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.     

Microsatellite loci for Paspalum atratum (Poaceae) and cross-amplification in other species

? Premise of the study: Paspalum atratum is a perennial, cespitose, tropical grass native to Central and South America. This species belongs to a polyploid complex (Plicatula group) little known at the genetic level. The characterized microsatellite markers provide new informative tools for further studies of the hybridization, mating systems, and structure of the population. ? Methods and Results: Using the microsatellite-enriched library method, we isolated and characterized 19 microsatellite markers from P. atratum. Eleven of them were polymorphic, showing a variable degree of variation, while eight were monomorphic in the samples analyzed. Additionally, the transferability of these microsatellite markers was tested in other species. ? Conclusions: These results suggest that the characterized markers have enough discriminatory potential to be used in genetic characterizations of Paspalum taxa, which are based on an understanding of their mating systems and genetic structure, as well as in understanding the evolutionary processes involved in the evolution of groups of Paspalum.     

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 of microsatellite markers for the dove tree, Davidia involucrata (Nyssaceae), a rare endemic from China

? Premise of the study: Microsatellite primers were developed for the endangered Davidia involucrata to assess the population genetics and infer its evolutionary history. ? Methods and Results: Using both the modified magnetic bead hybridization method and the dual-suppression PCR method, we isolated and characterized 12 polymorphic microsatellite loci using 134 individuals from five populations in southwestern China. The number of alleles per locus ranged from six to 21 (mean = 10.8). The expected heterozygosity per locus ranged from 0.404 to 0.918 and observed heterozygosity ranged from 0.015 to 0.821. ? Conclusions: All of the 12 microsatellite markers developed for D. involucrata are polymorphic, and lay a solid foundation for further studies of the population genetics of this famous tree.     

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology

? Premise of the study: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. ? Methods and Results: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. ? Conclusions: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.     

Microsatellite markers for Duperrea pavettifolia (Rubiaceae)

? Premise of the study: The development of microsatellite primers for Duperrea pavettifolia will be the foundation for mating system analysis and conservation research. ? Methods and Results: Nineteen microsatellite markers were developed and characterized in two wild populations by using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol. Polymorphisms were evaluated in 24 individuals from two natural populations. Eleven of these primers generated polymorphic loci. ? Conclusions: These microsatellite markers will be useful in future investigations into the population genetics and mating system of D. pavettifolia.     

Isolation and characterization of microsatellite markers from Clematoclethra scandens (Actinidiaceae)

? Premise of the study: Microsatellite makers were developed for Clematoclethra scandens to investigate its population genetics and speciation. ? Methods and Results: A total of 36 microsatellite markers were isolated using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. Their polymorphisms were assessed in two natural populations. The results showed that 30 markers displayed prominent polymorphisms and six markers were monomorphic. ? Conclusions: These microsatellite loci will facilitate further studies on population genetics and speciation of C. scandens.     

Microsatellites for Tetracentron sinense (Trochodendraceae), a Tertiary relict endemic to East Asia

? Premise of the study: Tetracentron sinense (Trochodendraceae) is a Tertiary relict endemic to East Asia. Microsatellite markers were developed and characterized to investigate the population genetics of the species. ? Methods and Results: Microsatellite markers were isolated from the genome of T. sinense using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). Eight polymorphic microsatellite markers were assessed in 44 samples collected from three wild populations. The number of alleles observed for each locus ranged from two to five. The observed and expected heterozygosities ranged from 0.0000 to 0.9375 and 0.0000 to 0.7681, respectively. ? Conclusions: The microsatellite markers will be helpful in further studies of the population genetics and phylogeography of T. sinense.     

Microsatellite markers for the relictual dove tree, Davidia involucrata (Cornaceae)

? Premise of the study: Microsatellite markers were developed for the dove tree, Davidia involucrata (Cornaceae), a Tertiary relict currently endemic to China, to investigate its population genetics and phylogeography. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, nine polymorphic microsatellite loci were identified and screened in 44 individuals from three wild populations of D. involucrata. The number of alleles per locus ranged from two to three, while the observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.6000 and from 0.0000 to 0.6323, respectively. ? Conclusions: These new microsatellite loci will facilitate further studies of the population genetics and phylogeography of D. involucrata, as well as of the evolutionary history of the plant and other Tertiary relicts endemic to East Asia.     

