Polymorphic microsatellite DNA markers for a relictual angiosperm Cercidiphyllum japonicum Sieb. et Zucc. and their utility for Cercidiphyllum magnificum

The genus Cercidiphyllum is considered to be a primitive, relictual angiosperm, with two extant species occurring in limited areas of East Asia. We developed seven microsatellite markers for Cercidiphyllum japonicum, of which, five showed a clear, strong single band for each allele. The average number of alleles and observed heterozygosity were 26.2 and 0.82, respectively. Three pairs of polymerase chain reaction primers successfully amplified the DNA of the other species, Cercidiphyllum magnificum.     

Development of 12 microsatellite markers for Platycrater arguta (Hydrangeaceae) endemic to East Asia

? Premise of the study: Microsatellite markers were characterized in Platycrater arguta, a rare endemic shrub from eastern China and southern Japan, to investigate its population genetic structure, phylogeographic patterns, and breeding system. ? Methods and Results: Using both the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol and the compound microsatellite marker technique, we developed 12 microsatellite markers. All primers showed polymorphism when assessed in 43 individuals from two populations in eastern China and southern Japan. Overall, the number of alleles ranged from five to 21, with an average of 14.91 alleles per locus. ? Conclusions: These markers can be used in further studies on population genetics, phylogeography, and breeding system of P. arguta.     

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae)

? Premise of the study: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. ? Methods and Results: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. ? Conclusions: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.     

Development of microsatellite markers for the moss Ptychomitrium gardneri (Ptychomitriaceae)

? Premise of the study: Microsatellite markers were developed for Ptychomitrium gardneri to study population genetics of this eastern Asian-North American disjunct moss. ? Methods and Results: A total of 13 microsatellite markers were developed in Chinese populations of P. gardneri, using the Fast Isolation by AFLP of Sequences COntaining Repeats protocol. Eight of the markers showed polymorphism when assessed in a sample of four populations of 29 individuals from China. These markers amplified three to four alleles per locus. Five primers also amplified in P. linearifolium and P. wilsonii. ? Conclusions: These markers may be useful for further investigation of population genetics of P. gardneri.     

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.     

Microsatellite primers for the endangered aquatic herb, Ottelia acuminata (Hydrocharitaceae)

? Premise of the study: Microsatellite primers were developed in the endangered aquatic herb, Ottelia acuminata, to characterize its genetic diversity and understand its population structure. ? Methods and Results: Eight polymorphic microsatellite markers were developed from two populations of O. acuminata in China. The number of alleles per locus ranged from one to 15; the observed and expected heterozygosities ranged from 0 to 0.885 and from 0 to 0.888, respectively, in the two populations. Selected loci also amplified successfully in O. sinensis. ? Conclusions: These microsatellite markers will facilitate further studies on the conservation genetics and evolutionary history of O. acuminata.     

Characterization of polymorphic microsatellites for the invasive grass Microstegium vimineum (Poaceae)

? Premise of the study: Microsatellite markers were developed for the invasive plant Microstegium vimineum (Poaceae) to assess its population structure and to facilitate tracking of invasion expansion. ? Methods and Results: Using 454 sequencing, 11 polymorphic and six monomorphic microsatellite primer sets were developed for M. vimineum. The primer sets were tested on individuals sampled from six populations in the United States and China. The polymorphic primers amplified di-, tri-, and tetranucleotide repeats with three to 10 alleles per locus. ? Conclusions: These markers will be useful for a variety of applications including tracking of invasion dynamics and population genetics studies.     

Development of polymorphic microsatellite markers for Cymbidium goeringii (Orchidaceae)

? Premise of the study: Microsatellite markers were developed in Cymbidium goeringii to investigate its genetic diversity and population genetic structure. ? Methods and Results: From a microsatellite-enriched genomic library, 21 novel polymorphic microsatellites were isolated. The polymorphic patterns were verified in four populations (East Korea, West Korea, China, and Japan). The number of alleles per locus ranged from 11 to 29 with a mean of 20.29. The observed and expected heterozygosities varied from 0.272 to 0.799 and from 0.461 to 0.911, respectively. ? Conclusions: These microsatellite markers will be useful tools for understanding genetic variation and population ecogenetic structure in C. goeringii.     

