Genetic diversity and structure in a natural Elymus caninus population from Denmark based on microsatellite and isozyme analyses

 Genetic diversity in a natural Elymus caninus population from Denmark was assessed using isozyme and microsatellite markers. A total of 119 individuals from 46 maternal plants were assayed. Microsatellite loci are shown to display higher levels of variation than isozyme loci. The mean number of alleles per locus was 1.04 for isozymes and 1.38 for microsatellites. The percentage of polymorphic loci for isozymes and microsatellites was 4.7% and 23.6% across the maternal plant, respectively. The genetic diversity at population level was 0.1 for isozymes, and 0.63 for microsatellites. The mean genetic diversity at maternal plant level was 0.027 for isozyme loci and 0.117 for microsatellite loci. The average of total allozyme diversity (H_T) was 0.22. The average of total microsatellite diversity was 0.56. Isozyme and microsatellite variation showed the same pattern of differentiation between maternal plants. More than 75% total genetic diversity was found among maternal plants. About 25% total genetic diversity was detected within maternal plants. Ten (22.7%) maternal plants produced heterozygous offspring at allozyme loci, and 30 (68.2%) maternal plants gave heterozygous offspring at microsatellite loci. Both types of markers revealed a relatively high genetic diversity in this population. Received November 7, 2000 Accepted February 15, 2001     

Isolation and characterization of microsatellite loci in the threatened wild toad lily Tricyrtis flava (Liliaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from the threatened toad lily Tricyrtis flava. The number of alleles ranged from three to 17, and the observed and expected heterozygosities ranged from 0.35 to 0.96 and from 0.50 to 0.93, respectively, in a population of T. flava. We also tested cross‐species amplifications of the markers in three other species of Trycyrtis sect. Flavae. Most of 12 markers developed for T. flava were successfully amplified in the species of sect. Flavae and will be useful for population genetic studies of these species.     

Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated “core” sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50–3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m²)] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89–0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F_st (0.32–0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.     

The effect of geographic range and dichogamy on genetic variability and population genetic structure in Tricyrtis section Flavae

Populations of each of the four species of Tricyrtis sect. Flavae were sampled using enzyme electrophoresis to examine the effect of geographic range and dichogamy on the genetic diversity of the species. The most widespread species, T. nana, had the lowest level of genetic diversity at both the population and the species level. The depauperate genetic diversity at the population level of T. nana appears to result from the high self-fertilization of the species. The low genetic diversity at the species level of T. nana probably resulted from the bottleneck effect during the speciation process in which this species diverged from the progenitor species, T. flava. Genetic differentiation among populations was high in both adichogamous T. nana and protandrous T. flava. High self-fertilization in T. nana and the colonizing nature of T. flava are likely the main factors causing the differentiated population genetic structure. In contrast to a previous study on chloroplast DNA (cpDNA) variation in Tricyrtis sect. Flavae, T. nana was most closely related to T. flava, which corresponds to the morphological resemblance of both species.     

Analysis of microsatellite variation in the rainbow trout <Emphasis Type="Italic">Parasalmo (Oncorhynchus) mykiss</Emphasis> from Kamchatka

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.     

Variability of allozyme and cpSSR markers in the populations of Siberian spruce

The variability of 21 allozyme and three microsatellite loci of chloroplast DNA (cpDNA) was studied in the populations of Siberian spruce (Picea obovata Ledeb.) from Irkutsk oblast, Magadan oblast, Buryatia, and Mongolia. It was demonstrated that the highest level of genetic diversity among the examined populations at both allozyme and microsatellite loci was observed in the Tulyushka population from Irkutsk oblast. The lowest level of genetic diversity was observed in marginal isolated populations of Bogd Uul and Magadan. In the relict spruce population from Olkhon Island, differing from the other populations in the lowest allelic diversity of both types of markers, no expected decline of expected heterozygosity and haplotype diversity was observed. In this population, the variability parameters mentioned were close to the population mean. The obtained intrapopulation and intraspecific variability parameters of allozyme and microsatellite loci of chloroplast DNA and the data on the population differentiation at these loci indicate that the given markers can be used for the analysis of the population structure of Siberian spruce.     

