Isolation and characterization of trinucleotide microsatellites in African malaria mosquito Anopheles funestus

We developed microsatellite markers for an important African malaria mosquito Anopheles funestus Giles. The microsatellite‐enriched genomic library was constructed and screened with single‐strand oligonucleotides [(CCT)₁₇, (AAT)₁₇, (CAG)₁₇ and (GA)₂₅] as probes. Among the 47 pairs of polymerase chain reaction primers screened, 31 produced successful and consistent amplification. Although only a few A. funestus individuals from one geographical location were used to screen microsatellite marker polymorphism, 27 markers were found polymorphic and four markers monomorphic. Most polymorphic markers are trinucleotide markers. Isolation of polymorphic microsatellite markers provide useful tools for A. funestus population genetic studies and genome mapping.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.     

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti

Rousettus leschenaulti is an abundant species in many countries of South‐East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84–94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.     

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite‐enriched sub‐genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species.     

Isolation and characterization of 12 microsatellite loci for cross-species amplification in the cyprinid gudgeons Squalidus chankaensis and S. japonicus

Twelve dinucleotide markers were successfully isolated and characterized from a microsatellite‐enriched genomic library obtained for the gudgeon Squalidus chankaensis biwae. These markers were also available for the congeners S. c. tsuchigae and S. japonicus from Japan, which had five to 46 alleles and an expected heterozygosity ranging from zero to 0.946. Linkage equilibrium was observed at all loci, and most loci did not show significant deviation from Hardy–Weinberg equilibrium. The isolated microsatellite markers will be useful for genetic diversity studies of Squalidus populations.     

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.     

Identification of microsatellite markers in the red flour beetle,Tribolium castaneum

We isolated and characterized microsatellite markers for the red flour beetle Tribolium castaneum, an important model species for studies in various areas of evolutionary biology and ecology. A microsatellite‐enriched genomic library was constructed and screened with single stranded oligonucleotide probes [(CCT)₁₇, (AAT)₁₇ and (CAG)₁₇]. Forty‐five primer pairs were designed of which 19 pairs produced successful amplification. Polymorphism screening involved beetles from five beetle strains and revealed 15 polymorphic and four monomorphic markers. The development of polymorphic microsatellite markers will facilitate future ecological and genetic studies involving T. castaneum beetles.     

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.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

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.     

Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.     

Isolation and characterization of twelve polymorphic microsatellite markers in the endangered Hopea hainanensis (Dipterocarpaceae)

Microsatellite markers were isolated and characterized for Hopea hainanensis Merrill & Chun, an endangered tree species with scattered distribution in Hainan Island and northern Vietnam. Twenty‐six microsatellite markers were developed based on next‐generation sequencing data and were genotyped by capillary electrophoresis on an ABI 3730xl DNA Analyzer. Twelve markers were found to be polymorphic in H. hainanensis. GENODIVE analyses indicated that the number of alleles ranged from 2 to 6 per locus, and the observed and expected heterozygosity varied from 0 to 0.755 and from 0.259 to 0.779, respectively. Primer transferability was tested with Hopea chinensis Hand.‐Mazz. and Hopea reticulata Tardieu, in which 3 and 7 microsatellite markers were found to be polymorphic, separately. The results showed that H. reticulata and H. hainanensis had similar levels of genetic diversity. A neighbor joining dendrogram clustered all individuals into two major groups, one of which was exclusively constituted by H. hainanensis, while the other consisted of two subgroups, corresponding to H. reticulata and H. chinensis, respectively. The 12 polymorphic microsatellite markers could be applied to study genetic diversity, population differentiation, mating system, and fine‐scale spatial genetic structures of H. hainanensis as well as its close relatives, facilitating the conservation and restoration of these endangered but valuable Hopea species.     

