No genetic variation detected within isolated relict populations of Saxifraga cernua in the Alps using RAPD markers

Genetic variation in seven relict populations of Saxifraga cernua from three regions of the Alps was investigated using RAPD (random amplified polymorphic DNA) markers. No variation, either within the populations or within the regions, could be demonstrated. Nevertheless, each alpine region was characterized by a unique RAPD phenotype. Absence of genetic variation in these relict populations is attributed to population bottlenecks and founder effects during or following the ice ages. Contrasting hypotheses about the history of these populations, either as survivors of the glacial period or as products of postglacial immigration, are discussed in the light of the data presented.     

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.     

Genetic enrichment of the arctic clonal plant Saxifraga cernua at its southern periphery via the alpine sexual Saxifraga sibirica

Isolation of populations at the margins of a species range may lead to decreasing genetic diversity via genetic drift and inbreeding. Hybridization between peripheral populations of two species can, however, counteract genetic impoverishment. The mainly clonal, polyploid plant Saxifraga cernua has a wide arctic distribution but also extends southwards into alpine sites. In the Ural Mountains, its peripheral distribution overlaps with that of its sexually reproducing, diploid relative Saxifraga sibirica, and fertile polyploids of more or less intermediate appearance are found in this overlap zone. We used amplified fragment length polymorphism (AFLP) analysis to address the potential impact of interspecific gene flow on genetic diversity in the peripheral populations. A total of 149 plants from 17 populations along a 1650 km south-north gradient were analysed for 253 markers. The results suggest that three Middle Ural populations containing fertile and morphologically more or less intermediate plants have been affected by hybridization. All of these plants formed a strongly supported (100%) group with S. cernua in a neighbour-joining tree, but their AFLP phenotypes assigned either to S. cernua or to artificial (simulated) F(1) hybrids between S. cernua and S. sibirica in multilocus assignment tests. The three populations were highly diverse with virtually every plant representing a distinct AFLP phenotype, providing additional evidence for formation of later-generation hybrids and/or backcrossing to S. cernua. In contrast, other peripheral populations of S. cernua were typically monoclonal, suggesting that hybridization with S. sibirica can increase genetic diversity in S. cernua at its southern periphery.     

Clonality and recombination in the arctic plant Saxifraga cernua

The circumarctic clonal plant Saxifraga cernua reproduces efficiently via bulbils, largely depends on insects for pollination and appears to set seed very rarely. However, high levels of genotypic variation observed at small spatial scales in the arctic archipelago of Svalbard have been taken as evidence of occasional sexual reproduction. Here we assess the relative contributions of mutation and recombination to random amplified polymorphic DNA variation in four populations in East Greenland and re-analyse the Svalbard data. Greater variation due to recombination was predicted in Greenland than in Svalbard, because the higher summer temperatures and longer growing season likely increase the chances for sexual reproduction. Although we observed higher levels of genotypic diversity in Greenland than in Svalbard, matrix incompatibility and linkage disequilibrium measures provided no evidence of more sexuality, suggesting differences in glacial/postglacial history. The genetic structure and spatial distribution of clones suggest that clonal migration may increase variability in local populations, which is consistent with frequent large-scale migration in this species inferred from a circumarctic analysis of chloroplast DNA haplotypes. We conclude that a combination of somatic mutations and sexual reproduction has contributed to the observed patterns of genotypic diversity in the Greenland and Svalbard populations of S. cernua , and that sexual reproduction is important in maintaining genotypic diversity, despite the rarity of observations of seed setting.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 209–217.     

Low temperature acclimation of guard cell chloroplasts by the arctic plant Saxifraga cernua L.

