Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)

A method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy-Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub-population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.     

RAPD analysis of natural populations of Acanthopanax brachypus

Random amplified Polymorphic DNA(RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population.The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan an,Shanxi Province.Through more than 2,000 PCRs,deep-going RAPD analysis was carried out on DNA samples from 49 inviduals.The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2%,18.6% and 5.4%;the average genetic distances within population,0.055,0.026 and 0.008;the average genetic distances between populations (I-II),(I-III) and (II-III),0.105,0.096 and 0.060.The genetic diversity of A.brachypus within and between populations was found,for the first time,to be rather poor,thus revealing innate factors as the cause contributing to its endangered status.In addition,our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.     

长筒石蒜居群遗传多样性RAPD分析

利用RAPD标记对长筒石蒜3个居群的遗传多样性及分化程度进行了研究。12条随机引物扩增出94个可分析位点,多态位点比率(PPB)为65.96%,表明长筒石蒜具有比较高的遗传多样性。经POP-GENE32分析表明:Nei’s基因多样性指数(h)为0.1897,香农多样性指数(Ⅰ)为0.2945,基因分化系数(GST)为0.1191,基因流(Nm)为3.6980。经WINAMOVA分析表明:居群内遗传变异占71.75%,而居群间只占28.25%。遗传多样性分析表明,各居群的遗传多样性水平由高到低为琅琊山居群>宝华山居群>盱眙居群。遗传分化表明:长筒石蒜各居群间遗传分化程度较低;大部分遗传变异存在于居群内部,表明其具有较强的进化潜力,自然情况下不会处于濒危状态,野生种质资源的破坏,主要来自于人为干扰。     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifica tions showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.     

Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae)

Genetic variation among 12 populations of the American chestnut (Castanea dentata) was investigated. Population genetic parameters estimated from allozyme variation suggest that C. dentata at both the population and species level has narrow genetic diversity as compared to other species in the genus. Average expected heterozygosity was relatively low for the population collected in the Black Rock Mountain State Park, Georgia (He = 0.096 +/- 0.035), and high for the population in east central Alabama (He = 0.196 +/- 0.048). Partitioning of the genetic diversity based on 18 isozyme loci showed that ~10% of the allozyme diversity resided among populations. Cluster analysis using unweighted pair-group method using arithmetric averages of Rogers' genetic distance and principal components analysis based on allele frequencies of both isozyme and RAPD loci revealed four groups: the southernmost population, south-central Appalachian populations, north-central Appalachian populations, and northern Appalachian populations. Based on results presented in this study, a conservation strategy and several recommendations related to the backcross breeding aimed at restoring C. dentata are discussed.     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

华山新麦草自然居群沿海拔梯度的遗传分化

华山新麦草为我国特有种 ,仅分布于陕西华山 ,是国家一级珍稀濒危植物和急需保护的农作物野生亲缘种。应用 RAPD技术 ,选取 8条引物对华山新麦草自然分布区的 3个山峪(种群 ) 1 1个样方 (亚居群 ) 79个华山新麦草个体的总 DNA进行随机扩增 ,共得到 65个RAPD位点。统计分析表明 ,在华山的 3个山峪 (居群 )中 ,黄埔峪 (居群 )与其它 2个山峪 (居群 )发生较显著的遗传分化 ;华山峪 6个亚居群的个体平均 RAPD位点数有随海拔的升高而下降的趋势 ;6个亚居群间的相似性系数也有随海拔的升高而下降的趋势 ;华山峪的高海拔亚居群和低海拔亚居群间表现出明显差异。主成分分析结果进一步证明了黄埔峪居群与华山峪居群和仙峪居群间、以及华山峪的高海拔亚居群与低海拔亚居群间已发生一定程度的分化。研究结果暗示海拔差异是导致华山新麦草自然居群遗传分化的主要因素 ,海拔差异造成的有限的基因流可能才是影响居群和亚居群遗传分化的主要因子 ,而亚居群内遗传变异程度则与该亚居群的所处的特定生境有关     

利用RAPD对稻蝗属昆虫亲缘关系的研究

通过20个随机引物的PCR扩增,得到了日本主要稻蝗的随机扩增多态性DNA（RAPD）图谱,根据扩增结果,计算了种间相似系数和遗传距离,建立了UPGMA系统树。结果表明,分布没有重叠、种间容易交配、能产生杂种的中华稻蝗台湾亚种与小翅稻蝗的亲缘关系最近;分布重叠的日本稻蝗与中华稻蝗台湾亚种、日本稻蝗和小翅稻蝗的亲缘关系较近。小稻蝗与其它3种稻蝗的亲缘关系较远。     

