Characterization of 22 polymorphic microsatellite loci for the black-chested spiny-tailed iguanas (Ctenosaura melanosterna) and cross-amplification success in four other Ctenosaura species

The Ctenosaura palearis complex (C. bakeri, C. melanosterna, C. palearis and C. oedirhina) comprises four endangered species. We isolated 22 polymorphic microsatellites from C. melanosterna and evaluated them for amplification success and polymorphism in the source population, two non-source populations, the three remaining C. palearis complex taxa, and C. similis. We observed ??90% amplification success in the source population and discovered 2?C9 alleles per locus with genetic diversity estimates (observed heterozygosity) ranging from 0.18 to 0.81. During cross amplification, 91% of the screened markers amplified for all groups, the proportion of polymorphic loci ranged from 0.55 to 0.95, and 35% of the markers were polymorphic for all groups. The markers presented here provide useful tools for population-level studies of the taxa in the C. palearis complex.     

Isolation and characterization of twenty-eight polymorphic microsatellite markers in Odontobutis potamophila and cross-amplification in other Gobioidei

Odontobutis potamophila is widely distributed in river systems of southeast China. Twenty-eight microsatellite loci were isolated and characterized for this species. Number of alleles ranged from 2 to 19 per locus, and the observed and expected heterozygosity were 0.161–0.967 and 0.207–0.928, respectively. No evidence of linkage disequilibrium was detected. The significant departures from Hardy–Weinberg expectations at three loci suggested the presence of null alleles. These microsatellite loci will be useful for studying population genetic variation. Cross-amplification of these microsatellite loci indicated that many are polymorphic in four closely related species of Gobioidei.     

Development of 39 novel polymorphic microsatellite markers for the giant mottled eel Anguilla marmorata and cross-amplification in other eel species

In this study, we have developed microsatellite markers for the giant mottled eel (Anguilla marmorata) from expressed sequence tag (EST) sequences using next-generation sequencing. Consequently, a total of 39 novel polymorphic microsatellite markers were identified in 32 individuals from Wangquan River wild population area. The range of observed heterozygosity, expected heterozygosity and polymorphism information content were 0.044–1.000, 0.285–0.946 and 0.3783–0.9016, respectively. In addition, eight loci were deviated from Hardy–Weinberg equilibrium and modified by Bonferroni correction, and the results showed no significant linkage disequilibrium between the locus pair. The amplification of cross-species was conducted in Anguilla bicolor pacifica and Anguilla japonica, which revealed the applicability of EST-SSR primers. These EST-SSR markers can provide sufficient polymorphism for population genetic studies, pedigree and genome mapping of these three kinds of closely related eels.     

Characterization of 15 polymorphic microsatellite loci in Rimicaris exoculata, and cross-amplification in other hydrothermal-vent shrimp

Rimicaris exoculata is an alvinocarid shrimp endemic to the hydrothermal vents of the Mid-Atlantic Ridge. In order to study genetic variability and connectivity in this species, we developed fifteen polymorphic microsatellite markers. The markers were tested on one population and, except for one, all showed no departure from Hardy?CWeinberg equilibrium, with an average overall observed heterozygosity of 0.63. Two primer pairs revealed possible linkage disequilibrium, and 14 cross-amplified at least one of the three co-occurring shrimp species tested (Alvinocaris muricola, Alvinocaris markensis and Mirocaris fortunata). These markers therefore open perspectives for population genetic studies of hydrothermal vent shrimp species in order to unravel connectivity and evolution of populations, and to add information on possible future impact studies.     