Development and characteristics of microsatellite markers in Pinus merkusii

Pinus merkusii is an important industrial species that is distributed only in Southeast Asia. We isolated 10 microsatellite markers from this species using a dual‐suppression‐polymerase chain reaction technique. Of these markers, five loci were codominant and polymorphic. The number of alleles per locus ranged from three to six and the expected heterozygosity ranged from 0.389 to 0.728. These microsatellite markers will be available for analysis on population genetics and mating patterns.     

Development of microsatellite markers in black locust (Robinia pseudoacacia) using a dual‐supression‐PCR technique

Black locust (Robinia pseudoacacia) is an economically and ecologically important tree species in the world. We isolated seven polymorphic microsatellite loci from R. pseudoacacia using a dual‐suppression‐PCR technique. These loci provided microsatellite markers with high polymorphism ranging from three to 12 alleles per locus and expected heterozygosity between 0.538 and 0.944. The markers are now available for more detailed investigation of population genetic structure and pollen and seed dispersal.     

Polymorphic microsatellite markers for the Eucalyptus fungal pathogen Colletogloeopsis zuluensis

Nine polymorphic microsatellite markers for the phytopathogenic fungus Colletogloeopsis zuluensis, the causal agent of an important stem canker disease of Eucalyptus, were isolated and characterized. Two methods, random amplified microsatellite sequences (RAMS) and fast isolation by AFLP of sequences containing repeats with modifications (M‐FIASCO), were used to isolate the microsatellites. Primers for 28 prospective microsatellite regions were designed and nine of these were polymorphic for C. zuluensis. Allelic diversity ranged from 0.12 to 0.80 with a total of 37 alleles. These markers will be used in future to determine the population genetic structure of C. zuluensis isolates and to monitor their global movement.     

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.     

Novel microsatellite markers for Dalechampia scandens (Euphorbiaceae) and closely related taxa: application to studying a species complex

We developed novel microsatellite markers for D alechampia scandens L. (Euphorbiaceae). The target plants belong to a distinct, but undescribed, species in the D . scandens species complex, characterized by small resin‐producing glands. In total, 110 alleles over 36 novel markers were identified across 39 individuals from three populations. The number of alleles varied from one to seven, with an average of 3.06 ± 0.26 alleles per locus. The developed markers, along with previously developed ones for a large‐glanded D . scandens species, were tested for amplification in 11 additional species of the genus D alechampia. Four markers did not produce any detectable allele in 37 individuals from two populations of the large‐glanded species. Average expected heterozygosity across all small‐ and large‐glanded specific loci was 0.36 and 0.15, for the small and large glanded populations, respectively. Cross‐species amplification showed that 89% of all markers were successfully amplified in at least one of the 11 other D alechampia species. These microsatellite markers may be useful for detecting undescribed species in the D . scandens species complex, and can be used for comparative analyses of genetic structure, mating system and phylogeography of other D alechampia species.     

Isolation,characterization and cross‐species amplification of eight microsatellite DNA loci in the migratory locust (Locusta migratoria)

Eight polymorphic di‐ and trinucleotide microsatellite loci suitable for population genetic analysis were developed in Locusta migratoria from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.45 to 0.97, with the observed allele numbers varying between nine and 45. The overall microsatellite cloning efficiency in L. migratoria is 14%, suggesting that in migratory locusts, microsatellite sequences are abundant and should provide a valuable and easily accessible source of nuclear markers for genetic studies. These microsatellite loci were highly Locusta‐specific, with only very limited cross‐species applicability.     

Isolation and characterization of microsatellite markers in pangolins (Mammalia,Pholidota, Manis spp.)

Thirty‐four polymorphic dinucleotide microsatellite loci were developed in the Malayan pangolin Manis javanica. Of the 34 markers, 32 and 18 were also amplified, respectively, in the Chinese pangolin (Manis pentadactyla) and the African tree pangolin (Manis tricuspis). Analysis of 24 Malayan, 12 Chinese and 2 African tree pangolins showed high levels of variability (heterozygosity ranging from 0.321 to 0.708). These are the first available microsatellite markers in Pholidota and will be an invaluable tool for evolutionary and conservation genetic studies in pangolins.