Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji

To understand the reproduction of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji, Japan, the in situ genet dynamics of sporocarps were analysed. Sporocarps of the two Laccaria species were sampled at fine and large scales for 3 and 2 consecutive years, respectively, and were genotyped using microsatellite markers. In the fine-scale analysis, we found many small genets, the majority of which appeared and disappeared annually. The high densities and annual renewal of Laccaria genets indicate frequent turnover by sexual reproduction via spores. In the large-scale analysis, we found positive spatial autocorrelations in the shortest distance class. An allele-clustering analysis also showed that several alleles were distributed in only a small, localised region. These results indicate that Laccaria spores contributing to sexual reproduction may be dispersed only short distances from sporocarps that would have themselves been established via rare, long-distance spore dispersal. This combination of rare, long-distance and frequent, short-distance Laccaria spore dispersal is reflected in the establishment pattern of seeds of their host, Salix reinii.     

Patch establishment and development of a clonal plant,Polygonum cuspidatum,on Mount Fuji

Microsatellite analysis was used to investigate the patch establishment and development of Polygonum cuspidatum Sieb. et Zucc, a clonal herbaceous plant that dominates the primary succession on the southeast slope of Mount Fuji. Genotypes of P. cuspidatum in 155 patches at the study site differed from each other. This indicates that P. cuspidatum patches are initially established by seed dispersed on the bare scoria field, and not by clonal rhizome extension. Genetic differentiation was estimated using the FST values between subpopulations at the study site. There was almost no genetic differentiation between subpopulations, indicating the presence of massive gene flow. The pollen fathers of seeds and maternal genets of current-year seedlings were inferred from the microsatellite allele composition by a simple exclusion method. The wide, random distribution of pollen fathers suggests that pollen dispersal occurs over a broad area. Maternal analysis showed a tendency for seed dispersal to be biased to the area nearby and down slope from the mother plants. Patch establishment under massive gene flow may result from such pollen and seed dispersal. To understand the process of patch development, aerial photographs taken from 1962 to 1999 were compared, and then genets in each of 36 patches were identified from the microsatellite genotypes of P. cuspidatum shoots. The comparison of aerial photographs showed that most of the patches enlarged each year and that some neighbouring patches combined during growth. Genet analysis demonstrated a high correlation between patch area and the area of the largest genet within it, and that new genets were recruited at the patch periphery. These findings indicate that both vegetative and sexual reproduction, i.e. rhizome extension and the establishment of new seedlings, contribute to the development of P. cuspidatum patches.     

Development of microsatellite markers in polyploid Salix reinii

Salix reinii Franch. et Savat., a crawl shrub, is one of the most dominant pioneer trees on the southeastern slope of Mt. Fuji, on which an early stage of the volcanic desert succession has been underway since the last eruption in 1707. To investigate the population genetic structure within and among patches of S. reinii, seven polymorphic microsatellite markers were developed by an enrichment procedure. The multiple banding patterns amplified by developed microsatellite markers showed the polyploidy in S. reinii growing on the southeastern slope of Mt. Fuji.     

DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis)

Uniparentally inherited mitochondrial (mt)DNA and chloroplast (cp)DNA microsatellites (cpSSRs) were used to examine population genetic structure and biogeographic patterns of bird-dispersed seed and wind-disseminated pollen of whitebark pine (Pinus albicaulis Engelm.). Sampling was conducted from 41 populations throughout the range of the species. Analyses provide evidence for an ancestral haplotype and two derived mtDNA haplotypes with distinct regional distributions. An abrupt contact zone between mtDNA haplotypes in the Cascade Range suggests postglacial biogeographic movements. Among three cpSSR loci, 42 haplotypes were detected within 28 cpSSR sample populations that were aggregated into six regions. Analysis of molecular variance (amova) was used to determine the hierarchical genetic structure of cpSSRs. amova and population pairwise comparisons (FST ) of cpSSR, and geographical distribution of mtDNA haplotypes provide insights into historical changes in biogeography. The genetic data suggest that whitebark pine has been intimately tied to climatic change and associated glaciation, which has led to range movements facilitated by seed dispersal by Clark's nutcracker (Nucifraga columbiana Wilson). The two hypotheses proposed to explain the genetic structure are: (i) a northward expansion into Canada and the northern Cascades in the early Holocene; and (ii) historical gene flow between Idaho and the Oregon Cascades when more continuous habitat existed in Central Oregon during the late Pleistocene. Genetic structure and insights gained from historical seed movements provide a basis on which to develop recovery plans for a species that is at risk from multiple threats.     

