Isolation and characterization of polymorphic microsatellite loci in the Chagas’ disease vector Triatoma infestans (Hemiptera: Reduviidae)

Microsatellites were identified and characterized from Triatoma infestans, the principal vector of Chagas’ disease in the Southern Cone of South American countries. Ninety‐three microsatellite loci were isolated from partial genomic libraries, of which 30 were amplified and 10 were selected for genotyping. The degree of intrapopulation variation in these loci was determined using 34 specimens from the locality of Chancaní (Córdoba, Argentina). The number of alleles per locus ranged from five to 19 and the expected heterozygosity ranged from 0.608 to 0.941. The variability of these microsatellite markers provides a valuable molecular tool for population genetic studies in T. infestans.     

Isolation and characterization of simple sequence repeat loci in Rubus hochstetterorum and their use in other species from the Rosaceae family

The identification of microsatellite loci in Rubus hochstetterorum provides an important tool for the characterization and conservation of wild populations of this species. Cross‐species amplification of markers may be of particular interest for the study of other Rubus species. In this study, 41 simple sequence repeat markers were identified in a genomic library of R. hochstetterorum. Fifteen of the identified microsatellite loci were characterized in a set of 30 samples and revealed to be polymorphic with three to 19 alleles per locus. All the identified markers allowed cross‐species amplification in at least one of the other three tested species from the Rosaceae family.     

Isolation and characterization of microsatellite loci from Macadamia

Thirty‐three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies.     

Characterization of polymorphic microsatellite loci for the polychaete tubeworm Hobsonia florida

Eight dinucleotide microsatellite loci were isolated and characterized from Hobsonia florida, a tube‐dwelling ampharetid polychaete. The identified loci were highly polymorphic, with allelic diversity ranging from six to 11 alleles. Levels of expected heterozygosity were 0.52 or greater in all cases, averaging 0.78 across the complete set of loci. Cross‐species amplification was successful in three of the eight loci for one or both of the other species (Melinna cristata and Ampharete acutifrons) tested. Although these novel loci were designed for immediate utility in H. florida population‐level research, these results indicate they may prove useful in studies of other related taxa.     

Microsatellite DNA markers for population-genetic studies of Labeo dyocheilus (McClelland, 1839)

Fifty‐four primers published for six cyprinid fishes were tested to amplify homologous microsatellite loci in Labeo dyocheilus. Fifteen primers yielded successful amplification and seven were polymorphic with 3–9 alleles. To evaluate utility of the identified loci in population genetic study, 84 samples were analysed. The samples were collected from four rivers viz. Beas, Satluj, Yamuna and Jiabharali. The four sample sets displayed different diversity levels, with observed heterozygosity from 0.34 to 0.53. Significant genotype heterogeneity (P < 0.001) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine‐scale population structure analysis of L. dyocheilus.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Isolation and characterization of microsatellite loci in the Pacific pleasure oyster,Crassostrea corteziensis,and their cross‐species amplification in four other oyster species

Microsatellite loci were isolated in Crassostrea corteziensis using (GT)_n, (CT)_n and (CTGT)_n‐enriched genomic libraries. Within each of 45 sequenced clones, an average of three microsatellite regions (156 total) were observed. Thirty‐three primers were designed, from which 11 microsatellite loci amplified. Ten of those were polymorphic, with a range of two to 30 alleles. Three loci were not in Hardy–Weinberg equilibrium, and linkage disequilibrium was found for six pairs of loci. These microsatellite loci will be further tested for segregation distortions and null alleles to establish a set for population genetic studies of the species in the Northwest coasts of Mexico, and for optimization of aquaculture development. Seven of the microsatellite loci cross‐amplified in Crassostrea palmula, a sympatric species, and will be useful in further genetic studies.     

New microsatellite resources for groupers (Serranidae)

We characterized nine microsatellite loci and identified an additional 60 genomic regions containing microsatellites in the red hind grouper, Epinephelus guttatus. The nine loci were highly polymorphic, and primers designed from red hind genomic DNA produced a strong amplification product in a test panel of 16 other groupers in the genera Epinephelus and Mycteroperca collected from across the world. Most of the amplified regions were homologous to the red hind locus and a well‐defined microsatellite repeat was generally evident. The nine loci, together with the 60 uncharacterized microsatellite‐containing regions, provide a powerful tool for ecological and evolutionary studies in groupers.     

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.     

Identification of 15 polymorphic microsatellite loci in the Príncipe seedeater (Serinus rufobrunneus) and assessment of their utility in nine other Serinus species (Fringillidae,Aves)

We tested 74 passerine microsatellite loci for cross‐amplification in the Príncipe seedeater (Serinus rufobrunneus), and identified 15 loci that were both polymorphic and easy to score. In a sample of 113 individuals, the number of alleles ranged between three and 71. Three loci deviated from Hardy–Weinberg equilibrium after correcting for multiple tests, and one locus had high estimated null allele frequency. These 15 loci were highly successful in amplifying polymorphic products also in nine other Serinus species.     