Population differentiation in randomly amplified polymorphic DNA of red-cockaded woodpeckers Picoides borealis

Random amplified polymorphic DNA (RAPD) phenotypes generated by 13 primers were scored for 101 individuals in 14 populations of the endangered red-cockaded woodpecker Picoides borealis. Although no population-specific markers were found, the frequencies of several markers differed significantly among populations. Application of the recently developedamova method (analysis of molecular variance; Excoffier, Smouse & Quattro 1992) showed that more than 90% of phenotypic variance occurred among individuals within populations; of the remaining variance, half was attributed among groups of geographically adjacent populations and half among populations within those groups. The statistical significance of these patterns was supported by Monte Carlo sampling simulations and permutation tests. Estimation of allele frequencies from phenotypes provided somewhat weaker evidence for population structure, although among-population variance in allele frequencies was detectable (F_st= 0.19; x²₁₆₉= 509.3, P < 0.0001). Upgma cluster analyses based on Rogers' (1972) genetic distance revealed grouping of some geographically proximate populations. A Mantel test indicated a positive (r = 0.16), although not significant, correlation between geographic and genetic distances. We compared a subset of our RAPD data with data from a previous study that used allozymes (Stangel, Lennartz & Smith 1992). RAPD (n= 75) and allozyme (n= 245) results based on samples from the same ten populations showed similar patterns. Our study indicates that RAPDs can be helpful in differentiating populations at the phenotypic level even when small sample sizes, estimation bias, and inability to test for Hardy-Weinberg equilibrium complicate the genotypic interpretation. Lack of large differences among populations of red-cockaded woodpeckers may allow flexibility in overpopulation translocations, provided factors such as habitat preference, latitudinal direction of translocation, and status of donor populations are considered.     

A preliminary examination of genetic variation in a peripheral population of Blanding's turtle, Emydoidea blandingii

Random amplified polymorphic DNA (RAPD) was used to compare the Nova Scotia population of Blanding's turtle (Emydoidea blandingii) with several populations from the species' main range. The Nova Scotia population is believed to have been isolated from the main range for 4000-8000 years. Cluster analysis using a neighbour-joining algorithm produced a dendrogram showing the Nova Scotia population clustering separately from those populations in the main range. Analysis of molecular variance shows 34.28% of total variance to be accounted for between the Nova Scotia population and populations in the main range. While this study is preliminary, the results suggest that the Nova Scotia population of Blanding's turtle may be important to the maintenance of genetic diversity in the species.     

Evidence for the hybrid origin of Nuphar xrubrodisca (Nymphaeaceae)

Plants intermediate in appearance between Nuphar microphyllaand N. variegata (Nymphaeaceae) have long been assumed to bethe result of hybridization. The evidence for this is based primarilyon field observations of morphology, poor fruit production, closegeographical proximity of presumed parent species, and limited pollensterility data. Fertile populations of the same plants have also beendocumented. We employed multivariate analyses of morphology, pollenfertility studies, and random amplified polymorphic DNA (RAPD) markersto test the hypothesis that Nuphar × rubrodiscarepresents a natural interspecific hybrid between N.microphylla and N. variegata. Examination of 15morphological characters demonstrated the intermediacy of N.× rubrodisca between N. microphylla and N.variegata, and the pollen data revealed a markedly lower meanpollen viability in N. × rubrodisca (23%)compared to the other two species (91 and 86%, respectively). Eight 10-mer primers produced 13 species-specific RAPD markers forN. microphylla and nine for N. variegata, with all 22markers present in N. × rubrodisca. The datafrom RAPDs are concordant with morphology in implicating N.microphylla and N. variegata as parents of N.×rubrodisca.     

Patterns of genetic variation detected by RAPDs suggest a single origin with subsequent mutations and long-distance dispersal in the apomictic fern Dryopteris remota (Dryopteridaceae)

Debates on speciation processes in pteridophytes have revived. In order to study the evolutionary origin of an apomictic fern species, we investigated the genetic variation in the strictly agamosporous Dryopteris remota. We determined the genotypes of 22 individuals from many different locations within the species' European distribution and of 20 individuals from a Swiss population. A previous study on isozyme variation showed no intraspecific genetic variation in a similar sample set (Schneller and Holderegger, 1994, American Fern Journal 84: 94-98). In contrast to this, four out of 12 random amplified polymorphic DNA (RAPD) primers tested revealed low genetic diversity among individuals of D. remota from different locations. Intrapopulational genetic variation was also very low, but in the single population studied, a unique multiband genotype could be detected. The geographic distribution of genetic variation found in D. remota was best explained by the assumption of a single origin, the accumulation of somatic mutations during spread, and occasional, but effective, events of dispersal over large distances. The present study thus stresses the importance of long-distance dispersal in evolutionary processes and biogeography of ferns.     

