Identification and characterization of 17 microsatellite primers for the tick,Ixodes ricinus,using enriched genomic libraries

Ixodes ricinus is the main vector for important infectious diseases in both humans and in animals. Microsatellite loci were isolated from a dinucleotide‐enriched library made from I. ricinus sampled in Norway. Seventeen polymorphic microsatellites were further characterized among 24 individuals sampled from an island in the Oslofjord region. The number of observed alleles ranged from two to 17 and the observed heterozygosities between 0.10 and 0.83. Analysis of family materials gives evidence of non‐Mendelian inheritance of several of the characterized loci, among which most could be explained by presence of null alleles.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Isolation and characterization of highly polymorphic microsatellite loci for the garden tiger moth Arctia caja (Lepidoptera: Arctiidae)

A set of microsatellite markers was isolated from Arctia caja, a species that has been declining in UK for several decades. As has been found with other Lepidoptera species, this proved to be a difficult task. Eight enriched libraries identified 103 positive clones, and these yielded only seven polymorphic microsatellites. Allelic diversity ranged from 10 to 23 alleles per locus in a population of 30 individuals. Significant heterozygosity deficits were shown by three of these loci, presumably due to null alleles. The remaining four loci were in Hardy–Weinberg equilibrium and will be used to investigate the population genetic structure of A. caja.     

Isolation and characterization of polymorphic microsatellite loci in Hemibarbus labeo (Cyprinidae) using PCR‐based isolation of microsatellite arrays (PIMA)

A total of 10 polymorphic microsatellite loci from Hemibarbus labeo were isolated and characterized using an optimized protocol to construct a microsatellite‐enriched genomic library. The analysis of variability was performed in 24 specimens of mainland China. The mean number of alleles across loci was 3.10 ± 1.10 and the level of expected heterozygosity varied from 0.0417 to 0.7482. Frequencies of null alleles of the 10 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population. Five primer pairs cross‐amplify the microsatellites in other species, indicating transportability of the markers within the family Cyprinidae.     

Isolation and characterization of microsatellite loci in Pseudoplatystoma corruscans (Siluriformes: Pimelodidae) and cross‐species amplification

A total of five polymorphic microsatellites loci from Pseudoplatystoma corruscans were isolated and characterized. A population survey involving 43 specimens resolved a large number of alleles (range seven to eight among loci) and high observed heterozygosity (0.500–0.615), indicating its usefulness in population genetics studies. Cross‐species amplification was successful in four other Pimelodidae species.     

Twenty-one new microsatellite markers for the ecologically important midge Heterotrissocladius marcidus,and their use in studies of alpine lakes

Heterotrissocladius marcidus (Walker, 1856) (Diptera: Chironomidae) inhabits aquatic habitats across the Holarctic and Oriental regions, including remote alpine glacial lakes. It is thus a promising species for studies of population structure and genetic diversity in these highly vulnerable habitats. Here, 21 polymorphic microsatellites for H. marcidus are described, obtained using 454 GS-FLX pyrosequencing. The number of alleles per locus varied from 2 to 9 and no significant linkage disequilibrium was observed. To test applicability, 40 individuals were used from two lakes in the Tatra Mts (Western Carpathians). Observed and expected heterozygosity varied from 0 to 0.9 and 0.102 to 0.888, respectively. In both populations, three loci deviated significantly from Hardy–Weinberg equilibrium after Bonferroni correction, probably due to presence of null alleles or undetected biological processes. Application of microsatellites was tested on six alpine lake populations. Bayesian cluster analysis assigned individuals to lakes of their origin and revealed limited gene-flow between them. Five loci were successfully cross-amplified in the related midge Macropelopia sp. (Tanypodinae) and two in Pseudodiamesa branickii (Nowicki, 1873) (Diamesinae). The microsatellites described herein proved to be useful for genetic studies of alpine populations, and can provide important data for management and conservation of these threatened habitats.     

Characterization of 17 polymorphic microsatellite loci in the Anise swallowtail,Papilio zelicaon (Lepidoptera: Papilionidae), and their amplification in related species

Fifteen polymorphic dinucleotide and two trinucleotide microsatellite loci were identified in the Anise swallowtail, Papilio zelicaon, from DNA genomic libraries enriched for simple sequence repeats. Allele numbers varied from eight to 29, with an excess of homozygotes observed for nine loci. This homozygosity is a feature of other lepidopteran microsatellites and is probably due to null alleles. Sixteen markers were amplified successfully in other representatives of Papilio with 11 loci retaining polymorphism in at least one species. These results suggest that the microsatellites reported here may be appropriate for measuring population genetic structure in a number of Papilio species.     

Habitat and occurrence of ixodid ticks in the Liguria region,northwest Italy

Questing ticks were collected during monthly dragging sessions (March–August 2011) in three provinces of the Liguria region, north-western Italy, to evaluate the species occurrence, spatial distribution and relative abundance. A total of 1,464 specimens were collected in 94 dragging sites. Ixodes ricinus was the most abundant species (81.3 % of collected ticks), followed by Haemaphysalis punctata (10.9 %), Dermacentor marginatus (5.5 %), Ixodes frontalis (1.3 %), and Rhipicephalus spp. (0.9 %). Ixodes frontalis is reported for the first time in Liguria. An aggregation of I. ricinus positive sites was observed in inland areas characterized by dense forests dominated by deciduous trees (Castanetum and Fagetum phytoclimatic zones), especially in the west of the region where the differences in the Normalized Difference Vegetation Index (NDVI) were higher between inland and coastal sites. Negative binomial regression for repeated measures was used to model the associations of NDVI and season with counts of host-seeking I. ricinus nymphs. The NDVI was a good predictor of I. ricinus nymphs abundance, and confirmed its utility in discriminating habitat suitability for this vector in north-western coastal Italy, where dry habitat conditions may limit the distribution of this species.     

