Microsatellites for the mangrove tree Avicennia germinans (Acanthaceae): Tools for hybridization and mating system studies

? Premise of the study: We developed a new set of microsatellite markers for the black mangrove Avicennia germinans, to provide new informative tools for further studies of the mating system, interspecific hybridization, and population genetics. ? Methods and Results: We used the microsatellite-enriched library approach to isolate and characterize 25 new primer pairs. Sixteen of them are polymorphic, showing a variable degree of variation in A. germinans, while nine were monomorphic in the samples examined. Eight exhibited private alleles in A. schaueriana. ? Conclusions: These results indicate that these new microsatellite markers will be useful molecular tools for further studies of A. germinans and A. schaueriana population genetics, mating systems, and hybridization.     

Development of microsatellite markers for <Emphasis Type="Italic">Manilkara maxima</Emphasis> T.D. Penn. (Sapotaceae) and their use in conservation genetics

Manilkara maxima is an endemic tree species of the Atlantic Forest in southern Bahia, Brazil. It is considered important for forest conservation due to its mutualistic interactions with endemic and endangered animals. Our aim was to develop microsatellite markers to estimate genetic diversity in order to provide information for effectiveness of future conservation programs. We used next generation sequencing technology to develop the first specific microsatellite markers for M. maxima. Seventeen new microsatellite loci were applied in 72 individuals sampled in three natural populations. On average, the number of alleles per loci was 8.8. The expected heterozygosity varied between 0.72 and 0.77, indicating that the developed set of molecular markers is useful for genetic diversity studies. Additionally, the estimated value for the combined probability of exclusion (Q) was greater than 0.999, which indicates the powerful of these molecular tools for paternity and kinship analysis. Our results demonstrate that the set of microsatellites developed in this work is a powerful tool for population genetics, molecular ecology and conservation biology purposes.     

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.     

A set of microsatellite markers for Arrabidaea chica (Bignoniaceae), a medicinal liana from the Neotropics

? Premise of the study: Microsatellite markers were developed, optimized, and characterized for Arrabidaea chica (Humb. & Bonpl.) Verl. (Bignoniaceae), a Neotropical liana extensively used in folk medicine. The aim of this study was to develop molecular tools to investigate the genetic structure and diversity of natural populations and germplasm collections of this species. ? Methods and Results: Eight highly polymorphic microsatellite markers revealed a multibanded pattern, suggesting that the species is polyploid. The total number of bands per locus ranged from 9 to 17, revealing high levels of polymorphism. ? Conclusions: The high level of polymorphism detected with these markers indicates their utility in devising conservation strategies and rational exploitation of A. chica.     

Microsatellite markers for the endangered root holoparasite Dactylanthus taylorii (Balanophoraceae) from 454 pyrosequencing

? Premise of the study: Microsatellite loci were isolated and developed as polymorphic markers for the New Zealand endemic root holoparasite Dactylanthus taylorii for use in population and conservation genetics studies. ? Methods and Results: Shotgun 454 pyrosequencing was performed on genomic DNA pooled from three individuals of D. taylorii. From 61709 individual sequence reads, primers for 753 microsatellite loci were developed in silico and 72 of these were tested for consistent amplification and variability. Ten microsatellite loci were found to be polymorphic and consistently scorable when screened in 44 individuals from five geographically distant populations. The number of alleles per locus ranged from four to 16 with an average of 9.7, and average observed heterozygosity per locus was between 0.182 and 0.634. ? Conclusions: These polymorphic microsatellite markers establish an important resource for ongoing conservation initiatives and planned population genetic studies of D. taylorii.     

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.     

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.     

Isolation and characterization of novel microsatellite markers for Avena sativa (Poaceae) (oat)

? Premise of the study: A new set of microsatellite primers was developed for Avena sativa and characterized to assess the level of genetic diversity among cultivars and wild genotypes. ? Methods and Results: Using an enrichment genomic library, 14 simple sequence repeat markers were identified. The loci of these markers were characterized and found to be polymorphic in size among 48 genotypes of oat from diverse geographical locations. The number of alleles per locus ranged from two to eight, while the observed heterozygosity ranged from 0.031 to 0.75. ? Conclusions: These newly identified microsatellite markers will facilitate genetic diversity studies, fingerprinting, and genetic mapping of oat. Moreover, these new primers for A. sativa will aid future studies of polyploidy and hybridization in other species in this genus.     

