Novel Y‐chromosome short tandem repeats in Sus scrofa and their variation in European wild boar and domestic pig populations

Y‐chromosome markers are important tools for studying male‐specific gene flow within and between populations, hybridization patterns and kinship. However, their use in non‐human mammals is often hampered by the lack of Y‐specific polymorphic markers. We identified new male‐specific short tandem repeats (STRs) in Sus scrofa using the available genome sequence. We selected four polymorphic loci (5–10 alleles per locus), falling in one duplicated and two single‐copy regions. A total of 32 haplotypes were found by screening 211 individuals from eight wild boar populations across Europe and five domestic pig populations. European wild boar were characterized by significantly higher levels of haplotype diversity compared to European domestic pigs (H_D = 0.904 ± 0.011 and H_D = 0.491 ± 0.077 respectively). Relationships among STR haplotypes were investigated by combining them with single nucleotide polymorphisms at two linked genes (AMELY and UTY) in a network analysis. A differentiation between wild and domestic populations was observed (F_ST = 0.229), with commercial breeds sharing no Y haplotype with the sampled wild boar. Similarly, a certain degree of geographic differentiation was observed across Europe, with a number of local private haplotypes and high diversity in northern populations. The described Y‐chromosome markers can be useful to track male inheritance and gene flow in wild and domestic populations, promising to provide insights into evolutionary and population genetics in Sus scrofa.     

Characterization of microsatellite loci in the western tarnished plant bug,Lygus hesperus Knight (Hemiptera: Miridae)

A microsatellite‐enriched partial genomic DNA library of Lygus hesperus was generated and screened by sequencing. Ten polymorphic microsatellite marker loci were characterized by genotyping 92 insect samples. The observed number of alleles ranged from three to seven with an average of 4.5 (SE ± 0.45), while the effective number of alleles ranged from 1.21 to 3.02 with an average of 2.14 (SE ± 0.20). Significant deviations from Hardy–Weinberg expectations were detected at three loci. Significant linkage disequilibrium was also detected between the loci LhMS2‐54 and LhMS3‐32. Seven of the L. hesperus markers could be transferred to Lygus lineolaris.     

Characterization of eight polymorphic microsatellite markers in the tarnished plant bug,Lygus lineolaris (Palisot de Beauvois)

A partial genomic library of the tarnished plant bug, Lygus lineolaris, enriched for microsatellite sequences was screened to identify marker loci. Eight polymorphic loci suitable for population genetic studies were identified by screening 192 field‐collected insects. The observed number of alleles ranged from four to 21 with an average of 12.25 (SE ± 1.94) while the effective number of alleles ranged from 1.23 to 11.05 with an average of 4.49 (SE ± 1.15). No linkage disequilibria or significant deviations from Hardy–Weinberg expectations were detected at any of the loci. Seven of the eight L. lineolaris microsatellite loci were transferable to Lygus hesperus.     

Characterization of microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L., is a cosmopolitan species with a capacity to transmit human pathogens. Here, we report on the development of polymorphic microsatellite loci for house flies and present preliminary results from four house fly subpopulations from Manhattan, Kansas. Twenty‐four microsatellite loci were isolated and characterized using DNA enriched for repeat sequences. Forty individuals from four locations in Kansas were assayed to identify for polymorphic loci. Eight loci were polymorphic with the number of alleles ranging from three to six.     

Development of sequence‐tagged microsatellites for the barley net blotch pathogen,Pyrenophora teres

A modified sequenced‐tagged microsatellite (STM) profiling procedure was used to develop 80 STMs for the barley net blotch pathogen, Pyrenophora teres. Of these, 60 STMs amplified 67 loci in one or both of the spot (P. teres f. maculata) and net (P. teres f. teres) forms of the pathogen. When screened on six field‐sampled isolates of each pathogen form, 25 STMs revealed 26 polymorphic loci, with an average of 3.2 ± 1.0 alleles and mean gene diversity of 0.59 ± 0.12.     

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Isolation,characterization and cross‐salmonid amplification of 31 microsatellite loci in the lake whitefish (Coregonus clupeaformis,Mitchill)

Coregonine fish represent the most successful evolutionary lineage of salmonids with Coregonus as the most speciose salmonid genus inhabiting numerous postglacial lakes across the northern hemisphere. We isolated and characterized 31 polymorphic microsatellite loci in Coregonus clupeaformis with an average number of 5.3 alleles per locus (range three to eight) and an overall expected heterozygosity of 0.74 ± 0.11. Two loci revealed significant linkage associations through analyses of mapping families. Six additional salmonid taxa assessed for cross‐species amplification revealed between 18 and 26 positive amplifications and between two and 12 polymorphic loci per species.     

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.     

Novel polymorphic microsatellite markers for paternity analysis in the red‐capped robin (Petroica goodenovii: Aves)

Seven microsatellite loci were isolated and characterized from the red‐capped robin Petroica goodenovii, using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Five loci showed no evidence of null alleles and were variable [mean heterozygosity (H_E) = 0.440, mean number of alleles = 8]. Cross‐amplification using primers for microsatellites in Phylloscopus occipitalis and Emberiza schoeniclus yielded another two polymorphic loci. The combined set of five red‐capped robin and two cross‐amplified loci are suitable for paternity assignment (exclusion probability for seven unlinked loci = 0.9760).     

