Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.     

Historic DNA reveals genetic consequences of fragmentation in an endangered,endemic mustard

Understanding how anthropogenic disturbance affects genetic diversity is essential to appropriately incorporating genetic considerations into conservation plans. Unfortunately, we rarely have information about a population’s genetic diversity before it becomes imperiled. Here we reconstruct the historic range of the naturally rare annual mustard Streptanthus glandulosus subsp. niger (Sgn) and use herbarium specimens to quantify pre-disturbance genetic diversity. We compare this to the genetic diversity in the contemporary plant populations and to plants in the seed bank. We conclude that Sgn was recently a single, panmictic population composed of orders of magnitude more plants than exist today but experienced recent and abrupt declines following housing development. Today Sgn persists as two disjunct populations, the larger of which has retained historic levels of diversity although there is a downward trend in all measures. The smaller population has lost 21–28% of the diversity that was present only 50 years ago with an N_e?~?5–16. The contemporary populations have differentiated from each other due to drift. The seed bank contained no novel alleles and had high levels of homozygosity, indicating that it is incapable of providing genetic rescue. This novel combination of hDNA, the aboveground plant population and the seed bank can be used to design high impact conservation plans that appropriately incorporate genetic diversity for this and other imperiled species.

     

Microsatellite loci in Bactris gasipaes (Arecaceae): their isolation and characterization

We constructed a microsatellite‐enriched genomic library for Bactris gasipaes, an economically important, domesticated palm. We developed 18 polymorphic microsatellite markers, and found an average of seven alleles per locus in a sample of 14 individuals selected from a germplasm bank. Cross‐species amplification was evaluated in six other Bactris species. The loci detected will permit population studies and germplasm characterization, and can be used for genetic analyses in related species.     

Non‐timber Forest Product Harvest does not Affect the Genetic Diversity of a Tropical Tree Despite Negative Effects on Population Fitness

The level of genetic diversity in a population can affect ecological processes and plant responses to disturbance. In turn, disturbance can alter population genetic diversity and structure. Populations in fragmented and logged habitats often show reduced genetic diversity and increased inbreeding and differentiation. Long‐term harvesting of wild plants (for foliage, bark, and roots), can affect population genetic diversity by altering individual fitness and genetic contribution. Our understanding of these changes in genetic diversity due to the harvesting of plant organs is still limited. We used nine microsatellite markers to study the effect of long‐term bark and foliage harvest by Fulani people on the genetic diversity and structure of 12 populations of African mahogany (Khaya senegalensis) in Benin. We sampled 20 individuals in each population to test the effect of harvesting. For each population, we divided the samples equally between seedling and adults to test if the effects are stronger in seedlings. We found moderate genetic diversity (H_e = 0.53 ± 0.04) and weak but significant differentiation among local populations (F_ST = 0.043, P < 0.001). There was no significant effect of harvest on genetic diversity or structure, although previous work found significant negative effects of harvest on the reproduction of adults, offspring density, and population fitness. Our results suggest that demographic responses to disturbance precede a detectable genetic response. Future studies should focus on using parentage analysis to test if genotypes of harvested parents are directly represented in the offspring populations.     

Assessment of genetic stability of the germplasm lines of medicinal plant <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi (Huang-qin) in long-term,in vitro maintained cultures

An approach of combining flow cytometry (FCM) analysis with morphological and chemical profiling was used to assess the genetic stability and bioactive compound diversity in a Scutellaria baicalensis Georgi (Huang-qin) germplasm collection that was clonally maintained in in vitro for a period of over 6 years. Based on the FCM analysis of nuclei samples from young shoots, the nuclear DNA content of S. baicalensis was calculated as 0.84 pg/2C. FCM analysis showed no significant variation in the nuclear DNA contents and ploidy levels in the long-term in vitro maintained germplasm lines. Germplasm lines, acclimatized to ex vitro conditions, exhibited distinctive plant growth and bioactive compound production capacities. The high level of genetic stability observed in in vitro maintained S. baicalensis lines opens up a variety of opportunities such as allowing long-term aseptic preservation and easy distribution of well-characterized germplasm lines of this medicinal plant species. This study represents a novel approach for continuous maintenance, monitoring, and production of medicinal plant tissues with specific chemistry.     

