Development of 11 polymorphic microsatellite loci in the small abalone (Haliotis diversicolor Reeve)

Eleven polymorphic microsatellite loci for Haliotis diversicolor were isolated and characterized from (CT)(n) - and (AC)(n) -enriched library. They were tested in 24 individuals from a natural population. All of them were polymorphic, with the number of alleles varying between three and 10. The observed heterozygosities and expected heterozygosities ranged from 0.083 to 0.913 and from 0.159 to 0.856, respectively. The 11 isolated microsatellite loci, except SA-JMU6 and SA-JMU12, followed Hardy-Weinberg expectations. Seven sets of primers also amplify in closely related species, H. ovina and H. asinine. These 11 polymorphic microsatellites will be useful for analysing the population structure and genetic diversity in H. diversicolor.     

Characterization of nine polymorphic microsatellite markers in sprat (Sprattus sprattus L.)

Nine polymorphic microsatellites were isolated from sprat (Sprattus sprattus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(12) probe. The loci showed different variation patterns in a Baltic Sea population (44 individuals) with mean number of alleles at 12.7 and mean observed heterozygosity at 0.78. These microsatellite loci are expected to be used for taxonomic considerations in sprat, stock differentiation and population genetic analysis.     

Isolation and evaluation of 18 microsatellite markers in Siniperca chuatsi (Basilewsky)

Eighteen novel microsatellite markers were isolated in the Chinese perch Siniperca chuatsi (Basilewsky) using the FIASCO method, from (AC/TG)(n) , (AG/TC)(n) , (AT/TA)(n) , (GATA/CTAT)(n) and (GATT/CTAA)(n) repeat genomic libraries. The number of alleles per locus ranged from two to eight in a sample of 30 individuals from a wild population. Observed heterozygosity was between 0.100 and 0.737. Seven loci showed significant deviation from Hardy-Weinberg equilibrium, null alleles were suggested at nine loci but no linkage disequilibrium between loci was detected. These loci could be useful in the population genetic study of S. chuatsi.     

Development of 18 microsatellite loci for the freshwater snail Bellamya aeruginosa (Mollusca, Gastropoda)

Although it is a major freshwater gastropod species, genetic diversity of Bellamya aeruginosa was completely unknown. Eighteen microsatellite loci were isolated and characterized from (AC)(15)-enriched genomic libraries of the freshwater snail B. aeruginosa. Most of the 18 loci were successfully amplified and high polymorphic information content values were found, ranging from 0.244 to 0.792 (mean 0.541). The number of alleles per locus ranged from 5 to 13 (mean 8.8), the expected heterozygosity varied from 0.347 to 0.950 (mean 0.815) and the observed heterozygosity varied from 0.087 to 0.782 (mean 0.431). Eight loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni's correction and no significant genotypic linkage disequilibrium was detected between most locus pairs, except for TXH79-TXH97 and TXH113-TXH121. These 18 polymorphic microsatellite loci should be useful for population genetics analysis and species identification of Bellamya.     

Microsatellite isolation and linkage group identification in the yellow fever mosquito Aedes aegypti

Microsatellites have proved to be very useful as genetic markers, as they seem to be ubiquitous and randomly distributed throughout most eukaryote genomes. However, our laboratories and others have determined that this paradigm does not necessarily apply to the yellow fever mosquito Aedes aegypti. We report the isolation and identification of microsatellite sequences from multiple genomic libraries for A. aegypti. We identified 6 single-copy simple microsatellites from 3 plasmid libraries enriched for (GA)(n), (AAT)(n), and (TAGA)(n) motifs from A. aegypti. In addition, we identified 5 single-copy microsatellites from an A. aegypti cosmid library. Genetic map positions were determined for 8 microsatellite loci. These markers greatly increase the number of microsatellite markers available for A. aegypti and provide additional tools for studying genetic variability of mosquito populations. Additionally, most A. aegypti microsatellites are closely associated with repetitive elements that likely accounts for the limited success in developing an extensive panel of microsatellite marker loci.     