Assessment of population genetic structure in common wild rice Oryza rufipogon Griff. using microsatellite and allozyme markers

The genetic structure of five natural populations of common wild rice Oryza rufipogon Griff. from China, was investigated with 21 microsatellite loci and compared to estimates of genetic diversity and genetic differentiation detected by 22 allozyme loci. Microsatellite loci, as expected, have much higher levels of genetic diversity (mean values of A = 3.1, P = 73.3%, Ho = 0.358 and He = 0.345) than allozyme loci (mean values of A = 1.2, P = 12.7%, Ho = 0.020 and He = 0.030). Genetic differentiation detected by microsatellite loci ( FST = 0.468, mean I = 0.472) was higher than that for allozyme loci ( FST =0.388, mean I = 0.976). However, microsatellite markers showed less deviation from Hardy-Weinberg expectation (Wright's inbreeding coefficient FIS = -0.069) than do allozymes ( FIS = 0.337). These results suggest that microsatellite markers are powerful high-resolution tools for the accurate assessment of important parameters in population biology and conservation genetics of O. rufipogon, and offer advantages over allozyme markers.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

RAPD and Allozyme Analysis of Genetic Diversity in Panax ginseng C.A. Meyer and P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

GENETIC STRUCTURE OF POPULATIONS OF GEOGRAPHICALLY RESTRICTED AND WIDESPREAD SPECIES OF ASTRAGALUS (FABACEAE)

Evolutionary theory predicts that species with small ranges and few individuals will exhibit low levels of genetic polymorphism. We investigated the population genetic structure of two locally endemic and two geographically widespread species of Astragalus. To facilitate direct comparisons among these congeners, three populations of each species were sampled in a consistent manner and scored for allozyme polymorphisms at 12 loci. Genetic polymorphism was lower in restricted A. linifolius and A. osterhouti than in widespread A. pectinatus. However, the restricted species do exhibit a moderate level of isozyme variation, comparable to that of widespread A. pattersoni and higher than has been detected in several other rare plant taxa. As measured by Wright's F statistics, there were no consistent differences between the restricted and widespread taxa with respect to the organization of genetic variation. F_ST values were low in all taxa, indicating little heterogeneity among populations.     

Development of microsatellites DNA markers in the cultivated seaweed,Gracilaria chilensis (Gracilariales,Rhodophyta)

The red algae Gracilaria chilensis is extensively cultivated for agar production. In spite of its commercial significance as the first algal resource in Chile, no information is available on the pattern of genetic diversity. In this paper, we isolated six polymorphic microsatellite markers from a G. chilensis‐enriched DNA library. Genetic diversity was assessed in two natural populations revealing relatively low levels of heterozygosity ranging from 0.00 to 0.51. The six loci developed here are good candidates to assess the level of genetic resources within this species, which probably suffered from over‐exploitation in several localities along the Chilean coast.     

Polymorphic microsatellite loci for tiger salamanders,Ambystoma tigrinum

Microsatellites have been developed for few amphibian species. However, developing genetic markers for population genetic studies in amphibians is critical because amphibians are declining globally. The tiger salamander, Ambystoma tigrinum, is widespread throughout the United States and includes the endangered subspecies, A. t. stebbinsi. We present primers and amplification conditions for 10 polymorphic microsatellite loci that have produced successful results in three subspecies of A. tigrinum. Number of alleles per locus ranged from one to 11 and heterozygosity ranged from 0 to 0.815 depending on the subspecies and locus analysed. These markers should prove useful for future studies of genetic diversity and population subdivision.     

Microsatellite polymorphism in natural populations of wild emmer wheat,Triticum dicoccoides,in Israel

