Isolation and characterization of twenty-one polymorphic microsatellite loci in the Tibetan macaque (<Emphasis Type="Italic">Macaca thibetana</Emphasis>)

Twenty-one microsatellite loci were isolated from AC-enriched library of Tibetan macaque (Macaca thibetana). The number of alleles at the 21 microsatellite loci ranged from 8 to 15, with an average of 12.2 per locus. Polymorphism information content (PIC) ranged from 0.805 to 0.910 with an average of 0.873. The observed and expected heterozygosities ranged from 0.208 to 0.792 and from 0.843 to 0.938, respectively. These microsatellite loci will be useful for future studies that relate to the genetic diversity and population structure of Tibetan macaque.     

Characterization of microsatellite DNA markers in the Emei moustache toads (<Emphasis Type="Italic">Leptobrachium boringii</Emphasis>)

We report ten microsatellite loci in the Emei moustache toads, Leptobrachium boringii. Markers were obtained by screening a genomic library enriched for microsatellite motifs. Twenty-four individuals from one breeding site were examined and ten loci were polymorphic. The number of alleles per locus ranged from 3–9 with an average of 6.3/locus. The expected heterozygosity and observed heterozygosity ranged from 0.3874 to 0.8432, and from 0.4583 to 0.9167, respectively. Cross-species amplification was tested in a closely related species L. leishanensis. These markers will be useful in future studies on characterizing the mating system of the species.     

Characterization of eight polymorphic microsatellite markers in the tarnished plant bug,Lygus lineolaris (Palisot de Beauvois)

A partial genomic library of the tarnished plant bug, Lygus lineolaris, enriched for microsatellite sequences was screened to identify marker loci. Eight polymorphic loci suitable for population genetic studies were identified by screening 192 field‐collected insects. The observed number of alleles ranged from four to 21 with an average of 12.25 (SE ± 1.94) while the effective number of alleles ranged from 1.23 to 11.05 with an average of 4.49 (SE ± 1.15). No linkage disequilibria or significant deviations from Hardy–Weinberg expectations were detected at any of the loci. Seven of the eight L. lineolaris microsatellite loci were transferable to Lygus hesperus.     

Microsatellite loci transferability from Theobroma cacao to Theobroma grandiflorum

Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Forty‐eight microsatellite loci developed for the congener Theobroma cacao were tested in cupuassu, and 29 (60.4%) produced robust alleles. The analyses of 216 cupuassu accessions using the 21 polymorphic microsatellite loci revealed a total of 113 alleles. The number of alleles per polymorphic locus ranged from two to 11, with an average of 5.38 alleles per locus. The average observed heterozygosity was 0.343, while the mean expected heterozygosity was 0.614. The successful transferability of T. cacao microsatellite primers to cupuassu was consistent with currently accepted phylogeny.     

Characterization of microsatellite loci in the western tarnished plant bug,Lygus hesperus Knight (Hemiptera: Miridae)

A microsatellite‐enriched partial genomic DNA library of Lygus hesperus was generated and screened by sequencing. Ten polymorphic microsatellite marker loci were characterized by genotyping 92 insect samples. The observed number of alleles ranged from three to seven with an average of 4.5 (SE ± 0.45), while the effective number of alleles ranged from 1.21 to 3.02 with an average of 2.14 (SE ± 0.20). Significant deviations from Hardy–Weinberg expectations were detected at three loci. Significant linkage disequilibrium was also detected between the loci LhMS2‐54 and LhMS3‐32. Seven of the L. hesperus markers could be transferred to Lygus lineolaris.     

New polymorphic microsatellite markers for the summer flounder, Paralichthys dentatus

Given the ecological and commercial importance of the summer flounder (Paralichthys dentatus), there is a surprising paucity of information on the molecular genetics of this species. Some studies published to date are concentrated on the reproduction biology. To address this shortcoming, a microsatellite-enriched genomic DNA library of P. dentatus was generated and screened by sequencing. Twelve dinucleotide microsatellite loci were characterized by genotyping 24 samples. The observed number of alleles ranged from three to thirteen with an average of 8.25, while the effective number of alleles ranged from 2.21 to 8.28 with an average of 5.06. The observed and expected heterozygosities ranged from 0.0833 to 0.9583 and from 0.5594 to 0.8980, respectively. Significant deviations from Hardy–Weinberg expectations were detected at three loci and linkage disequilibrium between two loci was significant after applying Bonferroni correction. In cross-species amplification, three species showed at least two polymorphic loci. The 12 highly polymorphic microsatellite markers represent a powerful molecular tool, which will allow for detailed population genetic analyses on this important marine fish.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Identification and characterization of eight microsatellite loci in Machilus pseudokobu (Lauraceae), an endemic species of the Bonin Islands

Eight microsatellite loci were identified and characterized for the endangered Machilus pseudokobu (Lauraceae), an endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from 1 to 20 with an average of 6.2, and the expected heterozygosity ranged from 0.00 to 0.83 with an average of 0.47. All eight loci were screened in cross-amplification tests for two other endemic Machilus species that also inhabit the Bonin Islands. All loci were successfully amplified in these species.     