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.     

基于叶绿体微卫星研究鄂报春谱系遗传结构

鄂报春Primula obconica作为一种广泛栽培的园艺植物,其野生居群的遗传多样性及遗传结构的研究还少见报道。本文通过叶绿体微卫星分析了17个鄂报春野生居群(共278个个体),共发现4个多态性位点(16个等位基因),得到14个单倍型。结果表明鄂报春具有很高的总基因多样性(HT=0.971)和极低的居群内基因多样性(HS=0.028);分子方差分析(AMOVA)显示98%的变异存在于居群间。这些结果说明早期的生境片断化及有限的种子传播能力是造成当前遗传结构的重要原因。Nst值显著大于Gst值,表明关系相近的单倍型会出现在相同的地区内,同时最小生成树(MST)的分析结果证实了这样的结论。我们在最小生成树的两个组中推断出一些古老单倍型,并推测在冰期时湖北和我国的西南地区可能是该物种的避难所。     

New insights into the intraspecific cytoplasmic DNA diversity,maternal lineages classification and conservation issues of Tunisian pearl millet landraces

Tunisian pearl millet (Pennisetum glaucum L.) landraces are still growing in contrasting agro-ecological environments and are considered potentially useful for national and international breeders. Despite its genetic potential, the cropping areas of this species are still limited and scattered which increases the risk of genetic erosion. The chloroplast DNA polymorphism and maternal lineages classification of forty nine pearl millet landraces representing seven populations covering the main distribution area of this crop in Tunisia were undertaken based on informative cpSSR molecular markers. A total of 21 alleles combining to 9 haplotypes were detected with a mean value of 3.5 alleles per locus and a haplotype genetic diversity (Hd) of 0.82. The number of chloroplast haplotypes per population ranged from 1 to 4 with an average of 1.28. The haplotypes median-joining network and UPGMA analyses revealed two probable ancestral maternal lineages with a differential pearl millet seed-exchange rate between the investigated areas. Northern and Central populations presented unique genetic backgrounds while historical farmers’ practices in the South-East area resulted in the isolation of their own local landraces. The genetic evidences strongly support at least two introduction origins of pearl millet in Tunisia, one in the North and the other in the South followed by distinct local dispersal histories. Complementary in-situ and ex-situ conservation strategies taking into account the conservation of the maternal lineage cytoplasmic diversity are required. The investigated chloroplast SSRs provide useful molecular markers which could be used in further genetic studies and breeding surveys of pearl millet genetic resources.     

Absence of geographical structure of chloroplast DNA variation in sallow, Salix caprea L

In the present study, we have used PCR-RFLP markers to investigate the chloroplast DNA variation in 24 European populations of Salix caprea L. A subset of these populations has also been analysed with chloroplast microsatellites. The main feature of both markers is the absence of a clear geographic structure (G(ST(PCR-RFLP))=0.090, G(ST(microsatellites))=-0.017) and high levels of variation within populations. This lack of phylogeographic structure in S. caprea is suggested to be the consequence of the joint action of several factors: (i) presence of intermediate latitude refugia with large population sizes during the last glacial maximum, (ii) a high speed of recolonisation and dispersal ability, (iii) a high mutation rate and (iv) extensive hybridisation with other willow species. In addition to the S. caprea samples, a limited number of individuals from several other Salix species were also analysed with PCR-RFLP: S. cinerea, S. aurita, S. purpurea, S. atrocinerea, S. appendiculata, S. elaeagnos, S. fragilis and S. alba. Many of the haplotypes found in Salix caprea were also detected in S. cinerea, S. aurita, S. purpurea, S. atrocinerea and/ or S. appendiculata but not in S. alba, S. elaeagnos or S. fragilis. Our data suggest that hybridisation and gene flow have occurred within these two groups but not between them.     

Isolation of compound microsatellite loci in the herbaceous perennial Cirsium purpuratum (Maxim.) Matsum

The herbaceous perennial Cirsium purpuratum is a pioneer on the southeast side of Mount Fuji in Japan. For genetic analysis of reproduction in this species, we developed polymorphic compound microsatellite markers using an adaptor-ligated library method and a simpler method called the intercompound microsatellite method. The latter method was an effective method for developing compound simple sequence repeat markers. In total, 11 polymorphic, codominant microsatellite markers were developed and characterized for this species. These polymorphic markers had three to 20 alleles per locus, a range of observed heterozygosity from 0.25 to 0.90, and were considered effective for genetic analysis.     

