Isolation and characterization of highly polymorphic microsatellite loci in the bladder campion,Silene vulgaris (Caryophyllaceae)

This study reports the isolation and characterization of seven highly polymorphic microsatellite loci in Silene vulgaris (Caryophyllaceae). The loci were isolated from two libraries constructed from genomic DNA enriched for CA and GA repeats. These markers yielded nine to 40 alleles per locus (mean 22.1) in a survey of 45 individuals from a single population located in the western Swiss Alps. Average observed heterozygosity ranged from 16.2 to 77.4%. These microsatellite loci should be valuable tools for studying fine‐scale genetic structure.     

Evolutionary dynamics of mitochondrial plasmids in natural populations of Silene vulgaris

Although plasmid-like mitochondrial DNA molecules have been investigated in a number of cultivated plant species, knowledge about their occurrence and behavior in natural plant populations is scarce. In the bladder campion, Silene vulgaris, a common weed of northern Europe, mt-plasmids of three different sizes were detected in a survey of S. vulgaris populations in southern Sweden. Two of the three plasmids usually occurred together within individual plants and showed large variation in frequency between populations. From F(ST)-estimates of plasmids, mitochondrial markers, and nuclear markers it was concluded that the plasmids are predominantly maternally inherited in their natural habitat, as observed in greenhouse experiments. The association between mt-plasmids and mitochondrial haplotype was strong, but not complete, in the natural material. These results indicate that the mt-plasmids of S. vulgaris have evolved toward almost strict maternal inheritance.     

Molecular and quantitative genetic differentiation in European populations of Silene latifolia (Caryophyllaceae)

BACKGROUND AND AIMS: Among-population differentiation in phenotypic traits and allelic variation is expected as a consequence of isolation, drift, founder effects and local selection. Therefore, investigating molecular and quantitative genetic divergence is a pre-requisite for studies of local adaptation in response to selection under variable environmental conditions. METHODS: Among- and within-population variation were investigated in six geographically separated European populations of the white campion, Silene latifolia, both for molecular variation at six newly developed microsatellite loci and for quantitative variation in morphological and life-history traits. To avoid confounding effects of the maternal environment, phenotypic traits were measured on greenhouse-reared F(1) offspring. Tests were made for clinal variation, and the correlations among molecular, geographic and phenotypic distances were compared with Mantel tests. KEY RESULTS: The six populations of Silene latifolia investigated showed significant molecular and quantitative genetic differentiation. Geographic and phenotypic distances were significantly associated. Age at first flowering increased significantly with latitude and exhibited a Q(st) value of 0.17 in females and 0.10 in males, consistent with adaptation to local environmental conditions. By contrast, no evidence of isolation-by-distance and no significant association between molecular and phenotypic distances were found. CONCLUSIONS: Significant molecular genetic divergence among populations of Silene latifolia, from the European native range is consistent with known limited seed and pollen flow distances, while significant quantitative genetic divergence among populations and clinal variation for age at first flowering suggest local adaptation.     

The hierarchical spatial distribution of chloroplast DNA polymorphism across the introduced range of Silene vulgaris

Silene vulgaris was introduced into North America sometime prior to 1800. In order to document the population structure that has developed since that time, collections were made from 56 local populations distributed among 9 geographical regions in eastern North America. Individual plants were characterized for chloroplast DNA (cpDNA) haplotype by restriction fragment size analysis of four noncoding regions of cpDNA amplified by polymerase chain reaction. A total of 19 cpDNA haplotypes were detected using this method. The overall gene diversity of 0.85 is quite similar to the diversity detected in these same regions of cpDNA in a previously published sample of S. vulgaris taken from across much of Europe. The spatial distribution of the North American cpDNA diversity was quantified by hierarchical F-statistics that partitioned the genetic variance into variation among local populations within regions, and variation among regions. The average FST among populations within regions was 0.66 and the FST among regions was 0.09. The among-region variation was due to both differences among regions in the frequency of two most common haplotypes, and to the presence of a number of region-specific haplotypes. In order to test for isolation by distance at the regional level, FST values were calculated for all possible pairs of regions, and regressed against the geographical distance between those regions. There was no evidence for isolation by distance. It is suggested that the local population structure is generated by recent extinction/colonization dynamics, and that the among-region structure reflects demographic events associated with range expansion following introduction to North America.     

