Genotyping the clonal structure of a gorgonian coral, Junceella juncea (Anthozoa: Octocorallia), using microsatellite loci

The identification of different clones is fundamental to the study of population structure among organisms with mixed reproductive modes such as cnidarians. However, due to the low genetic variation of coral mtDNA and contamination by zooxanthellate DNA, very few molecular markers are available for studying the clonal structure of cnidarians. Herein we used four polymorphic loci of microsatellite DNA isolated from a zooxanthellae-free octocoral, Junceella juncea, to study its clonal structure in seven populations collected from three localities in Taiwan. In total, 40 multilocus genotypes were found among 152 colonies, and the number of genotypes (clones) identified in the seven populations ranged from 2 to 16. Each of the 40 multilocus genotypes was restricted to a single population, even where adjacent populations were only 100 m distant. The ratio of observed to expected genotypic diversity (G_o:G_e) ranged from 0.217 to 0.650, and G_o showed a significant departure from G_e (p<0.05) at each site indicating that asexual fragmentation may play a major role in the maintenance of established populations. Mean relatedness (R) values showed that genotypes within reefs were more closely related than those between regions. The results indicate that microsatellites are useful for discerning the clonal structures among and within populations at different spatial scales. Electronic supplement: Unique multilocus genotypes (clones) revealed by 4 polymorphic loci for Junceella juncea colonies collected from Xiashuijui (Reefs A, B, C, Transplant, Transect), Nanwan and Shicheng     

Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae)

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.     

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations ('clone mates') within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.     

Microsatellite evidence for high clonality and limited genetic diversity in Ziziphus celata (Rhamnaceae), an endangered, self-incompatible shrub endemic to the Lake Wales Ridge, Florida, USA

Genetic data are often crucial for designing management strategies for rare and endangered species. Ziziphus celata is an endangered sandhill shrub endemic to the Lake Wales Ridge of central Florida. This self-incompatible clonal species is known from only 14 wild populations, most of which are small (under 100 plants). Focusing on the five populations discovered in 2007, we evaluate the level of genetic diversity and identify clonal lineages within the wild populations of the species with a set of microsatellite loci. To account for somatic mutations and genotyping errors, we identified clonal lineages using a threshold cutoff for pair-wise genetic distances among samples. The microsatellites had up to 18 alleles/locus, and, consistent with outcrossing, samples were highly heterozygous (average population level H _o  = 0.69). Most populations of Z. celata consist of a single clone, and the most diverse population has only 10 clones. Overall Z. celata comprises 41 multi-locus genotypes, and 30 clonal lineages. With nearly 1,000 recorded plants (595 genotyped) and only 30 clonal lineages, Ziziphus celata is highly clonal: clonal richness, R = 0.049. The pair-wise distance method facilitates identification of clonal lineages, avoiding overestimation of clonal diversity. In most cases, the samples that grouped into a lineage were one to four plants differing from a surrounding genotype by a single microsatellite repeat insertion/deletion mutation, consistent with these having arisen via somatic mutations. Our data will enable managers to incorporate extant diversity from wild populations into ex situ collections. Additionally, our research demonstrates the utility of microsatellites for conservation of imperiled species, identifying genotypes of high priority for preservation.     

微卫星标记在种群生物学研究中的应用

微卫星是以几个碱基 (一般为 1～ 6个 )为重复单位组成的简单的串联重复序列 ,具有丰度高、多态性高、共显性标记、选择中性、可自动检测等优点。本文着重介绍了微卫星在种群生物学研究中的应用。微卫星位点可以提供具高分辨率的遗传信息 ,这一特点使微卫星既适合于个体水平上的研究 ,又适合于种群水平上的研究。在个体水平上包括个体识别、交配系统和亲本分析、基因流等研究。微卫星是常用的个体识别手段 ,但在克隆植物遗传结构研究方面的应用还很有限 ;微卫星提高了交配系统和亲本分析、基因流等研究的准确性。在种群水平上微卫星可用于遗传结构、有效种群大小、种群的系统发育重建等研究。微卫星在很多物种 (包括珍稀物种 )的遗传结构研究中得到应用 ;利用微卫星标记确定有效种群大小、检测有效种群大小的波动可以促使我们正确理解种群遗传结构动态和种群进化过程 ;微卫星在种群的系统发育重建研究方面有很大的应用潜力。然而微卫星并不是研究所有问题的唯一选择。文中还讨论了在实际工作中应如何正确利用分子标记等问题     

