RAPD Profiling in Detecting Genetic Variation in Endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA (RAPD) markers were used to measure genetic diversity of Coelonema draboides (Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P.R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% (161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.     

Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Φ _ST comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory θ_B) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.     

Consistency of population genetics parameters estimated from isozyme and RAPDs dataset in species of genus <Emphasis Type="Italic">Prosopis</Emphasis> (Leguminosae,Mimosoideae)

Genetic variability, population structure and differentiation among 17 populations of 5 species and 2 natural interspecific hybrids of section Algarobia of genus Prosopis were analyzed from data of 23 isozyme and 28 RAPD loci. Both markers indicated that the studied populations are highly variable. P. alba populations in average showed lower values of genetic variability estimates from isozyme data, but this trend was not observed for RAPD markers. The hierarchical analyses of the distribution of genetic variability showed that the highest proportion of variation occurred within populations, the differentiation among species was intermediate and the lowest component was observed among populations within species. The consistency between results from both dataset implies that they are not biased and reflect the actual genetic structure of the populations analyzed. The matrices of Euclidean distances obtained from the two sets of markers were highly correlated according to Mantel test. In both cases the corresponding phenogram and MDS plot tended to cluster conspecific populations while hybrid populations were not intermediate between putative parents. Some disagreements between isozyme and RAPD phenograms were observed mainly in the affinities of hybrid populations. Such inconsistencies might result from reticular rather than dichotomic evolutionary relationships. The phenetic associations retrieved gave no support to the division of the section Algarobia into series.     

The genetic structure of taro: a comparison of RAPD and isozyme markers

Germplasm characterization and evolutionary process in viable populations are important links between the conservation and utilization of plant genetic resources. Here, an investigation is made, based on molecular and biochemical techniques for assessing and exploiting the genetic variability in germplasm characterization of taro, which would be useful in plant breeding and ex situ conservation of taro plant genetic resources. Geographical differentiation and phylogenetic relationships of Indian taro, Colocasia esculenta (L.) Schott, were analyzed by random amplified polymorphic DNA (RAPD) and isozyme of seven enzyme systems with specific reference to the Muktakeshi accession, which has been to be proved resistant to taro leaf blight caused by P. colocasiae. The significant differentiations in Indian taro cultivars were clearly demonstrated by RAPD and isozyme analysis. RAPD markers showed higher values for genetic differentiation among taro cultivars and lower coefficient of variation than those obtained from isozymes. Genetic differentiation was evident in the taro accessions collected from different regions of India. It appears that when taro cultivation was introduced to a new area, only a small fraction of genetic variability in heterogeneous taro populations was transferred, possibly causing random differentiation among locally adapted taro populations. The selected primers will be useful for future genetic analysis and provide taro breeders with a genetic basis for selection of parents for crop improvement. Polymorphic markers identified in the DNA fingerprinting study will be useful for screening a segregating population, which is being generated in our laboratory aimed at developing a taro genetic linkage map.     

Landscape conservation genetics of <Emphasis Type="Italic">Dipteryx alata</Emphasis> (“baru” tree: Fabaceae) from Cerrado region of central Brazil

In this paper random amplified polymorphic DNA (RAPD) was used to evaluate the degree of among-population differentiation and associated spatial patterns of genetic divergence for Dipteryx alata Vogel populations from Cerrado region of central Brazil, furnishing support for future programs of conservation of this species. We analyzed patterns of genetic and spatial population structure using 45 RAPD loci scored for 309 trees, sampled from five different regions with two populations each. Genetic structure analysis suggested that panmixia null hypothesis can be rejected, with significant among-population components of 15%. Hierarchical partition by Analysis of Molecular Variance (AMOVA) shows that 5% of genetic variation is within regions, whereas 10% of variation is among regions, and these results were confirmed by a Bayesian analyses on HICKORY. The Mantel correlogram revealed that this divergence is spatially structured, so that local populations situated at short geographic distances could not be considered independent units for conservation and management. However, genetic discontinuities among populations were found in the northwest and southeast parts of the study area, corresponding to regions of recent socio-economic expansion and high population density, respectively. Taking both geographic distances and genetic discontinuities into account it is possible to establish a group of population to be conserved, covering most of D. alata geographic distribution and congruent with previously established priority areas for conservation in the Cerrado region.     

