Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.     

岛屿植物舟山新木姜子居群遗传多样性的RAPD分析

基于随机扩增多态 DNA(RAPD)方法分析了舟山群岛濒危植物舟山新木姜子 (N eolitsea sericea) 6个居群的遗传多样性及分化程度。 10条随机引物扩增出 84个可分析位点 ,多态位点百分比 (PPL)为 3 8.10 %。经 POPOGENE分析发现 ,舟山新木姜子居群平均水平的多态位点百分比 (PPL )为 2 3 .18% ,Nei' s基因多样度 (HE)为 0 .0 793 ,Shannon信息指数 (H )为 0 .12 0 1,与其它岛屿植物比较具有中等偏低水平的遗传多样性 ;岛屿各居群间遗传分化程度较高 (Gst=0 .3 646) ;地理距离与遗传距离之间具有显著相关性 (r=0 .7697,P=96.62 % ) ,岛屿隔离效应是导致居群间遗传分化的重要因素。结合居群遗传多样性及UPGMA聚类分析 ,推测普陀山岛舟山新木姜子部分个体可能为大猫岛迁入的后裔 ,而朱家尖岛舟山新木姜子则由人为移植自普陀山岛。基于舟山新木姜子的物种保护及资源利用 ,建议加强现有自然居群的就地保护 ,促进居群自然更新 ;建立种质资源库 ,收集不同岛屿的种源进行混合繁殖 ,促进基因交流 ;选育优良品系用于海岛植被恢复及园林观赏     

Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA (RAPD) markers

Hippophae rhamnoides is an outcrossing pioneer plant species with a severely fragmented distribution. Random amplified polymorphic DNA (RAPD) marker variation was analysed in 10 populations of ssp. rhamnoides and in one population of ssp. mongolica to estimate the amount and distribution of genetic variability. No less than 89.7% of the scorable markers were polymorphic, but few of these were fixed and populations consequently differed mainly by frequency variation of individual markers. Within-population gene diversity was somewhat low for an outcrossing plant species: 0.192 or 0.159 for ssp. rhamnoides , depending on whether it was based on all 156 polymorphic RAPDs or on only those 63 RAPDs that fulfilled the 3/ N criterion. Analysis of molecular variance applied to the ssp. rhamnoides showed only 15% between-population variability, indicating a relatively restricted population differentiation as expected in outcrossing species and shown in several other AMOVA studies. The tendency for island populations to be somewhat more differentiated, and to have less within-population diversity than mainland populations, may indicate an effect of population fragmentation. Genetic distance estimates among populations, obtained with and without pruning of RAPD loci on the basis of the 3/ N criterion, were generally in very good agreement. Cluster analyses and principal coordinate analyses showed populations of ssp. rhamnoides to be rather close, but quite isolated from the single ssp. mongolica population. Genetic and geographical distances between the ssp. rhamnoides populations were not associated, indicating that large-scale geographical and ecotypic differentiation was not reflected in the RAPD profiles.     

Genetic structure of the endangered plant Neolitsea sericea (Lauraceae) from the Zhoushan archipelago using RAPD markers

BACKGROUND AND AIMS: The Zhoushan archipelago is the largest archipelago in China. It separated from the mainland about 9000 years ago due to rising sea levels and climate change. Because of the long-term influences of human activities, the original forest vegetation on the large islands has been badly damaged and its plant diversity reduced. METHODS: Levels and patterns of genetic diversity in 114 individuals from six natural populations and four cultivated populations of the insular endangered plant Neolitsea sericea (Lauraceae) on the Zhoushan archipelago were assessed using random amplified polymorphic DNA (RAPD) markers. KEY RESULTS: A total of 99 discernible loci were obtained for all populations using ten primers, 50.5 % of which were polymorphic [percentage of polymorphic bands (PPB)=50.5 %]. Despite being a woody, long-lived, perennial, outcrossing and insect-pollinated plant, N. sericea exhibited low levels of genetic variation. The cultivated populations (PPB=18.9 %, HE=0.060, S=0.092) were genetically less diverse than the natural populations (PPB=23.1 %, HE=0.082, S=0.123). Based on analysis of molecular variance, a high degree of among-population differentiation was revealed for both natural (0.387) and cultivated populations (0.598). CONCLUSIONS: Removal of plants from the wild for horticulture purposes has eroded the level of genetic variation of N. sericea. Low levels of genetic diversity and a high degree of population differentiation indicate that management strategies should include conservation of natural habitats occupied by all six wild populations, and sampling of germplasm resources from multiple seed sources.     

