Optimization of novel polymorphic microsatellites in the endangered Sumatran rhinoceros (Dicerorhinus sumatrensis)

Loss of habitat and poaching have led to a drastic reduction in numbers of the Sumatran rhinoceros (Dicerorhinus sumatrensis). To aid in the conservation management of this species, we isolated and optimized 10 polymorphic Sumatran rhinoceros microsatellite loci. A survey of six individuals yielded a mean number of alleles of 3.7, mean expected heterozygosity of 0.551 and probability of identity of 3.46 × 10^?8. Although this estimate is similar to estimates of microsatellite variability in the Black, Indian and White rhinoceroses, such a conclusion is premature as locus purity, sample size and number of loci surveyed vary significantly among studies.     

Isolation and characterization of polymorphic microsatellite loci in Hemibarbus labeo (Cyprinidae) using PCR‐based isolation of microsatellite arrays (PIMA)

A total of 10 polymorphic microsatellite loci from Hemibarbus labeo were isolated and characterized using an optimized protocol to construct a microsatellite‐enriched genomic library. The analysis of variability was performed in 24 specimens of mainland China. The mean number of alleles across loci was 3.10 ± 1.10 and the level of expected heterozygosity varied from 0.0417 to 0.7482. Frequencies of null alleles of the 10 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population. Five primer pairs cross‐amplify the microsatellites in other species, indicating transportability of the markers within the family Cyprinidae.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Polymorphic microsatellite loci in the endangered Indian rhinoceros,Rhinoceros unicornis

Eleven novel polymorphic microsatellite loci are presented for the highly endangered Indian (or greater one horned) rhinoceros Rhinoceros unicornis (Mammalia: Rhinocerotidae). These will be used to analyse the genetic variability within and between the two remaining large populations of the Indian rhinoceros and to manage captive breeding.     

Comparison of whole mitochondrial genome sequences of northern and southern white rhinoceroses (<Emphasis Type="Italic">Ceratotherium simum</Emphasis>): the conservation consequences of species definitions

The probable extinction of the last confirmed population of northern white rhinoceros (Ceratotherium simum cottoni) in the world has ignited debate regarding its species status compared to the southern white rhinoceros (Ceratotherium simum simum). Previous studies, based on partial mitochondrial sequences, have reported conflicting results regarding the species status of the northern white rhinoceros. We use whole mitochondrial genomes obtained using Next Generation Sequencing of four northern white rhinoceros and three southern white rhinoceros using novel primers in three overlapping fragments. Phylogenetic relationships were constructed, using Maximum Likelihood, and recovered monophyletic clades for northern white rhinoceros and southern white rhinoceros. The divergence time between the two mitochondrial DNA lineages was estimated to be between 0.46 and 0.97 million years ago using Bayesian inferences. Since there are currently only three surviving northern white rhinoceros individuals these results put into sharp focus the ongoing debate regarding the methods of species definition, and in particular the consequences of such definitions in conservation management of endangered species and subspecies. We conclude that the designation of sub-species status is more applicable to northern and southern white rhinoceros.     

Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.     

Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

Polymorphic CA/GT and GA/CT microsatellite loci for Ostrinia nubilalis (Lepidoptera: Crambidae)

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short (n = 10–12). Ten microsatellites, PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range six to 20), and six of 10 loci showed heterozygote deficiency. Amplification of eight loci was observed in the sister species O. furnicalis.     

Isolation and characterization of 13 microsatellite loci in Rhinolophus pusillus (least horseshoe bat) with cross-amplification in five related species

We used the enriched genomic library method to isolate and characterize dinucleotide microsatellite loci in the least horseshoe bat, Rhinolophus pusillus. Seventeen loci were obtained and tested on 31 individuals sampled from Guangxi Province in southern China. Thirteen of these markers were polymorphic with expected heterozygosity ranging from 0.821 to 0.909. A total of 164 alleles were detected and the number of alleles per locus ranged from 9 to 16 (mean 12.6). These polymorphic markers will be used to assess population structure in R. pusillus. In addition, successful cross-amplification in five congeneric bat species suggests most of these markers will also be useful for studying related species.     

Isolation and characterization of microsatellite loci in Ononis repens,Leguminosae

Ten polymorphic microsatellites were isolated from Ononis repens, using a microsatellite enrichment protocol and selective hybridization with a mixed synthetic dinucleotide repeat probe. Primers for DNA amplifications using polymerase chain reaction (PCR) were designed and synthesized. The loci amplified in both O. repens and O. spinosa. All 10 loci were polymorphic in each of four populations studied. These microsatellite markers will be useful for population genetic analysis and biodiversity studies of O. repens and O. spinosa.     

Isolation and characterization of microsatellite loci in Hexagrammos otakii based on 2b-RAD method

In this study, we isolated 20 novel microsatellites loci associated with the growth of Hexagrammos otakii (Fat greenling) by using 2b-RAD sequencing method. The characteristics of 20 microsatellite loci were amplified in 105 H. otakii individuals which came from four different groups and tested by Capillary electrophoresis. The number of allele of the 20 microsatellite loci ranged from 8 to 26 with an average of 19.95. PIC value ranged from 0.1659 to 0.9227 with an average of 0.7555. Observed and expected heterozygosity varied from 0.0667 to 0.8571 and from 0.1710 to 0.9314, respectively. Highly polymorphic characteristics were observed in each microsatellite loci of the study except HO6. Further research showed that 10 microsatellite loci had transferability in Hexagrammos agrammus. In this study, high polymorphism and genetic diversity using the 15 polymorphic microsatellite loci suggest that they are suitable for investigating the fine-scale population structure, genetic relationship and further evaluating the artificial reproduction strategy of H. otakii.     