Genetic structuring in wild populations of two important medicinal plant species as inferred from AFLP markers

Systematic analysis of germplasm diversity and genetic relationship among cultivars is critical for development of appropriate conservation and breeding strategies. This approach has been applied to gain an insight about the molecular variance that exists in wild population of two important medicinal plant species of India that have a long history of therapeutic usage in herbal medicine. Adhatoda vasica and Andrographis paniculata, members of the family Acanthaceae, have wide geographical and climatic distribution across India suggesting a large amount of genetic diversity available for resource management and breeding programs. In this study we have assessed the genetic diversity of both these species distributed in five varied geo-environmental regions, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting with selected primer combinations and statistical analysis. Cluster analysis and analysis of molecular variance also suggested a very high genetic variation. Detailed analyses of the results predict that the genetic variation found in A. vasica was more discrete that reflected strongly in the populations studied, whereas the genetic variation in A. paniculata was relatively uniform. Considering significantly large sample size and distinctive characteristics of the selected populations, this work contributes valuable insights that can be used to engineer conservation and utilization strategies for these species.     

Using minimum DNA marker loci for accurate population classification in rice (Oryza sativa L.)

Because of the rich diversity among rice accessions grown around the world in distinct environments, traditional methods using morphology, cross compatibility and geography for classifying rice accessions according to different sub-populations have given way to use of molecular markers. Having a few robust markers that can quickly assign population structure to germplasm will facilitate making more informed choices about genetic diversity within seedbanks and breeding genepools. WHICHLOCI is a computer program that selects the best combination of loci for population assignment through empirical analysis of molecular marker data. This program has been used in surveys of plant species, for fish population assignment, and in human ancestry analysis. Using WHICHLOCI, we ranked the discriminatory power of 72 DNA markers used to genotype 1,604 accessions of the USDA rice core collection, and developed panels with a minimum number of markers for population assignment with 99% or higher accuracy. A total of 14 markers with high discriminatory power, genetic diversity, allelic frequency, and polymorphic information content were identified. A panel of just four markers, RM551, RM11, RM224 and RM44, was effective in assigning germplasm accessions to any of five sub-populations with 99.4% accuracy. Panels using only three markers were effective for assignment of rice germplasm to specific sub-populations, tropical japonica, temperate japonica, indica, aus, and aromatic. Assignment to tropical japonica, temperate japonica, or indica sub-populations was highly reliable using 3–4 markers, demonstrated by the high correlation with assignment using 72 markers. However, population assignment to aus and aromatic groups was less reliable, possibly due to the smaller representation of this material in the USDA core collection. More reference cultivars may be needed to improve population assignment to these two groups. This study demonstrated that a small number of DNA markers is effective for classification of germplasm into five sub-populations in rice. This will facilitate rapid screening of large rice germplasm banks for population assignment at a modest cost. The resulting information will be valuable to researchers to verify population classification of germplasm prior to initiating genetic studies, maximizing genetic diversity between sub-populations, or minimizing cross incompatibility while maximizing allelic diversity within specific sub-populations.     

Fine‐scale Population Genetic Structure of Two Dioecious Indian Keystone Species,Ficus hispida and Ficus exasperata (Moraceae)

Although Ficus (Moraceae) is a keystone plant genus in the tropics, providing resources to many frugivorous vertebrates, its population genetic structure, which is an important determinant of its long‐term survival, has rarely been investigated. We examined the population genetic structure of two dioecious fig species (Ficus hispida and Ficus exasperata) in the Indian Western Ghats using co‐dominant nuclear microsatellite markers. We found high levels of microsatellite genetic diversity in both species. The regression slopes between genetic relationship coefficients (f_ij) and spatial distances were significantly negative in both species indicating that, on average, individuals in close spatial proximity were more likely to be related than individuals further apart. Mean parent–offspring distance (σ) calculated using these slopes was about 200 m in both species. This should be contrasted with the very long pollen dispersal distances documented for monoecious Ficus species. Nevertheless, overall population genetic diversity remained large suggesting immigrant gene flow. Further studies will be required to analyze broader scale patterns.     

Genetic diversity of six arable plants in relation to their Red List status

In Central Germany and throughout Europe, arable plants count among some of the most endangered plant species. Over the last few decades, the number and size of populations have been in sharp decline due to modern land use techniques, including the application of fertilizers, herbicide use and seed cleaning procedures. As arable plant species are underrepresented in population genetic studies, it is unknown whether agricultural intensification has affected the extant populations, and whether genetic structure varies among species with differing vulnerability in respect of their Red List status. We sampled 53 populations from 6 arable plant species throughout Central Germany. Random amplified polymorphic DNA analyses (RAPD) were applied to calculate measures of genetic diversity at the population level and genetic differentiation. Genetic diversity was found to be lowest in Bupleurum rotundifolium and Anagallis foemina, and highest in Consolida regalis and Nigella arvensis. The highest levels of genetic differentiation were observed among populations of An. foemina and B. rotundifolium but within populations in all other species. Φ_ST values differed strongly ranging between 0.116 for C. regalis and 0.679 for An. foemina. Patterns of genetic structure were related to the Red List status for all the species studied except An. foemina, for which it should consequently be raised. Our data confirm that even relatively recent threats are accompanied by detrimental genetic structure. As losses of populations and increased fragmentation have occurred in all common and uncommon species, the situation for arable plants could change for the worse in the following decades, highlighting the need for consistent monitoring.     

Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia

Aim Phylogeographical studies in the Brazilian Atlantic Forest (BAF) have mostly included species associated with forest habitats, whereas taxa associated with grassland and sand‐dune plant communities have so far been largely overlooked. This study examines the phylogeography of the orchid Epidendrum fulgens, which occurs on coastal sand dunes and granitic outcrops, in order to identify major genetic divergences or disjunctions across the range of the species and to investigate the genetic signatures of past range contractions and expansions. Location Southern and south‐eastern seashore vegetation along the BAF biome, and granitic and arenitic outcrops that occur in the subtropical grassland plant communities located south of the BAF. Methods Nine nuclear and four plastid microsatellite loci were used to genotype 424 individuals from 16 populations across the distributional range of E. fulgens. For both sets of markers, we estimated genetic diversity and population differentiation, testing for a north–south gradient of genetic diversity. The plastid haplotype network and a Bayesian assignment analysis of nuclear markers were used to infer population structure. Past demographic changes were investigated using a coalescence approach. Results A deep disjunction was found between northern populations within the BAF and southern populations outside the BAF that occur on granitic and arenitic outcrops. Recent demographic reductions were detected in northern populations on coastal sands. Such demographic changes were not expected for those populations, as previous studies with forest species had found evidence of population expansion in the same areas. Higher genetic diversity was found in southern populations on granite, in contrast to patterns observed in previous studies of forest species. Main conclusions The results are consistent with the long‐term persistence of E. fulgens. Bottlenecks were detected in populations from areas where population expansion events have been detected in other plant (and animal) species, suggesting that forest expansion after the Last Glacial Maximum played a role in the population fragmentation and decrease in genetic diversity in E. fulgens. A substantial genetic division in E. fulgens corresponds to the ‘Portal de Torres’, a region that demarcates the northern limits of subtropical grassland plant communities and the southern limits of the BAF.     

Genetic variation and structure in the Mediterranean shrubs Myrtus communis and Pistacia lentiscus in different landscape contexts

Studies concerning different habitat configurations can provide insights into the complex interactions between species’ life‐history traits and the environment and can help to predict patterns in population genetics. In this study, we compared patterns of genetic variation in two Mediterranean shrub species (Myrtus communis and Pistacia lentiscus) that co‐occur in populations within three contrasting landscape contexts: continuous, fragmented‐connected and fragmented‐isolated populations. Analysing variation at microsatellites loci, our results revealed weak responses to the landscape contexts. We rather found a population‐specific response in both study species. However, despite both study species sharing similar levels of genetic diversity, Myrtus displayed higher levels of homozygosity and genetic differentiation among populations, stronger patterns of within‐population spatial genetic structure, lower values of mutation‐scaled effective population size and stronger evidence for recent genetic bottlenecks than Pistacia. This result highlights the influence of past events (e.g. historical connectivity, fluctuations in population size) and local factors (e.g. microhabitat availability for recruitment, habitat quality, plant density, native fauna) and that the landscape configuration per se (i.e. fragment size and/or isolation) might not completely determine the species’ genetic patterns.     

Patterns of population genetic diversity in riparian and aquatic plant species along rivers