Twenty new microsatellite loci for population structure and parentage studies of Parnassius apollo nevadensis (Lepidoptera; Papilionidae)

We characterized 20 new microsatellite loci isolated from Parnassius apollo nevadensis. The 20 loci displayed 2–12 alleles and expected heterozygosities varied from 0.22 to 0.89; the markers were amplified in nine multiplex reactions. The combined non-exclusion probabilities of this set are 0.001084 and 0.000005 for the first and second parents, respectively. This set of markers will thus enable other studies on reproductive strategies and population genetics in this endangered and previously understudied species. We show, for the first time, evidence of multiple paternity and that females may successfully copulate with more than one male despite the presence of mating plugs. Future studies on this mating behavior and population genetics of the species using the 20 loci here presented will be very important to define its conservation status and management measures.     

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.     

Genetic diversity enhanced by ancient introgression and secondary contact in East Pacific black mangroves

Regional distribution of genetic diversity in widespread species may be influenced by hybridization with locally restricted, closely related species. Previous studies have shown that Central American East Pacific populations of the wide-ranged Avicennia germinans , the black mangrove, harbour higher genetic diversity than the rest of its range. Genetic diversity in this region might be enhanced by introgression with the locally restricted Avicennia bicolor . We tested the hypotheses of ancient hybridization using phylogenetic analysis of the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA and intergenic chloroplast DNA; we also tested for current hybridization by population level analysis of nuclear microsatellites. Our results unveiled ancient ITS introgression between a northern Pacific Central American A. germinans lineage and A. bicolor . However, microsatellite data revealed contemporary isolation between the two species. Polymorphic ITS sequences from Costa Rica and Panama are consistent with a zone of admixture between the introgressant ITS A. germinans lineage and a southern Central American lineage of A. germinans . Interspecific introgression influenced lineage diversity and divergence at the nuclear ribosomal DNA; intraspecific population differentiation and secondary contact are more likely to have enhanced regional genetic diversity in Pacific Central American populations of the widespread A. germinans .     

Development of nuclear microsatellite markers for the fonio, Digitaria exilis (Poaceae), an understudied West African cereal

? Premise of the study: We developed nuclear microsatellite primers to explore the genetic diversity, population genetic structure, and evolutionary history of the fonio (Digitaria exilis), an understudied cereal cultivated in West Africa. ? Methods and Results: We used a microsatellite-enriched library approach to isolate and characterize 38 nuclear primer pairs (31 di-, five tri-, and two tetranucleotide repeats), of which 21 were polymorphic and exhibited a clear pattern in 36 accessions from West Africa. The number of alleles per locus ranged from two to 22, with a mean of 4.71, and expected heterozygosity ranged from 0.03 to 0.93. ? Conclusions: The developed set of 21 polymorphic SSR markers will provide tools for population and evolutionary genetics studies of the cultivated fonio.     

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.     

Isolation and characterization of microsatellite markers in an endangered species Dracaena cambodiana (Liliaceae)

?Premise of the study: The development of microsatellite primers in the endangered species Dracaena cambodiana will be the foundation for genetic and conservation studies of D. cambodiana and several Dracaena species. ?Methods and Results: A total of 26 microsatellite markers were developed in Chinese populations of D. cambodiana, using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) protocol. Among them, sixteen primer pairs generated polymorphic loci (fourteen of them successfully amplified in other four Dracaena species) and ten primer pairs produced monomorphic loci. ?Conclusions: These microsatellite markers could be used in the further investigation of population genetics of D. cambodiana and other Dracaena species.     

Mining online genomic resources in Anolis carolinensis facilitates rapid and inexpensive development of cross-species microsatellite markers for the Anolis lizard genus

Online sequence databases can provide valuable resources for the development of cross-species genetic markers. In particular, mining expressed tag sequences (EST) for microsatellites and developing conserved cross-species microsatellite markers can provide a rapid and relatively inexpensive method to develop new markers for a range of species. Here, we adopt this approach to develop cross-species microsatellite markers in Anolis lizards, which is a model genus in evolutionary biology and ecology. Using EST sequences from Anolis carolinensis, we identified 127 microsatellites that satisfied our criteria, and tested 49 of these in five species of Anolis (carolinensis, distichus, apletophallus, porcatus and sagrei). We identified between 8 and 25 new variable genetic markers for five Anolis species. These markers will be a valuable resource for studies of population genetics, comparative mapping, mating systems, behavioural ecology and adaptive radiations in this diverse lineage.     

Isolation and characterization of microsatellite loci in Schizolobium parahyba (Leguminosae)

Primer sequences for 16 microsatellite loci were developed from Schizolobium parahyba, a tropical tree species. Twelve loci were found to be polymorphic after screening diversity in individuals from Belize. A total of 39 alleles were found at nine loci. The markers are invaluable tools for studying the population genetics and mating system of the species.     

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.     

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.     

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.