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 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Microsatellite markers for population studies of the rice blast fungus,Magnaporthe grisea

We developed nine new microsatellite markers for rice blast (Magnaporthe grisea) population studies. These markers were used in addition to nine microsatellite markers previously developed by our group for mapping purpose. Altogether, the 18 markers were used in multiplex PCR (polymerase chain reaction) to characterize six populations from different geographical origins. The average number of alleles per locus across populations ranged from 1.2 to 7 and the total number of alleles detected from 2 to 19. Based on this large range of polymorphism, this set of markers is expected to be useful for different kind of population studies at different geographical scales.     

Development and characterization of 60 microsatellite markers in the abalone Haliotis diversicolor

The abalone, Haliotis diversicolor, is one of the most important mariculture species in southern China. We developed 60 new polymorphic microsatellite markers for H. diversicolor and characterized them in 30 individuals from a cultured population in Sanya, China. All 60 markers were found to be polymorphic. The number of alleles ranged from two to nine per locus, with an average of 4.12/locus. The expected and observed heterozygosities ranged from 0.10 to 0.88 and from 0.07 to 0.87, respectively. Forty-four loci were in Hardy-Weinberg equilibrium. These 44 microsatellite markers should be useful for genome mapping and population genetic studies.     

Characterization of nine novel microsatellite markers from Brandt's vole (Lasiopodomys brandtii)

Highly polymorphic microsatellite markers are the most powerful tools to infer kinship relations. In this study, a library enriched for (AC)(n) (AG)(n) and (AGAT)(n) was constructed for screening microsatellite markers in Brandt's vole (Lasiopodomys brandtii), and nine novel polymorphic microsatellite markers were developed and characterized. The number of alleles ranged from 4 to 11 per locus and the mean polymorphism information content was 0.7535. The observed and the expected heterozygosity values averaged 0.760 (0.554-0.908) and 0.7914 (0.718-0.845), respectively. Average nonexclusion probability for one candidate parent varied from 0.485 to 0.716. These nine novel markers are highly polymorphic and powerful enough for our future kinship analysis.     

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.     

Eighteen new tetranucleotide microsatellite DNA markers for Coregonus lavaretus cloned from an alpine lake population

We developed 18 polymorphic microsatellite markers for Coregonus lavaretus from genomic libraries enriched for (GACA)(n) and (GATA)(n) repeat sequences. Emphasis was placed on developing highly polymorphic, perfect repeats. These loci were screened in 69 individuals from two alpine populations in Austria. Allelic variation was high with nine to 37 alleles per locus and expected heterozygosities ranging from 0.37 to 0.95. The high level of polymorphism revealed by these loci will be relevant for population studies in context to the evolutionary history of this species.     

Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons)

The black muntjac (Muntiacus crinifrons) is a vulnerable species found in the mountains of eastern China, about which little is known. Here we develop 11 polymorphic microsatellite loci from a (CA)(n) -enrichment library for the animal. In the analyses of 25 individuals sampled, unbiased expected heterozygosity levels varied from 0.686 to 0.876 and the number of alleles per locus varied from five to nine. Results that 11 microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as parentage, mating system and population genetic structure of M. crinifrons.     

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals

? Premise of the study: Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. ? Methods and Results: Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. ? Conclusions: Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.     

Identification and characterization of 18 novel polymorphic microsatellite makers derived from expressed sequence tags in the Pacific oyster Crassostrea gigas

We report the development of 18 new polymorphic microsatellite DNA markers derived from Crassostrea gigas expressed sequences tags. Genotyping of 48 wild adult oysters sampled from Marennes-Oléron bay (France) revealed 12 to 48 alleles per locus. Observed and expected heterozygosity levels ranged from 0.64 to 1 and from 0.77 to 0.97, respectively. The development of these new markers creates a useful complementary tool for population genetics studies, parentage analysis and mapping in Pacific oyster, a species of major aquacultural and ecological importance.     

Microsatellite DNA library for Caiman latirostris

New genetic markers were characterized for the broad-snouted caiman (Caiman latirostris) by constructing libraries enriched for microsatellite DNA. Construction and characterization of these libraries are described in the present study. One microsatellite marker was developed from a (ACC-TGG)(n)enriched microsatellite DNA library, and 12 microsatellite markers were developed from a (AC-TG)(n)enriched microsatellite DNA library. These markers were tested in wild-caught animals, and these tests resulted in ten new polymorphic microsatellites for C. latirostris.     

Microsatellite markers for assessing genetic diversity of the medicinal plant Paris polyphylla var. chinensis (Trilliaceae)

Paris polyphylla var. chinensis is a perennial herb with medicinal properties that is widely used in traditional Chinese medicine. However, this plant has been on the edge of extinction during the last few decades because of excessive deforestation based on the intense ethnopharmaceutical interest. We isolated 12 microsatellite loci from a (CT)(n)-enriched genomic library of P. polyphylla var. chinensis. The polymorphism at each locus was analyzed by screening 30 individuals from a natural population. The number of alleles ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.000 to 0.467, with a mean of 0.247, and from 0.383 to 0.662 with a mean of 0.537, respectively. Six loci (Pp1, Pp3, Pp6, Pp7, Pp9, and Pp12) were found to significantly deviate from Hardy-Weinberg equilibrium. This may be due to the small population size, inbreeding or null alleles. Five of the pairwise comparisons (Pp1 and Pp4, Pp2 and Pp5, Pp2 and Pp9, Pp2 and Pp12, Pp11 and Pp12) exhibited significant linkage disequilibrium (P < 0.05). We conclude that these microsatellite markers will be useful for population genetic studies of P. polyphylla var. chinensis.     

Isolation and characterization of microsatellite loci for the analysis of genetic diversity in Whitmania pigra

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra. Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae)

? Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. ? Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). ? Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.     

Development and multiplex PCR amplification of new microsatellite markers for the freshwater fish,ayu (Plecoglossus altivelis)

Multiplex polymerase chain reaction (PCR) technique has recently been applied to gain many advantages in molecular genetics. The present study focused on the development of 15 new microsatellite markers with multiplex PCR systems in ayu, Plecoglossus altivelis, an important freshwater fish in Japan. All loci were followed Mendelian inheritance in 27 F1 progeny except for the one locus. The number of alleles per locus ranged from nine to 44 and the observed heterozygosities ranged from 0.680 to 0.980 in 50 unrelated individuals. The results indicate that these new microsatellite markers are useful for studies of linkage mapping and population genetics for the species.     

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.     

Microsatellite size homoplasies and null alleles do not affect species diagnosis and population genetic analysis in a fungal species complex

The suitability of 13 microsatellite loci for species diagnosis and population genetics in 11 species of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) was assessed. Two data sets were compared to test possible biases in species typing and clone detection resulting from null alleles and size homoplasies. The first data set was based on fragment lengths derived from a multiplex polymerase chain reaction (PCR) assay and the second data set was received from singleplex PCR at lower stringency and sequencing. Most null alleles observed in the multiplex PCR assay could be amplified during singleplex PCR under less stringent conditions. Size homoplasies resulting from mutations in flanking regions and differences in microsatellite structures were observed. For example, Phialocephala uotolensis possessed a (CT)(13) in addition to the (GT)(x) motif at locus mPF_0644. Despite the occurrence of null alleles and size homoplasies, species diagnosis and population genetic analysis studies were not affected. These markers will facilitate studies on population biology, ecology and biogeography of PAC species.     

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.     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996