Polymorphic microsatellite loci for paternity analysis in the Madagascar paradise flycatcher (Terpsiphone mutata: Aves)

Nine polymorphic microsatellite loci from the Madagascar paradise flycatcher Terpsiphone mutata were isolated using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Seven polymorphic loci showed no evidence of null alleles and exhibited high levels of variation in 18 unrelated individuals (mean diversity = 0.80, mean number of alleles = 13.6). These loci are therefore suitable for analysis of population structure and paternity (exclusion probability for six unlinked loci = 0.9998).     

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations assessed by microsatellite markers

Microsatellites are powerful markers to infer population genetic parameters. Here, 13 microsatellite loci isolated from a genomic and a cDNA library of Cryphonectria parasitica were used to characterize the genetic diversity and structure of four French populations. Twelve of these loci were polymorphic within populations, and average gene diversity (H_e) was estimated to be 0.35. There was a lower genetic diversity in a south-eastern population relative to three south-western populations. In these three populations, microsatellite genotypic diversity was higher than vegetative compatibility type diversity. A high genetic differentiation (G_ST = 0.27) suggested a low gene flow and/or founder effects of French populations which are in agreement with low dispersal of spores and different introductions of this species in southern France. This study demonstrates the significance of these microsatellite loci to assess gene flow and reproductive system in this important pathogen.     

Characterization of novel microsatellite loci for red clover (Trifolium pratense L.) from enriched genomic libraries

Twenty‐seven polymorphic microsatellite markers were isolated from red clover (Trifolium pratense). Allelic variability and cross‐species amplification were assessed on 24 red clover and eight white clover (Trifolium repens) genotypes. The number of alleles detected in red clover ranged from two to 25. Observed and expected heterozygosities were high with average values of 0.71 and 0.88, respectively. Five of the 27 loci were also successfully amplified from white clover, where two to 13 alleles were detected. These highly polymorphic microsatellite loci provide powerful tools for population genetic studies as well as for marker‐assisted selection in this important forage legume species.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of spotted maigre (Nibea albiflora)

In the present study, we reported 13 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of Nibea albiflora. The number of alleles, observed and expected heterozygosity per locus in 44 individuals ranged from 2 to 13, from 0.0909 to 0.9773 and from 0.0886 to 0.9073, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. Analysis of linkage disequilibrium showed non-significant among the two pairs of loci. As a result, 13 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Nibea albiflora. Shichao Xing, Changwei Shao contributed equally to this work.     

Population genetic structure and phylogeography of cyprinid fish, <Emphasis Type="Italic">Labeo dero</Emphasis> (Hamilton, 1822) inferred from allozyme and microsatellite DNA marker analysis

We examined population structure of Labeo dero (Hamilton, 1822) from different riverine locations in India using 10 polymorphic allozyme and eight microsatellite loci. For analysis, 591 different tissue samples were obtained from commercial catches covering a wide geographic range. Allozyme variability (An = 1.28–1.43, Ho = 0.029–0.071) was much lower than for microsatellites (An = 4.625–6.125, Ho = 0.538–0.633). Existence of rare alleles was found at three allozyme (MDH-2*, GPI* and PGDH*) and at two microsatellite loci (R-3* and MFW-15*). Deviation from Hardy–Weinberg equilibrium (P < 0.05, after the critical probability levels were adjusted for sequential Bonferroni adjustment) could be detected at three loci (EST-1*, -2* and XDH*) whereas, after correction for null alleles, two microsatellite loci (MFW-1*,-15*) deviated from HWE in the river Yamuna. Fst for all the samples combined over all allozyme loci was found to be 0.059 suggesting that 5.9% of the total variation was due to genetic differentiation while microsatellite analysis yielded 0.019 which was concordant to mean Rst (0.02). Hierarchical partition of genetic diversity (AMOVA) showed that greater variability (approx. 95%) was due to within population component than between geographical regions. Based on distribution of genetic differentiation detected by both markers, at least five different genetic stocks of L. dero across its natural distribution could be identified. These results are useful for the evaluation and conservation of L. dero in natural water bodies.     

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (<Emphasis Type="Italic">Rhynchotus rufescens</Emphasis>, Tinamiformes,Aves) and cross-amplification in other tinamous

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2–12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy–Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10⁻⁵), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.     

Development of microsatellite markers in Rhizophora stylosa using a dual‐suppression‐polymerase chain reaction technique

Rhizophora stylosa is an ecologically important mangrove tree species that is found in tropical and subtropical regions. We isolated five polymorphic microsatellite loci from R. stylosa using a dual‐suppression‐polymerase chain reaction technique. These loci provided microsatellite markers with polymorphism of four alleles in each locus for overall population. The mean expected heterozygosities ranged from 0.113 to 0.473.     

Isolation and characterization of five microsatellite loci in the ponerine ant Pachycondyla inversa (Hymenoptera,Formicidae)

We characterized five polymorphic DNA microsatellite loci for the neotropical ponerine ant Pachycondyla inversa. The variability was initially tested in 19 workers from nine colonies from a Brazilian population. We found five to 12 alleles per locus, with observed heterozygosities between 0.72 and 0.95. The allele size ranged from 73 to 197 bp. The primers also successfully amplified DNA at all five loci in the closely related species P. villosa.