Low levels of polymorphism at novel microsatellite loci developed for bathyergid mole-rats from South Africa

Eight microsatellite markers were developed for African mole-rats using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol and pyrosequencing. The markers were developed in Bathyergus suillus and applied to a selection of individuals from seven related taxa: Bathyergus janetta, Cryptomys hottentotus hottentotus, C. h. pretoriae, Fukomys damarensis, F. darlingi, F. mechowii and Georychus capensis. The markers displayed low to moderate variation with allele numbers ranging between one and six per species. We propose that larger repeat numbers at di-, tri- and tetranucleotide repeat loci generally yield higher levels of polymorphism.     

Microsatellite repeat motif and amplicon length affect amplification success of degraded faecal DNA

Degradation reduces DNA quality and quantity in faecal samples and leads to low amplification success. We investigated the influence of repeat motif and amplicon length by comparing the amplification success of five dinucleotide, five tetranucleotide, and two compound microsatellite markers for African elephant (Loxodonta africana) faecal DNA samples. We found that both repeat motif and amplicon length influenced amplification success, and suggest the use of simple microsatellite markers containing alleles with small amplicon sizes to maximise amplification success of degraded DNA.     

Characterization of polymorphic microsatellite markers for the bearded goby Sufflogobius bibarbatus

Fourteen polymorphic microsatellite markers with tetranucleotide repeats were developed for the bearded goby (Sufflogobius bibarbatus) from partial genomic DNA libraries using a repeat enrichment protocol, and characterized using two putative populations from the northern Benguela. The average number of alleles per locus ranged from 4 to 34, and the observed heterozygosities across loci were between 0.237 and 0.983. We also tested the utility of these markers in two other marine gobies; sand goby Pomatoschistus minutus and two-spotted goby Gobiusculus flavescens. These polymorphic markers can be employed to investigate population structure and related questions of the bearded goby.     

Isolation and characterization of EST-based microsatellite markers for Scatophagus argus based on transcriptome analysis

In the present study, expressed sequence tag-based microsatellite markers were developed by 454 pyrosequencing for the marine fish Scatophagus argus. A total of 510,939 reads were obtained, and 4,322 di-nucleotides, 1,354 tri-nucleotides, 219 tetra-nucleotides, 30 penta-nucleotides and 9 hexa- nucleotides were detected. Among these repeated sequences, 51 microsatellites where repeat units were greater than 4 were randomly selected to test the variability in natural populations. 12 polymorphic markers were identified and characterized. The number of alleles per locus varies from 3 to 6, and observed and expected heterozygosities ranged from 0.102 to 0.658 and from 0.124 to 0.691, respectively. No genetic linkage and significant departure from Hardy–Weinberg equilibrium were detected among these loci. High-throughput pyrosequencing allows fast and efficient isolation of microsatellite markers on a large scale, and can provide enough microsatellite markers for population genetic studies and genetic mapping.     

Development of seventeen novel microsatellite markers of a wild and flowering bamboo Dendrocalamus membranaceus (Poaceae) and cross-amplification in Dendrocalamus genus

17 novel microsatellite markers were developed in Dendrocalamus membranaceus Munro, a declining and flowering wild bamboo in Lancang–Mekong River Valley, using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. The number of alleles per locus ranged from 2 to 13, with an average of 5.5 per allele. The observed and expected heterozygosities varied from 0.043 to 0.696 and from 0.479 to 0.926, respectively. Cross-amplifications were carried out for seven species of Dendrocalamus Nees, and resulted in a 41.2–100 % of successful amplification. These markers will be useful for detecting the gene flow in the flowering populations and studying its breeding system.     

Development of 15 microsatellite loci from mulloway,Argyrosomus japonicus (Pisces: Sciaenidae) using next generation sequencing and an assessment of their cross amplification in other sciaenids

Next generation sequencing was used to develop 15 new polymorphic microsatellite markers for the commercially and recreationally important fish mulloway (Argyrosomus japonicus). Improved knowledge of the genetic structure of the species is necessary for fisheries and environmental management. In a population of mulloway, we found that genetic variation and heterozygosity were high in most of our loci (mean number of alleles per locus = 9.46; mean heterozygosity = 0.660). To test the usefulness of our new markers for genetic research on other sciaenids, we cross amplified samples from eight other related sciaenid species, including three that form important fisheries and others that have significant conservation issues. We found that most of our new primers pairs produced scorable profiles in each of the other important sciaenids.     