Abstract. The potential for thermal acclimation of photosynthetic electron transport by guard cell chloropiasts (GC ch) was assessed in epidermal peels taken from the abaxial side of Saxifraga cernua leaves grown at 20°C and 10°C. Chlorophyll a fluorescence induction kinetics measured in pairs of guard cells in individual stomata from tissue grown at 10 °C demonstrated a rise in the fluorescence to a maximum and a larger amplitude in variable fluorescence when measured at temperatures below 18°C than was seen in GC ch from tissue grown at 20°C. The rates of fluorescence quenching in 10°C-grown tissue were also faster than in 20°C-grown tissue when measured at temperatures below 18°C. State 1-State 2 transitions by GC ch were measured at selected temperatures between 5 and 25 °C as changes in the magnitude of the fluorescence emission maxima at 685, 695 and 730nm (F685, F695 and F730) measured at 77K. At measuring temperatures of 5 and 10°C, GC ch in tissue grown at 10 °C showed a greater transition to State 2 (a larger F730/F695 ratio) than did GC ch in tissue grown at 20 °C. At measuring temperatures of 20 and 25 °C, there was no difference in either the kinetics or the magnitude of the State 1 to State 2 transition in the two tissues. The ultrastructure of GC ch from tissues grown at 10 and 20 °C was also examined using transmission electron microscopy. Less than half (48%) of the grana from the higher temperature grown tissue had more than nine thylakoids/grana. Grana in GC ch which had developed at 10 °C showed a dramatic reduction in stacking, such that 85% of the grana contained no more than two thylakoids. The reduction in grana stacking was also accompanied by a decrease in the degree of appression of thylakoid membranes. The results demonstrate a capacity for thermal acclimation of GC ch function to low temperatures. This acclimation is associated with alterations in the chloroplast ultrastructure.     

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.     

Promiscuity in populations of the cushion plant Saxifraga oppositifolia in the Swiss Alps as inferred from random amplified polymorphic DNA (RAPD)

Overviews on patterns of genetic variation within and among plant populations show that widespread, outcrossing species should have a high proportion of the total genetic variation within populations and a low proportion among populations, which results in little population differentiation. However, in Alpine areas, large–scale distribution barriers as well as small-scale habitat heterogeneity could lead to geographical and temporal isolation, respectively. We investigated the genetic variation of Saxifraga oppositifolia from 10 populations of the Alps in southeastern Switzerland using random amplified polymorphic DNA (RAPD). Based on the banding patterns of four RAPD primers, 84 polymorphic markers identified all 189 sampled individuals as being genetically different. The genetic variation was mainly found within populations (95%), whereas less than 5% was found among populations and among regions. Analyses of molecular variance ( AMOVA ) suggested that population differentiation was highly significant. However, grouping populations differently into regions did not appear to result in a clear correspondence of genetic and geographical relatedness. Genetic variation did not significantly differ between populations of two elevational levels. This coincides with results of former pollination experiments that revealed a breeding system of S. oppositifolia which remains the same irrespective of the elevation. We assume that the high outcrossing rate, rare clonal reproduction, and some long-distance dispersal even among topographically separated populations are the crucial determinants for the pattern of genetic variation found in the investigated area.     

Importance of clonal plants and plant species diversity in the Northeast China Transect

In plant communities, the internal (genet-level) control mechanisms on a spatio-temporal scale of clonal plants impose strong constraints on spatial pattern as well as on competitive relations and, thus, species coexistence. Therefore, the presence of clonal species within a plant community affects spatio-temporal dynamics and plant species diversity. We examined the distribution of plants with different clonal growth forms in the Northeast China Transect (NECT) and correlated plant species diversity with the importance of clonal plants, and the importance of phalanx and guerilla clonal plants. Phalanx clonal plants were more abundant in western communities where the altitude was higher and both the soil nitrogen contents and precipitation were relatively low. Whereas guerilla clonal plants were more abundant in the middle of the NECT where the precipitation, mean annual temperature and photosynthetically active radiation were relatively high. In the relatively productive temperate typical steppe, plant species diversity was negatively correlated with the importance of phalanx clonal plants and positively correlated with the importance of guerilla clonal plants. In relatively unproductive temperate desert steppe, plant species diversity was positively correlated with the importance of both phalanx and guerilla clonal plants.     

