Polymorphic microsatellites in a mangrove species,Rhizophora mangle L. (Rhizophoraceae)

Twenty‐six microsatellite loci were isolated and characterized from the mangrove species Rhizophora mangle using (GT)_n and (CT)_n repeats. Eighty‐four per cent of the clones contained microsatellite sequences; the most common dinucleotides were the (GA/CT) and (CA/GT) repeats. Ten primers were selected to investigate the polymorphism among individuals of R. mangle from two natural populations of the Colombian Pacific Coast. The observed heterozygosity per locus varied from 0.20 to 0.80, the power of discrimination was 0.32–0.84 and the power of exclusion was 0.03–0.75. This set of microsatellites offers an efficient tool for population genetics studies on this species.     

Isolation and characterization of microsatellite loci in Tripterygion delaisi

We isolated 49 microsatellite loci from a genomic library of Tripterygion delaisi×anthosoma enriched for CA and GA repeats. Ten loci were screened in 30 individuals with high numbers of alleles per locus (averaging 15.5 ± 2.86) and observed heterozygosity (averaging 0.765 ± 0.052). No deviations from Hardy–Weinberg expectations were detected. These highly polymorphic markers will be useful in determining the spatial patterns of genetic diversity between and within subspecies of Tripterygion delaisi.     

Ten polymorphic dinucleotide microsatellite markers of the noble scallop Chlamys nobilis

The primers flanking 22 microsatellites isolated from a genomic library enriched for (CA)_n and (GA)_n were designed in the noble scallop Chlamys nobilis. Ten primer pairs provided clear and polymorphic amplification products. Based on characterization with 48 individuals, the number of alleles ranged from three to six. The values of observed heterozygosity and expected heterozygosity varied from 0 to 0.88 and from 0.29 to 0.76, respectively. No significant linkage disequilibrium between pairs of loci was found and six of 10 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore potentially useful for studies of the population structure of the species.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

Nine polymorphic microsatellite loci for the fennel Pondweed Potamogeton pectinatus L.

Fennel (= Sago) pondweed (Potamogeton pectinatus L.) is a submersed macrophyte of nearly cosmopolitan distribution. The plant is of worldwide ecological importance as structuring component of shallow lakes, and as food for waterfowl. We developed nine polymorphic microsatellite primers for the population genetic analysis of P. pectinatus. The loci were identified using a GA/CT‐enriched genomic library using subtractive hybridization with magnetic particles. All nine loci were highly polymorphic with 6–9 alleles and heterozygosities ranging from 0.23 to 0.80 in a subset of N = 40 genotypes from five locations.     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

Isolation and characterization of microsatellite loci in the intertidal sponge Halichondria panicea

GA‐ and CA‐enriched genomic libraries were constructed for the intertidal sponge Halichondria panicea. Unique repeat motifs identified varied from the expected simple dinucleotide repeats to more complex repeat units. All sequences tended to be highly repetitive but did not necessarily contain the targeted motifs. Seven microsatellite loci were evaluated on sponges from the clone source population. All seven were polymorphic with 5.43 ± 0.92 mean number of alleles. Six of the seven loci that could be resolved had mean heterozygosities of 0.14–0.68. The loci identified here will be useful for population studies.     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Characterization of polymorphic microsatellite loci in the aphid species Macrosiphum euphorbiae (Hemiptera: Aphididae)

Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)₁₀ (GT)₁₀ and (GATA)₄. Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.     

Development and characterization of microsatellite markers in the sexual-apomictic complex Taraxacum officinale (dandelion)

 Microsatellite markers were developed in Taraxacum officinale to study gene flow between sexual and apomictic plants and to identify clones. Twenty five thousand genomic DNA clones were hybridized with a (CT)₁₂D probe. The density of (GA/CT) _n repeats was estimated at one every 61 kb in the T. officinale genome, which translates to 13 500 repeats per haploid genome. Ninety two percent of 110 positive clones sequenced contained at least one (GA/CT) _n≥5 repeat. Sixteen (CA/GT) _n≥5 and 11 (AT) _n≥5 arrays were also found in these sequences, suggesting some clustering of dinucleotide repeats. Among 50 PCR primer pairs tested, 32 produced bands and 28 of them were polymorphic. Of these polymorphic markers, 15 were putatively single-locus and the other 13 produced only polymorphic fingerprints. Six loci were further characterized for polymorphism and showed between 6 and 32 alleles per locus. Among eight primer pairs used to analyze the progeny of a sexual cross, seven were co-dominant single-locus Mendelian markers, but one (MSTA10) gave a dominant pattern in accordance with the hypothesis of a null allele segregating in a Mendelian fashion. Three pairs of loci among 28 showed significant linkages of 10, 21, and 39 cM. Observed and expected heterozygosities in two sexual populations indicate that null alleles may be present at two loci, including MSTA10. Received: 4 November 1997 / Accepted: 12 February 1998     

Isolation and characterization of fourteen novel microsatellite loci in the Hong Kong oyster, Crassostrea hongkongensis

Fourteen polymorphic microsatellite markers were isolated from the Hong Kong oyster, Crassostrea hongkongensis, with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂, (GA)₁₂, (ATG)₆ and (TAGA)₄. Polymorphism of these loci was assessed in a sample of 48 wild unrelated individuals. The average allelic number of these polymorphic loci was 6.36 per locus, with a range of 4–16. The observed and expected heterozygosity varied from 0.208 to 0.729 (averaging 0.502) and from 0.193 to 0.789 (averaging 0.615), respectively. After Bonferroni correction (P > 0.0036), 11 of the 14 loci accorded with Hardy–Weinberg equilibrium, and the rest three were detected significant departure from HWE. Additionally, two loci (Ch103 and Ch104) showed significant linkage disequilibrium (P < 0.01). This is the first set of microsatellite loci developed in this species and would be useful for studies of population genetics, stock management and other relevant research in C. hongkongensis.     

