Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Isolation and characterization of novel microsatellite markers for Cymodocea serrulata (Cymodoceaceae), a seagrass distributed widely in the Indo‐Pacific region

Cymodocea serrulata is a tropical seagrass species distributed widely in the Indo‐Pacific region. We developed 16 novel microsatellite (simple sequence repeat) markers for C. serrulata using next‐generation sequencing for use in genetic studies. The applicability of these markers was attested by genotyping of 40 individuals collected from a natural population in the Philippines. Of the 16 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from two to seven, and the observed and expected heterozygosities ranged from 0.131–1.000 and 0.124–0.788, respectively. These markers are useful tools for elucidating genetic diversity, connectivity, and structure in this foundational coastal species.     

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Isolation and characterization of fourteen polymorphic microsatellite markers in the viperine snake Natrix maura

Nineteen polymorphic microsatellite loci were identified and developed for Natrix maura. Polymorphism was assessed for 120 individuals sampled across four sampling sites from the French Pyrenees Mountains. The number of alleles per locus ranged from 3 to 15, and expected heterozygosity per locus ranged from 0.227 to 0.863. We tested for deviation from Hardy–Weinberg equilibrium and linkage disequilibrium and assessed the presence of null alleles for all loci, resulting in a selection of 14 high‐quality polymorphic markers. These markers will be extremely useful in identifying fine‐scale genetic structures and providing insight into conservation management plans of this 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.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

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.     

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

Eleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.     

EST‐SSR markers from the Pacific oyster,Crassostrea gigas

Ten polymorphic microsatellite repeat markers were identified from Crassostrea gigas, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from three to 18, expected and observed heterozygosities ranged from 0.071 to 0.738 and from 0.306 to 0.913, respectively. Marker transferability was tested on other two Crassostrea species and polymorphic products were detected at nine loci. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of C. gigas.     

Isolation and strategies of novel tetranucleotide microsatellites with polymorphisms from different chromosomes of the rhesus monkey (Macaca mulatta)

A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P?<?0.05). All 45 primers were not significant with regard to linkage disequilibrium (P?>?0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r?=?? 0.741, P?=?0.022), whereas the positive correlation with the number of the samples (r?=?0.847, P?=?0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or?≥?15 (P?=?0.7472?~?0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.

     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Microsatellite markers for Sachalin fir (Abies sachalinensis Masters)

We developed eight polymorphic microsatellite markers from the Sachalin fir (Abies sachalinensis Masters). The number of alleles per locus ranged from eight to 31, with an average of 21.4. The observed and expected heterozygosities ranged from 0.656 to 0.937 and from 0.710 to 0.945, respectively. These markers will be available for population genetic studies and parentage analysis.     

Characterization of microsatellite loci in the brown treecreeper (Climacteris picumnus) and cross‐species amplification in the white‐throated treecreeper (Cormobates leucophaeus)

Eight polymorphic microsatellite markers were developed for the brown treecreeper, Climacteris picumnus. The number of alleles ranged from three to 25 per locus with observed heterozygosities between 0.05 and 0.76. Seven of the eight primer pairs also amplified polymorphic microsatellite loci in the white‐throated treecreeper (Cormobates leucophaeus). These markers are likely to be useful for population genetic and parentage studies in any of the Australasian treecreepers (Climacteridae) and are the first genetic markers developed for any member of this passerine family.     

Development of microsatellite markers for the drywood termite Incisitermes minor (Hagen)

Ten polymorphic microsatellite markers were developed in the drywood termite Incisitermes minor (Hagen) by using a genomic DNA extracted from the heads of workers. The microsatellite markers obtained in this study produced between two and seven alleles per locus. The observed and expected heterozygosities among the 10 markers ranged from 0.16 to 0.83 and from 0.43 to 0.84, respectively. These markers were shown to be good molecular tools for identification of the genetic structure and parentage assessment in I. minor.     

Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

Development of microsatellite markers for Dryobalanops aromatica (Dipterocarpaceae), a tropical emergent tree in Southeast Asia

Eight polymorphic microsatellite markers were developed in Dryobalanops aromatica, an emergent tree in tropical rain forests in Southeast Asia, using an enriched library method. For the assessment of microsatellite variation, 36 individuals from a natural population were analysed. The number of alleles per locus ranged from three to 16, with observed heterozygosity of 0.056–0.833 and expected heterozygosity of 0.054–0.882. These microsatellite markers will be useful for studies of population genetics, reproductive ecology and regeneration dynamics of D. aromatica.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.