Genetic variation and clonal diversity in four clonal sedges (Carex) along the Arctic coast of Eurasia

We studied the structure of genetic variation (at both ramet- and genet-level) and clonal diversity within and among populations in the four closely related arctic clonal sedges Carex bigelowii, C. ensifolia, C. lugens and C. stans by use of allozyme markers. Compared to other sedges and arctic plants, the studied taxa all had high levels of genetic variation, both within populations and taxa. These taxa contained most of the total gene diversity (H(T)) within populations and a small part of the diversity among populations (G(ST) ranged 0.05--0.43). Carex bigelowii had genetic variation (H(S) = 0.173, mean for populations) at a comparable level to other outbreeding arctic plants and to other widespread, rhizomatous and mainly outbreeding Carex species. In contrast, C. ensifolia (H(S) = 0.335), C. lugens (H(S) = 0.339) and C. stans (H(S) = 0.294) had within-population variations that were higher than in most other studied Carex species and for arctic plants in general. Genetic variation was not related to any tested environmental variable, but it was lower in areas deglaciated only 10,000 years BP compared to areas deglaciated 60,000 years BP or not glaciated at all during the Weichselian. All the populations were multiclonal, except for two populations of C. stans that were monoclonal. In contrast to genetic variation, clonal diversity decreased with latitude and did not differ between areas with different times of deglaciation. In accordance with previous studies, C. bigelowii and C. lugens were found to be outbreeding, while C. ensifolia and C. stans had mixed mating systems.     

Detection and analysis of genetic variation in Hordeum spontaneum populations from Israel using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to analyse genetic diversity within and between Hordeum spontaneum populations sampled from Israel. Nei's index of genetic differentiation was used to partition diversity into within and between population components. Fifty-seven per cent of the variation detected was partitioned within 10 H. spontaneum populations. Using principal component and multiple regression analysis, part of the variation detected between populations was seen to be associated with certain ecogeographical factors. Fifty-eight per cent of the distribution of the phenotypic frequencies of three RAPD phenotypes detected using a single primer in 20 H. spontaneum populations could be accounted for by four ecogeographical variables, suggesting adaptive variation at certain RAPD loci.     

Molecular diversity and derivations of populations of Silene acaulis and Saxifraga oppositifolia from the high Arctic and more southerly latitudes

A survey of allozyme diversity within and between populations of Silene acaulis from Spitsbergen, Norway, Iceland and Scotland, showed that populations from the high Arctic (Spitsbergen, > 76°N) contained high levels of diversity and were genetically similar to populations from more southern locations. Indirect measures of gene flow (Nm), calculated from Wrigh's F indicated that there had been extensive gene flow between Spitsbergen and some Norwegian populations. A restriction site analysis of chloroplast DNA (cpDNA) in S. acaulis revealed that all populations contained a single identical cpDNA haplotype, except one population from Norway which also contained a second haplotype. In contrast, five different cpDNA haplotypes were distinguished in a more limited survey of cpDNA variation in Saxifraga oppositifolia, with all five haplotypes present in one of two Spitsbergen populations surveyed. The contrasting cpDNA results for the two species suggest that whereas high-Arctic populations of Silene acaulis have most likely been derived from immigrants which arrived from the south after the last glacial period, high-Arctic populations of Saxifraga oppositifolia may be derived, in part, from ancient northern stocks which survived the last glaciation in high-Arctic refugia.     

Molecular markers in medicinal plant biotechnology: past and present

Plant based medicines have gained popularity worldwide due to their almost negligible side effects. In India, the three traditional medicinal systems, namely homeopathy, Ayurveda and Siddha rely heavily on plants for medicinal formulations. To prevent the indiscriminate collection of these valuable medicinal plants and for their proper authentication and conservation, it is imperative to go for sustained efforts towards proper germplasm cataloguing and devising conservation strategies. For this purpose, molecular markers have a significant role, as they provide information ranging from diversity at nucleotide level (single nucleotide polymorphisms) to gene and allele frequencies (genotype information), the extent and distribution of genetic diversity, and population structure. Over the past twenty years, the molecular marker field has completely transformed the meaning of conservation genetics which has emerged from a theory-based field of population biology to a full-fledged pragmatic discipline. In this review, we have explored the transition and transformation of molecular marker technologies throughout these years.     