Heavy metal-induced accumulation of free proline in a metal-tolerant and a nontolerant ecotype of Silene vulgaris

Accumulation of free proline in response to Cu, Cd and Zn was studied in nontolerant and metal-tolerant Silene vulgaris (Moench) Garcke. In the nontolerant ecotype these metals induced a massive accumulation of proline, especially in the leaves. When compared at equimolar concentrations in the nutrient solution, Cu was the most effective inducer, followed by Cd and Zn, respectively. However, when compared at equal toxic strength, as estimated from the degree of root growth inhibition, proline accumulation decreased in the order Cd > Zn > Cu. The threshold exposure levels for proline accumulation coincided with the highest no-effect-concentrations for root growth. In the metal-tolerant ecotype the constitutive proline concentration in the leaves was 5 to 6 times higher than in the nontolerant ecotype. Exposure to Cu and Zn, however, was without any effect on the leaf proline concentration, even at exposure levels that caused a 50% root growth inhibition. Only Cd, when present at concentrations above the highest no-effect-concentration for root growth, induced a further increase of the leaf proline content. Reducing transpiration by placing the plants under a transparent polyethylene cover almost completely inhibited proline accumulation, even at metal accumulation rates in the leaves that caused a 10-fold increase of the proline level in leaves of uncovered plants. The results demonstrate that metal-induced proline accumulation depends on the development of a metal-induced water deficit in the leaves. Differential metal-induced proline accumulation in distinctly metal-tolerant ecotypes is a consequence, rather than a cause of differential metal tolerance.     

Consequences of inbreeding for offspring fitness and gender in Silene vulgaris,a gynodioecious plant

Abstract In gynodioecious plants, hermaphrodite and female plants co‐occur in the same population. In these systems gender typically depends on whether a maternally inherited cytoplasmic male sterility factor (CMS) is counteracted by nuclear restorer alleles. These restorer alleles are often genetically dominant. Although plants of the female morph are obligatorily outcrossing, hermaphrodites may self. This selfing increases homozygosity and may thus have two effects: (1) it may decrease fitness (i.e. result in inbreeding depression) and (ii) it may increase homozygosity of the nuclear restorer alleles and therefore increase the production of females. This, in turn, enhances outcrossing in the following generation. In order to test the latter hypothesis, experimental crosses were conducted using individuals derived from four natural populations of Silene vulgaris, a gynodioecious plant. Treatments included self‐fertilization of hermaphrodites, outcrossing of hermaphrodites and females using pollen derived from the same source population as the pollen recipients, and outcrossing hermaphrodites and females using pollen derived from different source populations. Offspring were scored for seed germination, survivorship to flowering and gender. The products of self‐fertilization had reduced survivorship at both life stages when compared with the offspring of outcrossed hermaphrodites or females. In one population the fitness of offspring produced by within‐population outcrossing of females was significantly less than the fitness of offspring produced by crossing females with hermaphrodites from other populations. Self‐fertilization of hermaphrodites produced a smaller proportion of hermaphroditic offspring than did outcrossing hermaphrodites. Outcrossing females within populations produced a smaller proportion of hermaphrodite offspring than did crossing females with hermaphrodites from other populations. These results are consistent with a cytonuclear system of sex determination with dominant nuclear restorers, and are discussed with regard to how the mating system and the genetics of sex determination interact to influence the evolution of inbreeding depression.     

Toxicity of arsenate in Silene vulgaris, accumulation and degradation of arsenate-induced phytochelatins

The results presented in this paper describe the short- and long-term toxicity of arsenate in Silene vulgaris . Short-term toxicity, measured as inhibition of root elongation, depended on phosphate nutrition, arsenate being much less toxic at high phosphate supply. At low phosphate levels more arsenic was taken up by the plants. Under chronic exposure, toxicity (measured as inhibition of biomass production) did not increase with time. In addition, the accumulation of phytochelatins (PCs) as a function of toxicity and duration of exposure was studied. Short-term PC accumulation (over a 3 d period) was positively correlated with exposure. Isolation of peptide complexes from prolongedly exposed plants showed that PC₂, PC₃ and PC₄ were present, although the latter not until at least 3 d exposure. Arsenic co-eluted mainly with PC₂ and PC₃. Fractions containing PC₄ were devoid of As, probably due to dissociation of the complexes during extraction or elution. The breakdown of PCs after arresting As exposure was very slow. This could explain the continuous accumulation of PCs throughout longer periods of As exposure.     