Aim The downstream hydrochoric spread of seeds of aquatic and riparian plant species, without upstream compensation, can be expected to result in downstream accumulation of population genetic diversity. This idea has been termed the ‘unidirectional dispersal hypothesis’ and is the genetic equivalent of the more generally known ‘drift paradox’. Our aim was to test this unidirectional diversity hypothesis, and to present a general synthesis of the patterns of population genetic variation across different riparian and aquatic plant species along rivers. Location The Meuse River (Belgium) and rivers world‐wide. Methods First, we used amplified fragment length polymorphism markers to compare patterns of within‐ and between‐population genetic diversity among three riparian plant species (Sisymbrium austriacum, Erysimum cheiranthoides and Rorippa sylvestris), typically occurring in different habitats along a gradient perpendicular to the Meuse River. Second, we performed a meta‐analysis on studies reporting on the population genetic structure of riparian and aquatic plant species along rivers. Results Along the Meuse River, we found significant genetic differentiation among populations of all three riparian species, and significant isolation by distance for one of them (R. sylvestris). There was no clear association between the typical habitat of a species and its population genetic structure. None of the three species provided evidence for the unidirectional dispersal hypothesis. The meta‐analysis, based on 21 data records, did not support the unidirectional dispersal hypothesis either. Average weighted population genetic differentiation across species was significant. Main conclusions Important mechanisms of upstream seed dispersal, probably through zoochory, together with higher seed recruitment opportunities in upstream habitats due to density dependence of recruitment, may explain the absence of downstream accumulation of genetic diversity. Also, it seems difficult to find consistent patterns in genetic variation in species from aquatic and riparian habitats. We argue that this is due to the recurrent extinctions and colonizations characteristic of these habitats, resulting in complex genetic patterns. Our results strongly support previous suggestions that stream ecology should consistently embrace metapopulation theory to be able to understand patterns of genetic diversity, as well as species diversity.     

Genetic comparisons between seed bank and <Emphasis Type="Italic">Stipa krylovii</Emphasis> plant populations

The soil seed bank represents the potential plant population since it is the source for population replacement. The genetic structure of a Stipa kryiovii (Roshev.) plant population and its soil seed bank was investigated in the Xilinguole Steppe of Inner Mongolia using random amplified polymorphic DNA (RAPD) analyses. The population was sampled at two sites that were in close proximity to each other (0.5 km apart). Thirty plants and 18 seed bank samples were taken from each site to determine the genetic diversity between sites and between sources (plant or seed). The material was analyzed using 13 primers to produce 92 loci. Eighty-six were multi-loci, of which 23 loci (26.74%) of allele frequencies showed significant differences (P ≤ 0.05). The genetic similarity between two seed bank sites was 0.9843 while the genetic similarity between two plant sites was 0.9619. Their similarities were all greater than that between the seed bank and plant populations. An analysis of their genetic structure showed that 87.86% of total variation was derived by two-loci. Genetic structures between plant and soil seed bank populations in S. krylovii were different due to the variance of mean gametic disequilibria and mean gene diversity. AMOVA results showed that the majority of variance (88.62%) occurred within sites, 12.75% was from between-groups. Further research is needed to investigate the selective function in maintaining the genetic diversity of Stipa krylovii plant populations.     

Genetic diversity and population structure of two important medicinal plant species <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and <Emphasis Type="Italic">Schisandra sphenanthera</Emphasis> revealed by nuclear microsatellites

Seven polymorphic and transferable nuclear microsatellites were used to investigate the population structure of genetic diversity of Schisandra chinensis and Schisandra sphenanthera for facilitating their conservation and sustainable utilization. High levels of gene diversity were revealed in these two medicinal species, the majority of genetic diversity was harbored within populations, and population structure was might due to restricted gene flow among populations. Isolation by distance was close to significance in S. chinensis but not in S. sphenanthera. In S. chinensis, null alleles were identified as a cause for excess of homozygotes at loci G24 and WGA60, but inbreeding might also be partly responsible for the positive F _IS values in this species. In contrast, null allele frequencies were high at all the seven loci in S. sphenanthera and resulted in overestimation of fixation index. The strategy for ex situ conservation of these two medicinal species is discussed based on the genetic results.     

Past,present and future of mountain species of the French Massif Central – the case of Soldanella alpina L. subsp. alpina (Primulaceae) and a review of other plant and animal studies

Aim Our goals were: (1) to investigate patterns of genetic variation in the French Massif Central (MC) of Soldanella alpina (Primulaceae), an alpine plant species that has only one known population in the region; (2) to analyse these patterns in order to deduce the Quaternary history of the population and to predict how current climatic warming may affect it; and (3) to review molecular analyses from the MC to evaluate the importance of the region for the conservation of genetic diversity. Location Europe, with a special focus on the French Massif Central and adjacent regions. Methods Amplified fragment length polymorphisms (AFLPs) were analysed for 192 individuals (nine populations) of S. alpina (subsp. alpina) representing the MC, Pyrenees and south‐western Alps. Population genetic diversity was assessed by various parameters (e.g. H_E, Shannon’s I). Neighbor‐Net and Bayesian approaches, and analysis of molecular variance (AMOVA) were used to infer population genetic relationships and structure. Results Individuals generally clustered according to populations within mountain regions. Hierarchical AMOVA indicated significant variation among mountain ranges (33.2% of the total variance), but there was also strong differentiation between populations (26.3%). The single population of S. alpina from the MC was identified as a distinct lineage of high genetic diversity. Our literature survey indicated that taxa with low and with high genetic diversity exist in the MC, and that genetic relationships to surrounding regions are diverse. Main conclusions The high genetic diversity and distinctiveness of S. alpina in the MC suggests the long‐term persistence of the single population in this region, which might have been favoured through elevational range shifts in response to past climatic change. This interpretation partly accords with other studies indicating that several plant and animal populations in the MC contain comparatively high genetic diversity, represent genetically independent lineages, and/or are likely descendants of populations that persisted in the MC throughout the Quaternary. These data underline the conservation importance of the MC as a key area for the long‐term persistence of species with often high levels of intraspecific genetic diversity.     