PRIMER NOTE. Using the incomplete genome of the ectomycorrhizal fungus Amanita bisporigera to identify molecular polymorphisms in the related Amanita phalloides

We describe the cross‐genomic isolation of 13 single nucleotide polymorphisms (SNPs) and one variable microsatellite from five loci for the death cap mushroom Amanita phalloides. Microsatellite repeats were identified by searching the partial Amanita bisporigera genome. Flanking primers were designed for 25 of these microsatellite loci and tested for cross‐amplification in A. phalloides. One locus contained an interrupted, compound microsatellite, and four loci contained one to six SNPs. These results demonstrate the usefulness of even an incomplete genome to identify molecular markers for population studies in nonmodel organisms.     

New polymorphic microsatellite loci,cross‐species amplification and PCR multiplexing in the black aphid,Aphis fabae Scopoli

Aphis fabae includes four morphological cryptic subspecies, which are mostly identified by their partially distinct secondary host range. To determine the extent of gene flow and isolation between these four taxa, we isolated and characterized 12 microsatellite loci from Aphis fabae fabae and tested cross‐species amplification of eight loci from the closely related species Aphis gossypii. Using eight previously described microsatellite loci, we have developed the polymerase chain reaction (PCR) multiplexing of 24 loci, which were separated in tree sets and five PCRs. These sets of microsatellite loci provide high throughput capacity for large‐scale population genetic studies at a minimum cost.     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Characterization of microsatellites in the fungal plant pathogen Crinipellis perniciosa

Microsatellite markers of Crinipellis perniciosa, with three and four repeats, were developed from sequence database and evaluated for their usefulness in detecting genetic polymorphism. Thirty‐three primers produced unambiguous amplification products of 28 microsatellite‐containing loci and 14 microsatellite‐like polymorphic loci, with two to seven alleles at each locus. Three loci were useful to distinguish isolates from different biotypes and isolates from different countries. Amplification of the markers in the closely related fungi Moniliophthora roreri indicates that their usefulness in population's studies may go beyond the present study of the C. perniciosa and may have applications in population genetics of M. roreri.     

Identification of microsatellite DNA markers for population structure analysis in Indian major carp, Cirrhinus mrigala (Hamilton-Buchanan, 1882)

Forty‐four primer sequences available for four cyprinid fishes were tested to amplify microsatellite loci in Indian major carp, Cirrhinus mrigala. A total of 12 loci were successfully amplified with clear scorable patterns and five thereof were polymorphic. Suitability of the identified polymorphic loci in population structure analysis of C. mrigala was assessed. Genetic variation was examined in 76 specimens collected from five different rivers. The mean observed heterozygosity ranged from 0.247 to 0.333. Significant heterogeneity in allele frequencies was detected, indicating that the samples analysed did not belong to homogenous populations. The identified microsatellite markers are promising for the analysis of intraspecific divergence in C. mrigala across its distribution range.     

Identification of 13 polymorphic microsatellite loci in the zebra finch,Taeniopygia guttata (Passeridae,Aves)

Polymorphic microsatellite loci were identified in order to determine paternity in a captive population of the zebra finch, Taeniopygia guttata. Primer sets from 93 published passerine microsatellite sequences were tested for cross‐species amplification. Thirteen loci were found to be polymorphic, of which, eight displayed null alleles and one locus (Ase50) was found to be Z‐chromosome linked.     

Isolation and characterization of microsatellite markers from three species of swallows in the genus Tachycineta: T. albilinea, T. bicolor and T. leucorrhoa

We describe 30 microsatellite loci developed from three species of swallows in the genus Tachycineta: T. bicolor (tree swallow), T. albilinea (mangrove swallow), and T. leucorrhoa (white‐rumped swallow). These commonly studied birds nest in secondary cavities and are distributed from Alaska to Argentina. Primer pairs were designed for each species individually and tested for cross‐amplification in 40–48 individuals of all three species. Polymorphism ranged from 5 to 65 alleles per locus (mean = 19.1). These markers will allow comparative studies of extra‐pair paternity rates among members of the genus as well as the assessment of population structure.     

What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper,Lissotriton helveticus,and Triturus cristatus) and comparison with Illumina‐based approaches

The development of microsatellite loci has become more efficient using next‐generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra‐nucleotide motif‐enriched species‐specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra‐nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species‐specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454‐sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.     

Microsatellite loci from the ectomycorrhizal basidiomycete Suillus pictus associated with the genus Pinus subgenus Strobus

Seven microsatellite loci were isolated and characterized from ectomycorrhizal basidiomycete Suillus pictus associated with soft pine species (Pinus subgenus Strobus) using a dual‐suppression‐PCR technique. Microsatellite variation was assessed using 38 isolates of S. pictus sampled from three different local populations in Japan. The number of alleles per locus ranged from three to 12 within populations. These microsatellite loci can be used for studying the allelic variation and genetic structure in/among local populations of S. pictus.     

Isolation of polymorphic microsatellite loci in three phantom midge species of the genus Chaoborus (Diptera: Chaoboridae)

Because of its widespread distribution in lakes and ponds Chaoborus is of great interest to many freshwater ecologists. Interestingly some species are restricted to small fish‐less water bodies, whereas other species live mostly in large lakes. To eventually test the genetic and evolutionary implications of these different lifestyles we identified microsatellite loci in three species in this preliminary study: C. obscuripes, C. crystallinus and C. flavicans. Using a biotin/streptavidin capture technique of repetitive sequences in a 96 well format, we obtained microsatellite‐enriched genomic libraries for all three species and identified six polymorphic microsatellite markers for each species.