Isolation and characterization of microsatellite loci in a clonal herb, Cardamine leucantha (Brassicaceae)

? Premise of the study: Microsatellite markers were developed to analyze the clonal diversity and population genetic structure of a clonal plant, Cardamine leucantha. ? Methods and Results: Microsatellite loci were isolated by using compound simple sequence repeat (SSR) primers, resulting in 25 pairs including 14 useful markers. Samples from 33, 20, and 17 ramets in three populations were identified as belonging to 23, 18, and 17 genotypic individuals, respectively. ? Conclusions: These SSR markers will be used for identifying genets and evaluating genetic diversity of the clonal plant C. leucantha.     

Isolation and characterization of microsatellite markers in the rare clonal plant, Spiraea virginiana (Rosaceae)

? Premise of the study: Microsatellite markers were developed in Spiraea virginiana, a federally threatened native shrub found along stream banks, to identify clonal genotypes and measure population genetic variability. ? Methods and Results: Eleven primer sets were developed using a non-radioactive protocol. These revealed a moderate level of genetic variation, as indicated by the number of alleles per locus (range = 1-4) and an average observed heterozygosity of 0.595. Select loci also amplified successfully in the related species Spiraea japonica. ? Conclusion: Development of the markers described here is critical for the genetic identification of clonal plants as a first step in demographic analyses, and is necessary for the future conservation of this rare species. Amplification of the markers in S. japonica suggests their potential utility in research regarding this species.     

新疆胡颓子属植物(Elaeagnus)分类探讨

在花果标本定株采集的基础上,对新疆地区胡颓子属植物种类的形态学分类依据进行了探讨,对目前记载的新疆胡颓子属种类进行了整理,共确定新疆地区分布有胡颓子属3种1变种:尖果沙枣(E. oxycarpa Schlecht.)、沙枣(E. angustifolia L.)、东方沙枣(E. angustifolia var. orientalis(L.) Kuntze)、大果沙枣(E. moorcroftii Wall. ex Schlecht.),并对其中部分种类的特征描述进行了补充。     

Development of microsatellite primers of the largest seagrass, Enhalus acoroides (Hydrocharitaceae)

? Premise of the study: Microsatellite primers were developed for the seagrass Enhalus acoroides to investigate genetic variation and identify clonal structure. ? Methods and Results: Four polymorphic loci and 32 monomorphic loci were developed in E. acoroides. Two to four alleles per locus were observed at the polymorphic loci across 60 individuals of two E. acoroides populations. The observed and expected heterozygosities within populations ranged from 0.100 to 0.5667 and from 0.0977 to 0.5079, respectively. ? Conclusions: Our study revealed very low polymorphism in E. acoroides, even at the polymorphic loci. Nevertheless, these primers are a useful tool to study genetic variation, clonal structure, and mating system.     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

Trade-offs between clonal and sexual reproduction in Sagittaria latifolia (Alismataceae) scale up to affect the fitness of entire clones

? Many plants combine sexual reproduction with vegetative propagation, but how trade-offs between these reproductive modes affect fitness is poorly understood. Although such trade-offs have been demonstrated at the level of individual shoots (ramets), there is little evidence that they scale up to affect genet fitness. For hermaphrodites, reproductive investment is further divided between female and male sexual functions. Female function should generally incur greater carbon costs than male function, which might involve greater nitrogen (N) costs. ? Using a common garden experiment with diclinous, clonal Sagittaria latifolia we manipulated investment in reproduction through female and male sex functions of 412 plants from monoecious and dioecious populations. ? We detected a 1?:?1 trade-off between biomass investment in female function and clonal reproduction. For male function, there was no apparent trade-off between clonal and sexual reproduction in terms of biomass investment. Instead, male function incurred a substantially higher N cost. ? Our results indicate that: trade-offs between investment in clonal propagation and sexual reproduction occur at the genet level in S.?latifolia; and sexual reproduction interferes with clonal expansion, with investment in female function limiting the quantity of clonal propagules produced, and investment in male function limiting the nutrient content of clonal propagules.     