Random amplified polymorphic DNA and amplified fragment length polymorphism assessment of genetic variation in Nicaraguan populations of Pinus oocarpa

Pinus oocarpa is the most widely distributed pine species of Mexico and Central America. The natural populations of Nicaragua have been affected by extensive human activities. As a consequence, their size has been reduced, and there is a serious threat to the development of mature woodland. Knowledge of population structures and the genetic diversity of the species is required for the design of sustainable use and conservation strategies. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation among 10 populations from three geographical regions of Nicaragua. Both markers revealed high levels of diversity in these populations. G(ST) values and analyses of molecular variance (AMOVA) found that most variation was within populations but there is still a significant differentiation between populations indicating that the populations sampled cannot be considered a single panmictic unit. The partitions created by AMOVA also showed that there was little differentiation between populations of different regions, although cluster analyses based on RAPDs and AFLPs indicated a closer relationship among most of the populations from a same geographical region. Management of P. oocarpa in Nicaragua should be aimed to maintain the high degree of genetic variation within individual populations that is still observed even in some of these highly degraded populations.     

Conservation of genetic diversity in the endangered plant Eriogonum ovalifolium var. vineum (Polygonaceae)

The purpose of his research was to describethe organization of genetic variation in thefederally endangered plant taxon Eriogonumovalifolium var. vineum using allozymes. Such information can help prioritize sites andmanagement choices for capturing andmaintaining genetic variation and can reducethe number of populations necessary to committo conservation, thus reducing costs andconflicts with competing land uses. Information on genetic diversity patterns alsoprovides insight into evolutionary anddemographic history of a taxon which canprovide means to assess future risk of erosionof diversity. Similar to other Eriogonumovalifolium varieties, E. ovalifoliumvar. vineum is diverse atboth the taxon (proportion of polymorphic loci[P] = 0.55 and alleles per locus [A] =5.45 [SD = 2.5]) and population (P = 0.56 [SD =0.11] and A = 2.68 [SD = 0.35]) levels. Gene diversity (H _e) withinpopulations averaged 0.19 (SD = 0.03). Wefound some evidence for limited clonalreproduction within populations. Populationswere moderately differentiated from one another(_S = 0.14) and showed moderatedeviations from Hardy-Weinberg equilibriumwithin populations (f = 0.14 and F =0.19). Mean Nei's genetic distance (D)among all pairs of populations was 0.02. Populations with high levels of multiplemeasures of genetic diversity, high levels ofdifferentiation, and low levels of apparentinbreeding are suggested as conservationpriorities.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis inAllium

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.     

Random amplified polymorphic DNA variation among remnant big bluestem (Andropogon gerardii vitman) populations from Arkansas' grand prairie

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity and genetic distinctiveness of Andropogon gerardii from remnant Arkansas prairies. Six oligonucleotide primers, which generated 37 RAPD bands, were used to analyse 30-32 plants from six Grand Prairie populations, Baker Prairie (Arkansas Ozarks), two Illinois prairies and two cultivars. Genetic diversity of the Arkansas remnants ranged from 82.7 to 99.3%, with 89% of the total genetic variation within and 11% among populations. The partitioning of genetic variation was consistent with that reported for other outcrossing perennial grasses, using the more conservative allozyme markers. Principal component analysis indicated a northern and southern association within Arkansas' Grand Prairie. Although there was no genetic structuring at the landscape level, the Illinois prairies and cultivars were different from all Arkansas prairies tested. There was significant within-population structuring in four of the seven Arkansas remnants, with a negative relationship between genetic similarity and geographical distance. The three nonstructured populations were from a linear railroad remnant, suggesting different population-level dynamics from nonlinear prairies. The results of this study indicated that small isolated remnant big bluestem populations were not genetically depauperate and that genetic relationships among populations could not be predicted solely on geographical proximity.     

随机扩增多态DNA（RAPD）标记用于丁香品种遗传分析及品种分类

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）标记分析了１５个丁香品种的ＤＮＡ扩增产物。研究选用了１６个随机引物,共扩增出９６条带,其中５５条带为可重复性条带,有价值条带大小多在５１７ｂｐ至１６３６ｂｐ之间。这些标记足以区分这些丁香品种。欧丁香（Ｓｙｒｉｎｇａｖｕｌｇａｒｉｓ）与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ间的相似系数为６１．５％,欧丁香与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４７．２％,Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４３．６％。结果表明,欧丁香与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ亲缘关系最近。应用ＲＡＰＤ资料分析讨论了一些品种的起源。ＲＡＰＤ技术为丁香品种分类鉴定提供了可靠方法。     

