Isolation and characterization of 22 nuclear and 5 chloroplast microsatellite loci in the threatened riparian plant Myricaria germanica (Tamaricaceae, Caryophyllales)

The German tamarisk (Myricaria germanica) is a riparian shrub threatened in Europe, where populations are largely confined to the upstream areas of rivers. To study gene movement in this species within and among catchments, we isolated and characterized 22 nuclear and 5 chloroplast microsatellite loci and tested their polymorphism based on 40 individuals of M. germanica and four individuals of Myricaria rosea. For M. rosea, 15 markers amplified, and of these, eight were polymorphic. All markers were polymorphic for M. germanica. The polymorphic markers allow the characterization of gene flow patterns at various spatio-temporal scales.     

Characterization of 9 polymorphic microsatellite loci in Lamellibrachia sp. 2, a tubeworm found at deep-sea hydrothermal vents and cold seeps

Lamellibrachia sp. 2 is a deep-sea vestimentiferan tubeworm found at hydrothermal vents on the Mid-Cayman Spreading Center and at hydrocarbon seeps in the Gulf of Mexico and off the coast of Barbados. Nine selectively neutral and unlinked polymorphic microsatellite loci were developed for this species. Eight of these loci conformed to Hardy–Weinberg expectations. Average observed heterozygosity ranged from 0.14 to 0.92. Microsatellites developed for Lamellibrachia sp. 2 are being deployed to study connectivity and gene flow among populations of this species.     

Isolation, characterization and cross-species amplification of new microsatellite markers for three opossum species of the Didelphidae family

The American opossums from the Didelphidae family (Order: Didelphimorphia) are on the IUCN Red List, and their threatened status varies from extinct to data deficient. Although microsatellites have already been developed for some species, only a small amount of population genetic analysis of the didelphid wild populations has been made using them. In this work, we developed new microsatellites and tested for cross-species amplification for one species from the subfamily Caluromyinae, Caluromys philander, and two from the subfamily Didelphinae, Didelphis aurita and Metachirus nudicaudatus, to determine the degree of gene flow and isolation of populations of those species in some fragments of the Atlantic Forest in Minas Gerais state, Brazil. Among all microsatellite loci amplified, 18 from C. philander, 9 from D. aurita, and 17 from M. nudicaudatus were polymorphic. Cross-species amplification of these polymorphic microsatellite loci were performed in these three species and in one additional related species Micoureus paraguayanus. Our data showed that microsatellites were not highly conserved among Didelphidae species.     

First report of the development of microsatellite markers for a tropical sea cucumber (Stichopus chloronotus)

We isolated new 10 polymorphic microsatellites from the tropical sea cucumber Stichopus chloronotus. These loci provide one class of variable genetic marker as the number of alleles ranged from 2 to 6 and the observed and expected heterozygosity ranged from 0.083 to 1.0 and from 0.081 to 0.724, respectively. We consider that these loci are potentially useful for revealing clones (resulting from asexual repro) and then participate in detailing the genetic structure and gene flow among S. chloronotus populations.     

Isolation and characterization of 15 microsatellite loci in the specialist butterfly Boloria eunomia

Boloria eunomia is a boreo-montane butterfly species suffering from habitat loss and isolation in the relictual part of its distribution range. Small populations persist in habitats scattered on plateaux or low mountains in western, central and southern Europe. Quantifying gene flow within and between these remnant populations is thus a crucial point to properly delineate metapopulations, to understand their dynamics and hence to design appropriate conservation plans for this butterfly species. We developed primers for the amplification of 15 microsatellites loci for B. eunomia. Thirteen loci were grouped in 2 multiplexes and amplified in 50 individuals from 5 populations to validate their use in population genetics. Gene diversity was on average 0.63 across populations. Null alleles and recurrent Hardy–Weinberg disequilibrium were detected in 7 loci. However, Fst estimates after correction for the presence of null alleles were highly correlated (0.91) to Fst estimates without correction. The loci developed here are thus usable for fine scale population genetic analyses and represent a very useful tool to quantify gene flow within and between metapopulations of B. eunomia.     