Genetic Differentiation of Pedunculate Oak Quercus robur L. in the European Part of Russia Based on RAPD Markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73–90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (G _ST = 0.098); gene flow N _e m was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within-population variation.     

RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.     

High RAPD but no cpDNA sequence variation in the endemic and endangered plant, Heptacodium miconioides Rehd. (Caprifoliaceae)

Heptacodium miconioides Rehd. is an endangered species endemic to China and has suffered rapid decrease of distribution range and population size. This species has been disappeared in central China where the modal specimen was collected. We analyzed the genetic variation of the remaining populations to reveal whether the genetic diversity also suffered decrease and to provide some suggestions for conservation. All the nine known remaining populations were sampled. Genetic variation was analyzed based on RAPD markers and two fragments of cpDNA sequence, intergenic spacers of petG-trnP and trnS-trnG. No variation was observed in the two fragments of cpDNA sequence. However, the species exhibited high level of RAPD variation compared to other threatened or rare plants. Measures of genetic diversity within populations were strongly related to the log of estimated population size, indicating that large populations usually have more genetic diversity than that of small ones. About 25% of the variation was partitioned among populations. Significant relationship was observed between differentiation and geographical distance, indicating a pattern of isolation-by-distance. Given for few populations remaining, all the populations should be protected and urgent efforts be paid on the small populations to avoid their local extinction.     

Genetic diversity analysis of Rhododendron aureum Georgi (Ericaceae) located on Changbai Mountain using ISSR and RAPD markers

Rhododendron aureum Georgi (Ericaceae) is a perennial alpine shrub endemic to Changbai Mountain in China. We used ISSR and RAPD markers to describe the diversity and genetic structure within and among four natural populations located at different altitudes. DNA from 66 individuals was amplified with ten ISSR markers and seven RAPD markers. High genetic diversity was observed by these two techniques at the species level. The genetic diversity of populations increased with altitudinal gradients from low to high. The coefficient of gene differentiation (G_ST 0.3652 in ISSR and 0.2511 in RAPD) and AMOVA analysis revealed that most genetic diversity was distributed within populations (61.96% in ISSR and 70.23% in RAPD). The estimate of gene flow based on G_ST was 0.8690 in ISSR and 1.4910 in RAPD. The UPGMA clustering results using ISSR and RAPD showed that all individuals from the same altitude were gathered together, and the two populations (TYD2a and YHLa) from middle altitudes always clustered together. Compared with populations from different altitudes, similar genetic diversity and low genetic differentiation were obtained from populations at the same altitudes, as revealed by ISSR markers. In addition to the reproductive strategy of R. aureum, these data highlight that local environmental conditions may play an important role in shaping the diversity and genetic structure of this species.     

Genetic differentiation of pedunculate oak Quercus robur L. in the European part of Russia based on RAPD markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73-90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (GST = 0.098); gene flow Nem was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within--population variation.     

Genetic diversity,differentiation and conservation in <Emphasis Type="Italic">Araucaria bidwillii</Emphasis>(Araucariaceae), Australia's Bunya pine

Habitat fragmentation has resulted in many species becoming geographically restricted, as dispersal among subsequent isolates becomes compromised. This study investigated the effects of historical fragmentation on the genetic diversity and differentiation of Australia's Bunya Pine (Araucaria bidwillii) using random amplified DNA (RAPD) markers. High diversity characterises all Bunya populations sampled, regardless of population size or degree of isolation, which may be either due to similar effective population size among populations, or the lagging effects of the detection of contemporary genetic signal in long-lived conifer species. Large genetic differentiation characterises the northern and southern populations, although all sampled populations are significantly differentiated from each other. The northern population, at Mt Lewis, is responsible for the majority of variation detected among populations, and as such represents a significant genetic reservoir. The conservation of the genotypes of the Mt Lewis population may be imperative for the future of the species, given predicted models of accelerated climate change over the coming decades.     

