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.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.     

Isolation and charaterization of polymorphic microsatellite loci from so-iuy mullet (Mugil soiuy Basilewsky 1855)

The first set of polymorphic microsatellite markers were developed from so-iuy mullet (Mugil soiuy Basilewsky 1855). From a (GT)n-enriched genomic library, 53 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. Ten of these loci were polymorphic in a test population of 24 individuals with alleles ranging from 3 to 9, and observed and expected heterozygosities from 0.2083 to 0.9167 and from 0.2651 to 0.8812, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Mullet. Genbo Xu and Changwei Shao Contributed equally.     

Isolation and charaterization of 12 dinucleotide microsatellite loci from Belenger’s jewfish (Johnius belengerii Cuvier 1830)

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.     

Isolation and characterization of novel polymorphic nuclear microsatellite markers from Ophrys fusca (Orchidaceae) and cross-species amplification

The present study reports the isolation and characterization of eight new polymorphic microsatellite loci from the sexually deceptive orchid Ophrys fusca. Microsatellites were isolated from two partially enriched genomic libraries using FIASCO (Fast Isolation by AFLP of Sequences COntaining repeats). Seventy-three loci were screened for primer design and primer pairs corresponding to eight different loci were selected for microsatellite characterization of two Portuguese populations. Total number of alleles per locus ranged from 10 to 32. All loci showed a high level of observed heterozygosity (H₀) ranging from 0.33 to 1 and were possible to amplify in 16 other species of Ophrys using the same primers. H. C. Cotrim and F. A. Monteiro have contributed equally to this work.     

Characterization of one trinucleotide and six dinucleotide microsatellite markers in bicolor damselfish, <Emphasis Type="Italic">Stegastes partitus</Emphasis>, a common coral reef fish

Fourteen primer pairs were designed from 47 sequences containing microsatellites isolated from a genomic library enriched for (GACA)₄ in Stegastes partitus. Seven of these were shown to be polymorphic and provided clean amplification products. These seven loci were further characterized using 38 individuals, yielding a range in number of alleles from 5 to 31 and observed and expected heterozygosity values ranging from 0.45–0.89 and 0.66–0.96 respectively. Six of seven loci conformed to Hardy-Weinberg equilibrium. Successful amplification was also achieved in other Pomacentridae.     

Limited cross‐species microsatellite amplification and the isolation and characterization of new microsatellite markers for the greater stick‐nest rat (Leporillus conditor)

The greater stick‐nest rat (Leporillus conditor) is a conilurid rodent whose recent history provides an opportunity to examine genetic changes in reintroduced populations. We trialled 63 known microsatellite primers from Rattus rattus and Mus musculus in L. conditor. Three primer pairs produced polymorphic loci (number of alleles = 2–3, mean, H_E = 0.42). Subsequently, we isolated and characterized 12 novel polymorphic microsatellites (mean number of alleles = 5–16, mean H_E = 0.76) from L. conditor from genomic libraries for use in population genetic studies.     

Polymorphic microsatellite loci in the Coffee Berry Borer,Hypothenemus hampei (Coleoptera,Scolytidae)

Microsatellite loci were isolated from the coffee berry borer, Hypothenemus hampei, which is regarded as the major pest in coffee cultures. Seven polymorphic loci were obtained from an enriched genomic library. A low to moderate genetic diversity was observed per locus, with an observed number of alleles ranging from two to five in the 39 unrelated individuals sampled from Ethiopia. A clear deficit of heterozygotes within the population (mean heterozygosities, H_O = 0.10/H_E = 0.50) and an extreme inbreeding (F_IS, 0.70–1.00) were demonstrated. Cross‐species amplifications showed that some of the markers could be useful in two closely related Hypothenemus species.     

Characterization of four tetranucleotide and six dinucleotide microsatellite markers for use in the tropical freshwater fish Telmatherina antoniae and related species

Ten primer pairs were designed from two genomic libraries enriched for (GACA)₄ and (GACA)₇ in the sailfin silverside Telmatherina antoniae. Characterization with 57 T. antoniae individuals revealed between three and 30 alleles, with observed and expected heterozygosity values ranging from 0.47 to 0.98 and from 0.46 to 0.93, respectively. Eight of the 10 loci conformed to Hardy–Weinberg equilibrium, with no significant linkage detected between loci pairs. These microsatellite markers are intended for use in population genetic studies of T. antoniae and related fishes of the family Telmatherinidae.     

Development of microsatellite markers for mango (Mangifera indica L.)