Ten polymorphic microsatellite loci for the Atlantic Silverside, Menidia menidia

Ten polymorphic microsatellite loci were isolated from the Atlantic Silverside (Menidia menidia) in order to test hypotheses regarding the role of adaptive phenotypic variation in structuring estuarine populations along coastal North America. Loci were amplified in three multiplex panels requiring a total of four individual PCRs. All loci were highly polymorphic in individuals screened from an estuary in Nova Scotia, and none exhibited significant departures from Hardy–Weinberg equilibrium. The results suggest that these loci will be sensitive to low levels of neutral divergence among populations across M. menidia populations.     

Isolation and characterization of polymorphic microsatellite loci from the golden mouse (Ochrotomys nuttalli)

Thirteen polymorphic microsatellite loci were developed for golden mouse using an enrichment protocol. The markers were tested on 37 golden mouse specimens collected from southern Illinois. Numbers of alleles ranged from 3 to 11 with observed heterozygosities ranging from 0.29 to 0.92. Three of the loci exhibit nominally significant excesses of homozygosity, but none were significant following a correction for multiple tests. None of the loci appear to be linked. Although golden mouse is not considered at risk in the core of its range, peripheral populations in several states are considered threatened or otherwise at risk due to low population densities. Because of the evolutionary distinctiveness of Ochrotomys, species-specific primers are needed to identify patterns of gene flow among core and peripheral populations.     

Characterization of 11 microsatellite loci for the brown smooth-hound shark, Mustelus henlei (Triakidae), discovered with next-generation sequencing

The brown smooth-hound shark, Mustelus henlei (Triakidae), is an endemic member of the eastern Pacific shark assemblage considered both commercially and recreationally important. Here, microsatellite loci for M. henlei discovered by next-generation sequencing (Roche 454 pyrosequencing) are described. All loci were polymorphic (3?C10 alleles) with observed heterozygosities between 0.24 and 0.89 and expected heterozygosities between 0.23 and 0.86. These loci are the first to be characterized explicitly for M. henlei and should be useful for the investigation of population structure and gene flow in this species and for other members of the Triakidae.     

Microsatellite primer development for the king ratsnakes Elaphe carinata (Colubridae)

The king ratsnakes (Elaphe carinata) is widespread in south-eastern China, North Vietnam, and Japan (Ryukyu Islands). The snake is listed as a vulnerable species according to the China Red Data Book of Endangered Animals. To develop effective conservation strategies for the snake, we need a better understanding of gene flow, population structure and evolutionary history of this species by using kinds of molecular markers such as mitochondrial DNA and microsatellite loci. Here, we characterize 12 polymorphic microsatellite loci isolated from E. carinata genomic libraries. One hundred and thirty-one individuals were collected from three geographically distant populations: Ji’an (Jiangxi, N26°59′30″, E114°58′58″), Quanzhou (Guangxi, N25°44′20″, E111°6′48″), Zhijiang (Hunan, N27°20′36″, E109°8′46″). These markers revealed a high degree of genetic diversity (6–20 alleles per locus) and heterozygosity (H _O ranged from 0.326 to 0.894, and H _E ranged from 0.542 to 0.918). No locus exhibited significant deviations from Hardy–Weinberg equilibrium. There was no evidence of linkage disequilibrium among pairs of loci. These microsatellite markers will be useful for the study of gene flow, population structure and evolutionary history of E. carinata.     

Isolation and characterization of 15 polymorphic microsatellite loci in Tetragastris panamensis (Burseraceae), a widespread Neotropical forest tree

Tetagastris panamensis is a tropical forest canopy tree that is broadly distributed across Central and South America. We isolated 15 microsatellite loci and characterized them in populations from Panama and Ecuador. The number of alleles per locus in 61 individuals ranged from 4 to 22, and expected heterozygosity ranged from 0.044 to 0.909. The high overall single-parent exclusion probability (P = 1.00) highlights the utility of these loci for parentage-based analyses of gene flow.     

