Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Polymorphic microsatellite loci in the Australian tree frog, <Emphasis Type="Italic">Litoria peronii</Emphasis>

Eleven novel polymorphic microsatellite loci developed from a microsatellite enriched genomic library, are presented for the Australian tree frog Litoria peronii. We screened 29 individuals from a single population and detected high levels of polymorphism for all 11 loci with the number of alleles/locus ranging from 9 to 24. Values of expected and observed heterozygosities ranged from 0.789–0.955 and 0.207–1.00, respectively. These microsatellite markers should prove useful in determining levels of genetic diversity, measuring gene flow and migration, assigning individuals to their most likely population of origin, and in the assignment of paternity.     

Biological activity of new flavonoid from <Emphasis Type="Italic">Hieracium pilosella</Emphasis> L.

Hieracium pilosella L. (Asteraceae) is a well-known plant used in ethno-medicine as its inflorescences are particularly rich in beneficial polyphenolics. This research aimed to elucidate the structure of a new flavone glycoside isolated from the inflorescences of Hieracium pilosella and evaluate its antioxidant, antimicrobial and antiproliferative activities. The chromatographic methods were successfully applied to isolate the new flavonoid. Its structure was determined by subsequent UV, NMR and MS experiments and identified as isoetin 4′-O-β-D-glucopyranoside. Free radical scavenging capacity was examined by measuring the scavenging activity of the new isoetin derivative on 2,2-diphenyl-1-picrylhydrazyl (DPPH). The compound was also screened for spectrum of antimicrobial activity using the agar well diffusion method. Minimum inhibitory concentration (MIC) for Pseudomonas aeruginosa ATCC 9027 was performed by the micro-dilution broth method. The antiproliferative effect of tested glycoside was assessed in two human tumor cell lines derived from lung (A549) and colon (HT-29) carcinoma and cell proliferation was determined by means of MTT method. The tested compound showed high antiradical activity, reducing the DPPH? with EC₅₀ 7.9 μM (3.7 µg/ml) and exhibited narrow antimicrobial spectrum among tested microorganisms. The compound was active against Pseudomonas aeruginosa ATCC 9027 (MIC 125 μg/ml) which is prone to causing infections that are difficult to treat due to it developing extremely rapid antibiotic resistance. In the antiproliferative studies, cell proliferation of the colon (HT-29) carcinoma cell line was significantly decreased after exposure to the compound. The results indicate that isoetin 4′-O-β-D-glucopyranoside possesses antioxidant capacity and very promising antibacterial activity and could have uses as an effective antipseudomonal agent as well a antiproliferative agent.     

Natural hybridisation between <Emphasis Type="Italic">Populus nigra</Emphasis> L. and <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">x canadensis</Emphasis> Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Development of ten microsatellite loci for <Emphasis Type="Italic">Gentiana crassicaulis</Emphasis> (Gentianaceae)

Genetiana crassicaulis is one of famous Chinese medicinal plant. The over-collection for its root has caused its dramatic reduction. In order to devise adequate conservation and management strategies for this species, it is important to characterize its genetic diversity and understand its population structure. Here, 10 polymorphic microsatellite markers have been developed. AC/TG microsatellite was enriched by combining biotin capture method. Polymorphism of each locus was assessed in 30 individuals from six populations. The number of alleles ranged from 2 to 9 and the expected heterozygosity ranged from 0.32 to 0.78.     

Development and characterization of microsatellite markers for parentage analyses of the coral reef damselfish (<Emphasis Type="Italic">Pomacentrus amboinensis</Emphasis>: Pomacentridae)

Five new polymorphic microsatellite loci were developed for the coral reef damselfish Pomacentrus amboinensis. Twenty-four individuals from two Great Barrier Reef populations were genotyped at the five loci, with numbers of alleles per locus ranging from 6–23 and observed heterozygosity between 0.42–0.92. In addition, the cross-species testing of six primers developed for Stegastes partitus revealed one primer (SpGATA40) that was also polymorphic for P. amboinensis. Due to high levels of polymorphism (≥14 alleles) in at least four of the six loci and a high proportion of tetranucleotide repeats, these microsatellite markers should be useful for parentage assignment as well as other investigations of individual relatedness.     

