Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

Development of microsatellite markers for the endangered Neotropical tree species Tibouchina papyrus (Melastomataceae)

We isolated and characterized 12 microsatellite loci for Tibouchina papyrus (Melastomataceae), an endangered species with narrow and disjunct range, endemics to a few localities in "cerrado rupestre" from Central Brazil. These microsatellites were obtained by sequencing of a genomic shotgun library for primer design. Leaves from 96 individuals collected in the three known local populations were genotyped using the 12 primers designed to analyze the polymorphisms at each locus. The number of alleles per locus ranged from one to six; two loci were monomorphic. Among the polymorphic loci, expected heterozygosities ranged from 0.161 to 0.714. Combined paternity exclusion probability was 0.957 and combined genetic identity (0.051) was high for studies on parentage. Tibouchina papyrus is a rare and endemic tree species of outcrop quartzite and sandstone soils, with highly isolated populations, which may have lead to the low degree of polymorphism that we detected. Also, motifs of most loci are larger than dinucleotide, which typically display lower levels of polymorphism.     

Development of eighteen polymorphic microsatellite markers in <Emphasis Type="Italic">Scylla paramamosain</Emphasis> by 5′ anchored PCR technique

The mud crab Scylla paramamosain plays a significant role in fishery resources in China. In this study, we developed 18 polymorphic microsatellite markers in this important crab by 5′ anchored PCR technique. A total of 125 alleles were detected in a single population of 32 individuals of S. paramamosain. The number of alleles per locus ranged from five to nine, with the allele size ranging from 166 to 316 bp. The polymorphism information content (PIC), observed and expected heterozygosity ranged from 0.39 to 0.88, from 0.33 to 0.92 and from 0.42 to 0.86, respectively. Three loci (Scypa13, Scypa14 and Scypa15) deviated significantly from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction (P < 0.0028), and no linkage disequilibrium was found between loci pairs. These polymorphic microsatellite markers will be useful for the study of population genetic structure, construction of genetic linkage maps and mapping of economically quantitative trait loci (QTL) in S. paramamosain.     

Characterization of microsatellite loci isolated in Mountain Plover (Charadrius montanus)

Primers for 15 microsatellite loci were developed for Mountain Plover, a species whose distribution and abundance have been reduced drastically in the past 30 years. In a screen of 126 individuals collected from four breeding locales across the species’ range, levels of polymorphism ranged from two to 13 alleles per locus. No two loci were found to be linked, although one locus revealed significant departures from Hardy–Weinberg equilibrium. These microsatellite loci can be used in population genetic studies, ultimately aiding in management efforts for Mountain Plover. Additionally, these markers can potentially be used in studies investigating the mating system of Mountain Plover.     

Development and characterization of 13 novel microsatellite loci from the flat-headed bat (Tylonycteris pachypus) with cross-species amplification in closely related taxa

13 novel microsatellite loci was isolated using the enriched library method from genomic DNA of the flat-headed bat (Tylonycteris pachypus). These loci showed high levels of genetic polymorphism testing on 54 individuals sampled from Guangxi province, China. The number of observed alleles per locus ranged from 2 to 15. The expected and observed heterozygosity values ranged from 0.170 to 0.900 and from 0.185 to 0.944, respectively. One loci departed significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no Linkage disequilibrium was found between any pairs of loci. In addition, these loci were tested in the sister species, Tylonycteris robustula, seven loci amplified successfully and were also polymorphic.     

中国特有单种属血水草的微卫星分子标记开发与评价

血水草(Eomecon chionantha Hance)是中国亚热带地区特有的单种属植物。本研究采用(AG)15、(AC)15两种生物素探针及磁珠富集法从血水草基因组中开发并筛选出13个微卫星位点,且在该物种中能进行稳定的PCR扩增并表现出多态性。利用筛选出的13个SSR多态性标记对血水草4个野生居群24个个体进行PCR扩增,结果显示每个微卫星位点的等位基因数为2~7个,观测杂合度(HO)和期望杂合度(HE)分别为0.125~0.792和0.299~0.691。说明这些SSR标记可用于血水草遗传多样性的后续研究,并揭示该物种地理分布格局形成的原因,有助于保护和利用中国亚热带地区的血水草野生资源。     

Isolation and characterization of nine polymorphic microsatellite loci in the endangered shrub Disanthus cercidifolius var. longipes (Hamamelidaceae)

Nine polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Disanthus cercidifolius var. longipes. Microsatellite polymorphism was investigated using 24 individuals from one natural population. The observed number of alleles per locus ranged from two to four. Observed and expected heterozygosities ranged from 0.17 to 0.92 and from 0.16 to 0.72, respectively. These polymorphic microsatellite loci provide useful tools for the ongoing population genetic studies of D. cercidifolius var. longipes.     