Diversity in 20 microsatellite loci of wild emmer wheat, Triticum dicoccoides, was examined in 15 populations (135 genotypes) representing a wide range of ecological conditions of soil, temperature, and water availability, in Israel and Turkey. An extensive amount of diversity at microsatellite loci was observed despite the predominantly selfing nature of this plant species. The 20 Gatersleben wheat microsatellites (GWM), representing 13 chromosomes of genomes A and B of wheat, revealed a total of 364 alleles, with an average of 18 alleles per GWM marker (range: 5–26). The proportion of polymorphic loci per population averaged 0.90 (range: 0.45– 1.00); genic diversity, He, averaged 0.50 (range 0.094– 0.736); and Shannon’s information index averaged 0.84 (range 0.166–1.307). The coefficients of genetic distance between populations were high and averaged D=1.862 (range 0.876–3.320), an indication of sharp genetic divergence over short distances. Interpopulation genetic distances showed no association with geographic distance between the population sites of origin, which ruled out a simple isolation by distance model. Genetic dissimilarity values between genotypes were used to produce a dendrogram of the relationships among wild wheat populations by the unweighted pair-group method with arithmetic averages (UPGMA). The results showed that all the wild emmer wheat populations could be distinguished. Microsatellite analysis was found to be highly effective in distinguishing genotypes of T. dicoccoides, originating from diverse ecogeographical sites in Israel and Turkey, with 88% of the 135 genotypes correctly classified into sites of origin by discriminant analysis. Our present microsatellite results are non-random and in agreement with the previously obtained allozyme and RAPD patterns, although the genetic-diversity values obtained with microsatellites are much higher. Significant correlates of microsatellite markers with various climatic and soil factors suggest that, as in allozymes and RAPDs, natural selection causes adaptive microsatellite ecogeographical differentiation, not only in coding, but most importantly in non-coding genomic regions. Hence, the concept of ”junk DNA” needs to be replaced by at least partly regulatory DNA. The obtained results suggest that microsatellite markers are useful for the estimation of genetic diversity in natural populations of T. dicoccoides and for the tagging of agronomically important traits derived from wild emmer wheat. Received: 27 February 2001 / Accepted: 22 March 2001     

Characterization of dinucleotide microsatellite markers in the parthenogenetic mourning gecko (Lepidodactylus lugubris)

We present 16 variable dinucleotide microsatellite markers to quantify genetic variation in the parthenogenetic gecko, Lepidodactylus lugubris. Genetic diversity at these loci was unusually high for an asexual species. Subsets of individuals produced identical genotypes across all loci. Individual loci revealed evidence of polyploidy and marked differences between observed and expected heterozygosity, indicating the presence of null alleles. These patterns conform to prior expectations based on the distant genetic relationships between the presumed sexual progenitors of L. lugubris. Comparisons with sexual relatives will be required to determine the sources of the observed genetic variation.     

Population genetic structure and phylogeography of cyprinid fish, <Emphasis Type="Italic">Labeo dero</Emphasis> (Hamilton, 1822) inferred from allozyme and microsatellite DNA marker analysis

We examined population structure of Labeo dero (Hamilton, 1822) from different riverine locations in India using 10 polymorphic allozyme and eight microsatellite loci. For analysis, 591 different tissue samples were obtained from commercial catches covering a wide geographic range. Allozyme variability (An = 1.28–1.43, Ho = 0.029–0.071) was much lower than for microsatellites (An = 4.625–6.125, Ho = 0.538–0.633). Existence of rare alleles was found at three allozyme (MDH-2*, GPI* and PGDH*) and at two microsatellite loci (R-3* and MFW-15*). Deviation from Hardy–Weinberg equilibrium (P < 0.05, after the critical probability levels were adjusted for sequential Bonferroni adjustment) could be detected at three loci (EST-1*, -2* and XDH*) whereas, after correction for null alleles, two microsatellite loci (MFW-1*,-15*) deviated from HWE in the river Yamuna. Fst for all the samples combined over all allozyme loci was found to be 0.059 suggesting that 5.9% of the total variation was due to genetic differentiation while microsatellite analysis yielded 0.019 which was concordant to mean Rst (0.02). Hierarchical partition of genetic diversity (AMOVA) showed that greater variability (approx. 95%) was due to within population component than between geographical regions. Based on distribution of genetic differentiation detected by both markers, at least five different genetic stocks of L. dero across its natural distribution could be identified. These results are useful for the evaluation and conservation of L. dero in natural water bodies.     

Patterns of allozyme variation in relation to population size of the threatened plantMegaleranthis saniculifolia (Ranunculaceae) in Korea

Patterns of variation at 27 allozyme loci were investigated in the endangered endemic plantMegaleranthis saniculifolia. Levels of allozyme variation (A = 1.47,P = 40%,He = 0.088) were also compared with other endemic plant species. Genetic divergence between populations was very high (G ^st = 0.271 ), with moderate to high interpopulation differentiation, which probably arose through historical bottlenecks in a landscape of habitat fragmentation and/or human influence. The percentage of polymorphic loci, heterozygosity, and mean number of alleles per locus were positively related to population size, probably due to the stochastic loss of rare alleles in the smaller populations. Individuals in the small and marginal populations (TB, KD, and CJ) showed higher proportions of fixed loci. These ecologically marginal populations were typically more distant from the nearest neighboring population and were more genetically distinct from one another. The genetic structure of the current population ofM. saniculifolia is probably the result of local extinctions of intervening populations. This, in turn, is due to the Pleistocene climatic change and increased habitat destruction. A positive association appears to exist between genetic diversity and population size. Although these small population sizes are more sensitive to stochastic events, securing a certain number of individuals from the three larger populations (SB, JB, and TG) could be accomplished as part of a conservation strategy. In addition, it is important to prioritize populations in different regions in order to limit population declines caused by large-scale environmental catastrophes.     