Development of polymorphic microsatellite markers in Camellia chekiangoleosa (Theaceae) using 454-ESTs

? Premise of the study: A set of microsatellite markers for Camellia chekiangoleosa was developed and characterized using 454 sequencing technology to study the population genetic structure and the diversity of germplasm collections. ? Methods and Results: Eighteen polymorphic microsatellite markers were identified and tested in 150 individuals from three natural populations of C. chekiangoleosa. Alleles numbered from two to seven, and the observed and expected heterozygosities ranged from 0.100 to 0.760 and 0.133 to 0.809, respectively. ? Conclusions: These markers will potentially be conducive to further genetic studies on C. chekiangoleosa.     

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.     

Development of microsatellite markers in Parakmeria nitida (Magnoliaceae)

? Premise of the study: We developed microsatellite makers for Parakmeria nitida to investigate its population structure and conservation genetics. ? Methods and Results: A total of 25 microsatellite primer pairs were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, and polymorphism was assessed in three natural populations of P. nitida. Among these markers, 11 were monomorphic and 14 showed polymorphism. ? Conclusions: These markers are potentially useful for future population genetic analyses of P. nitida and will serve as an important tool for conservation efforts.     

Development and characterization of 43 microsatellite markers for the critically endangered primrose Primula reinii using MiSeq sequencing

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean=3.2) and 0.13 to 0.82 (mean=0.45), respectively. All loci were in HardyeWeinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

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.     

Microsatellite marker development for the threatened orchid Masdevallia solomonii (Orchidaceae)

? Premise of the study: Microsatellite markers for Masdevallia solomonii were developed to serve as a tool in future population genetic studies of this threatened species from the Bolivian Yungas. ? Methods and Results: Thirteen microsatellite primers were characterized by cloning an intersimple sequence repeat (ISSR) library. From these, 10 loci presented considerable variation in allele number (3-10), expected heterozygosity (0.537-0.865), and polymorphic information content per locus (0.500-0.848). ? Conclusions: The markers obtained for M. solomonii are the first in the genus and subtribe. The observed polymorphism will make it possible to assess genetic diversity and structure of this species and will serve to propose effective conservation actions.     

Development and characterization of 14 novel microsatellite markers from the golden pheasant (Chrysolophus pictus)

Fourteen polymorphic microsatellite loci were isolated from the golden pheasant (Chrysolophus pictus). The average allele number of these microsatellites was 11.14 per locus, ranging from two to seventeen in a group of 31 individuals. The mean observed and expected heterozygosities were 0.763 and 0.722, respectively. The average polymorphic information content value, cumulative discrimination power, overall probability of exclusion with both unknown parents or with only unknown sire for these markers were 0.741, 1.0, 0.9999, and 1.0, respectively. This set of microsatellite markers would provide useful tools for conservation genetic studies of the golden pheasant.     

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 microsatellites in Machilus thunbergii (Lauraceae), a warm-temperate coastal tree species in Japan

? Premise of the study: Microsatellite markers were developed and characterized in a typically coastal, widespread, and dominant tree species of the evergreen broadleaf forests, Machilus thunbergii, for comparison of the genetic diversity and structure of inland populations surrounding the ancient Lake Biwa and coastal populations in Japan. ? Methods and Results: Eighteen polymorphic microsatellites of this species were isolated using an improved technique for isolating codominant compound microsatellite markers. These isolated loci provided compound simple sequence repeat (SSR) markers with polymorphisms of three to 19 alleles per locus, with an average of 10.9. The expected and observed within-population heterozygosities ranged from 0.16 to 0.86 and from 0.13 to 0.72, respectively. ? Conclusions: These markers may be useful tools for further investigation of the population genetic structure and biogeographic history of M. thunbergii in the warm-temperate zone of East Asia.     

Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n?=?40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n?=?140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1–25.7%) with high allele diversity (mean number of alleles/locus?=?8.67?±?1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate?=?0.09; false allele rate?=?0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (P_ID), we suggest (i) a panel of five microsatellite markers (cumulative P_ID?=?1.24?×?10^–5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.