Isolation and characterization of 30 novel polymorphic microsatellite loci from Japanese halfbeak, Hyporhamphus sajori (Temminck et Schlegel, 1846)

The construction of genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the study, 60 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese halfbeak (Hyporhamphus sajori). And 30 polymorphic microsatellite loci were found to be polymorphic between 2 and 11 alleles. The number of observed, expected heterozygosity and polymorphism information content per locus in 24 individuals ranged from 0.1667 to 1.000, 0.1828 to 0.9220, 0.1828 to 0.8945, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 30 microsatellite loci probably should provide sufficient level of genetic diversity to investigate the fine-scale population structure, stock management and enhancement, genetic linkage map construction and molecular marker-assisted breeding in H. sajori.     

Isolation and characterization of polymorphic microsatellite markers from fireweed,Senecio madagascariensis Poir. (Asteraceae)

Ten polymorphic microsatellite loci were isolated and characterized from invasive fireweed (Senecio madagascariensis) populations in the Hawaiian archipelago. These loci provided markers with polymorphism of six to 24 alleles per locus within 96 individuals collected from two populations from the island of Maui. The expected and observed heterozygosities ranged from 0.31 to 0.91 and from 0.056 to 1. These markers should be useful to study the importance of genetic diversity in invasion success of this species.     

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.     

Rapid isolation and characterization of polymorphic microsatellite loci in the mud crab, Scylla paramamosain (Portunidae)

Scylla paramamosain is a widespread and commercially important species of coastal marine crab. We identified 13 polymorphic microsatellite loci from a genome library constructed with 5'-anchored PCR method. Thirty-two S. paramamosain from the East China Sea were used to analyze the characteristics of these loci. The number of alleles per locus ranged from 3 to 8, with a mean of 5.923. Observed and expected heterozygosities ranged from 0.500 to 0.875 and from 0.500 to 0.859, respectively. Eleven of the 13 loci were highly polymorphic (polymorphic information content >0.5). All of the 13 novel loci were in Hardy-Weinberg equilibrium after Bonferroni's correction (P < 0.0038). There was no null allele, stuttering errors or evidence of allelic dropout in any of the loci analyzed by MICRO-CHECKER. According to pairwise tests, no significant linkage disequilibrium was found among the 13 loci (P < 0.0038, adjusted value). These novel developed microsatellites will be useful for studies of genetic variation, population structure, conservation genetics, and molecular-assisted selective breeding of S. paramamosain.     

引进美洲红点鲑群体遗传多样性微卫星的分析

为了解引进种美洲红点鲑种群遗传结构和种质资源现状,本研究利用15个微卫星标记对其养殖群体遗传多样性进行了分析。结果表明:在30个个体中,15对微卫星引物除1对扩增产物为单态外,其余14对在美洲红点鲑群体内扩增均出现了多态,14个多态性位点等位基因数目为3～7不等,共检测到等位基因数为69个,平均有效等位基因数为3.03;期望杂合度在0.540～0.809之间,平均期望杂合度为0.664;多态信息含量在0.360～0.719之间,平均多态信息含量为0.578,表明引进的美洲红点鲑遗传多样性水平较高,具有良好的选育潜力,可以作为良好的育种材料。     

Development of microsatellite markers for Aquilegia buergeriana var. oxysepala (Ranunculaceae), a vulnerable Japanese herb

Aquilegia buergeriana var. oxysepala is a vulnerable herb, for which 20 microsatellite markers were developed. The applicability of these markers was confirmed by genotyping of 66 individuals collected from four natural populations in Japan. Of the 20 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from 2 to 12, and the expected and observed heterozygosities ranged from 0.13 to 0.77 and 0.02 to 0.65, respectively. The markers described here are sufficiently polymorphic and informative to investigate the genetic diversity and population genetic structure of A. buergeriana var. oxysepala.     