The effects of regeneration by fragmentation upon clonal diversity in the tropical forest shrub Poikilacanthus macranthus: random amplified polymorphic DNA (RAPD) results

Poikilacanthus macranthus (Acanthaceae), like many shrubs in the neotropical cloud forest of Monteverde, Costa Rica, is capable of regeneration through stem and twig fragments. Our field sampling indicated that reproduction in P. macranthus is primarily through fragmentation in the Monteverde region. Random amplified polymorphic DNAs (RAPDs) were employed to examine clonal diversity in four populations of P. macranthus , and two primers yielded 11 variable markers that were used to identify genets. Sixty-eight multilocus genotypes were identified out of 277 plants sampled. Clonal diversity values were higher than the average for clonal plant species, as the number of genets per ramet was 0.25, while the mean D value was 0.87 and the mean E value was 0.84.     

Development and characterization of microsatellite markers in a clonal plant,Dioscorea japonica Thunb.

We developed 10 microsatellite loci from genomic DNA of a dioecious clonal plant, Dioscorea japonica. Out of 384 clones, 148 contained microsatellite repeats. Polymerase chain reaction primer pairs were designed for 95 of these clones from their sequence data, of which, 10 pairs produced successful amplification. Thirty‐eight individuals were genotyped for allelic diversity. We detected three to nine alleles per locus, and the expected heterozygosity ranged from 0.461 to 0.851.     

Assessing the nucleotide diversity of three aphid species by RAPD

A method is presented for the estimation of nucleotide diversity and genetic structure of populations from RAPD (random amplified polymorphic DNA) data. It involves a modification of the technique developed by Lynch and Crease (1990) for the case of restriction sites as survey data. As new elements the method incorporates (i) dominance correction, (ii) values of asexual reproduction of the populations sampled, and (iii) an analytical variance of the number of nucleotide substitutions per site. Sampling was carried out at two geographic scales for three aphid species. At a macrogeographic scale, populations of Rhopalosiphum padi did not show statistical genetic differentiation. Aphis gossypii and Myzus persicae, which were sampled at a microgeographic scale, showed a higher genetic differentiation than R. padi, it being statistically significant in M. persicae. The major sources of sampling variance within- and between-populations were found to be nucleotide (i.e., the number of alleles used as a function of the number of primers used) and population (i.e., sample size) sampling. Extremely low estimates of nucleotide diversity were obtained for the species studied here. This result is consistent with previous reports on genetic diversity for the same or other aphid species which were based on allozyme polymorphism, mitochondrial DNA variation and qualitative analyses of RAPDs.     

艾纳香野生种群克隆多样性及克隆结构研究

艾纳香是具有克隆生长习性的多年生宿根性草本植物,其广布于中国南部,为了更有效地保护和合理利用艾纳香资源,本文利用RAPD分子标记技术,对4个野生艾纳香种群进行了克隆结构和克隆多样性(单克隆种群或多克隆种群)进行了初步研究。结果表明:(1)10对10bp随机引物共检测到70条谱带,其中多态带为60条,占85.71%,检测到64个基因型,且全部为局限基因型;(2)与Ellstrand Roose(1987)总结的克隆多样性平均值(PD=0.17,D=0.62)相比艾纳香的种群克隆多样性水平稍高,Simpson指数平均为0.973,基因型比率PD平均为0.800;(3)遗传一致度和遗传距离分析表明,4个艾纳香野生种群被分成两组,一组是海南的所有种群,另外一组是云南类群。     

The relationship between respiration and temperature in leaves of the arctic plant Saxifraga cernua

