashmere goats are found in northern China and Mongolia. They are regarded as precious resources for their production of high quality natural fibre for the textile industry. It was the first time that the genetic diversity and population structure of nine Chinese cashmere populations has been assessed using 14 ISAG/FAO microsatellite markers. In addition, two Iranian populations and one West African goat population were genotyped for comparison. Results indicated that the genetic diversity of Chinese cashmere goats was rich, but less than those of the Iranian goat populations. All pairwise F_ST values between the Chinese cashmere goat populations reached a highly significant level (P < 0.001), suggesting that they should all be considered as separate breeds. Finally, clustering analysis divided Chinese cashmere goats into at least two clusters, with the Tibetan Hegu goats alone in one cluster. An extensive admixture was detected among the Chinese goat breeds (except the Hegu), which have important implications for breeding management.     

Microsatellite markers of genetic diversity and population structure of Carica papaya

We assessed the genetic diversity of 96 papaya accessions by molecular characterisation using microsatellite markers. Fifteen polymorphic primers were selected. Accessions, which were classified as Common, Formosa and Solo according to fruit types, were evaluated for allele frequency, heterozygosity, polymorphism information content (PIC), inbreeding coefficient (f) and the genetic diversity structure. Fifteen primers amplified 68 alleles with an average of 4.53 per locus. PIC values ranged from 0.19 to 0.69. The observed heterozygosity (H_O) was low for all selected microsatellites. High f estimates (0.58) and excess of homozygotes indicated inbreeding, mainly caused by the tendency to select hermaphrodite plants for succeeding generations. Analysis of molecular variance showed that most of the variation (98%) resides within subpopulation. The genetic analysis based on Bayesian statistics proved to be sensitive enough to detect relationships among the papaya accessions, grouping them into six clusters, irrespective of their classification types.     

Subpopulation genetic structure of a plant panmictic population of Castanea sequinii as revealed by microsatellite markers

Castanea squinii Dode,an endemic tree widely distributed in China,plays an important role both in chestnut breeding and forest ecosystem function.The spatial genetic structure within and among populations is an important part of the evolutionary and ecological genetic dynamics of natural populations,and can provide insights into effective conservation of genetic resources.In the present study,the spatial genetic structure of a panmictic natural population of C.sequinii in the Dabie Mountain region was investigated using microsatellite markers.Nine prescreened microsatellite loci generated 29-33 alleles each,and were used for spatial autocorrelation analysis.Based on Moran's I coefficient,a panmictic population of C.sequinii in the Dabie Mountain region was found to be lacking a spatial genetic structure.These results suggest that a high pollen-mediated gene flow among subpopulations counteract genetic drift and/or genetic differentiation and plays an important role in maintaining a random and panmictic population structure in C.sequinii populations.Further,a spatial genetic structure was detected in each subpopulation's scale (0.228 km),with all three subpopulations showing significant fine-scale structure.The genetic variation was found to be nonrandomly distributed within 61 m in each subpopulation (Moran's I positive values).Although Moran's I values varied among the different subpopulations,Moran's I in all the three subpopulations reached the expected values with an increase in distances,suggesting a generally patchy distribution in the subpopulations.The fine-scale structure seems to reflect restricted seed dispersal and microenvironment selection in C.sequinii.These results have important implications for understanding the evolutionary history and ecological process of the natural population of C.sequinii and provide baseline data for formulating a conservation strategy of Castanea species.     

Microsatellite markers reveal shallow genetic differentiation between cohorts of the common sea urchin Paracentrotus lividus (Lamarck) in northwest Mediterranean