Cross-species amplification of bovid microsatellites in central African duikers (genus Cephalophus) and other sympatric artiodactyls

The present study set out to evaluate cross-species amplification of 34 bovid microsatellites in six central African duikers: Cephalophus callipygus, C. monticola, C. silvicultor, C. nigrifrons, C. dorsalis and C. leucogaster. Of these loci, 16 amplified across all species and appeared polymorphic when initially tested in polyacrylamide gel electrophoresis. Twelve of these loci were subsequently assembled into three multiplex panels of four loci each. These multiplexes successfully amplified across all six duiker species in the present study and the sympatric artiodactyls Tragelaphus spekei and Hyemoschus aquaticus. The only exception was the locus BM848 that did not amplify from C. leucogaster. For species with sufficient sample sizes (C. callipygus and C. monticola), the number of alleles ranged from three to ten and four to fifteen, respectively. Three loci deviated from Hardy–Weinberg equilibrium in C. callipygus and five in C. monticola. We attribute the latter result to possibilities of local population substructuring or to an excess of homozygotes because of null alleles. These multiplex assemblies will greatly facilitate studies of individual identification, parentage analysis, population size estimation and fine-scale analyses of population genetic structure in central African artiodactyls.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Identification and characterization of ten polymorphic microsatellite loci in the red panda Ailurus fulgens

Ten polymorphic microsatellite loci were characterized from two genomic DNA-enriched libraries of the red panda (Ailurus fulgens). The number of observed alleles among 35 samples of red pandas ranged from five to 12. Observed and expected heterozygosities were 0.286–0.971 and 0.443–0.894, and the mean polymorphic information content was 0.712. All loci followed Hardy–Weinberg expectations except Aifu-14 and Aifu-16, which may due to the presence of inbreeding or null alleles. Three pairs of loci exhibited significant linkage disequilibrium after Bonferroni correction for multiple comparisons. These microsatellites would be useful to strengthen population management, genetic diversity exploration, and demographic history speculation of this species.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Characterisation of polymorphic microsatellite markers for skates (Elasmobranchii: Rajidae) from expressed sequence tags

Simple sequence repeat markers (SSRs, microsatellites) were characterised for skates (Elasmobranchii: Rajidae) from published expressed sequence tags (ESTs) of Leucoraja erinacea. These were tested in four European species (Raja clavata, Raja montagui, Dipturus batis, and Leucoraja naevus). Thirteen of the fourteen amplified loci were polymorphic in at least one species. Polymorphic loci possessed on average 4.5–5.9 alleles per species, and expected heterozygosity ranged from 0.05 to 0.88. Possible null alleles were detected at three loci, while one locus showed significant deviation from Hardy–Weinberg Equilibrium proportions. Three locus-pairs exhibited significant linkage disequilibrium in one or more species. This marker set will be valuable for population genetic analyses of the focal taxa, and may prove useful for studies of other skate species.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Isolation and characterization of polymorphic microsatellites in Parnassius apollo and Euphydryas aurinia (Lepidoptera)

Microsatellite loci were developed from Parnassius apollo and Euphydryas aurinia, two endangered Palaearctic butterfly species. Respectively, six and five polymorphic loci were characterized from an enriched partial genomic library. Genetic diversity range from three to 25 alleles for the first species and from seven to 21 for the second. Although the presence of null alleles is suspected, these polymorphic loci are likely to provide important information on the fine scale genetic structure among populations of these species.     

Isolation and characterization of 10 highly polymorphic di- and trinucleotide microsatellite markers in the mayfly Ameletus inopinatus (Ephemeroptera: Siphlonuridae)

We describe the isolation of ten polymorphic microsatellite loci from the mayfly Ameletus inopinatus. Loci had di‐ or trinucleotide repeat motifs and were highly variable with three to 17 alleles (mean = 7.15). Observed heterozygosity ranged from 0.143 to 0.905. One locus (Ami_202) showed significant deviation from Hardy–Weinberg equilibrium in one population, but no evidence for null alleles. One locus (Ami_73) was significantly linked with three other loci. The remaining nine loci should prove highly informative for population genetic studies.     

Development and characterization of the first microsatellite markers in the mangrove species Pelliciera rhizophorae Triana & Planchon

Pelliciera rhizophorae is a unique Neotropical mangrove species belonging to Pelliciera genus. We isolated eight microsatellite loci from this species. All loci were polymorphic and showed three to nine alleles per locus in Colombian Pacific and Caribbean populations. Polymorphic information content ranged from 0.46 to 0.69. Two loci (PeRh‐14 and PeRh‐19) showed null alleles on the Caribbean coast, which suggest genetic differentiation between Pacific and Caribbean populations of P. rhizophorae. Development of these microsatellite loci constitutes a new molecular tool to carry out studies in the genome of the species and to evaluate its population dynamics.     

Characterization of microsatellite loci isolated in midget faded rattlesnake (Crotalus viridis concolor)

Primers for five polymorphic microsatellite loci were developed for the midget faded rattlesnake (Crotalus viridis concolor), a rare subspecies of western rattlesnake (Crotalus viridus) found only in parts of Wyoming, Colorado, and Utah. Five polymorphic microsatellites were isolated, four of which had relatively high levels of diversity (eight to nine alleles). We found only two departures from Hardy–Weinberg equilibrium and they occurred in different loci, so null alleles are likely not a problem. Moreover, we found that no two loci were linked. These loci will be applicable for population genetic analysis and perhaps analysis of paternity and mating systems.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.