Successful worldwide invasion of the veined rapa whelk, <Emphasis Type="Italic">Rapana venosa</Emphasis>, despite a dramatic genetic bottleneck

Biological invasions represent an important component of global change, with potentially huge detrimental effects on native biological biodiversity and ecosystems. Knowledge about invasion history provides information about the invasion process and the origin and genetic composition of invading populations. To clarify the source and invasive routes of a successful world-wide invader, the veined rapa whelk, Rapana venosa, genetic variability of samples from five representative native populations from coasts of Japan and China and 13 worldwide invasive populations was analyzed using 11 nuclear microsatellite loci. A dramatic decrease of genetic variation was detected in the invasive populations compared with the native populations. The results demonstrated that R. venosa was capable of establishing itself in many areas despite a dramatic genetic bottleneck, suggesting that a remarkable reduction of genetic diversity is not a limiting factor for short-term success of this invasive species. Considering the lack of mitochondrial variation previously observed in the invasive populations, the dramatic genetic bottleneck and the allele distribution detected using microsatellites suggested that the original introduced Black Sea population could have been founded by very few individuals, perhaps only a single female and a single male. The initial invasive Black Sea population was likely an accidental introduction from Japan, and then invaded the Adriatic Sea by range expansion, which served as a source for subsequent invasive populations in Europe and America by various transport vectors. In addition, microsatellite alleles in the invasive populations showed a tendency to mutate with the addition or deletion of a single repeat, which is consistent with the stepwise mutation model. Our findings provide a good example of how an aquatic invader with a drastic genetic bottleneck and very low genetic diversity rapidly expands its geographical range.     

High somatic instability of a microsatellite locus in a clonal tree,<Emphasis Type="Italic"> Robinia pseudoacacia</Emphasis>

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13–71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.Communicated by H.F. Linskens     

Microsatellite loci in eelgrass Zostera marina reveal marked polymorphism within and among populations

Using an enriched genomic library, we developed seven (CT)n/(GA)n microsatellite loci for eelgrass Zostera marina L. Enrichment is described and highly recommended for genomes in which microsatellites are rare, such as in many plants. A test for polymorphism was performed on individuals from three geographically separated populations (N = 15/population) and revealed considerable genetic variation. The number of alleles per locus varied between five and 11 and the observed heterozygosities for single loci ranged from 0.16 to 0.81 within populations. Mean allele lengths were markedly different among populations, indicating that the identified loci will be useful in studying population structure in Z. marina. As the frequency of the most abundant multilocus genotype within populations was always < 1%, these loci have sufficient resolving power to address clone size in predominantly vegetatively reproducing populations.     

Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation

Microsatellites are short tandemly repeated DNA sequence motifs that are highly variable in most organisms. In contrast to mammals, long microsatellites (>15 repeats) are extremely rare in the Drosophila melanogaster genome. To investigate this paucity of long microsatellites in Drosophila, we studied 19 loci with exceptionally long microsatellite alleles. Inter- and intraspecific analysis showed that long microsatellite alleles arose in D. melanogaster only very recently. This lack of old alleles with many repeats indicated that long microsatellite alleles have short persistence times. The size distribution of microsatellite mutations in mutation-accumulation lines suggests that long alleles have a mutation bias toward a reduction in the number of repeat units. This bias causes the short persistence times of long microsatellite alleles. We propose that species-specific, size-dependent mutation spectra of microsatellite alleles may provide a general mechanism to account for the observed differences in microsatellite length between species.     

An unusually low microsatellite mutation rate in Dictyostelium discoideum, an organism with unusually abundant microsatellites

The genome of the social amoeba Dictyostelium discoideum is known to have a very high density of microsatellite repeats, including thousands of triplet microsatellite repeats in coding regions that apparently code for long runs of single amino acids. We used a mutation accumulation study to see if unusually high microsatellite mutation rates contribute to this pattern. There was a modest bias toward mutations that increase repeat number, but because upward mutations were smaller than downward ones, this did not lead to a net average increase in size. Longer microsatellites had higher mutation rates than shorter ones, but did not show greater directional bias. The most striking finding is that the overall mutation rate is the lowest reported for microsatellites: approximately 1 x 10(-6) for 10 dinucleotide loci and 6 x 10(-6) for 52 trinucleotide loci (which were longer). High microsatellite mutation rates therefore do not explain the high incidence of microsatellites. The causal relation may in fact be reversed, with low mutation rates evolving to protect against deleterious fitness effects of mutation at the numerous microsatellites.     

