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We developed 31 microsatellite loci for a specialist hyperparasitoid wasp, Mesochorus cf. stigmaticus that parasitizes the parasitoid Hyposoter horticola, in Åland islands of southwestern Finland. The microsatellites were developed from 454 sequencing of the enriched libraries. The details of primers and the polymerase chain reactions for these microsatellite markers are described. All the loci tested on 24 samples were polymorphic with on average 4.1 alleles per locus (range 2-13). Genetic diversities as measured by average expected heterozygosity (H) and allelic richness (A) were generally high (H ≈ 0.10-0.80; A ≈ 1.9-11.5). These markers will be useful in population genetic studies of this parasitoid and other related taxa. [ABSTRACT FROM AUTHOR]

Egyptian clover (Trifolium alexandrinum), a multi-cut forage legume, is widely grown as an annual feed crop. Genetic improvement in this legume is largely hampered due to limited availability of genomic resources. Hence, a genomic library enriched for GA/CT motif was constructed to develop SSR primers. As a result, 667 SSR primer pairs were developed and 111 were validated in T. alexandrinum cv. Wardan s-99-1. Among these, 45 primer pairs were used for the analysis of genetic diversity among 86 Trifolium accessions, resulting in 220 alleles with an average of 4.88 alleles per locus. Polymorphic information content for these markers ranged from 0.040 to 0.819 with an average of 0.445. The observed and expected heterozygosity for individual loci ranged from 0.39 to 0.89 and 0.368 to 0.80 with an average of 0.675 and 0.659, respectively. The markers exhibited varied levels of cross-species transferability ranging from 93.33 % across T. resupinatum to 40.0 % across T. balansae. Phylogenetic relationships were analyzed using the unweighted pair group method with arithmetic averages (UPGMA)-based dendrogram which was able to establish the uniqueness of each genotype and grouped them into two major clusters. To the best of our knowledge, the SSR markers reported here are among the first to be reported in this important yet neglected forage legume. They were assessed to be highly efficient and polymorphic and found to be useful for distinguishing the closely related genotypes of Trifolium. This large resource of SSR markers would therefore facilitate new avenues for T. alexandrinum genomics. [ABSTRACT FROM AUTHOR]

Lentil ( Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2-5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement. [ABSTRACT FROM AUTHOR]

Limited availability of validated, polymorphic microsatellite markers in mung bean ( Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard's similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume. [ABSTRACT FROM AUTHOR]

We identified 14 informative microsatellite loci from two taxa, Grevillea aquifolium (6) and G. infecunda (8), belonging to the Pteridifolia Group, Aspleniifolia/ Hookeriana Subgroup (Proteaceae). Loci were screened for variability among 20 individuals of their source species. The number of alleles observed per locus for G. infecunda ranged from 1 to 3. Subsequent chromosome counts revealed between 20 (normal diploid) and 30 (triploid) chromosomes in different individuals. High levels of variability were observed with the number of alleles per locus ranging from 3 to 17, with a mean of 9.8 across all loci. Nei's genetic diversity ( H) ranged from 0.399 to 0.992 with a mean of 0.783. All loci amplified in a further 9-13 taxa from the same subgroup and under the same amplification conditions. These new markers provide useful tools for evolutionary and conservation studies assessing clonality and polyploidy in this important Australian genus. [ABSTRACT FROM AUTHOR]

Turbot ( Scophthalmus maximus) is a flatfish species commercially important for aquaculture. In this study, we generated a microsatellite-enriched genomic DNA library for Scophthalmus maximus, and then isolated and characterized 45 microsatellite loci by genotyping 30 individuals. The observed number of alleles ranged from 2 to 19 with an average of 6.24, while the effective number of alleles ranged from 1.30 to 11.11 with an average of 3.66. The expected heterozygosities varied from 0.235 to 0.925 4 and Polymorphie information content ranged from 0.2044 to 0.903 3, with an average of 0.622. Twelve loci deviated significantly from Hardy-Weinberg equilibrium, and no significant linkage disequilibrium was observed between any pair of loci after Bonferroni correction. In cross-species amplification, five flatfish species ( Paralichthys lethostigma, Verasper moseri, platichthys stellatus, Hippoglossoides dubius and Cynoglossus semilaevis) showed at least one Polymorphie locus. These Polymorphie microsatellite loci should prove useful for Population analysis of turbot and other related species. [ABSTRACT FROM AUTHOR]

The pomegranate ( Punica granatum L.) is a temperate climate species requiring high temperatures for proper and complete ripening. The species is consumed as a fresh fruit, but also can be used to obtain transformed products such as juice, jam, or preserve. It is a fruit tree species with a high degree of diversity, but the identification of cultivars by morphological traits is very difficult. Thus, the characterization of genotypes through molecular markers is of great value for germplasm preservation, genetic studies, and plant breeding. The number of simple sequence repeat (SSR or microsatellite) markers developed for this genus is too low, so in this work we report the development of 117 microsatellite loci from a CT/AG-enriched pomegranate genomic library. In order to check their utility, eleven accessions were analyzed. The polymorphism information content (PIC) value across all loci ranged between 0.09 and 0.71, with an average of 0.37. These markers will facilitate genetic diversity studies, mapping, and genotyping of pomegranate. [ABSTRACT FROM AUTHOR]