Geographic limits of a clonal population of wheat yellow rust in the Mediterranean region

Most plant pathogens present complex life cycles, in which the clonal reproduction may impede the delimitation of population entities. By studying the genetic structure of the wheat yellow rust caused by Puccinia striiformis f.sp. tritici (PST), we highlighted difficulties impeding population delimitation in highly clonal species. Despite the high dispersal potential of PST, southern France isolates were shown to be divergent from a northwestern European population. A 2-year survey was performed in the Mediterranean region to assess the geographic distribution of southern isolates: 453 isolates collected in 11 countries were genotyped using 15 simple sequence repeat markers. A subsample was analysed for virulence against 23 resistance genes. The dominant strain in the western Mediterranean region was further studied with amplified fragment length polymorphism markers to test for a geographic substructure. Both 'individual'- and 'population'-centred analyses of polymorphism markers revealed two south-specific groups: a predominant group, with a broad variability and a wide distribution in both western and eastern Mediterranean countries, and a minor group in the western Mediterranean. The east–west gradient of genetic diversity suggested gene flow from the Middle East with subsequent founder effects and genetic divergence, and demonstrated the local survival of a western Mediterranean population. The high frequency of the resistance gene Yr8 observed in cultivars from Tunisia and Algeria may contribute to maintain the north/south structure observed in France. In addition to migration and local adaptation, the dynamics of clonal lineage diversification and replacement should be considered to define population entities in strongly clonal species.     

Salinity tolerance and genetic variability in freshwater and brackish Iris hexagona colonies

? Premise of the study: Saltwater intrusion is one of the most widespread environmental threats to freshwater wetlands. Iris species worldwide are important members of these plant communities. Wetland irises reproduce clonally and sexually, which permits populations to spread and disperse in benign and stressful conditions. The ability of iris populations to tolerate and adapt to elevated salinity can play an important role in determining the long-term health of wetland ecosystems. ? Methods: We used microsatellite markers to evaluate population structure and genetic diversity, and we performed a common garden experiment to examine the effect of salinity on the growth and reproduction of wild Iris hexagona collected from freshwater and brackish wetlands. ? Key results: Colonies were genetically distinct, with average to high heterozygosity (0.55-0.66) for a clonal species. Salinity had negative linear effects on leaf mass (g), clonal growth (g), root mass (g), and flower numbers, and it had nonlinear effects on seed numbers and seed mass (mg). The greatest sexual reproduction occurred in the intermediate-salinity (4 parts per thousand) treatment. Flowering phenology was delayed for 5 days in the highest-salinity treatment. ? Conclusions: We hypothesized that irises from brackish habitats would tolerate salinity better than freshwater irises would, but no difference in iris performance existed between the two habitats. The observed salinity tolerance and genetic diversity of I. hexagona indicate that populations will persist despite moderate increases in environmental salinity.     

Microsatellite markers show decreasing diversity but unchanged level of clonality in Dryas octopetala (Rosaceae) with increasing latitude

? Premise of the study: Average arctic temperatures have increased at almost twice the global average in the past 100 years. Most studies on biodiversity along latitudinal gradients have focused on species richness or genetic diversity at lower latitudes, and only a few studies have inferred genetic diversity within a species along a latitudinal gradient at higher latitudes, even though these areas might be most affected by recent climate changes. Here, intraspecific genetic diversity of the arctic-alpine Dryas octopetala (Rosaceae) is studied along a latitudinal gradient to test the hypotheses that genetic diversity decreases and vegetative clonal growth increases with latitude. ? Methods: Ten microsatellite markers have been developed for D. octopetala and analyzed with population genetic methods in five populations along a latitudinal transect spanning from 59.0°N to 79.9°N. ? Key results: The nine microsatellites that were used in the final analyses resulted in a resolution high enough to distinguish between ramets while providing useful information at a larger geographical scale. Three genetic clusters were indicated, a southern Norway group, a northern Norway group, and a Svalbard group, with corresponding decreasing genetic diversity. No trend was found with regard to clonality along the gradient. ? Conclusions: The newly developed microsatellite markers provide a useful tool for further genetic studies of D. octopetala and its close relatives, addressing population structure as well as phylogeographic patterns. The results of this study support the hypothesis of decreasing genetic diversity with increasing latitude, which may have implications for future adaptability to climate change.     

