Alternative approach to species identification of Actaea racemosa L. (syn. Cimicifuga racemosa (L.) Nutt., black cohosh) herbal starting material: UV spectroscopy coupled with LDA

The North American Actaea racemosa L. (syn. Cimicifuga racemosa (L.) Nutt., commonly known as black cohosh), considered to be a more “natural” alternative to conventional therapies, is used to relieve menopausal symptoms. The high demand for plant material has led to problems with substitution/adulteration of raw material of wholesale origin. The authenticity of the starting material is crucial for the herbal product’s efficacy and safety, and tests on identity and substitution are integral parts of cGMP guidelines. Consequently, there is a need for economical and easy-applicable test procedures. The aim of this study is to reveal the capability of the well-established UV spectroscopy coupled with a multivariate classification procedure to serve as a tool for the identification of A. racemosa. We built a classification model applying linear discriminant analysis (LDA) to distinguish between A. racemosa and its common substitutes. The model showed a high level of accuracy predicting 100% of the samples correctly. Our results indicate that UV spectroscopy shows potential for the development of possible additional authentication methods for this herbal starting material.     

The pollination ecology of Actaea spicata (Ranunculaceae)

Pollination ecology of Actaea spicata L. (Ranunculaceae) was studied at localities ranging from deciduous to spruce forest. The species is self–incompatible and possibly facultatively apomictic. No anemogamy seems to occur. In deciduous forest, the plant is primarily pollinated by pollen–foraging and mate–searching Byturus ochraceus (Scriba) (Coleoptera). It flowers during a period between emergence of the beetle and onset of anthesis of the prime pollen source and only larval host plant, Geum ur–banum L. (Rosaceae). The beetles sit on the suitably long anther filaments or on top of the stigma. Floral morphology promotes pollination–performing movements by the beetle. The complex floral fragrance seems to induce close–range behaviour, i.e. alighting and/or foraging. Flies become abundant visitors only shortly before anthesis is over, and their contribution is small. Large syrphids are relatively effective as pollinators, since they grasp the stamens near the base and often touch the stigma with pollen–dusted parts of their bodies. Due to lack of its food–plant, B. ochraceus is absent at spruce forest localities and northwards. At these localities flowering is delayed until the main fly appearance, and the flowers are heavily pollinated by several syrphids and muscids. At three deciduous–forest localities a second flowering took place with new, juxtaposed inflorescences on separate stalks. The distribution of such inflorescences within the population along with the distribution and abundance of pollinators among the two generations of flowers suggest that these inflorescences occur as a response to low rate of pollination in the first anthesis.     

Development, characterization, and transferability of microsatellite markers for Kirengeshoma palmata (Hydrangeaceae)

? Premise of the study: Microsatellite markers were developed for Kirengeshoma palmata to assess the population genetics and mating pattern of this critically endangered species. ? Methods and Results: A total of 24 microsatellite markers were developed for K. palmata using an enrichment protocol. These markers were screened in 37 individuals from four populations in China and Japan, and twelve were found to be polymorphic, with the number of alleles per locus ranging from two to eight. All of these primers also amplified in K. koreana. ? Conclusions: These microsatellite markers provide a useful tool to investigate the mating system, gene flow, parentage, and population dynamics of Kirengeshoma.     

Development, characterization and transferability of microsatellite markers for Cullen australasicum (Leguminosae)

The development of eight polymorphic microsatellite markers for use in the Australian native legume Cullen australasicum is described. The number of alleles per locus ranged from 3 to 8. Observed heterozygosity ranged from 0.208 to 0.667. The cross-species amplification of these eight markers and a ninth marker, which is monomorphic in the populations examined, but may be used to distinguish between species, was also tested in five other species. These microsatellite markers will be useful for investigating the population structure of natural populations of C. australasicum and other Cullen species which may be susceptible to genetic contamination via pollen mediated gene flow from planted pastures of C. australasicum.     

Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons)

The black muntjac (Muntiacus crinifrons) is a vulnerable species found in the mountains of eastern China, about which little is known. Here we develop 11 polymorphic microsatellite loci from a (CA)(n) -enrichment library for the animal. In the analyses of 25 individuals sampled, unbiased expected heterozygosity levels varied from 0.686 to 0.876 and the number of alleles per locus varied from five to nine. Results that 11 microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as parentage, mating system and population genetic structure of M. crinifrons.     

Development and characterization of microsatellite markers for Berberis thunbergii (Berberidaceae)

? Premise of the study: Microsatellite markers were isolated and characterized in Berberis thunbergii, an invasive and ornamental shrub in the eastern United States, to assess genetic diversity among populations and potentially identify horticultural cultivars. ? Methods and Results: A total of 12 loci were identified for the species. Eight of the loci were polymorphic and were screened in 24 individuals from two native (Tochigi and Ibaraki prefectures, Japan) and one invasive (Connecticut, USA) population and 21 horticultural cultivars. The number of alleles per locus ranged from three to seven, and observed heterozygosity ranged from 0.048 to 0.636. ? Conclusions: These new markers will provide tools for examining genetic relatedness of B. thunbergii plants in the native and invasive range, including phylogeographic studies and assessment of rapid evolution in the invasive range. These markers may also provide tools for examining hybridization with other related species in the invasive range.     