By combining biotin-enrichment protocol and next generation pyrosequencing, through 454 GS-FLX Titanium technology, 55 polymorphic microsatellites loci with perfect motif were isolated from the Rhone streber ( Zingel asper), a critically endangered European fish species. Eight multiplex PCR kits were optimised in order to genotype a total of 58 polymorphic loci, including three previously published loci. The level of genetic diversity was assessed for 68 Z. asper, 30 Sander lucioperca, 33 Perca fluviatilis and four Gymnocephalus schraetzer individuals. Amplification success was also assessed on Romanichthys valsanicola and Zingel streber using single individuals. These markers will be useful to investigate the population structure of the highly fragmented Rhone streber. They represent a powerful tool for conservation issues and evolutionary approaches of this endemic species. Moreover, part of our markers demonstrated applicability to other percid species, allowing for potential applications to fisheries and aquaculture management. [ABSTRACT FROM AUTHOR]

Ten tetranucleotide microsatellite loci were isolated from GATA-enriched library of rock carp ( Procypris rabaudi). The loci were tested in 25 individuals from four populations. The number of alleles per locus ranged from 3 to 6. Polymorphism information content (PIC) ranged from 0.460 to 0.788. Observed and expected heterozygosities ranged from 0.375 to 0.739 and from 0.531 to 0.833, respectively. These microsatellites will facilitate the study of population genetics of rock carp. [ABSTRACT FROM AUTHOR]

A new set of 11 microsatellite markers was isolated and characterized in the large yellow croaker (Pseudosciaena crocea Richardson) from a CA-enriched genomic library. Of 35 microsatellite markers screened, 11 microsatellite markers are highly polymorphic in one hatchery stock which contained 58 individuals. The high genetic variability was represented mainly by the number of alleles, which ranged from 7 to 16, and the observed heterozygosity, which ranged from 0.43 to 0.79. These newly isolated markers increase the available molecular resources that can be used to analyze genetic diversity and population structure of large yellow croaker. [ABSTRACT FROM AUTHOR]

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5' end. which promotes efficient adenylation of the 3' ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy. [ABSTRACT FROM AUTHOR]

Twelve new microsatellites have been developed in olive. For that purpose, a genomic library of the olive cultivar ‘Arbequina’ was enriched for GA, GT and ACT repeats. Two methods of screening yielded 27 sequences containing microsatellites out of the 119 clones sequenced. The GA repeat seems to be the most abundant motif. Among sequences containing microsatellites, 4 (14.8%) were redundant, 1 (3.7%) was previously described in the literature and 12 (44.4%) could not be used for primers design because the repeat motifs were incomplete. Suitable primer pairs were obtained for the remaining 10 (37.0%) sequences plus an additional 14 recovered from a formerly developed library. For the 24 primer pairs designed, 4 failed to amplify, 8 produced a complex bands pattern and 12 succeeded in giving amplification products. Considering these 12 primer pairs, 10 showed single locus amplification, whereas the other 2 revealed two loci each. This was demonstrated by studying allele segregation in two olive progenies. Sixty-eight alleles were detected for the 12 microsatellites when 51 olive cultivars were analysed. The number of alleles per locus ranged from 1 to 13. The expected heterozygosity varied between 0 and 0.83. All pairs of cultivars could be distinguished using only three microsatellites due to their great discrimination power value. The data coming from genotyping the 51 olive cultivars for 7 out of the 12 new microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice similarity coefficient. Cultivar association according to their geographical origin was observed. [ABSTRACT FROM AUTHOR]

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’. [ABSTRACT FROM AUTHOR]

Ten microsatellite markers were isolated and characterized for piau-verdadeiro ( Leporinus obtusidens), an important Characiform fish species in South American basins. The markers were obtained from an enriched microsatellite genomic library, and polymorphisms were evaluated in a sample of 44 individuals caught in the upper São Francisco River. The number of alleles varied from 4 to 39. The observed and expected heterozygosities ranged from 0.614 to 0.977 and from 0.541 to 0.977, respectively. All markers were in Hardy-Weinberg equilibrium and no null alleles were detected, thus making them suitable for assessing the diversity structure of the populations of this species. [ABSTRACT FROM AUTHOR]

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1–5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided. [ABSTRACT FROM AUTHOR]

Poecilia vivipara is a small fish found throughout the coastal lowlands of South America. We report the isolation and characterization of 25 microsatellite loci in this tropical fish. In 24 individuals from two populations, 18 loci were polymorphic. The loci had 2-12 alleles, expected heterozygosities of 0.194-0.836, observed heterozygosities of 0.208-0.792, and discriminatory powers of 0.202-0.872. Four loci showed significant deviations from Hardy-Weinberg equilibrium. The microsatellite loci will be useful for investigating genetic diversity and population genetic structure, which will contribute to the development of efficient conservation programs for coastal lowlands where P. vivipara occurs. [ABSTRACT FROM AUTHOR]

