Development of 21 novel microsatellite markers for conservation genetic studies of Brachymystax lenok tsinlingensis,an endangered species in Korea,using next-generation sequencing

Using a next-generation sequencing approach, we have developed 21 novel microsatellite markers in Brachymystax lenok tsinlingensis, an endangered freshwater fish species in Korea. All new microsatellite markers were successfully genotyped using 30 samples from 2 tributaries (BA and DMZ) of the Han River. These new microsatellite markers produced 102 alleles, varying from 2 to 9 alleles per locus in each tributary. No significant deviation from Hardy–Weinberg equilibrium or linkage disequilibrium in marker-pair/population was observed between markers, except for BleDi55. These markers developed can be valuable tools to conservation genetics of this species across its distributional range.     

Isolation and characterization of microsatellite loci for the oval pigtoe mussel, Pleurobema pyriforme (Lea 1857)

We isolated nine microsatellite loci from Pleurobema pyriforme, a freshwater mussel species currently protected under the Endangered Species Act. Eight loci were polymorphic with 2–15 alleles and observed heterozygosities of 0.130–0.957. All loci conformed to Hardy–Weinberg expectations and all pairs of loci showed no significant genotypic disequilibrium. These are the first isolated microsatellite loci for this species and will serve in ongoing conservation efforts of P. pyriforme.     

Twelve tetranucleotide microsatellite loci for westslope cutthroat trout Oncorhynchus clarki lewisi (Salmonidae)

We isolated 12 tetra-nucleotide microsatellite loci from westslope cutthroat trout (Oncorhynchus clarki lewisi). These loci were tested against 58 individuals from a single creek for polymorphism. The number of alleles ranged from 2 to 8, with an average of 4.3. The expected heterozygosity ranged from 0.12 to 0.79, with an average of 0.52. Ten of the twelve loci conformed to Hardy–Weinberg expectations. These microsatellite loci will be useful for describing population structure in westslope cutthroat trout.     

Characterization of ten new microsatellite loci from the endangered endemic rodent, Reithrodontomys spectabilis

Reithrodontomys spectabilis is an endemic rodent inhabiting Cozumel island in the Mexican Caribbean. This species is categorized as Threatened by Mexican law and as Critically Endangered by the IUCN. We report 10 polymorphic microsatellite loci, which were tested in 30 individuals trapped on the island. Results show a number of alleles ranging from 3 to 14, an observed heterozygosity of 0.200–0.967 and expected heterozygosities from 0.396 to 0.899. Two loci showed deviation from Hardy–Weinberg equilibrium. None of the loci showed linkage disequilibrium. These new microsatellite loci for R. spectabilis are vital to investigate different aspects of genetic diversity and help conservation of this endemic, threatened species.     

Development of polymorphic microsatellite markers for the critically endangered and endemic Indian dipterocarp, Vateria indica L. (Dipterocarpaceae)

Vateria indica (Dipterocarpaceae) is an economically and ecologically important canopy tree endemic to the Western Ghats, India. The species has undergone extensive habitat loss and overexploitation and is therefore listed as ‘critically endangered’ on the 2012 IUCN Red List. We developed ten polymorphic microsatellite loci for V. indica. In addition, we confirm cross amplification and variation in two loci isolated from the closely related but geographically disjunct species Vateriopsis seychellarum, previously published by Finger et al. Conserv Genet Resour, 2 (S1):309–311, (2010). The twelve microsatellite primers screened on 48 adult samples of V. indica had 5–11 alleles per locus (mean of 8.5 per locus) with an average polymorphic information content of 0.64 across loci. Expected heterozygosity ranged from 0.44 to 0.84. These markers will enable us to quantify population genetic diversity in habitat fragments and to study fine scale spatial genetic structure and contemporary gene flow.     

Characterization of ten microsatellite loci for the dark pomfret, Pampus cinereus (Stromateidae, Perciformes)

Ten polymorphic microsatellite loci were isolated in the dark pomfret (Pampus cinereus), an important commercial foodfish species. The mean number of observed alleles per locus was 21 (range 13–33). Observed and expected heterozygosity values ranged from 0.73 to 0.98 and from 0.83 to 0.97 respectively. Two loci showed significant departure from Hardy–Weinberg equilibrium and significant linkage disequilibrium was found between P15-2 and P230. These highly informative microsatellite markers should be useful for population genetic analyses of P. cinereus and other pomfret species.     

Isolation, characterization and predicted genome locations of Eurasian eagle-owl (Bubo bubo) microsatellite loci

We isolated 134 eagle-owl (Bubo bubo) microsatellite sequences. Eight of these newly isolated loci were characterized in 38 Eurasian eagle-owls in a northern European population. Sequence homology was used to assign a predicted chromosome location for the eight loci. We also redesigned primers for four previously isolated eagle-owl sequences and cross-amplified two published primer sets previously characterized in other owl species. These 14 loci were amplified in three multiplex PCR sets and displayed 2–9 alleles with expected and observed heterozygosities ranging from 0.34 to 0.85 and from 0.42 to 0.97, respectively. Estimated frequencies of null-alleles were low and only one locus deviated from Hardy–Weinberg equilibrium. After correcting for multiple tests, linkage disequilibrium was found for a single pair of loci. The combined probability of identity for the 14 loci was 3.5 × 10^?12. These microsatellite loci are expected to be useful for genetic monitoring, parentage analysis and population genetic studies.     