Isolation and characterization of microsatellite loci in tiger frog (Hoplobatrachus rugulosus)

Nine novel microsatellite loci were developed from the tiger frog (Hoplobatrachus rugulosus) using enrichment methods. The number of alleles ranged from 6 to 11 with an average of 8.56 alleles per locus. The observed and expected heterozygosity ranged from 0.571 to 0.935 (average 0.796) and 0.765 to 0.888 (average 0.837) respectively. These polymorphic loci can be used in population structure, gene flow, and population differentiation.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

Isolation and characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant, Schisandra sphenanthera

Schisandra sphenanthera (Schisandraceae) is a traditional Chinese medicinal plant. In this study, nine polymorphic microsatellite loci were developed and characterized for the species. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 36 individuals sampled from a natural population. The observed number of alleles ranged from 2 to 10 with an average of 5.2. Expected and observed heterozygosity ranged from 0.30 to 0.82 and 0.33 to 0.97, respectively. These microsatellites have been directly applied to both population and conservation genetic studies of S. sphenanthera.     

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.

     

Characteristics of 13 polymorphic microsatellite markers in the corn earworm,Helicoverpa zea (Lepidoptera: Noctuidae)

Thirteen polymorphic microsatellite loci suitable for population genetic studies of Helicoverpa zea were discovered by screening partial genomic libraries enriched for microsatellite sequences. Insects collected (N = 96) in Stoneville, Mississippi were used to characterize these markers. The observed and effective number of alleles per locus ranged from two to nine (average 4.46) and from 1.07 to 2.45 (average 1.81), respectively. Fisher exact tests detected significant deviations from Hardy–Weinberg equilibrium at three loci, probably due to inbreeding, null alleles, or Wahlund's effect. Significant genotypic disequilibrium was not detected between any pair of loci.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

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.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.     

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species. Received: 6 October 2000 / Accepted: 13 March 2001     

A first set of gene-derived polymorphic microsatellite markers in half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important marine fish species in China. A set of type I microsatellite markers were identified through bioinformatic mining of the GenBank database. Thirteen of these markers showed polymorphisms through genotyping a sample of 30 individuals. A total of 47 alleles were detected, with an average of 3.6 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to five, from 0.14 to 0.93 and from 0.27 to 0.77, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0038) and no significant linkage disequilibrum between pairs of loci was found. The markers identified in this study will contribute to construction of genetic linkage maps and mapping of quantitative trait loci (QTL) of C. semilaevis.     

Twelve novel polymorphic microsatellite loci developed from the Asiatic black bear (<Emphasis Type="Italic">Ursus thibetanus</Emphasis>)

Since the number and range of Asiatic black bear (Ursus thibetanus) are declining due to habitat loss and illegal trade, it is essential to take effective actions to reinforce the conservation of the remaining bear populations. In order to aid such conservation efforts, we developed 12 novel polymorphic microsatellite loci of Asiatic black bear from genomic DNA-enriched libraries in this paper. The number of alleles per locus in 24 individuals ranged from 3 to 10, the average observed heterozygosity per locus from 0.214 to 0.950, and the average expected heterozygosity per locus from 0.243 to 0.891. Eight loci followed Hardy–Weinberg expectations after Bonferroni correction for multiple comparisons. No significant linkage association was found among all these loci. The 12 polymorphic microsatellite loci will be helpful to the conservation of the Asiatic black bear.     

Characterization of ten polymorphic microsatellite markers for the protected Spanish moon moth <Emphasis Type="Italic">Graellsia isabelae</Emphasis> (Lepidoptera: Saturniidae)

Ten polymorphic microsatellite loci were developed for Graellsia isabelae. Polymorphism was assessed for 20 individuals from a Spanish population (Els-Ports-de-Beseit, Catalonia) and 39 more individuals from one population in the French Alps and six other Spanish localities. Overall, the number of alleles per locus ranged from 5 to 24. Els-Ports-de-Beseit showed an average number of alleles per locus of 9.80 (SD = 4.32), observed heterozygosity was 0.71 (SD = 0.226), and expected heterozygosity was 0.788 (SD = 0.146). Genotypic frequencies conformed to Hardy–Weinberg equilibrium at the Catalonian population, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among populations of this highly protected moth. Several loci amplified and resulted polymorphic in two related species: two loci in Actias neidhoeferi, and three loci in A. luna.