Temporal variability was studied in the common sea urchin Paracentrotus lividus through the analysis of the genetic composition of three yearly cohorts sampled over two consecutive springs in a locality in northwestern Mediterranean. Individuals were aged using growth ring patterns observed in tests and samples were genotyped for five microsatellite loci. No reduction of genetic diversity was observed relative to a sample of the adult population from the same location or within cohorts across years. F _ST and amova results indicated that the differentiation between cohorts is rather shallow and not significant, as most variability is found within cohorts and within individuals. This mild differentiation translated into estimates of effective population size of 90–100 individuals. When the observed excess of homozygotes was taken into account, the estimate of the average number of breeders increased to c . 300 individuals. Given our restricted sampling area and the known small-scale heterogeneity in recruitment in this species, our results suggest that at stretches of a few kilometres of shoreline, large numbers of progenitors are likely to contribute to the larval pool at each reproduction event. Intercohort variation in our samples is six times smaller than spatial variation between adults of four localities in the western Mediterranean. Our results indicate that, notwithstanding the stochastic events that take place during the long planktonic phase and during the settlement and recruitment processes, reproductive success in this species is high enough to produce cohorts genetically diverse and with little differentiation between them. Further research is needed before the link between genetic structure and underlying physical and biological processes can be well established.     

Microsatellite markers for population and conservation genetics of tropical trees

We have developed microsatellite or simple sequence repeat (SSR) genetic markers for the tropical tree Pithecellobium elegans (Mimosoideae). The frequency of this class of marker is estimated and the level and distribution of variability at these markers is assessed and contrasted to that found at isozyme markers in the same populations. The results indicate that SSR loci are powerful tools for the analysis of population structure and that, in these populations, they provide a means of accurately examining two important parameters in conservation biology, gene flow and paternity.     

Population genomics of an outbreak of the potato late blight pathogen,Phytophthora infestans,reveals both clonality and high genotypic diversity

An outbreak of the potato late blight pathogen Phytophthora infestans in Denmark was characterized in order to resolve the population structure and determine to what extent sexual reproduction was occurring. A standard set of microsatellite simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP) markers generated using restriction site-associated DNA sequencing (RAD-seq) were employed in parallel. A total of 83 individuals, isolated from seven different potato fields in 2014, were analysed together with five Danish whole-genome sequenced isolates, as well as two Mexican individuals used as an outgroup. From a filtered dataset of 55 288 SNPs, population genomics analyses revealed no sign of recombination, implying clonality. In spite of this, multilocus genotypes were unique to individual potato fields, with little evidence of gene flow between fields. Ploidy analysis performed on the SNPs dataset indicated that the majority of isolates were diploid. These contradictory results with clonality and high genotypic diversity may suggest that rare sexual events likely still contribute to the population. Comparison of the results generated by SSRs vs SNPs data indicated that large marker sets, generated by RAD-seq, may be advised going forward, as it provides a higher level of genetic discrimination than SSRs.     

Microsatellite analysis of genetic variation among and within Alpine marmot populations in the French Alps

The genetic structure of the Alpine marmot, Marmota marmota, was studied by an analysis of five polymorphic microsatellite loci. Eight locations were sampled in the French Alps, one from Les Ecrins valley (n = 160), another from La Sassière valley (n = 289) and the six others from the Maurienne valley (n = 139). Information on social group structure was available for both Les Ecrins and La Sassière but not for the other samples. The high levels of genetic diversity observed are at odds with the results obtained using microsatellites, minisatellites and allozymes on Alpine marmots from Germany, Austria and Switzerland. Strong deficits in heterozygotes were found in Les Ecrins and La Sassière. They are caused by a Wahlund effect due to the family structure (i.e. differentiation between the family groups). The family groups exhibit excess of heterozygotes rather than deficits. This may be caused by outbreeding and this is compatible with recent results from the genetics of related social species when information on the social structure is taken into account. The observed outbreeding could be the result of females mating with transient males or males coming from neighbouring colonies. Both indicate that the species may not be as monogamous as is usually believed. The results are also compatible with a male-biased dispersal but do not allow us to exclude some female migration. We also found a significant correlation between geographical and genetic distance indicating that isolation by distance could be an issue in marmots. This study is the first that analysed populations of marmots taking into account the social structure within populations and assessing inbreeding at different levels (region, valley, population, and family groups). Our study clearly demonstrated that the sampling strategy and behavioural information can have dramatic effects on both the results and interpretation of the genetic data.     

