Genetic differentiation among three neighboring Brazil-cherry (Eugenia uniflora L.) populations within the Brazilian Atlantic rain forest

Eugenia uniflora L. (pitanga) is widely distributed in tropical areas. It is present in coastal vegetation from Ceara, in northeastern Brazil, to Rio Grande do Sul, at the southern tip of the country. Eugenia uniflora is of ecological importance, both as colonizing species on disturbed land and as food supplier for a wide variety of insects, birds and mammals. Pitanga plays a role in the maintenance of shrubby coastal ecosystems, especially at disturbed sites, and in 'restinga' ecosystems, at the interface between low forest and strand vegetation. To investigate the genetic diversity residing within the species, three neighboring populations at a distance of less than 24 km from each other, with varying degrees of human impact, were studied. The level of genetic diversity within and between populations was assessed with amplified fragment length polymorphism (AFLP) methodology. A total of 532 AFLP markers were analyzed in 66 individual trees. The polymorphism level varied from 61.2 to 96% depending on the primer combination used. Intra- and inter-population genetic diversity analysis showed that more than 88% of the variation resided within the populations, with a G_st of 0.123. Nevertheless, using neighbor joining (NJ) and principal component analysis (PCA), on the genetic distance (GD) data, permitted the three analyzed populations to be differentiated.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Microsatellite variation and assessment of genetic structure in tea tree (Melaleuca alternifolia– Myrtaceae)

Analysis of five microsatellite loci in 500 Melaleuca alternifolia individuals produced 98 alleles that were useful for population genetic studies. Considerable levels of observed heterozygosity were recorded (HO = 0.724), with approximately 90% of the variability being detected within populations. A low level of selfing (14%) was suggested to be the principal cause of excess homozygosity in a number of populations (overall FIS = 0.073). This study showed low levels of inbreeding in certain populations as well as a significant isolation-by-distance model. Only two groups of populations (Queensland and New South Wales) constituted different genetic provenances as a result of geographical isolation. The M. alternifolia data suggest that microsatellite loci did not always arise by a stepwise mutation process but that larger jumps in allele size may be involved in their evolution.     

AFLP分子标记技术在昆虫学研究中的应用

AFLP分子标记技术是一种建立在PCR技术和RFLP标记基础上的新的DNA指纹分析技术 ,具有多态性丰富、结果稳定可靠、重复性好、所需DNA量少、可以在不知道基因组序列的情况下进行研究等特点 ,现已广泛用于构建遗传图谱、遗传多样性研究、系统进化及分类学、遗传育种和品质鉴定以及基因定位等方面。该文介绍了AFLP标记技术的原理以及在昆虫学研究中的应用。     

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii ( Plumbaginaceae ) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii . Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.     

Patterns of genetic diversity in southern and southeastern Araucaria angustifolia (Bert.) O. Kuntze relict populations

Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five "genetic clusters" (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three "genetic clusters". These results are in agreement with the pattern of geographic distribution of the studied populations.     

Extensive spatial genetic structure revealed by AFLP but not SSR molecular markers in the wind-pollinated tree, Fagus sylvatica

Studies of fine-scale spatial genetic structure (SGS) in wind-pollinated trees have shown that SGS is generally weak and extends over relatively short distances (less than 30-40 m) from individual trees. However, recent simulations have shown that detection of SGS is heavily dependent on both the choice of molecular markers and the strategy used to sample the studied population. Published studies may not always have used sufficient markers and/or individuals for the accurate estimation of SGS. To assess the extent of SGS within a population of the wind-pollinated tree Fagus sylvatica, we genotyped 200 trees at six microsatellite or simple sequence repeat (SSR) loci and 250 amplified fragment length polymorphisms (AFLP) and conducted spatial analyses of pairwise kinship coefficients. We re-sampled our data set over individuals and over loci to determine the effect of reducing the sample size and number of loci used for SGS estimation. We found that SGS estimated from AFLP markers extended nearly four times further than has been estimated before using other molecular markers in this species, indicating a persistent effect of restricted gene flow at small spatial scales. However, our SSR-based estimate was in agreement with other published studies. Spatial genetic structure in F. sylvatica and similar wind-pollinated trees may therefore be substantially larger than has been estimated previously. Although 100-150 AFLP loci and 150-200 individuals appear sufficient for adequately estimating SGS in our analysis, 150-200 individuals and six SSR loci may still be too few to provide a good estimation of SGS in this species.     