On the evolution of clonal plant life histories

Clonal plant life histories are special in at least four respects: (1) Clonal plants can also reproduce vegetatively, (2) vegetative reproduction can be realised with short or long spacers, (3) and it may allow to plastically place vegetative offspring in benign patches. (4) Moreover, ramets of clonal plants may remain physically and physiologically integrated. Because of the apparent utility of such traits and because ecological patterns of distribution of clonal and non-clonal plants differ, adaptation is a tempting explanation of observed clonal life-history variation. However, adaptive evolution requires (1) heritable genetic variation and (2) a trait effect on fitness, and (3) it may be constrained if other evolutionary forces are overriding selection or by constraints, costs and trade-offs. (1) The few studies undertaken so far reported broad-sense heritability for clonal traits. Variation in selectively neutral genetic markers appears as pronounced in populations of clonal as non-clonal plants. However, neutral markers may not reflect heritable variation of life-history traits. Moreover, clonal plants may have been sampled at larger spatial scales. Empirical information on the contribution of somatic mutations to heritable variation is lacking. (2) Clonal life-history traits were found to affect fitness. However, much of this evidence stems from artificial rather than natural environments. (3) The relative importance of gene flow, inbreeding, and genetic drift, compared with selection, in the evolution of clonal life histories is hardly explored. Benefits of clonal life-history traits were frequently studied and found. However, there is also evidence for constraints, trade-offs, and costs. In conclusion, though it is very likely, that clonal life-history traits are adaptive, it is neither clear to which degree this is the case, nor which clonal life-history traits constitute adaptations to which environmental factors. Moreover, evolutionary interactions among clonal life-history traits and between clonal and non-clonal ones, such as the mating system, are not well explored. There remains much interesting work to be done in this field – which will be particularly interesting if it is done in the field.     

Microsatellite markers for the cushion rock jasmine, Androsace tapete (Primulaceae), a species endemic to the Qinghai-Tibetan Plateau

? Premise of the study: Microsatellite markers were developed in an alpine plant endemic to the Qinghai-Tibetan Plateau, Androsace tapete, to investigate its spatial genetic structure, gene flow, and mating patterns. ? Methods and Results: Using the combined biotin capture method, 16 microsatellite primer sets were isolated and characterized. Fifteen of these markers showed polymorphism, and the number of alleles per locus ranged from three to 13 across 56 individuals from six Tibetan populations. ? Conclusions: These markers provide a useful tool to investigate the spatial genetic structure, gene flow, and mating system of A. tapete.     

Development of microsatellite markers for the moss Ptychomitrium gardneri (Ptychomitriaceae)

? Premise of the study: Microsatellite markers were developed for Ptychomitrium gardneri to study population genetics of this eastern Asian-North American disjunct moss. ? Methods and Results: A total of 13 microsatellite markers were developed in Chinese populations of P. gardneri, using the Fast Isolation by AFLP of Sequences COntaining Repeats protocol. Eight of the markers showed polymorphism when assessed in a sample of four populations of 29 individuals from China. These markers amplified three to four alleles per locus. Five primers also amplified in P. linearifolium and P. wilsonii. ? Conclusions: These markers may be useful for further investigation of population genetics of P. gardneri.     

Clonal diversity in the Japanese silver crucian carp,Carassius langsdorfii inferred from genetic markers

