Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder (Platichthys stellatus) and cross-species amplification

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500 to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in P. stellatus. Guidong Miao and Changwei Shao have contributed equally.     

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.     

Highly polymorphic microsatellite loci for the Parsley frog (Pelodytes punctatus): characterization and testing for cross-species amplification

A microsatellite library was developed using genomic DNA of the Parsley frog, Pelodytes punctatus, an amphibian species which inhabits Mediterranean temporary pond systems. Number of alleles and heterozygosity ranged from 10 to 25 and from 0.66 to 0.90, respectively. Cross-species amplification was successful for 13 of the 15 developed loci for the related species, Pelodytes ibericus. The high levels of polymorphism revealed by these loci will be extremely useful for characterizing the population genetic diversity and structure and to estimate levels of dispersal and gene flow in the species P. punctatus and P. ibericus.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Novel microsatellite markers obtained from Gansu zokor (Eospalax cansus) and cross-species amplification in Plateau zokor (Eospalax baileyi)

Zokor (Mysopalacinae) is a group of subterranean rodents. Although they are regarded as important components in the ecosystems they belong to, due to their underground lifestyle, very little is known about their ecology and behavior. Development of microsatellite markers can potentially assist in addressing behavioral and ecological questions such as dispersal, mating and social systems. Here we report the development of 11 polymorphic microsatellite loci from the genome of Eospalax cansus using 454 shot-gun sequencing. Genetic diversity was assessed using DNA samples of 47 individuals of E. cansus from a single location. Cross-amplification in Eospalax baileyi was also tested, with five polymorphic markers being amplified successfully. These markers are the first published microsatellites loci from E. cansus and will be invaluable for studies addressing ecological and behavioral questions involving E. cansus and E. baileyi, and potentially other species in Mysopalacinae.     

Reproductive isolation in the Aegean Ophrys omegaifera complex (Orchidaceae)

The orchid genus Ophrys operates a system of sexual deception by which high specificity of pollination is attained. Reproductive isolation in Ophrys mainly rests upon prezygotic isolation mechanisms. The level of genetic separateness of Ophrys taxa with different pollinators is therefore likely determined by the fidelity of pollinators. The present study employs genetic fingerprinting to investigate this in the east Aegean Ophrys omegaifera s.l. complex, also including O. dryis, a west Mediterranean species of this complex. Ophrys fleischmannii, O. basilissa, and the west Mediterranean O. dyris, are found to be well-separated genetic entities whereas O. omegaifera s.str. and the putative hybrid taxon, O. sitiaca, are found to be genetically inseparable across their entire range of co-occurrence. This suggests that specific pollinators have high enough fidelity to act as effective isolating factors in east Aegean O. omegaifera s.l. as a whole, but that the situation in the species pair of O. sitiaca and O. omegaifera is likely to be more complex.     

Development and characterization of polymorphic microsatellite DNA markers for Sasa senanensis (Poaceae: Bambuseae)

Sasa senanensis is a dwarf bamboo species distributed on the floors of cool temperate forests in Japan and adjacent regions. We isolated eight polymorphic microsatellite loci from this species. The number of alleles ranged from two to eight and the observed heterozygosity per locus from 0.13 to 0.74. Seven of the eight loci were also polymorphic in Sasa nipponica. Most of these markers were successfully amplified in other dwarf bamboo species. These markers will be useful for investigating clonal structure and population genetics in some dwarf bamboo species.     

Isolation and characterization of microsatellite markers from the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>: cross-species amplification in other Tephritidae species reveals a varying degree of transferability

The Mediterranean fruit fly, Ceratitis capitata, is a pest of major economic importance and has become a model for the development of SIT control programs for insect pests. Significant information has been accumulated on classical and population genetics of this species during the past 2 decades. However, the availability of molecular markers is limited. Here, we present the isolation and characterization of 159 microsatellite clones and the development of 108 polymorphic microsatellite markers for this insect pest. Mapping by in situ hybridization to polytene chromosomes of 21 microsatellite clones enriched the cytogenetic map that was previously constructed by our group. The enriched map provides a large number of STSs for future genome mapping projects. Cross-species amplification of these microsatellite loci in 12 Tephritidae species and sequence analysis of several amplification products indicated a varying degree of transferability and their possible usefulness as molecular and genetic markers in these species where genetic and molecular tools are limited. E. E. Stratikopoulos and A. A. Augustinos contributed equally to this work.     

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands. Number of alleles ranged from 3 to 13 (average of 7.33) and the values of observed heterozygosities from 0.32 to 0.80 (average of 0.61). Cross-species amplification on three other marine turtles, Chelonia mydas, Eretmochelys imbricata and Dermochelys coriacea, revealed polymorphism and variability at eight, eleven and three loci, respectively.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

Development and characterization of 13 novel microsatellite loci from the flat-headed bat (Tylonycteris pachypus) with cross-species amplification in closely related taxa

13 novel microsatellite loci was isolated using the enriched library method from genomic DNA of the flat-headed bat (Tylonycteris pachypus). These loci showed high levels of genetic polymorphism testing on 54 individuals sampled from Guangxi province, China. The number of observed alleles per locus ranged from 2 to 15. The expected and observed heterozygosity values ranged from 0.170 to 0.900 and from 0.185 to 0.944, respectively. One loci departed significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no Linkage disequilibrium was found between any pairs of loci. In addition, these loci were tested in the sister species, Tylonycteris robustula, seven loci amplified successfully and were also polymorphic.     

Development of polymorphic microsatellite markers from barfin flounder (Verasper moseri) and their cross-species amplification

Barfin flounder (Verasper moseri) is a rare fish species in the world. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of barfin flounder (Verasper moseri). The number of alleles, observed, and expected heterozygosity per locus in a test population ranged from 2 to 6, from 0.3333 to 1.0000, and from 0.4866 to 0.7774, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci in additional five fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Verasper moseri. Gui-Dong Miao and Chang-Wei Shao Contributed equally to this work.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification

Obscure puffer (Takifugu obscurus) is an anadromous fish species in China. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of T. obscurus. The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from four to 10, from 0.57 to 0.86 and from 0.68 to 0.90, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in T. obscurus.     

Characterization of microsatellite loci in the black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum): their use for cross-species amplification and differentiation between the two species

As the population sizes of the black and white rhinoceroses continues to decline, more efforts are needed in multiple areas to help with the conservation efforts. One area being explored is the use of genetic diversity information to aid conservation decisions. In this study, we designed 21 microsatellite primers for white and black rhinoceroses, 16 and 17 of which amplified bands in the white and black rhinoceros, respectively. Out of these primers all 16 were polymorphic in the white rhinoceros and 12 of the 17 were polymorphic in the black rhinoceros. The mean number of alleles was 3.31 and 2.12, the expected heterozygosities were 0.420 and 0.372, and the observed heterozygosities were 0.436 and 0.322 for the white and black rhinoceroses, respectively. Seven of the primers produced different allele sizes and variations that distinguished between black and white rhinoceroses. Further genetic analyses with larger wild population sample sizes and markers are recommended to obtain a better understanding of the genetic structure of the black and white rhinoceros populations in order to be useful in the conservation efforts of these critically endangered species. A. Kilbourn—In memoriam.     

Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes

In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.