苎麻叶绿体SSR引物筛选及分析

叶绿体微卫星标记为单亲遗传(除部分裸子植物外),有独立的进化路线,它在植物遗传多样性、群体遗传结构、系统发育分析及杂种鉴定等研究上用途广泛,是研究谱系地理的有效手段。苎麻的主产地和主要分布区均在我国,但其谱系地理研究目前尚未见有报道。该研究选择来自全国不同地区的52个苎麻样本,利用聚丙烯酰胺凝胶电泳法,在23对已知通用叶绿体SSR引物中筛选出适用于苎麻谱系地理研究的SSR引物,并利用筛选到的多态引物对52个苎麻样本进行聚类分析和单倍型网络图分析。结果表明:从23对通用引物中共筛选出16对适用于苎麻的多态引物,其平均多态信息含量为0.1053,虽然以上引物多态性较低,但能够用于野生苎麻的遗传分析研究;这52个苎麻样本聚为10支,分为8个单倍型,初步分析表明叶绿体SSR遗传变异速率较慢,不适用于苎麻种内的系统发育研究,但以上引物能够检测苎麻种内单倍型变异,可用于苎麻的谱系地理研究。     

Dissimilar patterns of Pinus heldreichii Christ. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis

In the present study we investigated the genetic structure and genetic diversity of Pinus heldreichii populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. We were interested in addressing the following questions: (1) can population structuring in Bosnian pine be detected via chloroplast microsatellite markers; (2) are there differences in population differentiation as determined by terpenes and microsatellites; and (3) how are the patterns of size variant frequencies and geographical distances related. Four provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using 6 primer pairs. Between 2 and 5 size variants were identified at each locus. A total of 16 size variants at the 6 loci were identified, 4 occurring at low frequencies. They were combined in 21 different haplotypes including 11 that were unique. AMOVA analysis revealed that 18.25% of the variation was found among populations, while 81.75% was expressed within populations. The cpSSR analysis divided Bosnian pine populations into two groups, the first represented by populations B, C and D located in the south and north-western part of the Pirin and Slavianka mountains, while the second group, represented by population A, is located in the north-eastern Pirin mountain. Terpene analysis revealed that on average, 59% of the monoterpene pool in P. heldreichii is accounted for by limonene (range 36–48%) followed by α-pinene (range 16–17%). The presence of two distinct groups (Pop-A, Pop-D and Pop-B, Pop-C) is more consistent with physical distances between populations. No significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance determined by terpenes was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in Bosnian pine populations is the consequence of historical and biogeographical processes.     

Stem anatomy of Rhipsalis (Cactaceae) and its relevance for taxonomy

We used paternally inherited chloroplast microsatellites (cpSSR) to study population genetic structure in the endemic and highly restricted Serbian spruce Picea omorika. Fragment size polymorphism at the five cpSSR regions that could be amplified out of the nine tested combined into only four different haplotypes in the seven populations studied. Mean total haplotypic diversity was H _T = 0.395, and mean within-population diversity was H _s = 0.279. Haplotypic variation was lower than in most conifers described so far and partitioned into two geographical groups. All northern P. omorika populations were fixed or nearly fixed for the common haplotype, while southern populations exhibited two to three haplotypes. We suggest that current P. omorika populations are shaped by extreme demographic bottleneck and random genetic drift linked to Quaternary glacial cycles. P. omorika thus belongs to the small group of genetically depauperate tree species.     

Fine‐scale asexual and sexual reproduction in Quercus crispula var. horikawae,a stunted shrub oak,on a mountain with deep snow in central Japan

Quercus crispula var. horikawae, a stunted shrub oak, occurs on mountains with deep snow on the Sea of Japan side of Japan. This oak generates patches of multiple creeping stems. It is unclear whether these patches are the result of asexual or sexual reproduction, or both. We therefore aimed to describe the clonal structure and gene flow in Q . crispula var. horikawae on Mount Nasu in central Japan by using nuclear microsatellites. Genotypes of 331 stems with no distinct connection with roots and creeping stems above the ground were determined using nine loci in two study plots, and 64 acorns from three mother genets in a plot were determined using eight loci. The results of the clonal identification indicated that the patches consisted of 51 genets; at least 85% of the stems may have been derived from asexual reproduction through sprouting and layering. The prominence of asexual reproduction may be a result of adaptation to the snowy environment. In contrast, 15% of the ramets in the study plots probably originate via sexual reproduction by seedling regeneration. Analyses of the spatial genetic structure and paternity showed that limited ability of the pollen and seeds to disperse might result in the spatial aggregations of closely related offspring at a relatively short distance (<10 m), and inbreeding, a factor that might reduce sexual reproduction, was not observed. Thus, sexual reproduction could be reduced by ecological rather than genetic factors, namely the hindrance of seedling regeneration by the dense coverage of dwarf bamboo (Sasa) on the forest floor.     