Microsatellite variation and fine-scale population structure in the wood frog (Rana sylvatica)

We investigated genetic population structure in wood frogs (Rana sylvatica) from a series of Prairie Pothole wetlands in the northern Great Plains. Amphibians are often thought to exist in demographic metapopulations, which require some movement between populations, yet genetic studies have revealed strong subdivision among populations, even at relatively fine scales (several km). Wood frogs are highly philopatric and studies of dispersal suggest that they may exhibit subdivision on a scale of approximately 1-2 km. We used microsatellites to examine population structure among 11 breeding assemblages separated by as little as 50 m up to approximately 5.5 km, plus one population separated from the others by 20 km. We found evidence for differentiation at the largest distances we examined and among a few neighbouring ponds, but most populations were strikingly similar in allele frequencies, suggesting high gene flow among all but the most distant populations. We hypothesize that the few significant differences among neighbouring populations at the finest scale may be a transient effect of extinction-recolonization founder events, driven by periodic drying of wetlands in this hydrologically dynamic landscape.     

Spatial genetic analyses reveal strong genetic structure in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae)

The development of spatial genetic structure (SGS) in seed plants has been linked to several biological attributes of species, such as breeding system and life form. However, little is known about SGS in ferns, which together with lycopods are unique among land plants in having two free‐living life stages. We combined spatial aggregation statistics and spatial genetic autocorrelation analyses using five plastid microsatellites and one nuclear gene to investigate SGS in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae). We assessed how the observed patterns compare with those estimated for other ferns and seed plants. Populations of A. firma exhibited strong SGS, spatial clustering of individuals, substantial clonal diversity and no inbreeding. SGS in ferns appears to be higher than in most seed plants analysed to date. Contrary to our expectations, an outcrossing breeding system, wind dispersal and an arborescent life form did not translate into weak or no SGS. In ferns, SGS is probably being affected by the life cycle with two free‐living life stages. The reproductive biology of ferns appears to be more complex than previously thought. This implies that SGS in ferns is affected by some factors that cannot be inferred from the study of flowering plants. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 439–449.     

Microsatellite variation within and among North American lineages of Phragmites australis

Over the past century, the spread of the common reed (Phragmites australis) has had a dramatic impact on wetland communities across North America. Although native populations of Phragmites persist, introduced invasive populations have dominated many sites and it is not clear if the two types can interbreed. This study compares patterns of differentiation in 10 microsatellite loci among North American and European Phragmites individuals with results obtained from sequencing of noncoding chloroplast DNA. Three population lineages (native, introduced and Gulf Coast) were previously identified in North America from chloroplast DNA and similar structuring was found in the nuclear genome. Each lineage was distinguished by unique alleles and allele combinations and the introduced lineage was closely related to its hypothesized source population in Europe. Size homoplasy and diagnostic base substitutions distinguishing lineages were evident at several loci, further emphasizing that native, introduced and Gulf Coast North American Phragmites lineages are genetically distinct. Gene flow between lineages was low and invasive introduced populations do not represent a hybrid population type.     

Analysis of genetic diversity and population structure of Chinese yak breeds （Bos grunniens） using microsatellite markers

Nine Chinese yak breeds (Maiwa,Tianzhu White,Qinghai Plateau,Sibu,Zhongdian,Pall,Tibetan High Mountain,Jiulong,and Xin-jiang) and Gayal were analyzed by means of 16 microsatellite markers to determine the level of genetic variation within populations,genetic relationship between populations,and population structure for each breed.A total of 206 microsatellite alleles were observed.Mean F-statistics (0.056) for 9 yak breeds indicated that 94.4% of the genetic variation was observed within yak breeds and 5.6% of the genetic variation existed amongst breeds.The Neighbor-Joining phylogenetic free was constructed based on Nei's standard genetic dis-tances and two clusters were obtained.The Gayal separated from the yaks far away and formed one cluster and 9 yak breeds were grouped together.The analysis of population structure for 9 yak breeds and the Gayal showed that they resulted in four clusters; one clus-ter includes yaks from Tibet Autonomous Region and Qinghai Province,one cluster combines Zhongdian,Maiwa,and Tianzhu White,and Jiulong and Xinjiang come into the third cluster.Pali was mainly in the first cluster (90%),Jiulong was mainly in the second cluster (87.1%),Zhongdian was primarily in the third cluster (83%),and the other yak breeds were distributed in two to three clusters.The Gayal was positively left in the fourth cluster (99.3%).     