Chloroplast microsatellite markers for the Neotropical orchid genus Epidendrum, and cross-amplification in other Laeliinae species (Orchidaceae)

One of the most significant challenges confronting orchid researchers is the lack of specific molecular markers, mainly for species in the Neotropics. Here we report the first set of specific chloroplast microsatellite primers (cpSSR) developed for Neotropical orchids. In total, nine polymorphic cpSSR loci were isolated and characterized in four species occurring in the Brazilian Atlantic Rainforest: Epidendrum cinnabarinum, E. denticulatum, E. fulgens and E. puniceoluteum. Levels of intraspecific polymorphism were characterized using two populations for each species, with 13–20 individuals each. Allele numbers varied from two to three per locus, while the number of haplotypes ranged from three to six per species. Extensive differentiation among the taxa was detected. All markers were successfully cross-amplified in eight other different genera. These cpSSRs markers will enable novel insights into the evolution of this important Neotropical genus.     

Isolation and characterization of polymorphic microsatellite loci for the gopher tortoise (Gopherus polyphemus) and cross-amplification in other species of Gopherus

All members of the tortoise genus Gopherus have some degree of conservation concern. We report on the development of thirty-two new microsatellite loci for gopher tortoise, which were tested on 31 individuals from Ft. Benning, Georgia, USA. These loci possessed an average number of 5.09 alleles, an average observed heterozygosity of 0.574 and an average expected heterozygosity of 0.589. We also tested these loci in three congeners: Gopherus morafkai, Gopherus flavomarginatus and Gopherus berlandieri. The new microsatellite loci should prove useful in conservation efforts of Gopherus polyphemus and other Gopherus species.     

Isolation and characterization of microsatellite markers in Rhaphiodon vulpinus (Cynodontidae, Characiformes) and their cross-amplification in other Cynodontinae species

Eleven microsatellite markers were developed for Rhaphiodon vulpinus, a widely distributed fish in the Amazon region. Recently, the Amazon fishes are being threatened by the construction of several dams, therefore genetic investigations are essential to further evaluate the impact of these dams on native species. Two R. vulpinus populations were analyzed and 3–20 alleles were found, with observed and expected heterozygosities ranging between 0.24 and 0.90 and between 0.22 and 0.90, respectively. After Bonferroni correction, no significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium were detected. These microsatellite loci were cross-amplified in others six Cynodontinae species and at least five loci demonstrated to be useful.     

Characterization of microsatellite loci in Sinilabeo rendahli and cross-amplification in four other Chinese cyprinid species

Twenty-eight microsatellite loci were isolated in Sinilabeo rendahli, an endemic cyprinid fish in China. In a test on a population of 48 individuals, 15 primer pairs exhibited polymorphism with 2–12 alleles per locus, while the rest 13 were monomorphic. Observed and expected heterozygosities ranged from 0.104 to 0.915 and 0.195 to 0.865, respectively. Four loci showed significant deviation from Hardy–Weinberg equilibrium and two of them had evidence of null alleles. Twenty-six markers successfully cross-amplified in at least one of the four additional Chinese cyprinid species (Garra pingi pingi, Pseudogyrincheilus procheilus, Discogobio yunnanensis and Onychostoma sima) examined in this study.     

Characterization of novel microsatellite loci for Hetaerina americana damselflies, and cross-amplification in other species

Hetaerina damselflies are distributed throughout the neotropics. We developed eleven microsatellite loci for the damselfly Hetaerina americana. Microsatellites were tested for polymorphism on a panel of 24 individuals. The number of alleles ranged from 2 to 6, observed heterozygosity from 0.080 to 0.701, and the fixation index from ?0.266 to 1.000. Cross-amplification was tested in 7 different species in the genus Hetaerina from the United States and Mexico. These microsatellite loci will be useful for studies of population structure and gene flow in H. americana.     

Characterization of 36 additional microsatellite loci in splittail (Pogonichthys macrolepidotus) and cross-amplification in five other native Californian cyprinid species

We characterized 36 new microsatellite markers for splittail (Pogonichthys macrolepidotus), an estuarine fish species endemic to the San Francisco Estuary. Genetic variation was assessed using 25 individuals from the Sacramento River and 16 individuals from the Petaluma River. Number of alleles per locus varies considerably, ranging from 3 to 19. One locus deviated significantly from Hardy?CWeinberg equilibrium and a single pair of loci was found to be in linkage disequilibrium. Twenty-four markers successfully cross-amplified and were polymorphic in at least one of the five additional California cyprinid species (Ptychocheilus grandis, Siphateles bicolor, Lavinia exilicauda, Orthodon microlepidotus & Mylopharodon conocephalus) examined in this study.     