Isolation and characterization of twenty tetranucleotide microsatellite DNA makers in a schizothoracin fish, Schizothorax prenanti (Tchang)

Prenant’s schizothoracin, Schizothorax prenanti (Tchang) is an important polyploidy fish in upper reaches of the Yangtze River. Given the extreme anthropogenic environmental alteration in the region, S. prenanti represents an excellent model organism to study historical and contemporary environmental changes. We developed twenty tetranucleotide microsatellite DNA markers from a tetranucleotide microsatellite enriched library. These highly polymorphic markers (2–23 alleles) will facilitate the population genetic analyses of this species.     

Characterisation of polymorphic microsatellite loci for the bryozoan Fredericella sultana,the primary host of the causative agent of salmonid proliferative kidney disease

The freshwater bryozoan Fredericella sultana is the most common primary host for the myxozoan parasite Tetracapsuloides bryosalmonae, causing proliferative kidney disease (PKD) in salmonid fish. An Illumina next-generation sequencing library containing genomic DNA from both T. bryosalmonae and F. sultana yielded 10,653 microsatellite motifs. Twenty-four loci were tested in both species and 12 novel polymorphic tri- and tetranucleotide microsatellite markers were developed for F. sultana. These markers provide a valuable resource for population genetic studies of F. sultana, whilst the apparent lack of microsatellites in the PKD agent suggests a low frequency of tri- and tetranucleotide repeats in the T. bryosalmonae genome.     

Isolation and characterization of 14 tetranucleotide microsatellite DNA markers in the elongate loach Leptobotia elongata (Cypriniformes: Cobitidae) using 454 sequencing technology

Anthropogenic factors have contributed to a severe decline of Leptobotia elongata. Conservation information of L. elongata will be required. Here, 14 tetranucleotide microsatellite DNA markers were developed from L. elongata through high-throughput (454) sequencing data. Twenty-four individuals of the species were genotyped using these markers. All of the loci displayed a high level of polymorphism with the number of alleles per locus ranged from 7 to 23, polymorphism information content ranged from 0.650 to 0.943. Observed and expected heterozygosities ranged from 0.609 to 1.000 and from 0.699 to 0.967, respectively. These markers provide an excellent toolkit to study population genetic structure and would benefit the conservation of this species.     

Development of eight polymorphic microsatellite markers by FIASCO-based strategy for a arsenic-hyperaccumulator Chinese brake fern

Chinese Brake fern (Pteris vittata) is the first identified and well-known arsenic-hyperaccumulator. It is widely distributed in areas of temperate zone as diploid and of subtropics-tropics zone as tetraploid. Screening 60 individuals from Southern China, eight polymorphic microsatellite markers were developed for the first time by employing fast isolation by AFLP of sequences containing repeats protocol (FIASCO). The number of alleles for each marker ranged from two to seven and one to four bands per individual. Furthermore, five of the loci possess more than two alleles per individual. The results suggested these microsatellite markers provide a useful tool for studying the ongoing genetic variability of this specie as well as mating systems.     

Isolation of 13 polymorphic microsatellite loci for slimy sculpin (Cottus cognatus)

Our research on slimy sculpins (Cottus cognatus), a benthic, freshwater fish, requires highly polymorphic genetic markers, and microsatellite loci developed for other Cottus species were insufficient for our needs. We therefore developed 13 polymorphic microsatellite loci from C. cognatus libraries enriched for tri- and tetranucleotide repeats. These loci had 2–22 alleles and observed heterozygosities ranging from 0.36 to 0.86 in a sample of 47 individuals from one population. There was no evidence of linkage disequilibrium; however, one locus had a putative null allele. Twelve loci also worked for mottled sculpin (Cottus bairdi) but only eight were polymorphic in a sample of seven individuals.     

Isolation and characterisation of new microsatellite markers for the stonefly Brachyptera braueri comparing a traditional approach with high throughput 454 sequencing

We developed thirteen microsatellite markers for the stonefly Brachytpera braueri, a rare aquatic insect. We compared a “traditional” approach yielding an enriched library by cloning of positive host cells, hybridising and Sanger sequencing of target fragments with a 454 next generation sequencing approach. From a total of 881 fragments containing a repeat motif thirteen polymorphic loci were developed and tested for two populations. Number of alleles ranged from 4 to 20 and values of heterozygosity varied from 0.17 to 0.91 (H_O) and 0.26 to 0.91 (H_E). The markers are essential to investigate the colonisation potential of freshwater insects.     