Development of polymorphic microsatellite markers for Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

Dalbergia nigra is an endangered tree restricted to the Brazilian Atlantic Forest. Ten polymorphic microsatellite loci were isolated and characterized in 47 trees from two populations. The total number of alleles per locus ranged from three to 12 alleles. The levels of observed and expected heterozygosities ranged from 0.304 to 0.740 and from 0.278 to 0.872, respectively. Significant deviations from Hardy-Weinberg were detected for only three loci in each population. No pair of loci exhibited significant linkage disequilibrium. These microsatellites provide an efficient tool to investigate genetic structure in forest remnants with the purpose of conservation of this species.     

Population genetic structure of the South American species Hypochaeris lutea (Asteraceae)

The genus Hypochaeris has a recent evolutionary history caused by long‐distance dispersal in conjunction with adaptive radiation in the South American continent. Hypochaeris lutea is a perennial herb that grows mostly at altitudes of around 1000 m in cold swamps of the southern regions of Brazil. We investigated the amplified fragment length polymorphism (AFLP) in 270 individuals representing 11 Brazilian populations of H. lutea to elucidate the population genetic structure of this species. The frequencies of polymorphic loci and gene diversity ranged from 83.42% to 91.66% and from 0.26 to 0.34, respectively. Analysis of molecular variance revealed that most of the genetic variability was found within (76.67%) rather than among (23.3%) populations, agreeing with the pattern of genetic distribution within and among populations observed in other allogamous species of Hypochaeris. A Mantel test showed no correlation between genetic and geographic distances when all populations were considered. Simulations performed using a Bayesian approach consistently identified two clusters with different admixture proportions of individuals, as also revealed by a UPGMA dendrogram of populations. The pattern of genetic structure observed in H. lutea is consistent with a process of successive colonization events by long‐distance dispersal resembling the rapid and recent radiation that has been proposed to explain the origin of the South American species of Hypochaeris.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened species.     

Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88.5%) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84.8%), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on phiST was moderate (Nm=1.395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species.     

中国石榴栽培群体遗传多样性的荧光AFLP分析

以中国山东、安徽、陕西、河南、云南和新疆6个栽培石榴群体的85个品种类型为试材,利用荧光标记AFLP对中国石榴群体遗传多样性进行了研究。结果表明：8对引物组合在种级水平扩增的多态性位点数范围从135～185个不等,平均为158.25个,多态位点百分比范围为62.5%～86.11%,平均为73.26%,说明中国石榴品种遗传多样性较为丰富;石榴品种种级水平遗传多样性大于群体水平,6个群体的遗传多样性依次为河南群体〉新疆群体〉陕西群体〉安徽群体〉山东群体〉云南群体,并且具有显著性差异;群体间的遗传分化系数GST为0.2018,说明石榴遗传变异主要存在群体内,群体间的遗传变异占总变异的20.18%,根据基因分化系数,测得的基因流Nm为1.9027,说明中国石榴群体间存在适当的基因交流;UPGMA聚类分析结果表明,同一群体的大部分品种都聚在一起,但同时存在部分基因交流。所有遗传参数表明,中国石榴栽培品种遗传多样性较为丰富,其中河南群体遗传多样性显著高于其他群体,在中国石榴品种选育中具有更为广阔的应用前景。     

Amplified Fragment Length Polymorphism (AFLP) as a source of genetic markers for red algae

A recent PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), was successfully applied to the red alga Chondrus crispus Stackh. This is apparently the first account to describe the application of AFLP methodology to an alga. Six isolates of C. crispus were analyzed by AFLP. A total of twenty-five primer pairs were screened and six primer pairs were selected for further investigation. Both conservative and variable markers were identified within and between populations; some markers were unique to individuals. As such, AFLP should prove useful as a source of genetic markers in algae for applications as diverse as genome mapping to population genetic investigations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L.) cultivars using AFLP

Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.     