The Japanese silver crucian carp, Carassius langsdorfii, is a naturally polyploid fish that reproduces by gynogenesis, resulting in a geographically clustered clonal population. To determine the amount of clonal diversity in this species, we used genetic markers to compare fish from several Japanese locations. Samples were collected from Lake Kasumigaura (Ibaraki Pref.), Lake Imba (Chiba Pref.), Lake Fukushimagata (Niigata Pref.), Lake Biwa (Shiga Pref.), Lake Koyama (Tottori Pref.), and the Niyodo and Monobe Rivers (Kochi Pref.). The genetic markers used were the microsatellite loci GF1(*), GF17(*), and GF29(*), as well as isozyme (creatinekinase CK(*)) and mtDNA (D-loop region) RFLP. In the seven locations sampled, 61 clonal lines were identified by combining the genotypes of the three microsatellite loci. Populations at all locations consisted of six to 28 clonal lines. Fourteen common clonal lines were detected in two or more locations, and 13 of these were observed in Lake Biwa. We suggest that these clonal lines have been widely distributed in Japanese freshwaters through the intentional stocking of fish species such as "gengorobuna" (C. cuvieri) and "ayu" (Plecoglossus altivelis altivelis) obtained from Lake Biwa and its adjacent waters. The CK(*) genotypes and mtDNA haplotypes of fish from Lake Kasumigaura differed from those of other locations.     

高寒草原优势种叶片结构变化与生态因子的关系

 为了揭示青藏高原高寒草原优势物种青藏苔草（Carex moorcroftii）和紫花针茅（Stipa purpurea）对高原特殊环境的适应性,该文研究了 它们的叶片结构在自然环境梯度下的变化以及这些变化与生态因子之间的关系。结果表明：这两个物种叶片的大多数结构特征在各个样地间变 化显著,其结构特征与环境因子间存在显著的相关关系。紫花针茅叶肉细胞大小随土壤有效K含量的增高而减小,下表皮细胞厚度和韧皮部面积 随生长季云盖度的增高而增加,单一导管半径和导管平均面积随生长季月均湿度的增加而增大;青藏苔草上表皮细胞厚度随生长季月均最低温 的降低而增厚,泡状细胞厚度（径向直径）随大陆度的增强而增加,上表皮细胞大小随土壤pH值的增大而增大,导管总数和韧皮部面积随土壤 速效P含量的增高而增加。青藏苔草的保护组织、光合组织以及综合指标变异系数明显大于紫花针茅,仅维管组织指标变异系数小于紫花针茅。     

Sex-specific pattern of spatial genetic structure in dioecious and clonal tree species, <Emphasis Type="Italic">Populus alba</Emphasis> L.

Fine-scale spatial genetic structure (SGS) has profound ecological and genetic consequences for plant populations, and some studies indicate that clonal reproduction may significantly enhance SGS. Clonality is widespread among dioecious species, but little is known about the relationship between clonal reproduction and SGS in the frame of sexual dimorphism. We asked the following questions: (1) Is there a sexually dependent pattern of SGS in white poplar population? (2) What is the relationship between clonal reproduction and SGS? and (3) Does this relationship have a sex-specific component? Using 16 microsatellite markers, genetic structure including fine-scale SGS and clonality of females and males of white poplar were investigated. Significant SGS was noted for both sexes at the ramet and genet levels. At the genet level, males had 2.7-fold higher SGS than that of females. Clonality significantly contributed to SGS only in females. A sibship structure revealed with pedigree analysis and clustering-based methods among males was likely the major factor of the observed SGS. The sexes differed in their clonal growth strategies. Spatial positioning of ramets in female clones suggested foraging behavior and/or avoidance of competition, while for male clones it indicated more expansion and space colonization. The obtained results led us to conclude that sexual dimorphism in life history traits may affect the course and rate of demo-genetic processes acting in natural populations of dioecious species. To our knowledge, this is the first study demonstrating a sex-specific pattern of SGS in natural populations of dioecious species.     

Microsatellite markers for the native Texas perennial grass, Panicum hallii (Poaceae)

? Premise of the study: We developed microsatellites for Panicum hallii for studies of gene flow, population structure, breeding experiments, and genetic mapping. ? Methods and Results: Next-generation (454) genomic sequence data were used to design markers. Eighteen robust markers were discovered, 15 of which were polymorphic across six accessions of P. hallii var. hallii. Fourteen of the markers cross-amplified in a P. capillare accession. For the 15 polymorphic markers, the total number of alleles per locus ranged from two to 26 (mean: 11.0) across six populations (11-19 individuals per population). Observed heterozygosity (mean: 0.031) was 13.7 times lower than the expected heterozygosity (mean: 0.426). ? Conclusions: The deficit of heterozygous individuals is consistent with P. hallii having a high rate of self-fertilization. These markers will be useful for studies in P. hallii and related species.     