Microsatellite markers indicate genetic differences between cultivated and natural populations of endangered Taxus yunnanensis

Taxus yunnanensis (Taxaceae) is an endangered plant known for its effective natural anti‐cancer metabolite, taxol. To assess the effectiveness of T. yunnanensis ex situ conservation in China, the genetic diversity and genetic structure of nine cultivated and 14 natural populations were compared using 11 polymorphic microsatellite markers. The purpose of this comparison was to determine whether the cultivated populations were genetic representatives of natural types. Among the 14 natural populations studied, three were from Tibet and 11 were from the Hengduan Mountains. There were fewer genetic variations among cultivated populations than among natural populations, but this difference was not significant. However, 23 unique alleles were detected in cultivated populations and 14 were detected in natural populations. Striking differences in genetic structure (G_ST = 0.365) was observed between them. STRUCTURE, AMOVA and cluster analyses also consistently showed a clear genetic separation between the 14 natural and seven cultivated populations. Only five natural populations from the south‐central Hengduan Mountains were genetically represented in two cultivated collections. The reason for the occurrence of distinct genetic differences between cultivated and natural populations and the loss of natural population‐specific alleles in the cultivated populations can be attributed to uneven selection during establishment. These results suggest that the genetic structure of T. yunnanensis may not have been conserved effectively. Based on our study, practical conservation strategies for ex situ preservation of T. yunnanensis have been developed. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 450–461.     

利用微卫星标记初步分析橘小实蝇4个地理种群的遗传多态性

橘小实蝇Bactrocera dorsalis(Bactrocera)(Hendel)是我国重要的检疫性害虫,对果蔬生产及其国际贸易造成很大影响。作者运用微卫星分子标记技术,用6对微卫星引物对采自我国福建、广东、云南3个地区及邻近国越南橘小实蝇种群间的遗传关系进行了初步分析,结果显示上述4个橘小实蝇地理种群间存在一定的遗传差异,地理隔离是造成这一差异的主要原因。     

Subpopulation genetic structure of a plant panmictic population of Castanea sequinii as revealed by microsatellite markers

Castanea squinii Dode, an endemic tree widely distributed in China, plays an important role both in chestnut breeding and forest ecosystem function. The spatial genetic structure within and among populations is an important part of the evolutionary and ecological genetic dynamics of natural populations, and can provide insights into effective conservation of genetic resources. In the present study, the spatial genetic structure of a panmictic natural population of C. sequinii in the Dabie Mountain region was investigated using microsatellite markers. Nine prescreened microsatellite loci generated 29–33 alleles each, and were used for spatial autocorrelation analysis. Based on Moran’s I coefficient, a panmictic population of C. sequinii in the Dabie Mountain region was found to be lacking a spatial genetic structure. These results suggest that a high pollen-mediated gene flow among subpopulations counteract genetic drift and/or genetic differentiation and plays an important role in maintaining a random and panmictic population structure in C. sequinii populations. Further, a spatial genetic structure was detected in each subpopulation’s scale (0.228 km), with all three subpopulations showing significant fine-scale structure. The genetic variation was found to be nonrandomly distributed within 61 m in each subpopulation (Moran’s I positive values). Although Moran’s I values varied among the different subpopulations, Moran’s I in all the three subpopulations reached the expected values with an increase in distances, suggesting a generally patchy distribution in the subpopulations. The fine-scale structure seems to reflect restricted seed dispersal and microenvironment selection in C. sequinii. These results have important implications for understanding the evolutionary history and ecological process of the natural population of C. sequinii and provide baseline data for formulating a conservation strategy of Castanea species. __________ Translated from Acta Phytoecologica Sinica, 2006, 30(1): 147–156 [译自: 植物生态学报]     