Development and cross-amplification of 16 polymorphic microsatellite markers in the Chestnut Bulbul Hemixos castanonotus

A set of primers to amplify 16 microsatellite DNA loci was developed for the Chestnut Bulbul Hemixos castanonotus and the cross-amplification of these loci was tested in a closely related species, the ashy bulbul Hemixos flavala. A total of 30 Chestnut Bulbul individuals were genotyped. An average of 6.5 alleles per locus (2–12 alleles) was detected. The expected heterozygosity values ranged from 0.218 to 0.888. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. No significant linkage disequilibrium was detected. Fifteen of 16 loci were successfully amplified in the ashy bulbul, with rich polymorphism in 15 individuals tested. These markers will be useful in population genetics, conservation and phylogenies of the chestnut bulbul.     

Hybridization and polymorphic microsatellite markers for two lagomorph species (Genus Sylvilagus): implications for conservation

Eastern cottontails (Sylvilagus floridanus) are a prolific and popular game species in North America but have declined within the core of their range. Swamp rabbits (S. aquaticus) are sympatric with eastern cottontails and are a species of concern at the northern fringe of their range in Illinois. Although hybridization between these species may be a potential factor affecting their declines, no studies have yet been conducted to address this issue. We examined the potential for hybridization between sympatric eastern cottontails and swamp rabbits in Illinois by sequencing a portion of the mitochondrial 12S gene in 17 individuals of each species. Additionally, we developed six microsatellite markers for the eastern cottontail (7–13 alleles/locus) that also amplify in swamp rabbits (3–5 alleles/locus). We found fixed differences between species with haplotypes forming well-differentiated clades indicating a lack of hybridization. Hybridization between eastern cottontails and swamp rabbits does not appear to be affecting their declines. Misidentification due to ambiguous morphological characteristics of the two species occurred in an earlier study and in our own. We suggest the mitochondrial 12S gene may be a convenient molecular marker for the resolution of species identity questions. Information on microsatellites, occurrence of natural hybridization, and a molecular marker for species resolution will be useful for developing management plans for lagomorphs and focusing further research on factors affecting population status.     

Characterization of eight polymorphic microsatellite loci for the California spiny lobster, Panulirus interruptus and cross-amplification in other achelate lobsters

Microsatellite sequences were isolated from both non-enriched and enriched genomic libraries of California spiny lobster, Panulirus interruptus. Eight consistently amplifying, scorable and polymorphic loci were characterized for 79 individuals collected at Santa Cruz and San Clemente Islands, California, and tested for cross-species amplification in four closely related Panulirus spp., as well as four other species of the order Achelata. The number of alleles observed per locus ranged from three to 54 and observed heterozygosities ranged from 0.57 to 0.98. Quality control testing shows that all loci were reliably scorable, independently segregating, inherited in Mendelian ratios, and had low to moderate (≤14.4%) frequencies of null alleles and high statistical power for detecting fine scale genetic structure.     

Development of microsatellite markers for blue duiker (Cephalophus monticola) using next-generation sequencing and cross-amplification in other duikers

We developed nine microsatellite markers for duikers by next-generation sequencing using a muscle tissue sample of blue duiker (Cephalophus monticola). For blue duikers, the number of alleles ranged from 4 to 14 (mean 9.4), and the observed and expected heterozygosities were 0.333–0.833 (mean 0.716) and 0.625–0.878 (mean 0.817), respectively. Cumulative probability of identity was low (1.5 × 10^?12), indicating that these markers are enough for individual discrimination. All nine markers were successfully amplified in three other duikers (C. dorsalis, C. ogilbyi and C. sylvicultor), and almost all loci were polymorphic. These markers will be useful for clarifying genetic structure of duikers.     