Genetic population structure, fitness variation and the importance of population history in remnant populations of the endangered plant Silene chlorantha (Willd.) Ehrh. (Caryophyllaceae)

Habitat fragmentation can lead to a decline of genetic diversity, a potential risk for the survival of natural populations. Fragmented populations can become highly differentiated due to reduced gene flow and genetic drift. A decline in number of individuals can result in lower reproductive fitness due to inbreeding effects. We investigated genetic variation within and between 11 populations of the rare and endangered plant Silene chlorantha in northeastern Germany to support conservation strategies. Genetic diversity was evaluated using AFLP techniques and the results were correlated to fitness traits. Fitness evaluation in nature and in a common garden approach was conducted. Our analysis revealed population differentiation was high and within population genetic diversity was intermediate. A clear population structure was supported by a Bayesian approach, AMOVA and neighbour-joining analysis. No correlation between genetic and geographic distance was found. Our results indicate that patterns of population differentiation were mainly caused by temporal and/or spatial isolation and genetic drift. The fitness evaluation revealed that pollinator limitation and habitat quality seem, at present, to be more important to reproductive fitness than genetic diversity by itself. Populations of S. chlorantha with low genetic diversity have the potential to increase in individual number if habitat conditions improve. This was detected in a single large population in the investigation area, which was formerly affected by bottleneck effects.     

Habitat distribution influences dispersal and fine-scale genetic population structure of eastern foxsnakes (Mintonius gloydi) across a fragmented landscape

Dispersal is a fundamental attribute of species in nature and shapes population dynamics, evolutionary trajectories and genetic variation across spatial and temporal scales. It is increasingly clear that landscape features have large impacts on dispersal patterns. Thus, understanding how individuals and species move through landscapes is essential for predicting impacts of landscape alterations. Information on dispersal patterns, however, is lacking for many taxa, particularly reptiles. Eastern foxsnakes (Mintoinus gloydi) are marsh and prairie specialists that avoid agricultural fields, but they have persisted across a fragmented region in southwestern Ontario and northern Ohio. Here, we combined habitat suitability modelling with population genetic analyses to infer how foxsnakes disperse through a habitat mosaic of natural and altered landscape features. Boundary regions between the eight genetic clusters, identified through assignment tests, were comprised of low suitability habitat (e.g. agricultural fields). Island populations were grouped into a single genetic cluster, and comparatively low F(ST) values between island and mainland populations suggest open water presents less of a barrier than nonsuitable terrestrial habitat. Isolation by resistance and least-cost path analysis produced similar results with matrices of pairwise individual genetic distance significantly more correlated to matrices of resistance values derived from habitat suitability than models with an undifferentiated landscape. Spatial autocorrelation results matched better with assignment results when incorporating resistance values rather than straight-line distances. All analyses used in our study produced similar results suggesting that habitat degradation limits dispersal for foxsnakes, which has had a strong effect on the genetic population structure across this region.     

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab （Eriocheir sinensis）

Chinese mitten crab (Eriocheir sinensis) has higher commercial value as food source than any other species of Eriocheir in China.To evaluate the germplasm resources and characterize the genetic diversity and population structure of the crabs in different water systems,two stocks and two farming populations were assessed with 25 polymorphic microsallite loci available in public GenBank.Basic statistics showed that the average observed heterozygosity (Ho) amongst populations ranged from 0.5789 to 0.6824.However,a remarkable presence of inbreeding and heterozygote deficiencies were observed.To analyze population structure,pairwise FST coefficients explained only ～10.3% variability from the subdivision of mitten crab populations,the remaining variability stems from the subdivision within subpopulations.Although the four populations had slight differentiation,different allelic frequencies resulted in distinct population structures.Two stocks and one farming population were clustered together to the phylogenetic branch of Yangtze crab,with an approximate membership of 95%.Whereas,another fanning population was clustered singly to the phylogenetic branch of the Liaohe crab,with a membership of 97.1%.The tests for individual admixture showed that Yangtze crab had probably been contaminated with individuals from other water systems.Genetic relationships between populations also supported the conclusion that Yangtze crab and Liaohe crab had different gene pools in spite of the origins of the same species.     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

Detection and analysis of genetic variation in Hordeum spontaneum populations from Israel using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to analyse genetic diversity within and between Hordeum spontaneum populations sampled from Israel. Nei's index of genetic differentiation was used to partition diversity into within and between population components. Fifty-seven per cent of the variation detected was partitioned within 10 H. spontaneum populations. Using principal component and multiple regression analysis, part of the variation detected between populations was seen to be associated with certain ecogeographical factors. Fifty-eight per cent of the distribution of the phenotypic frequencies of three RAPD phenotypes detected using a single primer in 20 H. spontaneum populations could be accounted for by four ecogeographical variables, suggesting adaptive variation at certain RAPD loci.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.