Isolation and characterization of novel tetranucleotide microsatellite DNA markers for the spotted salamander,Ambystoma maculatum

Fifteen tetranucleotide microsatellite loci were identified and characterized for spotted salamanders (Ambystoma maculatum) collected from three vernal pools in the south‐eastern USA. These markers revealed a high degree of genetic diversity (7–32 alleles per locus), heterozygosity (31.6–86.3%) and allelic heterogeneity (91% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.50 and 0.65, and all F_ST values (0.08–0.14) were statistically significant. Moreover, genotypic assignment tests correctly classified all individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and mating system.     

Novel tetranucleotide microsatellite DNA markers for the wood frog,Rana sylvatica

Fifteen tetranucleotide microsatellite loci were identified and characterized for wood frogs (Rana sylvatica) collected from three vernal pools in the southeastern US. These markers revealed a high degree of genetic diversity (nine to 34 alleles per locus), heterozygosity (30.6–92.3%) and allelic heterogeneity (69% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.40 and 0.55 and all F_ST values (0.02–0.05) were statistically significant. Genotypic assignment tests correctly classified 103 of 113 individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and metapopulation dynamics.     

Identification and characterization of nuclear microsatellite loci in the aquatic moss Platyhypnidium riparioides (Brachytheciaceae)

Eight microsatellite loci from the aquatic moss Platyhypnidium riparioides were identified using the method of microsatellite‐enriched libraries. Polymorphism was assessed in a sample of four populations of 20 individuals each from four streams of the Meuse hydrographic basin in southern Belgium. The markers amplified three to seven alleles per locus. Comparison of observed and expected heterozygosities as well as F‐statistics (F_ST = 0.62) reveals a significant genetic differentiation among populations. These markers will be useful for further investigation of population genetic structure and diversity at different nested spatial scales.     

Microsatellite DNA markers for the study of horseshoe crab (Limulus polyphemus) population structure

Twenty‐two microsatellite DNA loci were identified and characterized for horseshoe crabs (Limulus polyphemus) collected from two Atlantic coast and one Gulf of Mexico site. These markers revealed a high degree of genetic diversity (8–35 alleles per locus), heterozygosity (25.0% to 100.0%), and allelic heterogeneity (69.8% of comparisons). Considerable regional differentiation was observed as genetic distances (chord) ranged between 0.25 and 0.45, and all F_ST values (0.014–0.092) were significant. These preliminary findings are consistent with patterns of regional differentiation observed using allozyme variation and contradictory to findings of limited gene flow reported for sequence variation at the mitochondrial DNA COI region.     

Improved genetic markers for monitoring recruitment dynamics in the endangered Mary River cod (Maccullochella mariensis)

The Mary River cod (Maccullochella mariensis) is a large predatory freshwater fish identified as a potential flagship for freshwater ecosystem conservation in Australia. The species is endemic to the Mary River catchment in eastern Australia, and is listed as Endangered. Previous conservation genetic assessment of Mary River cod was based on a small set of microsatellite loci developed for congeneric Maccullochella species. Here we develop a novel set of 15 microsatellite loci specific to M. mariensis, and demonstrate that these markers exhibit higher variability than those used previously. Mean number of alleles per locus was 4 and mean expected heterozygosity was 0.57. We genotyped 35 Mary River cod larvae belonging to a single cohort using the 15 novel loci and eight previously used loci, and found 10 full‐sib family groups along with clear genetic differentiation between individuals collected from the two sub‐catchments – Tinana Creek and Mary River. Microsatellites presented here will be useful for cost‐effective monitoring of genetic diversity and recruitment dynamics in this endangered fish species.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

Assessment of genetic variation between reproductive ecotypes of Klamath River steelhead reveals differentiation associated with different run-timings

Seven microsatellite DNA loci were optimized to assess genetic differentiation in coastal steelhead (Oncorhynchus mykiss irideus) sampling groups from the lower Klamath River (California, USA). Genetic relationships among three winter‐run and two summer‐run groups were investigated. The different groups displayed high levels of allelic variation. Pairwise F_ST comparisons and Nei's genetic distance supported low, yet significant, genetic differentiation between summer and winter run‐timings similar to other studies of temporal variation in salmonids. Analysis of molecular variance showed that most of the genetic variation was at the individual level (97.9%), although significant genetic variation existed between timing of runs (2.59%). Additionally, at least one locus in each group was out of Hardy–Weinberg equilibrium due to a deficiency in heterozygotes, and significant F_IS values were observed in three temporal collections. Together, these results suggest stock admixture, caused by multiple populations of origin in each sampling group, better known as the Wahlund Effect. These observations are preliminary evidence for isolation by time between Klamath River steelhead runs during distinct periods.     

Isolation and characterization of a set of microsatellite loci in the submerged macrophyte,Vallisneria spinulosa Yan (Hydrocharitaceae)

Twenty‐six microsatellites were isolated and characterized from an AAG‐enriched genomic library of Vallisneria spinulosa, a dominant submerged macrophyte in the middle‐lower reaches of the Yangtze River. Microsatellite polymorphism was evaluated in a random population and 11 microsatellite loci were found polymorphic with 2–18 alleles per locus and observed heterozygosity ranging from 0.281 to 1.000. These polymorphic markers were expected to be valuable for population genetic and demographic analyses of V. spinulosa.     