Allozyme variation in endangered Castanea pumila var. pumila

Allozyme genetic variation in 12 populations of the endangered Castanea pumila var. pumila (Allegheny chinkapin), sampled across the natural range of the species in the United States, was evaluated using 11 loci from seven enzyme systems. At the species level, the percentage of polymorphic loci (Ps) was 72.7 %, the mean number of alleles per locus (As) was 1.9, the mean number of alleles per polymorphic locus (APs) was 2.3, the effective number of alleles per locus (Aes) was 1.5 and the genetic diversity (Hes) was 0.296. At the population level, Pp = 49.2 %, Ap = 1.5, Aep = 1.4, APp = 2.1 and Hep = 0.21. Most of the allozyme variation (70 %) in C. pumila var. pumila occurred within populations. Wright's gene flow rate [Nm(W)] was as low as 0.57. Population differentiation along the species range was not detected. Populations of C. pumila var. pumila in Florida had the most variable levels of genetic diversity, but populations in Virginia and Mississippi also showed high levels. Based on the results of this study, conservation management strategies are discussed.     

Random amplified polymorphic DNA (RAPD) analysis reveals genetic relationships among the annual Cicer species

 Random amplified polymorphic DNA markers were used to distinguish between nine different Cicer taxa representing the cultivated chickpea and eight other related annual wild species. Of the 75 random10-mer primers tested, only 8 amplified genomic DNA across all the species. A total of 115 reproducibly scorable RAPD markers were generated, all except 1 polymorphic, and these were utilized to deduce genetic relationships among the annual Cicer species. Four distinct clusters were observed and represented C. arietinum, C. reticulatum and C. echinospermum in first cluster followed by C. chorassanicum and C. yamashitae in the second cluster, while C. pinnatifidum, C. judaicum and C. bijugum formed the third cluster. Cicer cuneatum did not cluster with any of the species and was most distantly placed from the cultivated species. Except for the placement of C. chorassanicum and C. yamashitae, deduced species’ relationships agreed with previous studies. In addition, species-diagnostic amplification products specific to all the nine species were identified. The results clearly demonstrate a methodology based on random-primed DNA amplification that can be used for studying Cicer phylogeny and chickpea improvement. Received: 27 July 1998 / Accepted: 5 August 1998     

Direct evidence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints

The relevance of using dominant random amplified polymorphic DNA (RAPD) fingerprints for estimating population differentiation was investigated when typically small population sample sizes were used. Haploid sexual tissues were first used to determine genotypes at RAPD loci for 75 eastern white pines ( Pinus strobus L.) representing five populations. Dominant RAPD fingerprints were then inferred from genotypic data for each individual at each locus, and gene diversity estimates from both sources of data were compared. Genotypic information at RAPD loci indicated little or no differentiation among populations, similar to allozyme loci. However, estimates of population differentiation derived from dominant RAPD fingerprints according to various common methods of analysis were generally inflated, especially when all fragments were considered. Simulations showed that an increase in loci sampling and population sample sizes did not significantly alleviate the biases observed.     

Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L. (Caryophyllaceae): a random amplified polymorphic DNA analysis

Metal-contaminated sites can occur naturally in serpentine outcrops or as consequence of anthropogenic activities, such as mining deposits, aerial fallout from smelters and industrial processes. Serpentine outcrops are characterized by high levels of nickel, cobalt and chromium and present a typical vegetation which includes endemisms and plants which also live in uncontaminated soils. These latter metal-tolerant populations provide the opportunity to investigate the first steps in the differentiation of plant populations under severe selection pressure and to select plants to be used in the phytoremediation of industrially contaminated soils. In this report eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in noncontaminated soil in central Italy, were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the pattern of genetic variation. The genetic diversity observed in populations at copper mine deposits was found to be at least as high as that of the neighbouring serpentine populations. Analysis of molecular variance (AMOVA) of the RAPD markers gave high statistical significance to the groupings of populations according: (i) with their geographical location; and (ii) with the metals present in the soil of origin (copper vs. nickel), indicating that RAPD markers detected a polymorphism related to the soil contamination by copper. Finally, two RAPD bands exclusive to copper-tolerant populations were identified.     

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.     