Analysis of Genetic Variation in Rare Endemic Species Oxytropis chankaensis Jurtz. (Fabaceae) Using RAPD Markers

The method of polymerase chain reaction with random primers (RAPD) was used to assess genetic variation and population differentiation in the rare endemic plant Oxytropis chankaensis Jurtz. (Fabaceae). DNA samples from plants of two isolated populations were compared at 133 loci detected by use of ten primers. Both populations examined were characterized by high polymorphism levels (P ₉₅ = 72.9%; A = 1.92 and P ₉₅ = 74.4%; A = 1.88, respectively). They were also statistically significantly different in the frequencies of most of the amplicons. For each of the plants, unique multilocus RAPD phenotype was established using 17 to 20 RAPD markers. Diagnostic markers were not revealed. The populations were poorly differentiated. On average, the between-population component accounted for about 8% of the variation, while 92% of the variation was detected within populations. High variation along with the low degree of differentiation characteristic of two most geographically remote populations of O. chankaensis can have several explanations, among which a polyploid origin of the species seems to be most important.     

Genetic variation in the endangered <Emphasis Type="Italic">Anisodus tanguticus</Emphasis> (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H_t and H_sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H_e and H_pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei’s coefficient of differentiation (G_ST) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.     

Population variation and phylogeny in the endangered <Emphasis Type="BoldItalic">Chamaesyce skottsbergii</Emphasis> (Euphorbiaceae) based on RAPD and ITS analyses

Chamaesyce skottsbergii var. skottsbergii is federally listed as an endangered taxon, and is found in small and isolated populations restricted to calcareous soils in dry shrubland habitats on the Hawaiian islands of Oahu and Molokai. Concern over the genetic relationship among these disjunct populations arose as a result of threats to the habitat of the Oahu population. The populations were examined using random amplified polymorphic DNA (RAPD) markers and sequence analysis of the internal transcribed spacer (ITS) region of the rDNA cistron. Chamaesyce skottsbergii var. vaccinioides, a closely related variety found in several small populations on Molokai, was used for baseline comparison of the genetic divergence among populations. RAPD analysis demonstrated that variation within and among populations is the highest for any Hawaiian species examined. Polymorphism was greater than 95% within populations and was 99.4% at the species level. Similarly, measures of genetic similarity indicate that differentiation among these populations is higher than is known for some species. Both RAPD and ITS sequence analysis indicate that populations of C. skottsbergii var. skottsbergii on Oahu and Molokai are genetically distinct, and the extent of this genetic differentiation supports the recognition of these populations as distinct varieties. The Molokai population is in fact much more closely related to var. vaccinioides than to var. skottsbergii on Oahu, and thus should be recognized by the previously used variety name, C. skottsbergii var. audens. Further conservation measures for each of the varieties are addressed.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

Population Genetic Structure of Astyanax scabripinnis (Teleostei, Characidae) from an Urban Stream

Despite the great anthropogenic interference on urban streams, information is still scarce about the genetic variability and structure of native fish populations inhabiting such streams. In the present study, random amplified polymorphic DNA (RAPD) markers were used to analyze genetic variability and structure of populations assigned to the Neotropical fish species Astyanax scabripinnis from an urban stream located in Londrina, Paraná State, southern Brazil. Thirty individuals of this species were collected from three sites throughout the upper Cambé stream. A total of 10 primers amplified 159 loci, of which 128 (80.5%) were polymorphic. Each of the three populations showed very similar proportions of polymorphic loci, which ranged from 63.5 to 64.8%. Unbiased genetic distances varied from 0.0612 to 0.0646. Theta_p-test values indicated moderate to high genetic differentiation among individuals from different localities. The number of migrants varied from 1.34 to 1.46, suggesting a low gene flow between populations. The genetic similarity among all individuals studied ranged from 0.424 to 0.848. The results suggest that populations of A. scabripinnis in Cambé stream are undergoing genetic differentiation.     