Isolation and characterization of microsatellite markers from yellowfin grouper, Mycteroperca venenosa

Twenty-seven nuclear-encoded microsatellites were isolated from an enriched genomic library of yellowfin grouper, Mycteroperca venenosa, and characterized in 24 individuals. The microsatellites include 17 dinucleotide repeats, eight trinucleotide repeats (one imperfect), one tetranucleotide repeat, and one pentanucleotide repeat. Yellowfin grouper are susceptible to fishing pressure targeted towards their spawning aggregations; data pertaining to stock structure and levels of genetic variability will aid in future management of this species.     

Isolation and characterization of 57 novel polynucleotide microsatellites from yellow catfish (Pelteobagrus fulvidraco) genome for genetic analysis

Yellow catfish (Pelteobagrus fulvidraco) is one of the important economic freshwater fish in Eastern Asia. In recent years, the wild resources were decreased sharply due to overfishing and pollution. It is necessary to conserve the wild stocks for the sustainable use of the species. In this study, 200 microsatellite markers were isolated from yellow catfish genome using Roche 454 pyrosequencing method. Among these markers, 57 exhibited polymorphism in yellow catfish population collected from Songhua River in China. The number of alleles ranged from 2 to 13 and the number of effect alleles ranged from 1.112 to 8.980. The observed heterozygosity (H _o) and the expected heterozygosity (H _e) varied from 0.106 to 0.957 and from 0.102 to 0.898, respectively. Most loci conformed to Hardy–Weinberg equilibrium with the exclusion of five loci. These polymorphic loci will be valuable for population genetic structure and genetic conservation for the P. fulvidraco.     

Development of polymorphic microsatellite loci isolated from the Ancherythoculter nigrocauda

Twelve polymorphic microsatellite markers from Ancherythoculter nigrocauda were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. These loci were characterized by genotyping 48 individuals. The observed number of alleles per locus ranged from 4 to 14 with an average of 9.4. The expected and observed heterozygosities ranged from 0.185 to 0.907 and from 0.083 to 0.952, respectively. Among these polymorphic microsatellite loci, four of the loci (HWB01, HWB04, HWB12 and HWB16) significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.05). All of the microsatellite loci were in linkage equilibrium. These microsatellite markers would be useful for investigating the genetic diversity and population structure of A. nigrocauda.     

Tools for the management and conservation of genetic diversity in brook trout (Salvelinus fontinalis): tri- and tetranucleotide microsatellite markers for the assessment of genetic diversity, phylogeography, and historical demographics

We document isolation and characterization of 13 tri- and tetranucleotide microsatellite DNA markers in brook trout (Salvelinus fontinalis). These markers displayed moderate to high levels of allelic diversity (averaging 20.5 alleles/locus) and heterozygosity (averaging 53.5%) in a range-wide survey of more than 13,000 fish. A comparison of two geographically proximal populations located on opposite sides of the eastern continental divide in Maryland, USA, found no deviations from Hardy?CWeinberg equilibrium and minimal linkage disequilibrium. Microsatellite markers developed for S. fontinalis yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate phylogeographic structure; and (3) provide unique demographic perspectives of population sizes and historical demographics. This suite of markers also provided considerable cross-species amplification utility among related salmonids.     

Isolation and characterization of twenty-six microsatellite loci for the tetraploid fish Dabry’s sturgeon (Acipenser dabryanus)

To evaluate the population genetic diversity as a means of devising conservation strategies, we constructed (CA)n and (CT)n enriched genomic libraries for Acipenser dabryanus, a narrowly endemic and endangered species in China. Twenty-six polymorphic microsatellite markers were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Between 3 and 13 alleles per locus were observed. Mean expected heterozygosities (H _E) and Shannon-Wiener Diversity Indices (H′) per locus ranged from 0.303 to 0.806, and from 0.480 to 1.893, respectively. The microsatellite markers described here are valuable tools for the population genetics research of A. dabryanus.     

Development and characterization of fourteen novel microsatellite loci in Chinese muntjac (Muntiacus reevesi)

Fourteen novel polymorphic microsatellite markers were isolated and characterized for Muntiacus reevesi, a small deer species. An (AG)_n enriched library was created from two individuals following the FIASCO protocol (Fast Isolation by AFLP of Sequences COtaining repeats). 14 primers were designed from 98 microsatellite sequences and tested in 32 samples. The number of alleles per locus ranged from 2 to 13 and the expected heterozygosities from 0.123 to 0.916. The loci had an average polymorphic information content value of 0.676. Five loci showed significant deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These markers should be a useful tool for further population genetic studies of Muntiacus reevesi.