Genetic structure of Polytrichum formosum in relation to the breeding system as revealed by microsatellites

Microsatellite variation was determined for three Danish and three Dutch populations of the haploid moss species Polytrichum formosum to gain insight into the relative importance of sexual vs. asexual reproduction for the amount and structure of genetic variation. In general, low levels of microsatellite variation were observed within this species. Even when estimated for polymorphic loci only, the levels of microsatellite variability (P=90.6, A=4.3 and H_S=0.468) within populations were on average lower than those reported for most other plant species. In contrast, genotypic diversity was high within each of the examined populations, indicating that sexual reproduction is a very important determinant of the genetic structure of P. formosum within populations. In agreement with previous findings for allozyme data, no significant genetic differentiation (F_ST=0.028, R_ST=0.015) was observed neither between populations nor between regions approximately 450 km apart (Denmark vs. the Netherlands). These low levels of population differentiation observed for both types of genetic markers are probably best explained by a high level of effective spore dispersal (gene flow) between populations. Therefore, also on a large geographical scale sexual reproduction is the most important determinant of the genetic structure of P. formosum, despite the high potential to reproduce clonally.     

Novel Polymorphic Microsatellite Markers for Panulirus ornatus and their Cross-species Primer Amplification in Panulirus homarus

Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species.     

A comparative floral and pollination biology ofTricyrtis flava maxim.,T. nana yatabe andT. ohsumiensis

The floral and pollination biology of three closely related species,Tricyrtis flava, T. nana andT. ohsumiensis, were comparatively investigated. The primary pollinator wasBombus diversus in all the three species observed, andAmegilla sp. also acted forT. ohsumiensis. The flowers ofT. flava andT. ohsumiensis bloom for two days and are protandrous. Thus autogramy seems to be prevented in these species when the larger bees forage on them, though geitonogamy may also occur. On the other hand,T. nana appears to be a primarily self-pollinating species. The flowers of this species open only during one day and are homogamy. The stigmata seem to receive much pollen of their own flower by the visit of bumblebees. Moreover, many flowers ofT. nana fruited without any visit of pollinators.T. nana has many features characterizing the autogamous derivatives.     

Parallel microgeographic patterns of genetic diversity and divergence revealed by allozyme, RAPD, and microsatellites in Triticum dicoccoides at Ammiad, Israel

The levels of genetic diversity were compared by means of 35 allozyme, 60 RAPD, and 25 microsatellite (SSR) markers for 75–175 individuals of tetraploid wild emmer wheat (Triticum dicoccoides) collected in 1993 from a microgeographic microsite, Ammiad, north of the Sea of Galilee, Israel. This microsite included four major habitats, which showed highly significant differentiation in ecological factors, in particular with respect to rock cover, proximity and height, and surface soil moisture in the early growing season of T. dicoccoides. Higher within-subpopulation genetic diversity was found in the primarily non-coding DNA regions (RAPD and SSR) rather than in the protein-coding (allozymes) regions. However, much larger gene differentiation (G _ST) among the subpopulations was observed in the protein-coding allozymes than in the RAPDs and SSRs. Larger genetic distance was found at SSR loci, followed by allozyme and RAPD loci. The subpopulations in drier habitats tend to have higher allozyme, RAPD and SSR diversities (He), the relatively wet Karst subpopulation showed only about half He of the other relatively drier habitats. The subpopulations with larger difference of soil moisture between habitats tend to show larger genetic distances at allozyme, RAPD and SSR loci. These results suggest that climatic selection through aridity stress may be an important factor acting on both structural protein-coding and presumably partly regulatory non-coding DNA regions, resulting in microscale adaptive patterns, although hitchhiking and random drift may also intervene. These results have profound implications for genetic conservation both in situ and ex situ.