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.     

Development and characterization of microsatellite markers for genetic analysis of the Korean black scraper, Thamnaconus modestus

Black scraper is a commercially important oceanodromous fish in Korea. The commercial catch of this fish has decreased continuously since 1990. However, its genetic characteristics have never been studied. For population genetic analysis to assist effective aquaculture and stock management efforts, we isolated and characterized 19 microsatellite loci using an microsatellite-enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a natural T. modestus population in southern Korea were genotyped. All loci except two, KTm213 and KTm284, were polymorphic, with an average of 15.18 alleles per locus (range, 2–41). The mean observed and expected heterozygosities were 0.67 (range, 0.33–0.97) and 0.79 (range, 0.40–1.00), respectively. A significant deviation from Hardy-Weinberg equilibrium was observed at six loci (KTm142, KTm145, KTm158, KTm24, KTm221, and KTm250). This high variability indicates that these microsatellites may be useful for high-resolution studies of population genetics.     

Development and characterization of microsatellite markers in Populus euphratica (Populaceae)

In this study, we developed 12 microsatellite loci for Populus euphratica and checked their variability in 27 individuals. The number of alleles for 12 loci ranged from five to nine, the observed and expected heterozygosities ranged from 0.32 to 0.48 and from 0.53 to 0.67. Frequencies of null alleles of all loci are not significantly greater than zero. None of the loci showed significant deviation from Hardy-Weinberg equilibrium. No significant linkage disequilibrium was observed between pairs of studied loci. These markers are useful in studies of population genetic structure of P. euphratica.     

Development and characterization of microsatellite markers for lippia (Phyla canescens: Verbenaceae)

Lippia (Phyla canescens: Verbenaceae) is a serious weed of wetlands, riparian zones and floodplains, particularly in eastern Australia where many Ramsar wetlands are threatened by hydrological changes precipitated by soil-accreting lippia mats. Enriched genomic DNA libraries were used to develop nine informative microsatellite markers. These markers will be valuable tools to understand the genetic structure of the lippia populations in different regions throughout the world.     

Development and characterization of microsatellite markers in Zanthoxylum ailanthoides (Rutaceae)

Nine microsatellite markers were developed for Zanthoxylum ailanthoides, a typical pioneer tree. Averaged over the nine loci, the number of alleles per locus was 5.1. The observed and expected heterozygosities ranged from 0.233 to 0.833 and from 0.314 to 0.823, with averages of 0.606 and 0.641, respectively. No loci showed significant departures from Hardy–Weinberg equilibrium or linkage equilibrium after Bonferroni correction (P > 0.05). These markers will be useful for parentage analyses and studies of population genetic structure in the species.     

Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae)

Genomic libraries of Arenaria grandiflora enriched for di- and trinucleotide repeats were used for the development of novel microsatellite markers. The subset of 13 polymorphic markers was characterized on 40 individuals of A. grandiflora originating from lowland locations in France. The loci amplified 3 to 10 alleles per locus and expected heterozygosities ranged from 0.46 to 0.83. The newly developed markers will be used for population genetic studies and for assessing genetic composition of a restoration experiment of lowland A. grandiflora populations that are protected in France.     

Development of microsatellite markers for the white mangrove (Laguncularia racemosa C.F. Gaertn., Combretaceae)

We developed nine new microsatellite markers for the white mangrove (Laguncularia racemosa C.F. Gaertn.), a mangrove species widely distributed in the Western Hemisphere. The loci were developed from a sample collected in Baja California Sur, Mexico, and were screened for polymorphism in 108 individuals from the Pacific Coast of Mexico and Costa Rica. The level of polymorphism was relatively low, from two to four alleles. These are the first set of polymorphic markers described for L. racemosa and should be of value in studying regional genetic differentiation among natural populations of this species.     

Isolation and characterization of polymorphic microsatellite markers for the black rockfishSebastes inermis

Black rockfish (Sebastes inermis, Scorpaenidae) is a commercially important fisheries resource in Korea. However, no genetic diversity information is available for wild or released hatchery populations. To study the effects of stock enhancement programs and conservation biology, we developed microsatellite DNA markers fromS. inermis. We report the isolation and characterization of seven microsatellite loci, designated KSi221B, KSi239A, KSi25A, KSi252B, KSi193A, KSi259B, and KSi234, isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a naturalS. inermis population in Yeosu, Korea, were genotyped. All loci except one, KSi193A, were polymorphic with an average of 9.5 alleles per locus (range 2–20). The mean observed and expected heterozygosities were 0.71 (range 0.43–1.00) and 0.76 (range 0.41–0.90), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at one locus, KSi221B. Such high variability indicates that these microsatellites should provide useful markers for studies of kinship and population genetics.     

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.