Genetic diversity of the Andean tetraploid cultivated potato (Solanum tuberosum L. subsp. andigena Hawkes) evaluated by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are native farmer-selected important cultivars that form a primary gene pool of the common potato (Solanum tuberosum L. subsp. tuberosum). The genetic diversity of 185 Andigena accessions and 6 Chilean native potatoes (S. tuberosum subsp. tuberosum) was studied using chloroplast DNA (ctDNA) microsatellites and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Andigena potatoes had 14 ctDNA haplotypes and showed higher variability in the central Andes, particularly in Bolivia, whereas those in the northern regions of the distribution area were remarkably uniform with A1 ctDNA and Chilean subsp. tuberosum with T ctDNA. Most of 123 clearly scored RFLP bands using 30 single-copy probes were randomly distributed throughout the distribution area and proved the same gene pool shared among these widely collected accessions. Nevertheless, the geographic trend of the nDNA differentiation from north to south along the Andes and the correlated differentiation between nDNA and ctDNA (r = 0.120) could also be revealed by canonical variates analysis. These results suggest that the genetic diversity in Andigena was brought about primarily from cultivated diploid species but considerably modified through sexual polyploidization and intervarietal and (or) introgressive hybridization and long-distance dispersal of seed tubers by humans.     

Structure of Pinus sylvestris L. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: Provenance tests

In the present study we investigated the genetic structure and genetic diversity of Pinus sylvestris populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. We were interested in addressing the following questions: (1) can population structure in Scots pine be detected via chloroplast microsatellites markers and terpenes; (2) are there differences in population differentiation between the two analyses; and (3) how are the patterns related to geographic distances. Twelve provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using six primer pairs. Between 4 to 8 size variants were identified at each locus. A total of 35 size variants at the six loci were identified, 11 occurring at low frequencies (<1%). They were combined in 134 different haplotypes, of which seven represent 1/3 of the genetic structure. AMOVA analysis revealed that 10.99% of the variation was found among populations, while 89.01% was expressed within populations. The cpSSR analysis divided Scots pine populations into two groups, the first represented by populations located in the south-western part of the Rhodopes and Pirin mountains, while the second group is located in the northeast of Rhodopes and Rila mountains. Terpene analysis revealed that on average, 53% of the monoterpene pool in P. sylvestris was accounted for by -pinene (range 47–59%) followed by β-pinene (range 6–12%). The presence of two distinct groups is weekly consistent with physical distances between populations, similar significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance (determined by terpenes) was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in Scots pine populations is the consequence of historical biogeographic processes.     

Historical and contemporary mating patterns in remnant populations of the forest tree Fraxinus excelsior L

Genetic variation at microsatellite markers was used to quantify genetic structure and mating behavior in a severely fragmented population of the wind-pollinated, wind-dispersed temperate tree Fraxinus excelsior in a deforested catchment in Scotland. Remnants maintain high levels of genetic diversity, comparable with those reported for continuous populations in southeastern Europe, and show low interpopulation differentiation (E = 0.080), indicating that historical gene exchange has not been limited (Nm = 3.48). We estimated from seeds collected from all trees producing fruits in three of five remnants that F. excelsior is predominantly outcrossing (t(m) = 0.971 +/- 0.028). Use of a neighborhood model approach to describe the relative contribution of local and long-distance pollen dispersal indicates that pollen gene flow into each of the three remnants is extensive (46-95%) and pollen dispersal has two components. The first is very localized and restricted to tens of meters around the mother trees. The second is a long-distance component with dispersal occurring over several kilometers. Effective dispersal distances, accounting for the distance and directionality to mother trees of sampled pollen donors, average 328 m and are greater than values reported for a continuous population. These results suggest that the opening of the landscape facilitates airborne pollen movement and may alleviate the expected detrimental genetic effects of fragmentation.     