Polymorphic microsatellite loci for the Japanese anchovy Engraulis japonicus (Engraulidae)

We isolated and characterized 14 polymorphic microsatellite loci in the Japanese anchovy (Engraulis japonicus) using a (GT)(13)-enriched genomic library. The numbers of alleles per locus ranged from 6 to 31, with a mean of 17.8. The observed and expected heterozygosities ranged from 0.180 to 0.949 and from 0.172 to 0.966, with means of 0.731 and 0.825, respectively. All 14 loci were in Hardy-Weinberg equilibrium and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers will be useful for analyzing the population genetic structure and gene flow of E. japonicus.     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.     

Isolation and characterization of 12 polymorphic microsatellite markers for the frog Pelophylax hubeiensis

Twelve polymorphic microsatellite loci were isolated from an (AC)n- and (AG)n-enriched DNA library for the endemic Chinese frog Pelophylax hubeiensis (Ranidae). The number of alleles per locus ranged from two to eight, with a mean of 5.17. The observed and expected heterozygosities ranged from 0.226 to 0.839 and from 0.204 to 0.826, with means of 0.568 and 0.656, respectively. No significant linkage disequilibrium was detected among these loci. However, two significant deviations from HWE were discovered at loci Pehu-11 and Pehu-12 (P<0.05). MICRO-CHECKER tests showed that null alleles could be present at locus Pehu-12. These polymorphic microsatellite loci can be employed for exploring mating mechanisms, population genetic structure and other relevant genetic investigations of P. hubeiensis.     

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides (CT)10 and (GT)10 served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.     

Isolation and characterization of 11 polymorphic microsatellite loci in the whitegirdled goby (Pterogobius zonoleucus) and cross-species amplification in the serpentine goby (P. elapoides)

Eleven polymorphic microsatellite markers were developed from a (CA)(n) -enrichment library of the whitegirdled goby (Pterogobius zonoleucus). Polymorphism at these loci ranged from 2 to 12 alleles, and observed and expected heterozygosities from 0.05 to 0.90 and from 0.05 and 0.86, respectively. All loci conformed to Hardy-Weinberg equilibrium, with no significant linkage disequilibrium between all locus pairs. Cross-species amplification tests were successful in P. elapoides, and most loci were polymorphic. These microsatellite markers will be useful in further population genetic studies of both species.     

Isolation and characterization of microsatellites loci in the lemon (Citrus limon)

This study reports the isolation and characterization of seven polymorphic microsatellite loci in Citrus. The loci were isolated from two libraries constructed from genomic DNA nonenriched for TC and AC repeats and enriched for AC repeats. These markers yielded four to nine alleles per locus (mean 6.14) in a survey of 32 Citrus cultivars. Average observed heterozygosity ranged from 0.43 to 0.72. The levels of polymorphism found in this study suggest that these microsatellite loci can become an important tool for genetic studies in Citrus.     

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 of 52 new polymorphic microsatellite markers for the olive flounder, Paralichthys olivaceus

We characterized 52 new microsatellite markers isolated from (GT)(n) and (CT)(n) microsatellite-enriched genomic libraries of the olive flounder (Paralichthys olivaceus). All markers were polymorphic, with eight to 30 (mean 15.1) alleles detected in 30 individuals from a single natural population. Observed heterozygosity ranged from 0.20 to 1.0. Segregation analysis within a mapping family revealed non-amplifying null alleles at six loci. These results indicate that these new microsatellite markers will be useful for population genetic, parentage, and genome mapping studies.     