Development and characterization of microsatellite markers for an endangered shrub, <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> (Leguminosae) and cross-species amplification in <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis>

We developed 11 polymorphic microsatellite markers for an endangered shrub Ammopiptanthus mongolicus using the Fast Isolation by AFLP of Sequences Containing repeats protocol. Polymorphism of each locus was assessed in 24 individuals from five natural populations of A. mongolicus. The average allele number of these markers was 4.2, ranging from 2 to 7. The observed (H _O) and expected (H _E) heterozygosities were 0.044–0.956 and 0.044–0.827, respectively. Further assessment in the congeneric species A. nanus revealed that nine of eleven loci were successfully amplified, but only two loci showed polymorphism. These markers provide powerful tools for the conservation genetics studies of A. mongolicus.     

Genetic variation in island populations of tuatara (<Emphasis Type="Italic">Sphenodon</Emphasis> spp) inferred from microsatellite markers

Tuatara (Sphenodon spp) populations are restricted to 35 offshore islands in the Hauraki Gulf, Bay of Plenty and Cook Strait of New Zealand. Low levels of genetic variation have previously been revealed by allozyme and mtDNA analyses. In this new study, we show that six polymorphic microsatellite loci display high levels of genetic variation in 14 populations across the geographic range of tuatara. These populations are characterised by disjunct allele frequency spectra with high numbers of private alleles. High F _ST (0.26) values indicate marked population structure and assignment tests allocate 96% of all individuals to their source populations. These genetic data confirm that islands support genetically distinct populations. Principal component analysis and allelic sequence data supplied information about genetic relationships between populations. Low numbers of rare alleles and low allelic richness identified populations with reduced genetic diversity. Little Barrier Island has very low numbers of old tuatara which have retained some relictual diversity. North Brother Island’s tuatara population is inbred with fixed alleles at 5 of the 6 loci.     

Evaluation and selection of microsatellite markers for an identification and parentage test of Asian elephants (<Emphasis Type="Italic">Elephas maximus</Emphasis>)

Numbers of the Asian elephants (Elephas maximus) population are declining due to poaching, human-elephant conflicts, capture of wild calves for tourism and export and habitat destruction, which also may cause inbreeding in fragmented populations. In order to contribute to a reversal of this trend, we have developed an identification and parentage test by evaluation and selection of markers from 43 microsatellite loci that have been previously described for Asian or African elephants. Testing these markers on a panel of 169 Asian elephants comprising the 23 mother-offspring, 13 father-offspring and 13 parents-offspring pairs yielded 26 polymorphic markers. However, only 14 of these were found to be suitable for an analysis of molecular diversity, 12 of which will be implemented for an identification and parentage test to control the capture of wild calves in Thailand and neighboring countries.     

Development of 10 microsatellite loci for <Emphasis Type="Italic">Rheum tanguticum</Emphasis> (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource with R. tanguticum. These microsatellite markers could provide a useful tool for genetic and conservation studies of the rhubarb species.     