Isolation and characterization of fifteen polymorphic microsatellite loci in the threatened freshwater mussel Solenaia oleivora (Bivalvia: Unionidae)

Freshwater mussels are among the most endangered fauna in the world, while little is known about their genetic diversity. We isolated 15 polymorphic microsatellite loci in a threatened freshwater mussel, Solenaia oleivora. The characteristics of these loci were tested with 46 individuals. The number of alleles per locus ranged from 2 to 15, and the PIC value ranged from 0.250 to 0.880. The observed and expected heterozygosity ranged from 0.167 to 0.929 and from 0.297 to 0.900, with an average of 0.562 and 0.647, respectively. Eight loci deviated significantly from Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for the assessment of population genetic diversity and resource conservation of S. oleivora. Cross-species amplification demonstrated that six of 15 microsatellite loci amplified successfully in two other unionid species.     

Isolation and characterization of 13 microsatellite loci in the plateau pika (Ochotona curzoniae)

High quality jumping microsatellite libraries of the plateau pika (Ochotona curzoniae) were constructed; 231 out of the 288 clones contained microsatellite repeat motif and 120 pairs of primers were designed accordingly. Polymorphism was assessed for 48 individuals. Only 13 microsatellite loci were polymorphic with high polymorphism information content (PIC) ranging from 0.598 to 0.871, a condition probably resulting from individuals used for assessment being very closely related. This degree of polymorphism, however, is sufficient to conduct parentage analysis. Expected and observed heterozygosities ranged from 0.219 to 0.935 and 0.659 to 0.890, respectively. One locus was in linkage disequilibrium. This information provides efficient tools to allow future parentage studies. Ke-xin Li and Jia-ning Geng contributed equally to this paper.     

Polymorphic microsatellite loci for Japanese Spanish mackerel (Scomberomorus niphonius)

We isolated and characterized 21 polymorphic microsatellite loci in Japanese Spanish mackerel (Scomberomorus niphonius) using a (GT)(13)-enriched genomic library. Forty individuals were collected from Qingdao, China. We found 3 to 24 alleles per locus, with a mean of 8.8. The observed and expected heterozygosities ranged from 0.263 to 0.975 and from 0.385 to 0.946, with means of 0.655 and 0.685, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Two loci showed evidence for null alleles. These microsatellite markers will be useful for population genetic analysis of Japanese Spanish mackerel.     

Development and characterization of microsatellite markers for the lizardfish known as the Bombay duck, Harpadon nehereus (Synodontidae)

The Bombay duck, or bummalo (Harpadon nehereus), is a lizardfish native to the Arabian sea, but also common in the China sea. It is normally dried and salted before consumption and export. To provide molecular information on this economically important fish species, we developed and characterized microsatellite markers. Ninety positive clones from the (CA)(15)-enriched genomic library were sequenced; 62 sequences contained sufficient repeat motifs (di-, tri- and tetra-nucleotide). Twenty-eight primer pairs were designed and 21 were successfully amplified; five loci were polymorphic, but with a low number of alleles (three or four). The observed and expected heterozygosities ranged from 0.3500 to 0.8421 and from 0.5244 to 0.6244, respectively. All of the five polymorphic loci were at Hardy-Weinberg equilibrium (adjusted), while linkage disequilibrium between Hane-97 and Hane-175 (P< 0.05, adjusted P value = 0.01) was significant. The low degree of polymorphism of microsatellite markers may be due to the large size of the repeat motifs. These markers will be useful for genetic diversity analysis of H. nehereus.     

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of spotted maigre (Nibea albiflora)

In the present study, we reported 13 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of Nibea albiflora. The number of alleles, observed and expected heterozygosity per locus in 44 individuals ranged from 2 to 13, from 0.0909 to 0.9773 and from 0.0886 to 0.9073, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. Analysis of linkage disequilibrium showed non-significant among the two pairs of loci. As a result, 13 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Nibea albiflora. Shichao Xing, Changwei Shao contributed equally to this work.     

Isolation and characterization of microsatellite markers in the newly discovered invasive fruit fly pest in Africa, Bactrocera invadens (Diptera: Tephritidae)

We describe the isolation and characterization of 11 polymorphic microsatellite loci from the recently discovered fruit fly pest, Bactrocera invadens. The polymorphism of these loci was tested in individual flies from two natural populations (Sri Lanka and Democratic Republic of Congo). Allele number per locus ranged from three to 15 and eight loci displayed a polymorphic information content greater than 0.5. These microsatellite loci provide useful markers for studies of population dynamics and invasion history of this pest species.