Development of twenty-two polymorphic microsatellite loci in the noble scallop, Chlamys nobilis

Twenty-two polymorphic microsatellites were isolated from the noble scallop, Chlamys nobilis with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂ and (GA)₁₂. Polymorphism of these loci was assessed in a sample of 32 unrelated individuals. The average allele number was 7.23 per locus with a range of 3–19, and the observed and expected heterozygosity varied from 0.032–0.875 (averaging 0.452) and 0.315–0.939 (averaging 0.646), respectively. After Bonferroni correction (P > 0.0023), 11 of the 22 markers accorded with Hardy–Weinberg equilibrium, and the rest showed significant departure from HWE. Three pairs of loci showed significant linkage disequilibrium (P < 0.01). A poor transferability of these loci to other two cultured species, C. farreri and Patinopecten yessoensis was detected. These markers would be useful for studies of population genetics, linkage mapping and other relevant research in C. nobilis.     

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT)n and (CA/GT)n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty-six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT)n motif was much more abundant than the (CA/GT)n motif in these clones. The (GA/CT)n repeats also showed longer average repeat length (mean n=10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.     

Polymorphic microsatellite loci for the trematode Diplostomum pseudospathaceum

We present primers for five polymorphic microsatellite loci in the eye fluke Diplostomum pseudospathaceum (Trematoda), a widely distributed parasite with a complex life cycle used as a model for parasitology and fish immunology. The loci were identified using a GA/CT‐enriched genomic library by subtractive hybridization with magnetic particles. All five loci were highly polymorphic, displaying 17 to 61 alleles and heterozygosities ranging from 0.53 to 0.92. We isolated populations of parasites within the first (snail) and second (fish) intermediate host and found small but significant genetic differentiation (F_ST = 0.012) between the two life stages of the parasite.     

Polymorphic microsatellite loci for paternity analysis in the Madagascar paradise flycatcher (Terpsiphone mutata: Aves)

Nine polymorphic microsatellite loci from the Madagascar paradise flycatcher Terpsiphone mutata were isolated using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Seven polymorphic loci showed no evidence of null alleles and exhibited high levels of variation in 18 unrelated individuals (mean diversity = 0.80, mean number of alleles = 13.6). These loci are therefore suitable for analysis of population structure and paternity (exclusion probability for six unlinked loci = 0.9998).     

Isolation and cross-amplification of microsatellites in pink abalone (Haliotis corrugata)

Ten novel microsatellite loci were isolated in pink abalone, Haliotis corrugata, using (GT)₁₅ and (CT)₁₅ enriched genomic libraries. Two previously reported Haliotis kamtschatkana microsatellites cross‐amplified in H. corrugata. A set of 12 polymorphic microsatellites were evaluated in a wild population sample (N = 49). The number of alleles ranged from two to 55, and the observed and expected heterozygosities ranged from 0.104 to 0.939 and from 0.213 to 0.982, respectively. Significant deviations from Hardy–Weinberg equilibrium at three loci and no linkage disequilibrium were observed. Haliotis corrugata microsatellites cross‐amplified in other abalone species, two in H. fulgens, and seven in H. rufescens.     

Isolation and characterization of microsatellite loci in the Pacific pleasure oyster,Crassostrea corteziensis,and their cross‐species amplification in four other oyster species

Microsatellite loci were isolated in Crassostrea corteziensis using (GT)_n, (CT)_n and (CTGT)_n‐enriched genomic libraries. Within each of 45 sequenced clones, an average of three microsatellite regions (156 total) were observed. Thirty‐three primers were designed, from which 11 microsatellite loci amplified. Ten of those were polymorphic, with a range of two to 30 alleles. Three loci were not in Hardy–Weinberg equilibrium, and linkage disequilibrium was found for six pairs of loci. These microsatellite loci will be further tested for segregation distortions and null alleles to establish a set for population genetic studies of the species in the Northwest coasts of Mexico, and for optimization of aquaculture development. Seven of the microsatellite loci cross‐amplified in Crassostrea palmula, a sympatric species, and will be useful in further genetic studies.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Isolation and characterization of microsatellite loci from a commercial cultivar of Musa acuminata

A genomic library from the commercial diploid cultivar ‘Ouro’ (Musa acuminata), enriched for CT‐ and GT‐repeats, was used to isolate and characterize 23 microsatellite loci. These loci were tested in 10 Musa genotypes, representing various Musa genomic groups with distinct ploidy level. The number of alleles per locus ranged from one to seven, and 20 loci were highly informative. Four loci appeared to amplify B genome‐specific alleles, while three loci seemed to be absent in the B genome. The polymorphism revealed by these loci will be extremely useful for genetic mapping, marker‐assisted selection, germplasm characterization and evolutionary studies in Musa.