Dynamic grapevine clones��an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.     

An extensive study of the genetic diversity within seven French wine grape variety collections

The process of vegetative propagation used to multiply grapevine varieties produces, in most cases, clones genetically identical to the parental plant. Nevertheless, spontaneous somatic mutations can occur in the regenerative cells that give rise to the clones, leading to consider varieties as populations of clones that conform to a panel of phenotypic traits. Using two sets of nuclear microsatellite markers, the present work aimed at evaluating and comparing the intravarietal genetic diversity within seven wine grape varieties: Cabernet franc, Cabernet Sauvignon, Chenin blanc, Grolleau, Pinot noir, Riesling, Savagnin, comprising a total number of 344 accessions of certified clones and introductions preserved in French repositories. Ten accessions resulted in being either self-progeny, possible offspring of the expected variety or misclassified varieties. Out of the 334 remaining accessions, 83 displayed genotypes different from the varietal reference, i.e., the microsatellite profile shared by the larger number of accessions. They showed a similarity value ranging from 0.923 to 0.992, and thus were considered as polymorphic monozygotic clones. The fraction of polymorphic clones ranged from 2 to 75% depending on the variety and the set of markers, the widest clonal diversity being observed within the Savagnin. Among the 83 polymorphic clones, 29 had unique genotype making them distinguishable; others were classified in 21 groups sharing the same genotype. All microsatellite markers were not equally efficient to show diversity within clone collections and a standard set of five microsatellite markers (VMC3a9, VMC5g7, VVS2, VVMD30, and VVMD 32) relevant to reveal clonal polymorphism is proposed.     

Development of microsatellite markers for Dimorphandra mollis (Leguminosae), a widespread tree from the Brazilian cerrado

? Premise of the study: Microsatellite markers were developed for Dimorphandra mollis (Leguminosae), a widespread tree in the Brazilian cerrado (a savanna-like vegetation). ? Methods and Results: Microsatellite markers were developed from an enriched library. The analyses of polymorphism were based on 56 individuals from three populations. Nine microsatellite loci were polymorphic, with the number of alleles per locus ranging from three to 10 across populations. The observed and expected heterozygosities per locus and population ranged from 0.062 to 0.850 and from 0.062 to 0.832, respectively. ? Conclusions: These microsatellites provide an efficient tool for population genetics studies and will be used to assess the genetic diversity and spatial genetic structure of D. mollis.     

Clonal and fine-scale genetic structure in populations of a restricted Korean endemic, Hosta jonesii (Liliaceae) and the implications for conservation

BACKGROUND AND AIMS: In plant populations the magnitude of spatial genetic structure of apparent individuals (including clonal ramets) can be different from that of sexual individuals (genets). Thus, distinguishing the effects of clonal versus sexual individuals in population genetic analyses could provide important insights for evolutionary biology and conservation. To investigate the effects of clonal spread on the fine-scale spatial genetic structure within plant populations, Hosta jonesii (Liliaceae), an endemic species to Korea, was chosen as a study species. METHODS: Using allozymes as genetic markers, spatial autocorrelation analysis of ramets and of genets was conducted to quantify the spatial scale of clonal spread and genotype distribution in two populations of H. jonesii. KEY RESULTS: Join-count statistics revealed that most clones are significantly aggregated at < 3-m interplant distance. Spatial autocorrelation analysis of all individuals resulted in significantly higher Moran's I values at 0-3-m interplant distance than analyses of population samples in which clones were excluded. However, significant fine-scale genetic structure was still observed when clones were excluded. CONCLUSIONS: These results suggest that clones enhance the magnitude of spatial autocorrelation due to localized clonal spread. The significant fine-scale genetic structure detected in samples excluding clones is consistent with the biological and ecological traits exhibited by H. jonesii including bee pollination and limited seed dispersal. For conservation purposes, genetic diversity would be maximized in local populations of H. jonesii by collecting or preserving individuals that are spaced at least 5 m apart.