Characterisation and cross-amplification of polymorphic microsatellite loci in ant-associated root-aphids

Twenty-six polymorphic microsatellite loci were developed for four species of ant-associated root-aphids: Geoica utricularia, Forda marginata, Tetraneura ulmi and Anoecia corni. We found up to 9 alleles per locus, with an average of 4.8. We also report polymorphic cross-amplification of eleven of these markers between different pairs of study species. Furthermore, we tested previously published aphid microsatellites and found one locus developed for Pemphigus bursarius to be polymorphic in G. utricularia. These microsatellite markers will be useful to study the population structure of aphids associated with the ant Lasius flavus and possibly other ants. Such studies are relevant because: 1. L. flavus mounds and their associated flora and fauna are often key components in protected temperate grasslands, and 2. L. flavus and its diverse community of root-aphids provide an interesting model system for studying the long-term stability of mutualistic interactions.     

Characterization of 62 polymorphic EST-SSR markers in the blood clam (Tegillarca granosa) and their cross-amplification in Scapharca subcrenata

Next-generation sequencing technologies provide an excellent opportunity to mining large numbers of EST-SSR markers quickly and cost-effectively for non-model organisms. Here we isolated and characterized 62 polymorphic EST-SSR markers in the blood clam, Tegillarca granosa, from the ESTs generated by 454 sequencing. The number of alleles for the 62 SSR markers in 25 individuals varied from 2 to 9, with an average of 3.90 alleles per locus. The observed heterozygosity ranged from 0.040 to 1.000, while the expected heterozygosity varied from 0.040 to 0.844. Polymorphic information content ranged from 0.038 to 0.806. Interspecific transferability of the 62 markers revealed that 16 were polymorphic in Scapharca subcrenata, resulting in a transferability rate of 25.81?%. To our knowledge, this is the first report on the survey of SSR transferability in blood clams. These novel polymorphic EST-SSR markers should be particularly useful for further investigation of population and conservation genetics.     

Isolation and characterization of 11 microsatellite loci from cattle egret (Bubulcus ibis) and cross-amplification in other Ardeidae species

The cattle egret (Bubulcus ibis) is an invasive species across American continent. Understanding the population genetics of this species is necessary for population monitoring and the development of effective management strategies. Here we present 11 microsatellite loci isolated from B. ibis sampled in the southeastern Brazil. Of the 32 primer pairs that were tested on samples from 35 individuals, 11 (34?%) were polymorphic for population-level analyses. The number of alleles per locus ranged from 2 to 4. The expected heterozygosity ranged from 0.083 to 0.561. All loci conformed to Hardy?CWeinberg and linkage equilibrium. Cross-amplification was successful for 7 of the 11 B. ibis microsatellite loci that were tested in samples from eight Ardeidae species. These markers are the first published microsatellites for B. ibis and they will be useful to understand the population structure and colonization of the American continent by this species.     

Characterization of polymorphic microsatellite markers isolated from Dendrobenthamia japonica var. chinensis (Cornaceae)

Dendrobenthamia japonica var. chinensis is an evergreen, broad-leaved, woody species of the Cornaceae family. Here we isolated 14 codominant compound microsatellite markers from D. japonica var. chinensis using an improved technique. Overall, the number of alleles ranged from 2 to 13, with an average of 7.21 alleles per locus. These markers would be the useful tools for analysing questions concerning population genetic structure and genetic diversity of D. japonica var. chinensis.     

Characterization of 10 novel microsatellite loci and cross-amplification of two loci in the snapping turtle (Chelydra serpentina)

We use 454 (??shotgun??) sequencing to obtain a partial genomic library for the snapping turtle (Chelydra serpentina). We characterize ten microsatellite loci from these sequences and test cross-amplification of loci originally developed for the alligator snapping turtle (Macrochelys temminckii). We genotype 127 individuals from Ontario at twelve loci. The number of alleles per locus ranges from 1 to 14; heterozygosity ranges from 0.157 to 0.850. These loci will be used to study population genetic structure in this long-lived reptile and may cross-amplify in two closely related species.