Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis

Nine nuclear‐encoded microsatellites from an enriched genomic DNA library of the HAB (harmful algal bloom) dinoflagellate Karenia brevis were isolated and characterized. The microsatellites include five perfect (three dinucleotide and two trinucleotide) and four imperfect (two dinucleotide and two trinucleotide) repeat motifs. Gene (haplotype) diversity ranged from 0.153 to 0.750 among a sample of 13 isolates; the number of alleles among the isolates ranged from two to six and pairwise tests of genotypic disequilibria were nonsignificant. The microsatellites developed in this study will provide insight into the genetic diversity of this HAB species and tools that may prove useful in predicting source populations and physiological parameters of individual K. brevis blooms.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

Isolation and variability of polymorphic microsatellite loci in Aedes aegypti,the vector of dengue viruses

We isolated five microsatellite sequences from an enriched‐(CAA)_n library of 5000 recombinant clones in Aedes aegypti. Two polymorphic microsatellites from our library and four from other sequence databases were tested: we investigated their polymorphism and Mendelian inheritance in mosquito populations. Our results indicate that trinucleotide repeat markers could be used to differentiate Ae. aegypti populations, making them valuable tools for the study of population genetic structure.     

Novel microsatellites from the green swordtail (Xiphophorus hellerii) also display polymorphism in guppy (Poecilia reticulata)

The green swordtail (Xiphophorus hellerii; Poeciliidae) is a popular ornamental freshwater fish species. Fourteen microsatellites were isolated from a green swordtail genomic library enriched for CA‐repeats. Thirteen microsatellites were polymorphic in green swordtail; interestingly four of them were tetranucleotide‐type. Cross‐species amplification showed that 10 of the 14 microsatellites were polymorphic in guppy (Poecilia reticulata; Poeciliidae) as well. No significant directional difference of allele length was seen between the two species at any of the loci tested.     

Isolation and characterization of polymorphic microsatellite loci in Wuchang bream (Megalobrama amblycephala)

Wuchang bream (Megalobrama amblycephala) is an economically important fish in China. From a (GT)₁₃‐enriched genomic library, 20 microsatellites were developed. Nine of these 20 loci were polymorphic in a test population with allele numbers ranging from two to four, and the observed and expected heterozygosities ranging from 0.2609 to 0.7826 and from 0.3739 to 0.7546, respectively. In the cross‐species amplifications, six of these nine loci were also polymorphic in white amur bream (Parabramis pekinensis). These polymorphic microsatellite loci are potentially useful for population genetics of Wuchang bream and its closely related species.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

Novel polymorphic microsatellite markers for paternity analysis in the red‐capped robin (Petroica goodenovii: Aves)

Seven microsatellite loci were isolated and characterized from the red‐capped robin Petroica goodenovii, using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Five loci showed no evidence of null alleles and were variable [mean heterozygosity (H_E) = 0.440, mean number of alleles = 8]. Cross‐amplification using primers for microsatellites in Phylloscopus occipitalis and Emberiza schoeniclus yielded another two polymorphic loci. The combined set of five red‐capped robin and two cross‐amplified loci are suitable for paternity assignment (exclusion probability for seven unlinked loci = 0.9760).     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Isolation and characterization of microsatellite loci in the Caribbean gorgonian Pseudopterogorgia elisabethae

We report on the isolation and characterization of five polymorphic microsatellites in the gorgonian Pseudopterogorgia elisabethae from genomic DNA‐enriched libraries. Forty‐four microsatellites were screened from the libraries with the oligonucleotide probe (CA)₁₂. Five of the screened microsatellites were polymorphic. The levels of polymorphism found in 50 genotyped individuals from a single population suggest that microsatellites are useful tools for the study of genetic variation in gorgonians. These are the first microsatellite loci reported from any gorgonian species.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Newly developed polymorphic microsatellite markers in Job's tears (Coix lacryma‐jobi L.)

A microsatellite‐enriched library of Job's tears (Coix lacryma‐jobi L. var. Ma‐yuen Stapf) was constructed using a modified biotin–streptavidin capture method. After screening, 17 polymorphic microsatellites were used for diversity analysis among 30 Job's tears accessions. The number of alleles ranged from one to five alleles per locus with an average of 2.8 alleles. Expected heterozygosity (H_E) and polymorphism information content (PIC) ranged from 0 to 0.676 and from 0 to 0.666, respectively. The newly developed microsatellite markers are expected to be very valuable tools for evaluation of genetic diversity among large germplasm collection of Job's tears present in our Korean Gene Bank.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Isolation and characterization of polymorphic microsatellites in a sex‐reversal fish,rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a fish of economic importance in China and some Asian countries. From a (GT)_n‐enriched genomic library, 30 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Thirteen loci exhibited polymorphism with two to 13 alleles (mean 7.9 alleles/locus) in a test population and observed heterozygosity ranged from 0.3125 to 0.9688 (mean 0.7140). These loci should provide sufficient level of genetic variation to study the fine‐scale population structure and reproductive ecology of the species.     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

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.     

Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Trinucleotide (AGC)_n microsatellites are found as 3′ tails of the artiodactyl short interspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)_n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)_n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)_n microsatellites with 2–4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)_n microsatellites, Enrichment for (AGC), microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.     

Microsatellites from the burbot (Lota lota), a freshwater gadoid fish (Teleostei)

We developed 21 polymorphic dinucleotide microsatellite loci, (CA)_n and (CT)_n, for the Holarctic freshwater fish, Lota lota, using an enriched genomic library protocol. The species has an interesting life history because winter‐spawning adults migrate over long distances to form spawning aggregations, a behaviour which should maintain genetic homogeneity across large spatial scales. Availability of the reported microsatellites will facilitate the investigation of population genetic structure with regard to postglacial colonization history and conservation strategies. The primers were screened on 30 individuals from a natural population (Lake Constance, southern Germany), revealing three to 24 alleles per locus with expected heterozygosities ranging from 0.48 to 0.93.     

A new set of highly polymorphic microsatellites for the white and black anglerfish (Lophiidae)

A microsatellite‐enriched genomic library was obtained using individuals of the black anglerfish (Lophius budegassa) and eight polymorphic microsatellites were successfully optimized. The genetic analysis of 50 black anglerfish individuals captured in the Cantabrian coasts revealed high polymorphism with a mean of number of alleles per locus of N_a = 10.5 (5–28 alleles) and a mean expected heterozygosity of 0.740 (0.521–0.962). This microsatellite set was also functional with a sample from the white anglerfish (Lophius piscatorious) showing a high polymorphism.