RAPD variation within and among natural populations of outcrossing willow-leaved foxglove (Digitalis obscura L.)

 Random amplified polymorphic DNA (RAPD) markers were used to assess levels and patterns of genetic diversity in Digitalis obscura L. (Scrophulariaceae), an outcrossing cardenolide-producing medicinal plant species. A total of 50 plants from six natural populations on the Iberian Peninsula were analysed by six arbitrarily chosen decamer primers resulting in 96 highly reproducible RAPD bands. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to bands (35 of 96) whose observed frequencies were less than 1–3/n in each population. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of RAPDs (genotypic analysis) showed that most of the variation (84.8%) occurred among individuals within populations, which is expected for an outcrossing organism. Of the remaining variance, 9.7% was attributed to differences between regions, and 5.5% for differences among populations within regions. Estimates of the Wright, Weir and Cockerham and Lynch and Milligan F_ST from null-allele frequencies corroborated AMOVA partitioning and provided significant evidence for population differentiation in D. obscura. A non-parametric test for the homogeneity of molecular variance (HOMOVA) also showed significant differences in the amount of genetic variability present in the six populations. UPGMA cluster analyses, based on Apostol genetic distance, revealed grouping of some geographically proximate populations. Nevertheless, a Mantel test did not give a significant correlation between geographic and genetic distances. This is the first report of the partitioning of genetic variability within and between populations of D. obscura and provides important baseline data for optimising sampling strategies and for conserving the genetic resources of this medicinal species. Received: 7 September 1998 / Accepted: 28 November 1998     

Characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant,Gastrodia elata

Genetic characterization of germplasm resource is essential for the conservation and efficient utilization of a traditional Chinese medicinal plant, Gastrodia elata. Thirty‐two primer pairs flanking microsatellite repeats were designed and tested using 32 individuals from eight wild populations. A total of 13 microsatellite loci were found highly polymorphic, with three to 10 alleles per locus and gene diversity ranging from 0.400 to 0.841. These microsatellites have been directly applied to the ongoing population and conservation genetics studies.     

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid <Emphasis Type="Italic">Salix alba</Emphasis>–<Emphasis Type="Italic">Salix fragilis</Emphasis> complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.     

Can a seed bank provide demographic and genetic rescue in a declining population of the endangered shrub <Emphasis Type="Italic">Acacia pinguifolia</Emphasis>?

Many threatened species suffer reduced genetic diversity as a result of small population size and isolation. However, species with a persistent seed bank may be buffered against genetic loss as seed banks are expected to accumulate the reproductive output of many seasons. For fire-dependent species in decline, prescribed ecological burning may be a means to stimulate germination and recover genetic diversity stored in the seed bank, providing a demographic and genetic rescue effect. Here we investigated the effectiveness of this strategy in a small, isolated and inbred population of the endangered shrub, Acacia pinguifolia. We surveyed genetic diversity and structure of remnant populations of A. pinguifolia and monitored regeneration before and after burning. Germination was stimulated by fire, but seedling numbers 18 months post-fire were low and barely above the number of adults killed by the fire. Genetic diversity was marginally higher in the post-fire seedling cohort than the pre-fire adults (H_E = 0.1 vs. 0.09, respectively). Outcrossing rates of open-pollinated seed from surrounding plants suggested moderately high levels of self-fertilisation (t _m  = 0.65) and analysis of fine-scale genetic structure implied pollen and seed dispersal over distances of several metres, suggesting that restricted gene flow and inbreeding may act to limit genetic diversity in the seed bank. We conclude that prescribed burning has not been immediately successful as a recovery strategy for this relictual population of A. pinguifolia, though future monitoring may detect additional recruits. Alternative conservation strategies, including performing inter-population crosses, may be required to restore genetic diversity and ameliorate extinction risks.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.