Abstract Saxifraga cernua, a perennial herb distributed throughout the arctic and subarctic regions, shows high levels of dark respiration. The amount of respiration exhibited by leaves and whole plants at any temperature is influenced by the pretreatment temperature. Plants grown at 10°C typically show higher dark respiration rates than plants grown at 20°C. The levels of alternative-pathway respiration (or cyanide-insensitive respiration) in leaves of S. cernua grown at high and low temperatures were assessed by treating leaf discs with 0.25 mol m^?3 salicylhydroxamic acid during measurements of oxygen consumption. Alternative pathway respiration accounted for up to 75% of the total respiration. Tissues from 20°C-grown plants yielded a Q₁₀ of 3.37 for normal respiration, and of 0.97 for alternative-pathway respiration. Tissues from 10°C-grown plants yielded a Q₁₀ of 2.55 for normal respiration, and of 0.79 for alternative-pathway respiration. The alternative pathway does not appear to be as temperature sensitive as the normal cytochrome pathway. A simple energy model was used to predict the temperature gain expected from these high rates of alternative-pathway respiration. The model shows that less than 0.02°C can be gained by leaves experiencing these high respiration rates.     

Spatially dependent genetic diversity within and between colonies of wild raspberry Rubus idaeus detected using RAPD markers

In the Tayside region of Scotland, red raspberry Rubus idaeus exists both as extensive plantations of clonally propagated cultivars, and as wild populations that inhabit both the cultivated areas and uncultivated uplands to the north. In order to explore the genetic diversity of the wild populations and their degree of similarity to the plantation clones, individual plants in wild populations were sampled from four distinct sites along a 25-km transect northwards from the area of cultivation. The genetic material of each of 45 individuals and the commercial cv. Glen Clova were examined using six RAPD primers giving a total of 63 variable bands. Some identical banding patterns were observed, suggesting vegetative growth up to 20 m, but populations consisted mainly of genetically distinct individuals. Similarity matrices based on the marker bands showed, without exception, that plants were more similar within a site than between sites. None of the populations was closely related to the cv. Glen Clova. Although the most northerly site had the largest proportion of rare bands, there was no general trend between within-site diversity, or similarity to the cultivar, and position on the north-south line. All four sites had unique bands and bands not displayed by the cultivar. However, the genetic diversity of a site appeared to increase as the extent of the sampled area increased, implying that the genetic variation was spatially dependent. For example, maximum and minimum similarities were 100 and 80%, respectively, at interplant distances of 2 m; 100 and 60% at distances of 20 m; 85 and 55% at 200 m and 70 and 40% at 20 km.     

Detection of genetic diversity and selective gene introgression in coffee using RAPD markers

RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups — C. arabica var. typica and C. arabica var. bourbon — were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops.On study leave from: Universidad de San Carlos de Guatemala, Facultad de Agronomia, Ciudad Universitaria, Zona 12, Apartado Postal No. 1545, Guatemala, Central America     

Development and multiplexing of microsatellite markers using pyrosequencing in the clonal plant Comarum palustre (Rosaceae)

Microsatellites represent one of the most commonly used genetic markers for population genetic studies. Traditionally, their development is quite time consuming, requiring construction of a genomic library enriched for repeated motifs. Using pyrosequencing, a fast and cost-effective new generation sequencing technique, we produced 24,340,862 bases in 63,860 short fragment reads, including 1170 dinucleotide motifs with a minimum of six repeats and 1383 trinucleotide motifs with a minimum of four repeats for the Marsh Cinquefoil, Comarum palustre L., an endangered marsh pioneer species. We selected 58 loci with SSR (Short Sequence Repeat) segments (at least 10 repeats) for a preliminary screening. Out of them, we screened 29 loci on a capillary sequencer after ligation in a vector and PCR using T7 forward primer labelled with FAM fluorescent dye and the specific unlabeled reverse primers. This procedure allowed us to screen large number of candidate loci with the same labelled primer and unlabelled specific primers. Finally, we characterized 20 polymorphic microsatellite markers, nine dinucleotides and 11 trinucleotides. We used these markers to assess genetic diversity and clonal structure in two Belgian populations. All loci showed a maximum of two alleles per individual, suggesting that they are from a diploid genome. One genet was detected in a newly extending population while 53 different genets in a long-term ecologically managed population. The number of alleles per locus ranged from 6 to 14 in this old population with an expected heterozygosity, ranging from 0.5964 to 0.8278. These preliminary results show a genet size up to 7.2 m.