A preliminary study in the use of RAPD markers in detecting genetic differences in hatching patterns of Chirocephalus diaphanus Prévost, 1803 (Crustacea: Anostraca)

Randomly amplified polymorphic DNA (RAPD) was used to detect intra-clutch genetic differences of the anostracan Chirocephalus diaphanus Prévost, 1803. Thirty primers were tested on 130 nauplii from three different clutches. Two primers produced five repeatable and well-defined amplification products, that were used as genotype markers. Nauplii hatched after three successive dehydration and rehydration cycles. Consistent genetic differentiation was observed among nauplii from the same clutch. Significant association between presence or absence of three polymorphic amplification products and hatching after different stimuli was observed. These results suggest that RAPD markers might be related to the difference in nauplii hatching and provide reasonable support to the hypothesis that the source of the observed variation in hatching phenology are genetic as well as epigenetic.     

Microevolutionary Patterns and Molecular Markers: The Genetics of Geographic Variation in Ascaris suum

Molecular markers have been used only rarely to characterize the population genetic structure of nematodes. Published studies have suggested that different taxa may show distinct genetic architectures. Isoenzyme and RAPD markers have been used to investigate geographic variation of Ascaris suum at the level of infrapopulations (nematodes within individual hosts), within localities, and among geographic regions. Independent estimates of genetic differentiation among population samples based on isoenzyme and RAPD data showed similar patterns and substantial correlation. Heterozygote deficiencies within infrapopulations and large values for inbreeding coefficients among infrapopulations suggested that the composition of these populations was not consistent with a model of random recruitment from a large panmictic pool of life-cycle stages. Both isoenzyme and RAPD markers revealed moderate levels of genetic differentiation among samples representing infrapopulations and localities. Of total gene diversity, 9.4% (isoenzyme) and 9.2% (RAPD) was partitioned among infrapopulations. Geographic localities accounted for 7.8% (isoenzyme) and 6.2% (RAPD) of total diversity. Only infrapopulations from the same farm had low levels of differentiation.     

Analysis of the genetic diversity and relationships among and within species of Hippophae (Elaeagnaceae) based on RAPD markers

RAPD markers were used to assess the genetic diversity and inter- and intra-specific relationships of the genus Hippophae L. and to study the correlation between genetic distances and geographic distances among populations of H. rhamnoides ssp. sinensis. The results analyzed by the percentage of polymorphic loci and Shannon information index indicated that a high level of genetic diversity existed both among and within species of the genus Hippophae. In the UPGMA dendrogram, the species or subspecies were clustered into two main groups but not strictly grouped according to sect. Hippophae and sect. Gyantsensis Lian. The multiple regression analysis and Mantel test both indicated a significant correlation between genetic distance and altitude distance among populations of H. rhamnoides ssp. sinensis, and the cluster analysis suggested that the genetic variation among populations of H. rhamnoides ssp. sinensis was linked to their monophyletic origin. Moreover, some degree of genetic differentiation was found among samples collected at different times.     

Diffusion of the Nearctic leafhopper Scaphoideus titanus Ball in Europe: a consequence of human trading activity

Scaphoideus titanus Ball is a Nearctic leafhopper that was introduced for the first time in Europe probably at the beginning of the 20th century. In Europe, this species is a specialist on cultivated grapevines and is of great economic importance as the vector of Flavescence dorée (FD), a Grapevine Yellows disease caused by Candidatus Phytoplasma vitis. The Random Amplified Polymorphic DNA (RAPD) technique was employed to obtain genetic information about the diffusion and the structure of S. titanus populations. Two American and 14 European populations were analysed. A total of 188 reproducible bands, obtained from three arbitrary primers, were considered to assess the amount and the pattern of genetic variation within and among leafhopper populations. American populations showed high levels of intra-population polymorphism and dissimilarity and appeared to be the most isolated of all the tested samples. The results confirm the historical role of American samples as the sources for the more recently founded European populations. RAPD analyses revealed a weak genetic structure of European samples that could probably be explained invoking the human role in their diffusion. The non-natural spreading of S. titanus across Europe is in fact attributable to the exchange of grapevine canes and grafts carrying eggs that the insect laid under the bark to overwinter.