Directional evolution for microsatellite size in maize

Directional evolution in microsatellites is the tendency for microsatellites either to increase or to decrease in size over time between populations. We analyzed 99 microsatellite loci in a sample of 193 maize plants representing the entire pre-Columbian range of this crop for evidence of directional evolution. We took advantage of the known phylogeographic history of maize with the independent movement of maize from its ancestral location in Mexico to North and South America. We show that there is an increase in the average allele size of microsatellites in the geographically derived North and South American groups relative to the ancestral Mexican group. We also show that there is a negative correlation between average allele size and altitude in all three groups. Directional evolution in maize microsatellites can be explained by changes in the mutation rate over time and space, changes in the degree of mutational bias to a larger allele, demographic differences between the ancestral and geographically derived populations, and/or scenarios involving selection on microsatellite size. The occurrence of directional evolution for microsatellite size indicates that the estimation of population parameters with microsatellite data in maize should be done with caution.     

Cloning and Characterization of Microsatellite Loci in a Gorgonian Coral, Junceella juncea (Anthozoa; Octocorallia; Ellisellidae) and Its Application in Clonal Genotyping

We attempted to isolate microsatellites from a Symbiodinium-free gorgonian octocoral, Junceella juncea, using two methods, partial genomic library screening and enrichment. Among the 3856 clones screened by the partial library method, 10 possibly positive signals were found, and 3 of them could be used to design primers and amplified consistently. In contrast, only one locus isolated by the enrichment method gave reliable amplification and was useful. The results indicate that microsatellites are rare in Junceella juncea, as reported for other cnidarians. Overall, we obtained 4 polymorphic loci to test the feasibility in investigating clonal structure of J. juncea. A total of 40 multilocus genotypes were found among 152 colonies, and the number of genotypes (clones) identified at 7 reefs ranged from 2 to 16. The results of a nonmetric multidimensional scaling analysis indicated the recruitment of J. juncea populations mainly comes from self-retention. These novel microsatellite loci will provide a useful tool to study clonal structure and population genetics for J. juncea in the future.     

GENETIC POPULATION STRUCTURE AND MATING SYSTEM IN CHONDRUS CRISPUS (RHODOPHYTA)1

Chondrus crispus Stackh. has been intensely studied, yet no study to date has elucidated its population structure or mating system despite many populations in which there was a haploid bias and lack of male gametophytes. Therefore, 12 nuclear microsatellite loci were identified in this red alga. Microsatellite markers were developed and tested against a panel of specimens collected from two shore levels at two sites in Brittany, France: Pointe de Primel and Pointe de la Jument, Concarneau. Single locus genetic determinism was verified at eight polymorphic loci, as only one band was observed for haploid genotypes, whereas one or two bands were observed for diploids. These markers enabled the detection of unique genotypes within sampled populations, indicating that very few fronds shared the same multilocus genotype. This finding suggests that asexual reproduction was not the prevailing mode of reproduction. In addition, we explored the hierarchical population structure showing that gene flow is restricted at small spatial scales (<50 m) between upper and lower Chondrus‐range populations within a shore. Sexual reproduction predominated in the populations of C. crispus studied, but probably due to fine‐scale spatial substructuring, inbreeding was also significant. In conclusion, this study reveals that fine‐scale genetic variation is of major importance in C. crispus, suggesting that differences between microhabitats should be essential in understanding evolutionary processes in this species.     

Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites

Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide.     

Genetic heterogeneity among intertidal habitats in the flat periwinkle, Littorina obtusata

Comparisons among patterns exhibited by functionally distinct genetic markers have been widely used to infer the impacts of demography and selection in structuring genetic variation in natural populations. However, such multilocus comparisons remain an indirect evaluation of selection at particular candidate loci; ideally, the identification of a candidate gene by comparative genetic methodologies should be complemented by functional analyses and experimental manipulations of genotypes in the laboratory or field. We examined genotype frequency variation among replicated intertidal habitats at two spatial scales in the grazing snail Littorina obtusata. Both of the candidate allozyme markers varied predictably with environment, and these patterns were consistent at both spatial scales. Three of four reference loci were spatially homogeneous, but one microsatellite exhibited significant structure at both geographical and mesoscales. To initiate a direct examination of whether the observed genotype frequency variation at one of the candidate markers, mannose-6-phosphate isomerase (MPI), was impacted by differential survivorship of genotypes, we conducted a series of laboratory-based thermal stress assays using snails from two geographically disparate source populations. When snails were exposed to bouts of thermal/desiccation stress, patterns of mortality were nonrandom with respect to MPI genotype. Furthermore, patterns of mortality in the laboratory manipulation coincided with the observed distribution of genotypes in the field. The data suggest the operation of selection at the Mpi or a linked locus, but functional studies and further experimentation are required to establish the relationship between MPI genotype and fitness across heterogeneous intertidal environments.     