Variation in mutation rate and polymorphism among mitochondrial genes of Silene vulgaris

The prevailing wisdom of the plant mitochondrial genome is that it has very low substitution rates, thus it is generally assumed that nucleotide diversity within species will also be low. However, recent evidence suggests plant mitochondrial genes may harbor variable and sometimes high levels of within-species polymorphism, a result attributed to variance in the influence of selection. However, insufficient attention has been paid to the effect of among-gene variation in mutation rate on varying levels of polymorphism across loci. We measured levels of polymorphism in seven mitochondrial gene regions across a geographically wide sample of the plant Silene vulgaris to investigate whether individual mitochondrial genes accumulate polymorphisms equally. We found that genes vary significantly in polymorphism. Tests based on coalescence theory show that the genes vary significantly in their scaled mutation rate, which, in the absence of differences among genes in effective population size, suggests these genes vary in their underlying mutation rate. Further evidence that among-gene variance in polymorphism is due to variation in the underlying mutation rate comes from a significant positive relationship between the number of segregating sites and silent site divergence from an outgroup. Contrary to recent studies, we found unconvincing evidence of recombination in the mitochondrial genome, and generally confirm the standard model of plant mitochondria characterized by low substitution rates and no recombination. We also show no evidence of significant variation in the strength or direction of selection among genes; this result may be expected if there is no recombination. The present study provides some of the most thorough data on plant mitochondrial polymorphism, and provides compelling evidence for mutation rate variation among genes. The study also demonstrates the difficulty in establishing a null model of mitochondrial genome polymorphism, and thus the difficulty, in the absence of a comparative approach, in testing the assumption that low substitution rates in plant mitochondria lead to low polymorphism.     

Poly(γ-glutamylcysteinyl)glycines or phytochelatins and their role in cadmium tolerance of Silene vulgaris

Abstract. The effect of cadmium on growth of Cd-tolerant and -sensitive plants of Silene vulgaris and on the production of metal-binding compounds in both types of plants was studied. The Cd-content of the roots and the Cd-root/shoot ratio was higher in Cd-tolerant plants. A Cd-binding compound (Cd-BC) with an apparent molecular mass of 14.5 kD was isolated from the roots of Cd-tolerant and -sensitive plants, grown in 40 mmol m⁻³ Cd for 21 d. More than 60% of the total Cd in the roots was associated with this compound. Determination of the amino-acid content of the purified Cd-containing compound from both types of plants showed that they possessed a similar amino-acid composition to that of phytochelatins. Only the bis- and tris-forms were present. The amount of Cd and sulphide associated with phytochelatin was greater in tolerant plants than in sensitive ones suggesting that an increased sulphide content of complexes containing peptide, sulphide and Cd may form the basis of evolved Cd-tolerance in Silence vulgaris.     

Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L. (Caryophyllaceae): a random amplified polymorphic DNA analysis

Metal-contaminated sites can occur naturally in serpentine outcrops or as consequence of anthropogenic activities, such as mining deposits, aerial fallout from smelters and industrial processes. Serpentine outcrops are characterized by high levels of nickel, cobalt and chromium and present a typical vegetation which includes endemisms and plants which also live in uncontaminated soils. These latter metal-tolerant populations provide the opportunity to investigate the first steps in the differentiation of plant populations under severe selection pressure and to select plants to be used in the phytoremediation of industrially contaminated soils. In this report eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in noncontaminated soil in central Italy, were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the pattern of genetic variation. The genetic diversity observed in populations at copper mine deposits was found to be at least as high as that of the neighbouring serpentine populations. Analysis of molecular variance (AMOVA) of the RAPD markers gave high statistical significance to the groupings of populations according: (i) with their geographical location; and (ii) with the metals present in the soil of origin (copper vs. nickel), indicating that RAPD markers detected a polymorphism related to the soil contamination by copper. Finally, two RAPD bands exclusive to copper-tolerant populations were identified.     

Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance

The heavy metal zinc was administered to barley seedlings by increasing its concentration in the hydroponic medium. The most dramatic effect was a severe inhibition of root elongation with little effect on root biomass production. The growth of primary leaves was little affected although the zinc content of the primary leaves increased several-fold. A detailed compartment analysis was performed for 10-d-old barley primary leaves. Under low zinc nutrition (2mmol m ⁻³), highest zinc contents were observed in the cytoplasm of mesophyll protoplasts. At inhibitory zinc concentrations in the hydroponic medium (400 μmol m ⁻³), zinc levels dramatically and preferentially increased in the apoplastic space. Elevated zinc levels were also observed in the epidermal cells, and to a lesser extent, in mesophyll vacuoles. The cytoplasmic content of mesophyll protoplasts was unchanged, indicating perfect zinc homeostasis within the leaf. In order to understand the transport mechanisms underlying the steady-state distribution profile, we used ⁶⁵Zn to conduct uptake experiments with leaves whose lower epidermis had been stripped. The leaves were placed on zinc solutions of varying concentrations containing ⁶⁵Zn for 5 min to 6 h. After the incubation, the leaves were fractionated into mesophyll and epidermis protoplasts and residue, the latter mainly representing cell wall. Adsorption of Zn to the extracellular matrix was 100 times faster than Zn uptake into the cells. By far the largest portion taken up into the mesophyll protoplasts rapidly appeared in the vacuolar compartment. These results demonstrate the importance of compartmentation and transport as homeostatic mechanisms within the leaves to handle high, possibly toxic, zinc levels in the shoot.     

Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.     

超积累植物伴矿景天镉耐受基因SpMT2的分离及功能鉴定

超积累植物由于其对重金属具有地上部超积累以及超耐受等特性,不仅是研究植物离子转运及毒性耐受的理想模式,而且在植物修复的发展和应用中具有不可替代的作用。伴矿景天是近年在我国境内发现的一种景天科镉(Cd)/锌(Zn)超积累植物。为鉴定其富集和耐受Cd的关键基因,笔者构建了其酵母表达cDNA文库,利用酵母的遗传互补系统筛选到一个极大提高了酵母抗Cd能力的基因SpMT2。SpMT2属于富含半胱氨酸(Cys)的金属硫蛋白(Metallothionein)家族。亚细胞定位表明SpMT2表达于酵母细胞质中,并特异地提高酵母对Cd的抗性。进一步研究发现SpMT2的表达显著降低了酵母液泡中Cd含量,但酵母吸收的总Cd含量无显著变化。推测抗性增加是由于SpMT2在酵母细胞质中通过螯合Cd从而降低Cd对酵母的毒害。qRT-PCR分析表明SpMT2在伴矿景天的根和地上部都高丰度表达,且不受Cd诱导变化。鉴于SpMT2也定位于植物细胞质中,结合上述结果,推测SpMT2可能在伴矿景天细胞质中螯合Cd,在降低Cd毒害的同时可能还保持Cd在细胞质中的流动性,从而在Cd长途转运过程中也发挥重要作用。     

Transmission rates and phenotypic effects of mitochondrial plasmids and cytotypes in Silene vulgaris

We investigated the transmission properties and the phenotypic effects of two mitochondrial plasmids in a population of the bladder campion, Silene vulgaris. In reciprocal crosses between plasmid-free and plasmid-carrying plants, no cases of paternal transmission or loss during maternal transmission were recorded. Neither was any transmission via pollen observed when plasmid-carrying plants of S. vulgaris were used to pollinate plasmid-free plants of the closely related species Silene uniflora. The phenotypic effects of the plasmids were investigated by comparing germination rate, early growth properties, and the gender of plants grown from seeds with and without plasmids. A significant association between plasmid status, on the one hand, and germination propensity and offspring gender, on the other, was found. However, because all plants carrying plasmids in the experiment shared the same cytoplasmic background, the exact contribution of the plasmid to the phenotypic variation could not be determined. Taken together, our experiments show that in S. vulgaris the mt-plasmids are not currently involved in any strong genetic conflict, but that they evolve in close association with their mitochondrial host.