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.     

Isolation and characterization of 15 microsatellite loci in an endemic Chinese cyprinid fish, Pseudogyrincheilus prochilus, and their cross-species amplification in two related species

Fifteen polymorphic microsatellite loci were developed for an endemic Chinese cyprinid fish, Pseudogyrincheilus prochilus. The number of observed alleles per locus ranged from 2 to 17. The observed and expected heterozygosities ranged from 0.067 to 1.000 and from 0.066 to 0.932, respectively. No significant linkage disequilibrium was detected among the loci. Two loci showed significant deviation from Hardy–Weinberg equilibrium, and one of them had evidence for null alleles. Fourteen primer pairs cross-amplified in Garra pingi pingi, and 13 primer pairs cross-amplified in Onychostoma sima. These microsatellite loci should prove highly informative for future studies of genetic diversity conservation.     

Isolation and characterization of eleven microsatellite loci in the marbled rockfish,Sebastiscus marmoratus (Scorpaenidae)

The marbled rockfish, Sebastiscus marmoratus, is an important commercially near-shore fish inhabiting littoral rocky bottoms from Japan to the East China Sea. Eleven polymorphic microsatellite loci were isolated from S. marmoratus and the polymorphism of these loci was observed in 32 samples from one wild population. The number of alleles ranged from 2 to 8. The polymorphism information content ranged from 0.155 to 0.752. The levels of the observed and expected heterozygosity were 0.186–0.969 and 0.170–0.782, respectively. Only one locus (Sm3-63) deviated from Hardy–Weinberg equilibrium. These microsatellite markers can provide a useful tool for further study on genetic diversity and conservation of S. marmoratus.     

Characterization of microsatellite loci for a threatened species, the King Rail, Rallus elegans, using a next-generation sequencing protocol

The King Rail Rallus elegans (Audubon) has experienced population declines of 4.6 % per year on average since the 1960s. Wetland loss, most severely affecting inland marshes, has significantly reduced this species’ distribution to the coastal margins of its historic range. Polymorphic microsatellite markers were generated by 454 pyrosequencing of genomic DNA from King Rails, and Clapper Rails R. longirostris from Louisiana after AFLP enrichment and barcoding of restriction fragment cut sites across individuals. Of 1,419 microsatellite-containing sequences, 20 hypervariable microsatellite loci with up to 20 different alleles were identified at the alignment stage. We characterized nine loci, tested variability in 45 Atlantic coast King Rail samples, and detected 4–19 alleles per locus. Cross-species amplification revealed variability in the Virginia Rail, R. limicola, and Sora, Porzana carolina. These loci will be useful for studying secretive marsh rails, many of which are threatened or endangered.     

Development and characterization of 16 microsatellites for Hoffmann’s two-toed sloth, Choloepus hoffmanni

Hoffmann’s two-toed sloth (Choloepus hoffmanni) is an arboreal mammal found throughout the Neotropics. Due to its limited dispersal power and reliance on forested habitats, C. hoffmanni could serve as a model species for understanding the response of mammals to land cover change. To better understand sloth life history and their response to tropical forest fragmentation and loss, we developed and characterized 16 polymorphic microsatellite markers. We tested each locus with 16–23 C. hoffmanni individuals sampled in northeastern Costa Rica. The number of alleles per locus ranged from three to seven, while mean observed heterozygosity was 0.56 and ranged from 0.33 to 0.75. All loci met Hardy–Weinberg expectations and none of the loci exhibited significant linkage disequilibrium. The microsatellite markers developed herein will be used to investigate dispersal rates and gene flow among habitat patches in Costa Rica, as well as provide insights into the life history of two-toed sloths.     

Development and characterization of 21 polymorphic microsatellite markers for the barren-ground shrew, Sorex ugyunak (Mammalia: Sorcidae), through next-generation sequencing, and cross-species amplification in the masked shrew, S. cinereus

We used next generation shotgun sequencing to develop 21 novel microsatellite markers for the barren-ground shrew (Sorex ugyunak), which were polymorphic among individuals from northern Alaska. The loci displayed moderate allelic diversity (averaging 6.81 alleles per locus) and heterozygosity (averaging 70 %). Two loci deviated from Hardy–Weinberg equilibrium (HWE) due to heterozygote deficiency. While the population did not deviate from HWE overall, it showed significant linkage disequilibrium suggesting this population is not in mutation-drift equilibrium. Nineteen of 21 loci were polymorphic in masked shrews (S. cinereus) from interior Alaska and exhibited linkage equilibrium and HWE overall. All loci yielded sufficient variability for use in population studies.     