Development of microsatellite markers for the neotropical tree species Dipteryx alata (Fabaceae)

? Premise of the study: Microsatellite markers were developed for the population genetic analyses of the neotropical tree Dipteryx alata (Fabaceae). ? Methods and Results: Microsatellites were developed from a genomic shotgun library. Polymorphism at each microsatellite loci was analyzed based on 94 individuals from three populations. Eight loci amplified successfully and presented one to 10 alleles, and expected heterozygosities ranged from 0.097 to 0.862. Four loci also amplified in Pterodon emarginatus and presented similar polymorphism. ? Conclusion: The eight microsatellite primer pairs are potentially suitable for population genetic studies and successfully amplified in another Fabaceae species.     

Microsatellite markers for Grosmannia alacris (Ophiostomataceae, Ascomycota) and other species in the G. serpens complex

? Premise of the study: Polymorphic microsatellite markers were developed for the pine-infecting fungus, Grosmannia alacris. ? Methods and Results: Sixteen microsatellite markers were developed by using inter-simple sequence repeat (ISSR)-PCRs and 454 sequencing methods. Seven of these markers showed polymorphisms for a South African population of G. alacris, and 13 markers showed polymorphism when European isolates were included. Most of the primer pairs also amplified four closely related species: G. serpens, Leptographium gibbsii, L. castellanum, and L. yamaokae. ? Conclusions: These new markers will be useful for population studies of G. alacris and other species in the G. serpens complex.     

Development of microsatellite loci for the invasive weed Wedelia trilobata (Asteraceae)

? Premise of the study: Microsatellite markers were developed to help elucidate the population genetics of the invasive species Wedelia trilobata. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) repeats protocol, 23 sets of primers for amplifying microsatellite loci were identified in W. trilobata, 10 of which showed polymorphism (two to five alleles per locus) in samples of two populations of W. trilobata, one from China and one from Peru. Six of these loci were successfully amplified from samples of the native congener W. chinensis, with expected sizes. ? Conclusions: These markers may be useful for further investigation of population genetics of Wedelia trilobata and other congener species.     

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae)

? Premise of the study: We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. ? Methods and Results: We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. ? Conclusions: The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.     

Molecular data and ecological niche modelling reveal a highly dynamic evolutionary history of the East Asian Tertiary relict Cercidiphyllum (Cercidiphyllaceae)

? East Asia's temperate deciduous forests served as sanctuary for Tertiary relict trees, but their ages and response to past climate change remain largely unknown. To address this issue, we elucidated the evolutionary and population demographic history of Cercdiphyllum, comprising species in China/Japan (Cercdiphyllum?japonicum) and central Japan (Cercdiphyllum magnificum). ? Fifty-three populations were genotyped using chloroplast and ribosomal DNA sequences and microsatellite loci to assess molecular structure and diversity in relation to past (Last Glacial Maximum) and present distributions based on ecological niche modelling. ? Late Tertiary climate cooling was reflected in a relatively recent speciation event, dated at the Mio-/Pliocene boundary. During glacials, the warm-temperate C.?japonicum experienced massive habitat losses in some areas (north-central China/north Japan) but increases in others (southwest/-east China, East China Sea landbridge, south Japan). In China, the Sichuan Basin and/or the middle-Yangtze were source areas of postglacial northward recolonization; in Japan, this may have been facilitated through introgressive hybridization with the cool-temperate C.?magnificum. ? Our findings challenge the notion of relative evolutionary and demographic stability of Tertiary relict trees, and may serve as a guideline for assessing the impact of Neogene climate change on the evolution and distribution of East Asian temperate plants.     