Development of microsatellite markers for southern catfish (Silurus meriaionalis) and cross‐species amplification in northern sheatfish (Silurus soldatovi)

Forty microsatellite markers were developed from a (CA)_n enrichment library created from the DNA of southern catfish (Silurus meriaionalis). Also, the population structure of northern sheatfish (Silurus soldatovi) was examined by 24 microsatellite loci. They are polymorphic in at least one of the two geographically distant populations sampled from the Heilongjiang River and the Songhuajiang River in North China, respectively. Unbiased expected heterozygosity levels varied from 0.435 to 0.946 and number of alleles per locus varied from three to 20. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers.     

Microsatellite polymorphism and population subdivision in natural populations of European sea bass Dicentrarchus labrax (Linnaeus, 1758)

Polymorphism of microsatellite markers was used to study the genetic variability and structure in natural populations of European sea bass Dicentrarchus labrax. The data consisted of six microsatellite loci analysed for 172 individuals from three samples collected in the Golfe-du-Lion (France) and one sample collected in the Golfo-de-Valencia (Spain). Our goals were (i) to assess the level of genetic variability as revealed by these markers, (ii) to estimate the genetic differentiation among natural populations within a restricted area, and (iii) to evaluate how microsatellite loci fit the predictions of the two most widely used mutation models (the infinite allele model and the stepwise mutation model). As expected, our results indicate that the genetic polymorphism is very high when compared with previously used genetic markers, the mean expected heterozygosity per locus ranging between 0.69 and 0.93. We also found that all loci but one fitted the infinite allele model better. Using this model as a lower limit, we could extrapolate from the observed diversity effective population sizes on the order of 35 000 individuals. Our results also suggest that there may be a slight genetic differentiation between the two gulfs (F_ST= 0.007, P < 0.05), indicating that the corresponding populations are likely to be dynamically independent. This finding for a species with high dispersal abilities, if confirmed, has important beatings on fish-stock assessment.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

Microsatellite DNA primers for the endangered vermilion darter,Etheostoma chermocki,and cross‐species amplification in other darters (Percidae: Etheostoma)

The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Isolation of microsatellite loci from the common buzzard,Buteo buteo (Aves: Accipitridae)

We isolated 56 common buzzard (Buteo buteo) microsatellite loci from (AC)_n‐ and (AAAG)_n‐enriched genomic libraries. Eleven loci were tested on 90 individuals from Eastern Westphalia in Germany, yielding two to 17 alleles per locus (average 5.7) and expected heterozygosities ranging from 0.11 to 0.93 (average 0.53). These highly variable microsatellite markers provide a powerful means for investigating population genetic diversity in the common buzzard, a little‐understood aspect of this widespread species.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

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.     

Cost‐effective development of highly polymorphic microsatellite in Japanese quail facilitated by next‐generation sequencing

Next‐generation sequencing technologies permit rapid and cost‐effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild‐derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3–4.1 and 0.11–0.53 respectively. Compared with the wild‐derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (F_ST) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.     

Polymorphic microsatellite loci in the sponge Crambe crambe (Porifera: Poecilosclerida) and their variation in two distant populations

Seven polymorphic microsatellite loci were characterized in the marine encrusting sponge Crambe crambe from a partial genomic DNA‐enriched library. Preliminary data on allelic variation of these loci in two distant populations of C. crambe are presented to assess their potential utility as high‐resolution genetic markers for this species. The number of alleles per locus ranged between three and 16 and the distributions of allele frequencies differed considerably between the two populations, indicating a marked genetic differentiation between them. These are the first microsatellite loci reported from any species in the phylum.     

Novel Jefferson salamander,Ambystoma jeffersonianum,microsatellite DNA markers detect population structure and hybrid complexes

Twenty polymorphic microsatellite DNA markers were isolated and characterized in Ambystoma jeffersonianum collected from three vernal pools in the mid‐Atlantic region of the U.S. These markers revealed a high degree of genetic diversity (7–23 alleles per locus), heterozygosity (46.7% to 100%), and allelic heterogeneity (96% of comparisons were statistically significant). Genetic distances were greatest in comparisons between collections, intermediate within collections, and least among sibling pairs. Six markers were trisomic in A. jeffersonianum‐A. laterale hybrids. These microsatellite DNA loci should allow delineation of genetic structure within and among populations of the diploid A. jeffersonianum and provide an effective method for identification of triploid hybrid individuals.     

Development of eight polymorphic microsatellites for an Australasian rainforest tree species,Cryptocarya mackinnoniana (Lauraceae)

We developed eight microsatellite markers for Cryptocarya mackinnoniana to study the spatial genetic structure and ecological correlates of parentage in secondary rainforests in Australia's Wet Tropics. The microsatellite loci were screened in 99 trees, and 623 seedlings < 0.24 m tall, at a site extending from primary rainforest into adjacent 50‐year‐old secondary rainforest. The eight loci were polymorphic, exhibiting between three and 11 alleles, and gene diversity (H_E) from 0.25 to 0.84. For the trees, no departures from Hardy–Weinberg equilibrium were detected, except at locus Cm03, which had an estimated null allele frequency of 0.0903. No locus combinations exhibited linkage disequilibrium.