Isolation and characterization of microsatellite loci from forest musk deer (Moschus berezovskii)

This study reported the isolation and characterization of eight polymorphic microsatellite loci in endangered forest musk deer Moschus berezovskii. An improved enrichment protocol was used to isolate microsatellites, and polymorphism was explored with samples from wild musk deer population collected in Miyalo of Sichuan Province in China. Approximately 70% of clones from the genomic library constructed in current study contained dinucleotide (AC) repeats. Eight microsatellite loci amplified were highly polymorphic within forest musk deer population. The number of alleles per locus ranged from 6 to 14, and the observed and expected heterozygosities ranged from 0.41 approximately 1.0 and from 0.8 approximately 0.9, respectively. The average polymorphic information content (PIC) value for these markers was 0.82. This demonstrated that the eight microsatellite loci developed here are highly polymorphic, and can be used as genetic markers for further investigation of musk deer. Also, the results showed that the musk deer distributed in Miyalo had a relatively higher level of genetic variation.     

Characterization of nine novel microsatellite loci for the Venus clam (Cyclina sinensis)

The Venus clam, Cyclina sinensis, is one of the most important bivalves in China marine aquaculture. Using (CA)(15)-enriched genomic libraries of this species, nine novel polymorphic microsatellite loci were isolated and characterized. The mean number of observed alleles per locus was 16 (range 8-24). The observed and expected heterozygosity ranged from 0.119 to 0.872 and from 0.626 to 0.931, respectively. Three loci had significant departure from Hardy-Weinberg equilibrium and non-significant linkage disequilibrium was found among all nine loci. These highly informative microsatellite markers should be useful for population genetic analyses of C. sinensis.     

Development and characterization of microsatellite markers for the endangered Chinese yew Taxus chinensis var. mairei (Taxaceae)

We isolated and characterized 11 microsatellite loci for the Chinese yew, Taxus chinensis var. mairei, an endangered tree species in China, by constructing a (CA)(12)-enriched library. The number of alleles per locus ranged from 5 to 10. The observed heterozygosities ranged from 0.2500 to 0.8333 and the expected heterozygosities ranged from 0.5196 to 0.8680. No significant linkage disequilibrium was detected at these loci. However, four loci significantly deviated from Hardy-Weinberg equilibrium. The null alleles were found to be present at locus Tach9 and locus Tach11 by the Micro-checker test (P < 0.001). These polymorphic loci could be employed in research of gene flow and spatial genetic patterns of T. chinensis var. mairei.     

Eleven new microsatellites of the sea bass (Dicentrarchus labrax L.)

Eleven polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with an (AC)₁₂ probe. The loci showed different variation patterns in 21 unrelated sea bass individuals, with a mean number of alleles of 8.6 and a mean observed heterozygosity of 0.68. These microsatellite markers should be useful for population genetic analysis and biodiversity studies of sea bass.     

Isolation and characterization of 17 microsatellite loci for the Chinese longsnout catfish (Leiocassis longirostris)

We describe the isolation and development of 17 polymorphic dinucleotide microsatellite loci for the Chinese longsnout catfish (Leiocassis longirostris). All loci were polymorphic in the 30 individuals tested. The number of alleles per variable locus ranged from 6 to 18, with a mean of 11.71. Observed and expected heterozygosities ranged from 0.467 to 1.000 and from 0.540 to 0.929, respectively. Eight loci were found to have deviated from HWE in the sampled population after Bonferroni correction. Linkage disequilibrium tests revealed significant linkage between two loci (LLW5 and LL27). These microsatellite loci will be useful for revealing population structure, genetic diversity, and phylogeography of the Chinese longsnout catfish.     

Ten polymorphic microsatellite loci in Tibetan chicken, <Emphasis Type="Italic">Gallus gallus domesticus</Emphasis>

Through an improved enrichment protocol, a genomic library for (AC)₁₂ repeats was constructed and 34 microsatellite loci were isolated and characterized in an endangered animal, Tibetan chicken, Gallus gallus domesticus. In the 34 loci, ten loci showed a distinct allelic variation ranging from 4 to 14 alleles in 54 individuals tested. Polymorphism information content (PIC) ranged from 0.590 to 0.869 with an average of 0.713. Average observed and expected heterozygosities were 0.7988 (ranged from 0.310 to 1.000) and 0.7495 (ranged from 0.609 to 0.897), respectively. These ten microsatellites loci would be the valuable genetic markers for further investigation of Tibetan chicken.