Development of microsatellite markers for Ophiocordyceps sinensis (Ophiocordycipitaceae) Using an ISSR-TAIL-PCR method

? Premise of the study: Microsatellite primers were developed for Ophiocordyceps sinensis, an endangered medicinal fungus endemic to the Tibetan Plateau, to investigate its genetic diversity and population structure. ? Methods and Results: An inter-simple sequence repeat (ISSR)-thermal asymmetric interlaced (TAIL)-PCR method was established to develop microsatellite markers. A total of 30 perfect and imperfect microsatellites were identified in 48 individuals of O. sinensis from five provinces within China representing different populations. Seventeen loci were polymorphic with two to four alleles per locus, while 13 were monomorphic. ? Conclusions: The results indicate that the microsatellite markers developed here may be used in studies of population genetics and conservation biology of O. sinensis. Furthermore, the ISSR-TAIL-PCR method is a simple strategy for microsatellite marker development.     

Development and characterization of polymorphic microsatellite markers in Momordica charantia (Cucurbitaceae)

? Premise of the study: Polymorphic microsatellite markers were developed for Momordica charantia L. to investigate the genetic diversity and population structure within and between M. charantia and its four related species (Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L.). ? Methods and Results: Using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) method, 16 polymorphic microsatellite loci were identified in 36 individuals of M. charantia. Across all the M. charantia samples, the number of alleles per locus ranged from three to eight. Seven primers successfully amplified in the four related species. ? Conclusions: These markers will be useful to study population ecology and population differentiation among M. charantia species and its related species.     

Microsatellite markers for Bistorta vivipara (Polygonaceae)

? Premise of the study: Using genomic shotgun 454 sequencing, 50 candidate microsatellite markers were targeted for the arctic-alpine polyploid perennial herb Bistorta vivipara to distinguish between individual genets and ramets within a population. ? Methods and Results: Out of the 50 markers, 31 were polymorphic for seven test samples. We have developed a multiplex protocol for 16 of these microsatellite markers. ? Conclusions: Our results show that the microsatellite markers provide a powerful tool for the research on genetic variation of B. vivipara.     

Development of microsatellite loci for the endangered species Pityopsis ruthii (Asteraceae)

? Premise of the study: Microsatellite loci were developed for the endangered species Pityopsis ruthii and will permit genetic and conservation studies of the species. ? Methods and Results: A microsatellite-enriched library was used to develop 12 polymorphic microsatellite loci for P. ruthii. The loci amplified perfect and imperfect repeats with three to seven alleles per locus. Observed heterozygosity ranged from 0.05 to 0.80 and expected heterozygosity ranged from 0.23 to 0.75. ? Conclusions: These microsatellite loci provide a sufficient set of markers for further investigation of population genetics of P. ruthii.     

Development of microsatellite markers from Cercis chinensis (Fabaceae)

? Premise of the study: Microsatellite markers were developed to characterize the level of genetic diversity and population genetic structure of Cercis chinensis, a widely cultivated garden plant in China with congeneric species disjunctly distributed in East Asia, North America, and the Mediterranean. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, eight microsatellite markers were developed in C. chinensis. Seven of the markers displayed polymorphism, with the number of alleles ranging from one to four in four populations of C. chinensis. Four to six microsatellite loci exhibited interspecific transferability in C. glabra, C. chuniana, and C. chingii. ? Conclusions: These are the first microsatellite markers developed for C. chinensis, which will be further used in investigation of population genetic structure and phylogeographic pattern of C. chinensis and its congeneric species.     

Development and characterization of microsatellite markers for Emmenopterys henryi (Rubiaceae), a rare tree from China

? Premise of the study: Compound microsatellite primers were developed for Emmenopterys henryi, an endangered deciduous tree endemic to China, to assess its genetic diversity and population structure as well as its evolutionary history. ? Methods and Results: Using the compound microsatellite marker technique, 10 pairs of polymorphic microsatellite primers were isolated and characterized in E. henryi. Levels of polymorphism were tested across a total of 63 individuals from three natural populations. Allele numbers varied from 10 to 20 per locus, with an average of 14.50 alleles per locus. The observed heterozygosity per locus ranged from 0.125 to 0.962, and the expected heterozygosity ranged from 0.377 to 0.903. ? Conclusions: The highly polymorphic markers developed and characterized in this study will facilitate evolutionary and population genetic studies in E. henryi.     

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae)

? Premise of the study: We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. ? Methods and Results: By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. ? Conclusions: These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.