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.     

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.     

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.     

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.     

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 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.     

Random amplified polymorphic DNA in cattle and sheep: application for detecting genetic variation

The present study investigated the use of the random amplified polymorphic DNA (RAPD) method to detect genetic variation in cattle and sheep. The animals studied consisted of samples from five Finnish cattle breeds: native Eastern (18 animals), Northern (24), Western Finncattle (24), Finnish Ayrshire (24), and Finnish Friesian (18); as well as a white (6 animals) and a grey (9) colour type of Finnsheep. The cattle and sheep populations were analysed with 11 and 13 RAPD primers demonstrating the most repeatable amplification pattern. Two out of ten RAPD fragments tested by cross hybridization showed homology between the two species. The RAPD method did not prove efficient for finding new polymorphisms in either species, because we found only three polymorphic RAPD markers for cattle and seven markers for sheep with different allele frequencies between the breeds. Although there is a greater presence of polymorphic RAPD markers in sheep, according to the similarity indices the sheep populations showed a higher degree of homogeneity than the cattle breeds. However, the interbreed and intrabreed similarity indices for cattle did not suggest any significant differentiation of the Finnish breeds, contrary to earlier results based on blood group and protein polymorphism.     

Random amplified polymorphic DNA variation in the eggplant,Solanum melongena L. (Solanaceae)

RAPD analysis was carried out on 52 accessions of Solanum melongena (eggplant) and related weedy forms known as insanum. Twenty-two primers amplified 130 fragments. Solanum melongena exhibited 117 of the fragments, all of which were also present in insanum. Insanum displayed an additional 13 fragments not found in S. melongena. Overall, the insanum accessions were more diverse than those of S. melongena. The calculated similarity between them was 0.947. The RAPD results were closely concordant with the results of an electrophoretic isozyme survey performed on the same accessions. The concordance of the results shows that even though S. melongena and insanum are highly diverse morphologically, it is no longer appropriate to distinguish them taxonomically.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh

RAPD markers were used to assess genetic fidelity of 23 micropropagated plants of a single clone (L34) of Populus deltoides. Eleven arbitrary 10-base primers were successfully used to amplify DNA from in vivo and in vitro material. Of these, 5 distinguished a total of 13 polymorphisms common across 6 micropropagated plants. Apart from these 6 plants, the amplification products were monomorphic across all the micropropagated plants, the mother plant and 4 additional field-grown control plants. Our results show that RAPD markers can be used to gain rapid and precise information about genetic similarities or dissimilarities in micropropagation systems that might not be so easily evident from other commonly used techniques.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst.

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Extending a RFLP-based genetic map of rye using random amplified polymorphic DNA (RAPD) and isozyme markers

RFLP-based genetic map of rye, developed previously using a cross of lines DS2×RXL10 (F₂ generation), was extended with 69 RAPD and 12 isozyme markers. The actual map contains 282 markers dispersed on all seven chromosomes and spans a distance of 1,140 cM. The efficiency of mapping RAPD markers was close to ten loci per 100-screened arbitrary primers. A strong selection of polymorphic, intensive and reproducible fragments was necessary to reveal individual marker loci that could be assigned to rye chromosomes. Newly mapped markers cover a substantial part of the rye genome and constitute a valuable tool suitable for map saturation, marker-aided selection and phenetic studies. A specific nomenclature for the RAPD loci mapped on individual rye chromosomes, which could be helpful in managing of accumulating data, is proposed. Received: 8 May 2000 / Accepted: 17 October 2000     

Random amplified polymorphic DNA (RAPD) analysis reveals extensive natural chimerism in a marine protochordate

Random amplified polymorphic DNA (RAPD) analysis was applied to individual modules (zooids) of a colonial ascidian to investigate the presence and extent of chimerism, the parabiotic association of different genetic entities. The technique proved to be rapid and efficient for distinguishing different genotypes present in a colony, and revealed genetic mosaicism in wild material, as well as in laboratory cultures following planned fusion. Approximately one-third of colonies in the natural population studied possessed multiple genotypes, presumably as the result of fusion of different colonies. Furthermore, individual zooids of different genetic origin often intermingled after colony fusion, spreading each genotype throughout a larger total area.     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

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.     

Lack of genetic variability in the rare and endangered Limonium cavanillesii (Plumbaginaceae) using RAPD markers

Limonium cavanillesii is an extremely endangered plant species endemic to the east Mediterranean region of Spain. Regarded as extinct for several years, the recent discovery of a small population (only 29 individuals) has prompted the adoption of measures for its conservation by official agencies. As part of this effort, we have analysed genetic variation in this population by means of random amplified polymorphic DNA (RAPDs). The analysis of 29 individuals with 11 different primers produced 131 monomorphic bands. To our knowledge, this is the lowest level of genetic variation detected in plants using RAPD markers. This result could be explained both by the apomictic reproductive system of this species and by the passage through a severe bottleneck in recent times, after which there has been no chance for mutation to restore detectable genetic variation.