The genetic structure of the steppe plant Iris aphylla L. at the northern limit of its geographical range

Genetic diversity of Iris aphylla was examined in seven Polish populations located at the northern limit of the geographical range of the species. Using RAPD analysis, 491 DNA fragments were amplified, all of which were polymorphic. The peripheral populations were characterized by a low number of unique bands (one to three per population) and moderate values of genetic diversity parameters (percent of polymorphic loci = 30.6%, Shannon's index = 0.149 and Nei's gene diversity = 0.097, on average), which can be explained as being due to the recent expansion of the European biota and the extremely low frequency or absence of seed set and of juveniles, as well as limited pollen and seed dispersal. There was also no significant correlation between ecological properties (population size; number of flowering ramets and fruits) and genetic diversity parameters. Despite this fact, more fixed loci were noted in the smallest populations than were in the largest ones. All populations were significantly differentiated and geographically structured. Analysis of molecular variance between regions showed a low and nonsignificant value, but relatively high molecular variation was partitioned among and within populations. Only one group of south-eastern populations was structured according to isolation by distance pattern and neighbour-joining cluster analysis. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 245–255.     

Application of random amplified polymorphic DNA and inter-simple sequence repeat markers in the genus Crataegus

Hawthorn ( Crataegus spp.) has a long history as an ornamental and a source of medicine. We report the use of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers to determine genetic relationships in the genus Crataegus . Twenty-eight accessions, including eight species ( Crataegus pinnatifida , Crataegus bretschneideri , Crataegus maximowiczii , Crataegus kansuensis , Crataegus altaica , Crataegus songarica , Crataegus dahurica and Crataegus sanguinea ) and two botanical varieties ( C. pinnatifida var. major and C. maximowiczii var. ninganensis ) were analysed. Twelve RAPD primers reproducibly and strongly amplified 128 fragments of which 116 were polymorphic; similarly, 13 ISSR primers generated 127 products of which 119 were polymorphic. Dendrograms based on unweighted pair group method with arithmetic average analysis were constructed from both the RAPD and the ISSR data. Similarity coefficient based on RAPD and ISSR markers ranged from 0.22 to 0.98 and 0.23 to 0.98, respectively. The range in similarity coefficient indicated that the genus has a high level of genetic diversity. The Mantel test on the similarity matrices produced by RAPD and ISSR markers gave r  = 0.86, showing high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions. RAPD and ISSR appear to be reliable methods for the analysis of genetic relationships among hawthorns.     

The distribution of random amplified polymorphic DNA (RAPD) diversity amongst populations of Isotoma petraea (Lobeliaceae)

RAPDs were generated from plants of six populations of Isotoma petraea F. Muell. The species occurs on rock outcrops in southern and western Australia, with populations exhibiting different breeding systems, including complete autogamy, varying levels of outbreeding and complex hybridity. Non-metric multidimensional scaling (nMDS) analysis of the random amplified polymorphic DNA (RAPD) data set clearly resolved all populations. The Pigeon Rock population, which is home to both complex hybrid and structural homozygote plants, was divided into those two groups by the nMDS analysis. There was little diversity in highly autogamous populations, but levels were higher in the outbred Yackeyackine population. All complex hybrid populations and plants possessed numerous genetic system-specific RAPDs, some of which were shown to be held in fixed heterozygosity. Estimating G _ST using RAPDs has been problematical due to their dominance, and analytical methods usually rely on knowledge of the selfing rate or assume Hardy–Weinberg equilibrium. This assumption does not hold when populations exhibit fixed heterozygosity, and an alternative method, Shannon's Index, was used to partition genetic diversity. The distribution of genetic diversity fit expectations for an inbreeding species, with most of the variation (87.5%) occurring between populations. This compares to an average RAPD-based G _ST of 59.6% for inbreeding species generally and 15.5% for outbreeding species.     

Population genetic structure of two rare tree species (Colubrina oppositifolia and Alphitonia ponderosa,Rhamnaceae) from Hawaiian dry and mesic forests using random amplified polymorphic DNA markers

Hawaiian dry and mesic forests contain an increasingly rare assemblage of species due to habitat destruction, invasive alien weeds and exotic pests. Two rare Rhamnaceae species in these ecosystems, Colubrina oppositifolia and Alphitonia ponderosa, were examined using random amplified polymorphic DNA (RAPD) markers to determine the genetic structure of the populations and the amount of variation relative to other native Hawaiian species. Relative variation is lower than with other Hawaiian species, although this is probably not a consequence of genetic bottleneck. Larger populations of both species contain the highest levels of genetic diversity and smaller populations generally the least as determined by number of polymorphic loci, estimated heterozygosity, and Shannon's index of genetic diversity. Populations on separate islands were readily discernible for both species as were two populations of C. oppositifolia on Hawai'i island (North and South Kona populations). Substructure among Kaua'i subpopulations of A. ponderosa that were ecologically separated was also evident. Although population diversity is thought to have remained at predisturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Recovery efforts must focus on control of alien species if these and other endemic dry and mesic forest species are to persist.     

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.