Microsatellite markers for mango (Mangifera indica L.) were developed using a genomic library enriched for (GA)_n and (GT)_n dinucleotide repeats. A subset of 41 positive clones was sequenced and primers were designed. Twenty‐eight primer pairs produced polymorphic amplification products for a diversity sample including 15 mango cultivars and two accessions from the related species Mangifera laurina and Mangifera applanata. Nineteen simple sequence repeat (SSR) loci with clear scorable patterns were chosen to study diversity in the mango germplasm bank of Guadalupe (FWI). The number of alleles ranged from three to 13 with observed levels of heterozygosity ranging from 0.059 to 0.857.     

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.     

Isolation and characterization of microsatellite markers for the seagrass Cymodocea nodosa

In order to study the spatial patterns of genetic diversity of a clonal marine angiosperm, the seagrass Cymodocea nodosa, microsatellite markers were obtained by screening a genomic library enriched for the (CT) dinucleotide motif. Of 38 primer pairs defined, 15 amplified polymorphic microsatellites and are described. These loci identified a number of alleles ranging from two to seven, and showed expected heterozygosity ranging from 0.35 to 0.76, when a group of 40 individuals from Cadiz Bay in Spain was analysed. Additionally, we describe here the multiplexing conditions for 12 of these loci.     

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.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Development of microsatellite loci for Pelophylax plancyi and cross-amplification in other ranid species

Thirteen polymorphic microsatellite loci were obtained from the gold-striped pond frog, Pelophylax plancyi. The number of alleles per locus ranged from 5 to 13 with an average of 8.38. The observed and expected heterozygosities (H _O and H _E) ranged from 0.533 to 0.890 and from 0.620 to 0.897, with averages of 0.686 and 0.751, respectively. In order to assess interspecific amplification, all primer pairs were tested with the same PCR conditions on three other Pelophylax species: Pelophylax hubeiensis, Pelophylax fukienensis, Pelophylax nigromaculata and two species from the genus Hylarana and Hoplobatrachus, respectively. All of these loci can be amplified successfully in the Pelophylax species, with eight loci amplified in all species tested.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Isolation and characterization of microsatellite loci in the threatened brook lamprey Lethenteron sp. N

A microsatellite‐enriched genomic library was obtained for the threatened brook lamprey, Lethenteron sp. N, and from it 16 dinucleotide markers were successfully isolated and characterized. These markers were found to have between 1 and 6 alleles with heterozygosity ranging from zero to 0.79. Cross‐species amplification was successful, with eight loci amplifying products in the counterpart cryptic species, Lethenteron sp. S, as well as 15 loci in Lethenteron japonicum. One out of 10 primer pairs previously reported also amplified products in all the three species.     

Novel microsatellite DNA markers for the threatened African endemic tree species,Milicia excelsa (Moraceae), and cross‐species amplification in Milicia regia

Eleven microsatellite primer pairs were developed for the tropical African tree Milicia excelsa. Genomic DNA was enriched for dinucleotide (TC_n and TG_n) and tretranucleotide (GATA_n), and 188 random clones were sequenced from both orientations. We designed and tested 44 oligonucleotide primer pairs, which were evaluated using genomic DNA from 30 M. excelsa mature trees collected from a natural population in Benin. Eleven of the 44 markers showed good amplification and were polymorphic. The number of putative alleles for polymorphic primer pairs varied from three to seven, with expected and observed heterozygosities ranging from 0.10 to 0.64 and from 0.10 to 0.80, respectively. All 11 loci amplified the related species Milicia regia, indicating that these primers will be useful for population and ecology genetic studies in other species of the genus Milicia.     

Characterization of microsatellite markers in the mosquito Ochlerotatus caspius (Diptera: Culicidae)

Seven polymorphic microsatellite loci were developed for the mosquito species Ochlerotatus caspius, using an enriched genomic library. The number of alleles per locus varied between two and 11; the expected heterozygosity (H_E) ranged from 0.18 to 0.77. These microsatellite primers should prove useful for population genetic studies of this mosquito species.     

Development and characterization of simple sequence repeat DNA markers for Zelkova serrata

We developed 14 microsatellite loci from an enriched genomic DNA library of a broad‐leaved deciduous tree, Zelkova serrata. Of 198 clones from the library, 112 contained microsatellite repeat regions. The M13‐tailed primer method was used for economy. Sequence‐specific primer pairs were designed for 58 of 76 candidate clones. Fourteen of these primer pairs successfully amplified polymorphic single loci among 34 individuals collected from the Kanto breeding region in Japan. The expected heterozygosity for the 14 microsatellite markers ranged from 0.378 to 0.876, suggesting that these will prove valuable for breeding and ecological studies on Z. serrata.