Characterization of 10 polymorphic microsatellite loci in Munidopsis lauensis, a squat-lobster from the southwestern Pacific

Munidopsis lauensis is a deep-sea squat lobster commonly associated with hydrothermal vents at southwest Pacific back-arc spreading centers. Eight selectively neutral and unlinked polymorphic microsatellite loci were developed for this species and two additional loci from a related species were successfully cross-amplified. Eight of the ten total loci conformed to Hardy–Weinberg expectations. Average observed heterozygosity ranged from 0.23 to 0.64 (mean H _O  = 0.50, SD = 0.15). Seven of ten loci cross-amplified in Munidopsis antonii, a closely related species. Microsatellites developed for M. lauensis are being deployed to study connectivity among populations of this species that occur at hydrothermal vents in Manus and Lau Basins.     

Isolation and characterization of tetranucleotide microsatellite loci from the seaside sparrow (Ammodramus maritimus)

The seaside sparrow (Ammodramus maritimus; SESP) includes seven extant subspecies ranging from the northeastern coast of the United States to the Gulf of Mexico. Genetic diversity of SESP may be impacted since coastal marsh habitats are highly fragmented, thereby limiting gene flow among adjacent populations. Genetic analysis of SESP populations will provide insight for development of conservation and management strategies for the species. We describe development and characterization of 11 novel microsatellite markers for SESP. These loci will be useful for individual identification as well as studies of fine-scale genetic relatedness within populations and subspecies of SESP.     

Polymorphic microsatellite markers in the Neotropical forest tree Minquartia guianensis Aubl. (Olacaceae)

We present characteristics of eight microsatellite loci isolated from Minquartia guianensis Aubl (Olacaceae), an ecological and economically important forest tree species from Central and South America. Polymorphism was tested in two natural populations detecting 2 to 18 alleles per locus. The expected heterozygosities (H _E) range from 0.313 to 0.903. Linkage disequilibrium was found only at two locus combination in one population. High exclusion probability was estimated. These markers turn to be useful for population genetics and gene flow studies in this threatened timber species from the Neotropics.     

Microsatellite markers for Pseudoeurycea leprosa, a plethodontid salamander endemic to the Transmexican Neovolcanic Belt

We isolated and characterized 11 polymorphic microsatellite markers for the plethodontid salamander Pseudoeurycea leprosa to obtain population genetic data necessary for the proper management of this threatened species endemic to Central México. We tested polymorphism of these loci among 50 individuals from two populations (Texcalyacac and Calpan) in the states of México and Puebla, across the Transmexican Neovolcanic Belt. The number of alleles per locus ranged from three to 33 (mean; Na = 14.5). Observed and expected heterozygosities ranged from 0.20 to 0.88 and 0.22 to 0.93, respectively. We found deviations from Hardy–Weinberg equilibrium expectations for both populations at two loci (Plt028 and Plt066) and for Calpan population at locus Plt009. Evidence of significant linkage disequilibrium between pairs of loci was found only in one population. We found no evidence of large allele dropouts or stuttering, although null alleles could be present at loci Plt028 and Plt066, which showed significant excess of homozygotes. These markers will be useful for resolving fine-scale population genetic structure, gene flow, and population genetic diversity in Pseudoeurycea leprosa.     

Isolation and characterization of microsatellite loci in the Reevese’s butterfly lizard Leiolepis reevesii (Agamidae)

We characterize 25 polymorphic microsatellite loci isolated from Leiolepis reevesii genomic libraries. Thirty-four individuals were collected from two populations, eighteen from a mainland population in Xuwen, Guangdong, and the remaining 16 from an island population in Haikou, Hainan. These markers revealed a relatively high degree of genetic diversity (2?C18 alleles per locus) and heterozygosity (H _O ranged from 0.000?C0.938, and H _E ranged from 0.160?C0.954). Eight loci exhibited significant deviations from Hardy?CWeinberg equilibrium (HWE) after sequential Bonferroni correction due to heterozygote deficiencies, but only one locus (L311) with low heterozygosity in one population did significantly deviate from HWE in the other. Inbreeding may explain the deviations from HWE. There was no evidence of linkage disequilibrium among pairs of loci in the samples. These microsatellite markers will be useful for future studies focusing on gene flow, population structure and evolutionary history of L. reevesii.     