Genetic diversity assessment in Somali sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench) accessions using microsatellite markers

In the north-western region of Somalia, bordering Ethiopia, sorghum represents an important resources for human and animal nutrition. The critical situation of Somalia is threatening the preservation of this valuable resource and it becomes urgent to develop a strategy of correct evaluation of the sorghum germplasm in order to promote conservation and preservation programs. Microsatellites, also known as Simple Sequence Repeats (SSRs), are reproducible molecular markers useful in assessing the level of genetic diversity of plants. A total of 5 sorghum SSR-specific primer pairs were used to assess the genetic diversity of Somali sorghum landraces. Extensive variation was found at the microsatellite loci analysed, except for a locus that resulted in a monomorphic for some accessions. Considerable differences were found between total and effective number of alleles indicating non uniform allele frequency. Moreover allele frequency at a single locus significantly changed among accessions. Total gene diversity calculated for each locus ranged from 0.44 to 0.79. Most of the genetic diversity occurred within accessions demonstrating that accessions are not under selection processes and/or there is a continuous exchange of genes between sorghum populations. In any case, the patterns of clustering were significantly affected by the presence/absence of some alleles with high discriminant weight. Accessions Carabi, Abaadiro, Masego Cas and Masego Cad represent distinct genotypes confirming finding observed in previous phenotypic studies. The results highlight the central role of local farmers in maintaining and shaping local germplasm.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

Microsatellite markers reveal high genetic diversity in date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) germplasm from Sudan

Genetic diversity in date palm germplasm from Sudan representing 37 female and 23 male accessions was investigated using 16 loci of microsatellite (SSR) primers. Eight female accessions from Morocco were included as reference material. The tested SSR markers showed a high level of polymorphism. A total of 343 alleles were detected at the 16 loci. The number of alleles per marker ranged from 14 to 44 with an average of 21.4 per locus. A high level of expected heterozygosity was observed among Sudan cultivars (0.841), Morocco cultivars (0.820) and male accessions (0.799). The results indicate that the genetic groups of the Sudan cultivars and/or males do not follow a clear geographic pattern. However, the morocco group showed significant differentiation in relation to the Sudan groups, as measured by F (ST) values and genetic distances. The effect of the methods of pollination and cultivar selection on the genetic structure was clearly detected by the weak clustering association that was observed for the majority of accessions originating from Sudan and Morocco as well. This suggests the need for further investigation on the genetic diversity of Sudanese date palm germplasm. A deeper insight will be revealed by a detailed analysis of populations originating from different geographic locations.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Genetic diversity and structure of farm and GenBank accessions of cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Cameroon revealed by microsatellite markers

The genetic diversity of 400 accessions collected in cacao farms, 95 GenBank, and 31 reference accessions was analyzed using the 12 microsatellite markers. The GenBank and reference accessions were subdivided into 12 accession groups (AG) that belong to the traditional cacao genetic groups (GG) Lower Amazon Forastero (LA), Upper Amazon Forastero (UA), Trinitario, and Criollo (Cr). The 12-microsatellite loci revealed a total of 125 alleles, 113 of which were present in the farm accession group (FA). The within and between group variation for all AGs accounted respectively for 81% and 19% of the total molecular variation. The average F _is for the FA was 0.15 suggesting a moderate level of inbreeding. Significant differences for the level of gene diversity were found between the farm (0.50), GenBank (0.42 to 0.62), and reference (0.10 to 0.60) AGs. Genetic differentiation among AGs was variable with F _st values varying between 0.14 and 0.57 for the different AGs. Analysis using a Bayesian model-based method showed the existence of a high level of admixture for the farm accessions group. The LA genes were most represented in the FA (54%), followed by UA (33%) and Cr (7%). The genes of LA were also the most represented in the GenBank (48%), followed by UA (24%) and Cr (14%). Only 14% and 6% of the genes of the GenBank and farm accessions, respectively, could not be attributed to any of the reference GGs. The results suggest the predominating presence of LA genes in the Cameroon farm accessions and a high level of admixture, with apparent presence of genes of more than three GGs in most accessions. The traditional Trinitario types appear to have almost disappeared from farmers fields. The admixture must be the result of hybridization and recombination of these genes from the different GGs in seed gardens and in farmers’ fields. The use of selected farm accessions will depend on the GG that it belongs to and also on their level of heterozygosity. Further implications of the results for breeding and for introduction of new germplasm into the Cameroon GenBank are discussed.