Isolation and characterization of microsatellite loci in the redside dace, Clinostomus elongatus

We isolated and characterized eight polymorphic microsatellite loci for redside dace (Clinostomus elongatus), a colorful North American cyprinid that is threatened or endangered throughout most of its range. The number of alleles per locus ranged from three to eighteen, with observed heterozygosity ranging from 0.31–0.92. Cross-amplification revealed that these markers will also be useful for examining closely related and more distantly related species, including the rosyside dace (Clinostomus funduloides), Lahontan redside shiner (Richardsonius egregius), hornyhead chub (Nocomis biguttatus), and central stoneroller (Campostoma anomalum). These microsatellite loci will provide a valuable set of tools for examining fine and coarse scale population structure, exploring reproductive success, and testing outcomes of proposed conservation initiatives (e.g. captive breeding and translocation of wild individuals) for redside dace.     

Characterization of 14 microsatellite loci in the paired lamprey species Ichthyomyzon unicuspis and I. fossor and cross amplification in four other Ichthyomyzon species

We describe the isolation and development of 14 polymorphic microsatellite loci in silver lamprey (Ichthyomyzon unicuspis). The number of alleles ranged from 2 to 11 (with an average of 5.1 alleles per locus) in 23–37 lampreys and genetic diversity ranged from 0.036 to 0.837. All of these loci were also polymorphic (with 2–10 alleles, average 4.4) in the closely related northern brook lamprey (I. fossor), and at least eight were polymorphic in four other Ichthyomyzon species. These loci will aid in studying the population dynamics of these six Ichthyomyzon species.     

Development of microsatellite loci for the fish Poecilia vivipara (Cyprinodontiformes: Poeciliidae)

Poecilia vivipara is a small fish found throughout the coastal lowlands of South America. We report the isolation and characterization of 25 microsatellite loci in this tropical fish. In 24 individuals from two populations, 18 loci were polymorphic. The loci had 2–12 alleles, expected heterozygosities of 0.194–0.836, observed heterozygosities of 0.208–0.792, and discriminatory powers of 0.202–0.872. Four loci showed significant deviations from Hardy–Weinberg equilibrium. The microsatellite loci will be useful for investigating genetic diversity and population genetic structure, which will contribute to the development of efficient conservation programs for coastal lowlands where P. vivipara occurs.     

Isolation and characterization of microsatellite loci in the federally endangered fat threeridge mussel (Amblema neislerii)

We isolated and characterize 16 microsatellite loci from Amblema neislerii, a federally endangered freshwater mussel. Fifteen of 16 loci were polymorphic with an average of 11.62 alleles per locus and an average observed and expected heterozygosity of 0.63 and 0.75, respectively. Fourteen of 16 loci conformed to Hardy–Weinberg expectations and all pairs of loci showed no significant genotypic disequilibrium. These microsatellite loci will serve as important tools in ongoing conservation efforts of A. neislerii.     

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 characterization of microsatellite markers of an endangered tropical tree, Sideroxylon majus, and cross-species amplification in other Sapotaceae species

Sideroxylon majus (Sapotaceae) is an endangered endemic tree of La Réunion Island that has suffered from human actions. It is present in small and isolated populations that encounter severe difficulties to regenerate. To have powerful tools for population genetic studies, we have isolated and characterized 14 polymorphic microsatellite markers from S. majus. The 14 loci were tested on 57 individuals from 6 populations. The number of alleles per locus varied from 2 to 20, with an average of 11.8. The observed and expected heterozygosity levels ranged from 0.053 to 1.000, and 0.116 to 0.917, respectively. Six of the 14 loci deviated from Hardy–Weinberg equilibrium. These polymorphic microsatellite markers constitute new tools to study the genetic diversity and spatial genetic structure of S. majus. The cross-species amplifications indicate that most of these loci can be used to investigate population genetic structure in S. grandiflorum, S. boutonianum and S. sessiliflorum. These studies will provide useful results for the elaboration of effective conservation strategies.     

Development and characterization of microsatellite loci for two species of Beringian birds,rock sandpiper (Calidris ptilocnemis) and Pacific wren (Troglodytes pacificus)

Identification and assessment of small, endemic populations are priorities for conservation. We isolated and characterized 8 microsatellite loci from rock sandpiper (Calidris ptilocnemis) and 5 microsatellite loci from Pacific wren (Troglodytes pacificus), species with endemic populations of named subspecies that are of conservation concern. Eighteen to 20 individuals of each species from several locations in Alaska were screened for polymorphism. Loci for each species showed high polymorphism, with rock sandpiper ranging from 5 to 14 alleles per locus and 0.73–0.88 expected heterozygosity and Pacific wren ranging from 5 to 14 alleles per locus and 0.55–0.91 expected heterozygosity. Loci developed for rock sandpipers were also polymorphic in closely related taxa. These loci are the first developed for either species and will be used to identify and conserve endemic populations in the Bering Sea region.