Isolation and characterisation of sixteen microsatellite markers cross-amplifying in a complex of three African agricultural pests (Ceratitis rosa, C. anonae and C. fasciventris, Diptera: Tephritidae)

Sixteen new polymorphic microsatellite loci were developed for the three species of the Ceratitis FAR complex (C. rosa, C. fasciventris and C. anonae) and their optimal amplification conditions characterised. These markers were tested on six field populations of the three fruit flies sampled in different African countries. After Bonferroni corrections, allele frequencies supported Hardy–Weinberg equilibrium for most of the loci, with only two loci not being at the equilibrium in all populations. No linkage disequilibrium was detected among all pairs of loci tested. These new markers will be a useful tool to study the intra- and interspecific genetic structure of these African agricultural pests and will help disentangling their well-known classification issues.     

The discovery of microsatellite markers for Hardenbergia violacea (Fabaceae), using next-generation sequencing

Hardenbergia violacea is a climbing to prostrate shrub endemic to woodlands in the south east of Australia. We used next generation sequencing to develop a species-specific primer set. We obtained 106,529 reads in FASTA format with an average read length of 548?bp. DNA was successfully amplified for 20 markers and eight markers were characterized. The degree of polymorphism of these loci was tested on 95 samples from five populations. The total number of alleles ranged from two to 11 and observed and expected heterozygosities ranged from 0.0 to 0.807 and 0.0 to 0.933, respectively. Inbreeding coefficients were highly variable among populations, suggesting a mixture of selfing and outcrossing in nature. The characterized loci will be useful for studies of population structure, gene flow, mating systems, and the conservation and selection of provenances for restoration projects in degraded habitats.     

Novel polymorphic nuclear microsatellite markers for Pinus sylvestris L.

Scots pine (Pinus sylvestris L.) is one of the most widespread forest trees in the world, ranging from southern Mediterranean mountains to eastern Siberia. Ten polymorphic microsatellite loci were isolated from Scots pine cDNA sequences and were screened for variability in three natural populations. High levels of genetic variability were observed with effective number of alleles per locus ranging from 1.0 to 4.6 and average expected heterozygosity per population of 0.79. With only two exceptions, Hardy?CWeinberg expectations were confirmed. All loci were in linkage equilibrium and there was little evidence for confounding null alleles. These new markers will be used to resolve population structure and gene flow patterns in this major Eurasian forest tree.     

Development and characterization of microsatellite markers in the coffee-family herb Ophiorrhiza japonica (Rubiaceae)

Ophiorrhiza japonica (Rubiaceae) in Taiwan shows altitudinal morphological differentiation, suggesting altitudinal genetic differentiation. However, global warming and/or road building are possibly causing human-mediated altitudinal gene flow and genetic disturbance. To investigate this possibility, we developed the first nuclear microsatellite markers for the genus. Using the low-cost compound microsatellite marker technique, 17 markers were developed and nine markers were polymorphic. The number of alleles per polymorphic locus ranged from 2 to 13. At population level, observed (H _o ) and expected (H _e ) heterozygosities ranged 0.070–0.831 and 0.068–0.812, respectively. The polymorphic information content ranged 0.066–0.787. No null allele, significant deviation from Hardy–Weinberg equilibrium, or significant linkage disequilibrium was observed. The 17 marker loci were successfully PCR-amplified in three congeneric species except for three cases. The developed microsatellite markers will be used to investigate genetic structure and gene flow among O. japonica populations at different altitudes.     

Mogurnda adspersa microsatellite markers: multiplexing and multi-tailed primer tagging

A set of twelve microsatellite DNA loci were developed for the threatened Australian freshwater fish Mogurnda adspersa (Eleotridae). Primers were tailed with one of four 20-mer oligonucleotides for use in four-colour fluorescent detection and optimised for multiplex PCR. The loci were used to genotype individuals from two populations in the Pioneer River catchment of central Queensland, eastern Australia. Number of alleles per locus ranged from 2 to 33 and per locus heterozygosity ranged from 0.06 to 0.81. Successful cross-species amplification of all loci was achieved in the congener M. mogurnda. These markers will be used to estimate effective population size and to examine the relationship between flow regime and population demographic parameters.