Analysis of the Genetic Structure of Sclerotinia sclerotiorum (Lib.) de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and S hannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups,among populations within groups, and within populations were -0.96%, 51.48%, and 49.47%, respectively.The genetic differentiations among and within populations were highly significant (P ＜ 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm)was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.     

Identification and Assessing the Cultivars of Laminaria Lamx. （Phaeophyceae） with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars ofLaminariajaponica Aresch. used for breeding in China fell into one cluster. L.japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

Molecular differentiation of two morphological variants of Gracilaria salicornia

Xia in 1986 combined Gracilaria salicornia, G. canaliculata (G. crassa), G. cacalia and G. minor into one species: G.salicornia. Two morphological variants of G. salicornia were collectedfrom different localities in Malaysia. Variant A collected from Morib,Selangor grew on the roots of Avicennia. The samples showed absenceof main axis; segmented constrictions throughout; cylindrical or slightlycompressed thalli. Variant B was collected from the mudflats of TanjungTuan, growing on rocks, coral or forming mats on the mud. Plants showedabsence of main axis; segments were not constricted throughout the plant(if present only slightly articulated at the upper part), branching wasdichotomous or irregular; cylindrical or slightly compressed thalli. Thetechnique of Random Amplified Polymorphic DNA analysis (RAPD) wasused to investigate molecular characteristics of the two variants. Out ofsixty Operon primers that were screened, four primers, OPA 1, OPA 10,OPA 11 and OPK 7 were able to give polymorphism. The fingerprintsgenerated were stable and reproducible on repeated analysis. The DNAfingerprints generated were visually analysed and clustering analysis wascarried out using GelCompar 4.0. The matrix of similarities was based onthe Dice coefficients (S_D) and the cluster analysis was carried outusing the unweighted pair group method using arithmetic averages(UPGMA). DNA analysis showed that two primers (OPA 01, CAGGCCCTTC and OPK 07, AGCGAGCAAG) were able to differentiate the two variants.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA (RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China. A captive population has been established in the Beijing Zoo since 1999. In order to determine the kinship of the offsprings in 2001, randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo. To amplify the genomic DNA of each individual, 53 arbitrary primers were selected. The results of amplifications showed that 14 primers had clear and distinct RAPD patterns. Totally, 226 amplified fragments were generated by RAPD in this study. Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male). This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2005, 41(5): 507–509 [译自: 北京师范大学学报 (自然科学版), 2005, 41(5): 507–509]     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

The effects of regeneration by fragmentation upon clonal diversity in the tropical forest shrub Poikilacanthus macranthus: random amplified polymorphic DNA (RAPD) results

Poikilacanthus macranthus (Acanthaceae), like many shrubs in the neotropical cloud forest of Monteverde, Costa Rica, is capable of regeneration through stem and twig fragments. Our field sampling indicated that reproduction in P. macranthus is primarily through fragmentation in the Monteverde region. Random amplified polymorphic DNAs (RAPDs) were employed to examine clonal diversity in four populations of P. macranthus , and two primers yielded 11 variable markers that were used to identify genets. Sixty-eight multilocus genotypes were identified out of 277 plants sampled. Clonal diversity values were higher than the average for clonal plant species, as the number of genets per ramet was 0.25, while the mean D value was 0.87 and the mean E value was 0.84.     

Ex situ cultivation affects genetic structure and diversity in arable plants

Worldwide, botanical gardens cultivate around 80,000 taxa, corresponding to approximately one‐quarter of all vascular plants. Most cultivated taxa are, however, held in a small number of collections, and mostly only in small populations. Lack of genetic exchange and stochastic processes in small populations make them susceptible to detrimental genetic effects, which should be most severe in annual species, as sowing cycles are often short. In order to assess whether ex situ cultivation affects genetic diversity of annuals, five annual arable species with similar breeding systems were assessed with 42 in situ populations being compared to 20 ex situ populations using a random amplified polymorphic DNA (RAPD) analysis approach. Population sizes tended to be lower under ex situ cultivation and levels of genetic diversity also tended to be lower in four of the five species, with differences being significant in only two. Ex situ populations showed incomplete representation of alleles found in the wild. The duration of cultivation did not indicate any effect on genetic diversity. This implies that cultivation strategies resulted in different genetic structures in the garden populations. Although not unequivocally pronounced, differences nonetheless imply that conservation strategies in the involved gardens may need improvement. One option is cold storage of seeds, a practice that is not currently followed in the studied ex situ collections. This may reflect that the respective gardens focus on displaying living plant populations.