Chloroplast microsatellite diversity of Clintonia udensis (Liliaceae) populations in East Asia

Nineteen populations of Clintonia udensis Trautv. & Mey. were examined to quantify genetic diversity and genetic structure by chloroplast DNA microsatellites (cpSSR). Significant cpSSR genetic diversity (PPB = 63.64%) was detected in this species. Tetraploid populations demonstrated approximately the same level of genetic diversity as the diploid ones. A significant differentiation, however, was found between tetraploids and diploids. Most of the sixteen chloroplast haplotypes were limited to a single population. The level of haplotype diversity was high (Hd = 0.915). AMOVA, PCA and Bayesian clustering analysis revealed that there were significant genetic differences among populations. Inter-population genetic distances among population sites correlated significantly with geographic distances. These results indicate that the mixed-mating – breeding system, limited gene flow, environmental stress, and historical factors may be the main factors causing geographical differentiation in the genetic structure of C. udensis.     

Population genetic structure in European populations of Spiranthes romanzoffiana set in the context of other genetic studies on orchids

Spiranthes romanzoffiana Cham. is restricted in Europe to the British Isles, where it is recognised as a conservation priority species due to frequent extirpation of populations along with no evidence of seed set; vegetative reproduction has been invoked as the sole means of perpetuation and dispersal. To investigate the reproductive ecology of this species, 17 populations have been sampled for chloroplast microsatellites and amplified fragment length polymorphisms (AFLPs). These markers revealed a previously unsuspected genetic-geographic split in the species, which correlates with differences in patterns of within-population variation. Northern populations were fixed for one chloroplast haplotype but showed high levels of AFLP genotypic diversity consistent with sexual reproduction (proportion of genotypes distinguishable, PD = 0.98). More southerly populations showed fixed differences from the northern populations in their chloroplast haplotype and for 10 AFLP markers. They harboured only 12 unique multilocus genotypes among 113 individuals from six populations (PD = 0.11). These genotypes differed mostly by single bands, and none by more than 4/138 loci, with identical multilocus genotypes occurring in widely separated populations. This uniformity in southern populations is consistent with agamospermous or autogamous reproduction, and/or an extreme population bottleneck. Finally, the observed patterns of population differentiation in S. romanzoffiana are compared with other studies of orchids, revealing a wide range of values that belie recent contrasting published generalisations that claim that orchids show either higher, or lower, levels of population differentiation than other plant families.     

Using RAPD markers to identify genets of an arable grass weed, Arrhenatherum elatius ssp. bulbosum

RAPD (Random Amplified Polymorphic DNA) markers were used to identify genets within a weed infestation of Arrhenatherum elatius ssp bulbosum (“onion couch”). Three selected primers could discriminate between 18 sexually-derived plants of A. elatius ssp. elatius. Using these primers, RAPD phenotypes were obtained for ramets of ssp. bulbosum from a population of unknown sexual derivation, i.e. predominantly sexual or vegetative. Thirty three RAPD phenotypes were observed amongst 65 plants. Ramets with identical RAPD phenotypes were termed “genets”. Some genets of ssp. bulbosum were found in localised patches, whereas others had much wider ranges, indicating long-distance dispersal by farm machinery. RAPD markers provide a useful tool for discriminating between genets within an established plant population.     

Heterologous nuclear and chloroplast microsatellite amplification and variation in tea, Camellia sinensis.

The advantage of the cross transferability of heterologous chloroplast and nuclear microsatellite primers was taken to detect polymorphism among 24 tea (Camellia sinensis (L.) O. Kuntze) genotypes, including both the assamica and the sinensis varieties. Primer information was obtained from the closely related Camellia japonica species for four nuclear microsatellites, and from Nicotiana tabaccum for seven universal chloroplast microsatellites. All of the nuclear microsatellite loci tested generated an expected DNA fragment in tea, revealing between three and five alleles per locus. Four out of the seven chloroplast microsatellites primers amplified positively, and of these only one was polymorphic with three alleles, which is in agreement with the conserved nature of chloroplast microsatellites at the intraspecific level. A factorial correspondence analysis carried out on all genotypes and nuclear microsatellite alleles separated the assamica and sinensis genotypes into two groups, thus demonstrating the value of these markers in establishing the genetic relationship between tea varieties. Genetic diversity measured with nuclear microsatellites was higher than that measured with other types of molecular markers, offering prospects for their use in fingerprinting, mapping, and population genetic studies, whereas polymorphisms detected at a cpSSR locus will allow the determination of plastid inheritance in the species.