Isolation and characterization of microsatellite markers in the tetraploid birch,Betula pubescens ssp. tortuosa

The mountain birch, Betula pubescens ssp. tortuosa is a tetraploid tree species which forms the tree limit in northern Fennoscandia. We identified nine polymorphic microsatellite loci in order to characterize the genetic structure of populations at different elevations close to the treeline. The microsatellite loci were highly polymorphic, with 14–42 alleles per locus and an average expected heterozygosity of 0.73 ± 0.25 under random chromosome segregation and 0.68 ± 0.23 under random chromatid segregation.     

Isolation and characterization of microsatellite loci in Tripterygion delaisi

We isolated 49 microsatellite loci from a genomic library of Tripterygion delaisi×anthosoma enriched for CA and GA repeats. Ten loci were screened in 30 individuals with high numbers of alleles per locus (averaging 15.5 ± 2.86) and observed heterozygosity (averaging 0.765 ± 0.052). No deviations from Hardy–Weinberg expectations were detected. These highly polymorphic markers will be useful in determining the spatial patterns of genetic diversity between and within subspecies of Tripterygion delaisi.     

Isolation and characterization of microsatellites in yellow perch (Perca flavescens)

A total of 45 microsatellite loci from yellow perch, Perca flavescens, were isolated and characterized. Among the 45 microsatellite loci, 32 had more than two alleles. A wild population of P. flavescens (n = 48) was used to examine the allele range of the microsatellite loci. Mendelian inheritance of alleles was confirmed by examining the amplified products in pair‐mated families. The number of alleles for the 32 polymorphic loci varied from two to 16, and observed heterozygosity ranged between 0.024 (YP79) and 0.979 (YP60). Cross‐species polymorphic amplification in four other Percidae species was successful for 22 loci.     

Microsatellite loci for paternity assessment in the black swan (Cygnus atratus: Aves)

Seven polymorphic tetranucleotide microsatellite loci were isolated and characterized from the black swan Cygnus atratus Latham, using nonradioactive PCR‐based techniques to screen an enriched genomic library. These loci were highly variable (mean H _E  = 0.75, mean number of alleles = 9.1), and showed no evidence of linkage disequilibrium or null alleles. This locus set is suitable for detecting extra‐pair copulations (combined exclusion probability = 0.999) and for other population genetic applications.     

Fifteen microsatellite loci characterized in the golden eagle Aquila chrysaetos (Accipitridae,Aves)

Polymorphic microsatellite loci were identified in order to study golden eagle (Aquila chrysaetos) population fragmentation. Twenty‐six published Aquila and eight published Haliaeetus microsatellite loci were tested for polymorphism in A. chrysaetos. Fifteen loci were polymorphic with between two and six alleles detected per locus. Observed heterozygosity ranged from 0.15 to 0.77 among 177 unrelated individuals from Scotland. There was no evidence for null alleles. Two pairs of loci (Hal‐10 & Aa15 and Hal‐10 & Aa26) displayed linkage disequilibrium.     

Genetic diversity and molecular differentiation of Chinese toad based on microsatellite markers

Genetic diversity and population structure of 9 populations of Bufo gargarizans with total 111 samples in China were assessed using seven microsatellite loci. The analysed microsatellite markers produced 161 alleles, varied from 9 to 38 alleles each locus. The number of alleles per population per locus ranged from 4.43 to 10.29. Polymorphic information content showed that all seven loci were highly informative (mean = 0.810 ± 0.071). The average observed heterozygosity was less than the expected (0.353 ± 0.051 and 0.828 ± 0.067, respectively). All tested populations gave significant departures from Hardy–Weinberg equilibrium. Genetic differentiation among the populations was considerably high with the overall and pairwise F _ST values (mean = 0.160 ± 0.039), and showed fairly high level of inbreeding (indicated by a mean F _IS value of 0.504 ± 0.051) and global heterozygote deficit. In comparison to other amphibian studies; however, our results suggested that the level of genetic structuring in B. gargarizans was relatively low in the geographical scale of the study area. Interestingly, the speculated population bottleneck was found to be absent and the analyses provide only weak evidence for a recent contraction in size even though there was severe inbreeding (indicated by the F _IS value) in the Chinese toad populations.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Isolation of seven polymorphic microsatellites in Ophioblennius atlanticus atlanticus (Perciformes,Blenniidae)

We isolated and characterized seven polymorphic microsatellite loci of Ophioblennius atlanticus atlanticus (Valenciennes, 1836) using an optimized protocol to construct and screen a microsatellite‐enriched genomic library. The analysis of variability was performed in 16 specimens from Faial Island (Azores, Portugal). The mean number of alleles was 8.71 ± 2.43 and the level of expected heterozygosity ranged from 0.764 to 0.903. The total exclusionary probabilities using these loci for the first and the second parent were 0.985 and 0.998, respectively, suggesting that these microsatellites are a useful tool for large‐scale parentage analysis.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Isolation and characterization of polymorphic microsatellite loci in Acanthoscelides obvelatus Bridwell (Coleoptera: Bruchidae)

Five microsatellite loci were isolated from the bruchid Acanthoscelides obvelatus Bridwell (Coleoptera: Bruchidae). Each locus was polymorphic, with the number of alleles ranging from two to 15. We found high levels of within‐population variation at most loci, with heterozygosity ranging from 0.182 to 0.900. Cross‐species amplification of these loci was tested in two other species of the genus Acanthoscelides, A. obtectus Say and A. argillaceus Sharp.