Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species. Received April 10, 2000; accepted July 13, 2000.     

Isolation and characterization of 21 microsatellite loci in Lycium chinense and cross-amplification in Lycium barbarum

The present study reports isolation and characterization of 21 polymorphic microsatellite markers developed from Lycium chinense Mill. These markers produced a total of 86 alleles across 30 L. chinense accessions, with an average of 4.1 alleles per locus. Values for observed heterozygosity and polymorphism information content ranged from 0.03 to 0.81 (mean = 0.35) and from 0.03 to 0.78 (mean = 0.31), respectively. At the significance threshold (P < 0.05), 12 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 52 pairs of loci. All loci were successfully amplified for all L. barbarum accessions. These newly developed polymorphic microsatellite markers will be very useful for programming in the genetic conservation and classification of L. chinense and L. barbarum.     

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F₂ cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Microsatellite loci in the Propertius duskywing,Erynnis propertius (Lepidoptera: Hesperiidae), and related species

Fifteen polymorphic dinucleotide microsatellite loci were characterized for Erynnis propertius using an enrichment protocol. The number of alleles varied from nine to 28 for a sample of 24 individuals. Observed heterozygosities ranged from 0.25 to 0.96. Homozygote excess was detected for 10 loci. Twelve markers successfully amplified in related Erynnis species and eight loci were polymorphic in at least one other species.     

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 a marine fish species,the tusk (Brosme brosme)

We developed polymerase chain reaction primers for nine dinucleotide microsatellite loci in the marine deep‐sea fish, the tusk (Brosme brosme). All markers were obtained from a partial genomic DNA library, and characterized in 100 unrelated individuals from one putative population. The number of alleles ranged from two to 60 with an average of 15/locus. The observed heterozygosity ranged from 0.020 to 0.826 (average 0.438). Several of the markers amplified multiple alleles from the two other gadoid species tested.     

Characterization of 18 new microsatellite loci in Atlantic cod (Gadus morhua L.)

Eighteen new microsatellite loci consisting of 10 di‐, 5 tri‐, 2 tetra‐ and 1 heptanucleotide repeats are introduced for the Atlantic cod (Gadus morhua L.). All loci were co‐amplified in two polymerase chain reactions (plus two previously published microsatellites) and all products were typed clearly. The number of alleles per locus ranged from six (PGmo130) to 45 (PGmo76) and the observed heterozygosity ranged from 0.356 (PGmo130) to 0.957 (PGmo95). All loci except one followed Hardy–Weinberg expectations. Genetic linkage disequilibrium analysis between all pairs of loci did not yield any significant values.     

Polymorphic microsatellite markers for the Iberian rock lizard species,Iberolacerta cyreni,and cross‐species priming in other Iberolacerta species

Eight polymorphic microsatellite loci are described for the Iberian rock lizard species, Iberolacerta cyreni. Loci were isolated from a partial genomic library that had been enriched for AAAG repeat sequence. Number of alleles per locus ranged from two to 13 in a sample of 24 individuals from the Sierra de Guadarrama (central Spain). Observed heterozygosity ranged from 0.13 to 0.96. At least three loci were amplified and polymorphic in four other Iberian rock lizard species: Iberolacerta monticola, Iberolacerta bonnali, Iberolacerta aranica and Iberolacerta aurelioi. These markers will be used to study mating strategies and reproductive success in I. cyreni.     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

Microsatellite markers in Fijian crested iguanas (Brachylophus vitiensis) also amplify Fijian banded iguanas (B. fasciatus)

Five microsatellite markers were isolated from the Fijian crested iguana (Brachylophus vitiensis) using an enrichment technique. These loci also amplified the Fijian banded iguana (B. fasciatus). All five loci were polymorphic with between 2–7 and 4–7 alleles per locus in B. vitiensis and B. fasciatus, respectively. Heterozygosity (H_O) values ranged from 0.069 to 0.875. These markers were useful in resolving kinship relationships and will aid future endeavours by the captive management program to conserve genetic diversity.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

Isolation and characterization of microsatellite loci in the threatened wild toad lily Tricyrtis flava (Liliaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from the threatened toad lily Tricyrtis flava. The number of alleles ranged from three to 17, and the observed and expected heterozygosities ranged from 0.35 to 0.96 and from 0.50 to 0.93, respectively, in a population of T. flava. We also tested cross‐species amplifications of the markers in three other species of Trycyrtis sect. Flavae. Most of 12 markers developed for T. flava were successfully amplified in the species of sect. Flavae and will be useful for population genetic studies of these species.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

Identification and characterisation of nine new gene-associated microsatellite markers for Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.)

Nine polymorphic microsatellite markers were identified by screening of 2464 ESTs derived from a cDNA library of Atlantic cod (Gadus morhua L.). About 35 novel microsatellite loci were selected and characterised in 96 individual cod. Nine markers were successfully amplified with number of alleles from 3 to 18 per locus and the average heterozygosity was 0.57 in the panel examined (range 0.29–0.86). All loci followed the Hardy–Weinberg expectation and no significant linkage disequilibrium was found in a test including all pairwise combinations. The gene identity was determined at four of the loci, confirming the associated microsatellites as Type I markers.     

Isolation and characterization of six polymorphic microsatellite loci in the western flower thrips Frankliniella occidentalis (Insecta,Thysanoptera)

Highly polymorphic microsatellite markers can provide important demographic information on founder events and range expansion following initial introduction of invasive insect species. Six microsatellite loci were isolated from an enriched DNA library in order to study the invasion patterns of the western flower thrips, Frankliniella occidentalis. All loci tested were found to be polymorphic and successfully amplified in all individuals. The number of alleles per locus ranged from five to nine and heterozygosity ranged from 45 to 73%. Some of the loci were also successfully amplified in other thrips species.     

Relatedness of Indian Flax Genotypes (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.): An Inter-Simple Sequence Repeat (ISSR) Primer Assay

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03–0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard’s similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.     

Simple sequence repeat (SSR) markers in the ectomycorrhizal fungus Laccaria bicolor for environmental monitoring of introduced strains and molecular ecology applications

Simple sequence repeat (SSR) markers were developed from SSR-enriched genome libraries for the ectomycorrhizal basidiomycete Laccaria bicolor. Seven markers were single-locus and amplified unambiguously in L. bicolor. The seven SSR markers were further characterized using an array of 15 L. bicolor strains representative of diverse origins worldwide. The observed number of alleles per locus varied from 5–9 and the values of observed heterozygosity from 0.167 to 0.667. The seven SSR loci could be amplified from DNA extracted from root tips of L. bicolor inoculated pine seedlings. All the L. bicolor ectomycorrhizas analysed exhibited the same SSR multi-locus profile as that detected for the UAMH8232 inoculant strain. The set of markers described represents a potent tool for the monitoring of introduced strains of L. bicolor and for molecular ecology applications.     

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.     

Characterization of five microsatellite loci in the Pine Processionary Moth Thaumetopoea pityocampa (Lepidoptera Notodontidae Thaumetopoeinae)

Five microsatellite markers were developed for the lepidopteran species Thaumetopoea pityocampa using an enrichment protocol. All loci could be amplified with no evidence of null alleles and will be useful for population genetic studies. The number of alleles ranged from three to 12 for a population of 30 individuals. Observed heterozygosities ranged from 0.53 to 0.80. No significant heterozygote deficiency was detected. Four markers might be of interest for Th. wilkinsoni.