Investigation of the genetic diversity of Phytophthora capsici in China using a universal fluorescent labelling method

Phytophthora capsici is an important oomycete pathogen threatening the vegetable production in China, but very little is known about its population structure. The objective of the present study was to evaluate the genetic diversity of 49 P. capsici isolates obtained from 2007 to 2014 at nine provincial locations in China. Isolates were assessed for mating type, metalaxyl resistance and simple sequence repeat (SSR) genotype. Mating‐type analyses of the isolates showed that both mating types were present in all of the sampled production regions, and the mating‐type frequency in the total Chinese population did not deviate significantly from a 1:1 ratio. Responses of isolates to the fungicide metalaxyl indicated the presence of intermediate resistance to metalaxyl among the field population. A universal fluorescent labelling method was adapted in this study to improve the efficiency of SSR genotyping. Microsatellite genotyping of the isolates using seven SSR markers revealed 44 unique multilocus genotypes. Genetic analyses indicated the existence of two genetic clusters within Chinese P. capsici collection. Clonal reproduction may play a more prominent role in Yunnan Province, but non‐existence of repeated genotypes and existence of both mating types throughout all regions suggest outcrossing and sexual recombination likely play an important role in the overall epidemiology in China. Future studies would include expanded scale sampling at single regions over multiple years to better define the genetic diversity of P. capsici in China.     

Microsatellite variation within and among populations of Oryza officinalis (Poaceae), an endangered wild rice from China

Oryza officinalis Wall. ex Watt. is an agriculturally important but seriously endangered species of wild rice. To obtain more accurate estimates of population structure for improved conservation planning of the species, genetic variability at 14 microsatellite DNA loci was examined in population samples covering most of the species' range in China. Considerable genetic variability (overall Na = 1.886, P = 62%, HO = 0.056, HE = 0.216, and HS = 0.277) was detected at the 14 loci in 442 individuals of the 18 natural populations. The evaluation of partitioning of genetic variability (FST = 0.442) suggested high genetic differentiation among the Chinese O. officinalis populations. An overall value of Nm = 0.316 suggested limited gene flow occurred among the sampled populations. Most of the populations showed heterozygote deficits in tests of Hardy-Weinberg equilibrium and significantly positive FIS values. This could be due to some inbreeding occurring in this predominantly outcrossing species. For effective in situ conservation and restoration genetics, maintenance of significant historical processes is particularly important, including high outbreeding, considerable gene flow, and large population effective sizes. The high FST values detected among populations in this study are instructive for adopting a conservation plan that includes representative populations with the greatest genetic variation for either in situ conservation management or germplasm collection expeditions.     

Identifying the minimum number of microsatellite loci needed to assess population genetic structure: A case study in fly culturing

Small, isolated populations are constantly threatened by loss of genetic diversity due to drift. Such situations are found, for instance, in laboratory culturing. In guarding against diversity loss, monitoring of potential changes in population structure is paramount; this monitoring is most often achieved using microsatellite markers, which can be costly in terms of time and money when many loci are scored in large numbers of individuals. Here, we present a case study reducing the number of microsatellites to the minimum necessary to correctly detect the population structure of two Drosophila nigrosparsa populations. The number of loci was gradually reduced from 11 to 1, using the Allelic Richness (AR) and Private Allelic Richness (PAR) as criteria for locus removal. The effect of each reduction step was evaluated by the number of genetic clusters detectable from the data and by the allocation of individuals to the clusters; in the latter, excluding ambiguous individuals was tested to reduce the rate of incorrect assignments. We demonstrate that more than 95% of the individuals can still be correctly assigned when using eight loci and that the major population structure is still visible when using two highly polymorphic loci. The differences between sorting the loci by AR and PAR were negligible. The method presented here will most efficiently reduce genotyping costs when small sets of loci (“core sets”) for long-time use in large-scale population screenings are compiled.