Analysis of population clonal diversity using microsatellite markers in the salt marsh sedge Carex scabrifolia in western Japan

Carex scabrifolia, a perennial herb that commonly grows on sandbars in lagoons and tidal estuaries, is threatened by habitat reduction in some areas of Japan. Clonal diversity and the extent of gene flow among ten populations located along the Seto Inland Sea and the Japan Sea in western Japan were examined using six microsatellite loci. From 299 samples, we detected 77 multilocus genotypes. The mean number of alleles per population was 2.8 and the mean clonal diversity was 0.23. Many populations consisted of small patches, and the mean number of genets per patch was 2.0. The average number of alleles per locus and clonal diversity were positively correlated with the number of patches within a watershed. Gene flow was detected between the Ichikawa River and the Ohta River populations along the Seto Inland Sea, and weak differentiation among populations located along the Seto Inland Sea was observed. Our results suggest that effective conservation of C. scabrifolia populations should include maintaining all patches within a watershed regardless of population size, thus promoting genotype preservation.     

Assessing genetic diversity in clonal organisms: low diversity or low resolution? Combining power and cost efficiency in selecting markers

The increasing use of molecular tools to study populations of clonal organisms leads us to question whether the low polymorphism found in many studies reflects limited genetic diversity in populations or the limitations of the markers used. Here we used microsatellite datasets for two sea grass species to provide a combinatory statistic, combined with a likelihood approach to estimate the probability of identical multilocus genotypes (MLGs) to be shared by distinct individuals, in order to ascertain the efficiency of the markers used and to optimize cost-efficiently the choice of markers to use for deriving unbiased estimates of genetic diversity. These results strongly indicate that conclusions from studies on clonal organisms derived using markers showing low polymorphism, including microsatellites, should be reassessed using appropriate polymorphic markers.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Genetic diversity and population structure of the serpentine endemic Calystegia collina (Convolvulaceae) in northern California

We used enzyme electrophoresis to evaluate genetic diversity in 32 populations of Calystegia collina, a clonal plant species endemic to serpentine outcrops in northern California (USA). Of 34 loci examined 56% were polymorphic, but on average only 17% were polymorphic within local populations. Neither the total number of alleles nor the number of multilocus genotypes differed significantly between populations in small vs. large serpentine outcrops. Genetic and geographic distances between populations were positively correlated, but this relationship was not significantly affected by the isolation of serpentine outcrops. Populations were highly differentiated (F(st) = 0.417) and little genetic variation was explained by geographic region or serpentine outcrop.Observed heterozygosity within populations almost always exceeded Hardy-Weinberg expectations. In many populations, all 30 sample ramets were uniformly heterozygous at one or more loci yet were genetically variable at other loci. These results imply that many C. collina populations originate from one or a few genetic founders, with little recruitment from seeds. Genetic variation within uniformly heterozygous populations must be the product of multiple, closely related founders or somatic mutations within the population. We conclude that vegetative reproduction, perhaps coupled with somatic mutation, helps maintain genetic diversity in these isolated but long-lived populations.     

Clonality and host selection in the wheat pathogenic fungus Puccinia triticina

Clonal reproduction in Puccinia triticina, the cause of wheat leaf rust, has long been hypothesized but has never been demonstrated. Using a population genetics approach and microsatellite markers, we analysed genetic diversity of this fungus at each level of genome organisation. Sampling included isolates from two field populations growing on two cultivars carrying specific resistance genes, completed with isolates representing the main pathotypes identified from a national survey. For the two cultivars, populations differentiated according to the distribution of their genotypes and pathotypes. There was a high proportion of repeated genotypes, combined with a significant linkage disequilibrium and a strong negative value for FIS. These three factors, especially heterozygote excess, strongly support the hypothesis of a high rate of clonal reproduction. Each pathotype matched a unique multilocus genotype, except for a few isolates, which were taken to be mutants of the dominant genotype. We discussed the strong relationship between pathotypes and genotypes as the consequence of clonal reproduction combined with a strong selection exerted by host cultivars.