Nine microsatellite loci were isolated and characterized in the tire track eel, Mastacembelus favus by using library enriched for microsatellite region. Among 47 specimens collected from Mekong River, the number of alleles ranged from 2 to 17 while levels of observed and expected heterozygosities ranged from 0.021 to 0.873 and from 0.021 to 0.893, respectively. The primers were successfully tested in six closely related species, hence valuable for investigating population genetics and other related aspects of M. favus and its congener species. [ABSTRACT FROM AUTHOR]

Labeo fimbriatus, the fringe-lipped peninsula carp, is a commercially important fish species next to Indian major carps. We isolated and characterized polymorphic microsatellite markers to be used as a tool for delineation of genetic stock of this species. Partial genomic libraries enriched for CT and GT repeat motifs generated 103 positive clones out of which 16 loci were found with flanking regions enough for designing primers. Thirty-two individuals of L. fimbriatus collected from wild were used to characterize the polymorphism. All the 16 loci were polymorphic with allele numbers ranging from 3 to 9. The observed and expected heterozygosity ranged from 0.093 to 0.833 and from 0.146 to 0.843, respectively. Nine loci were in agreement with Hardy-Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers would be very useful for characterization of natural populations of this species. [ABSTRACT FROM AUTHOR]

Geelbeck ( Atractoscion aequidens) represents a valuable fishery resource in the Benguela Current region in southwestern Africa. Due to overexploitation the species is considered depleted in South Africa, and little information is available for the rest of its distribution in the region. Genetic studies of the species are essential to inform management plans for sustainable harvesting and conservation. To facilitate such studies 12 polymorphic microsatellite DNA loci were isolated from an enriched genomic library. Genotyping of 41 individuals revealed an average of 17.75 alleles per locus (range 2-37), and observed/expected heterozygosity per locus of 0.024-0.951 and 0.024-0.962, respectively. These loci constitute the first microsatellite markers isolated for geelbeck and are applicable to multiple areas of research on this species. [ABSTRACT FROM AUTHOR]

Thirteen tetranucleotide microsatellite loci were isolated from tetranucleotide microsatellite enriched libraries for the Tokay gecko ( gekko gecko). The species is endemic to Southeast Asia, known to be a habitat specialist, and exposed to severe hunting pressures in the mountainous areas of Southeast Asia. These highly polymorphic markers (6-25 alleles) will facilitate the population genetic analyses of this species. In particular, they will enable estimates of gene flow between the two types of the Tokay gecko: the black-spotted Tokay gecko and the red-spotted Tokay gecko-a critical element in assessing classification between the two. [ABSTRACT FROM AUTHOR]

Lychnophora ericoides is an endangered species, endemic to cerrado rupestre. Herein we report the development and characterization of 13 polymorphic microsatellite loci for the species, using enriched genomic library. Number of alleles per locus and expected heterozygosity ranged from 06 to 13 and 0.696 to 0.894, respectively. The high combined probability of genetic identity (2.69 × 10) and probability of paternity exclusion (0.9999) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of L. ericoides. [ABSTRACT FROM AUTHOR]

Ten novel microsatellite loci were isolated from Arctosa sancterosae, a white dune dwelling species of wolf spider. Diversity was assessed in 273 individuals sampled from 11 populations along the Northern coast of the Gulf of Mexico. These new genetic markers will be useful for the description and conservation of these limited populations. [ABSTRACT FROM AUTHOR]

We present characteristics of eight microsatellite loci isolated from Minquartia guianensis Aubl (Olacaceae), an ecological and economically important forest tree species from Central and South America. Polymorphism was tested in two natural populations detecting 2 to 18 alleles per locus. The expected heterozygosities ( H ) range from 0.313 to 0.903. Linkage disequilibrium was found only at two locus combination in one population. High exclusion probability was estimated. These markers turn to be useful for population genetics and gene flow studies in this threatened timber species from the Neotropics. [ABSTRACT FROM AUTHOR]

Bossiaea ornata is a member of the pea family that occurs in the forest understorey of south-western Australia. We constructed a genomic library and developed eight polymorphic loci to assess genetic variability and structure within the species. Polymorphism was investigated for these markers in 20 individuals from a single population located in the jarrah forest. Expected heterozygosity ranged from 0.765 to 0.889 with a mean of 0.842, and observed heterozygosity ranged from 0.550 to 0.900 with a mean of 0.742. The number of alleles per locus ranged from 7 to 16 and averaged 11.9. [ABSTRACT FROM AUTHOR]

Nine novel microsatellite loci were developed from the tiger frog (Hoplobatrachus rugulosus) using enrichment methods. The number of alleles ranged from 6 to 11 with an average of 8.56 alleles per locus. The observed and expected heterozygosity ranged from 0.571 to 0.935 (average 0.796) and 0.765 to 0.888 (average 0.837) respectively. These polymorphic loci can be used in population structure, gene flow, and population differentiation. [ABSTRACT FROM AUTHOR]

The long-snouted seahorse Hippocampus guttulatus is one of the two European seahorse species. We describe the isolation of the first 12 microsatellite loci in this threatened species. These new markers were tested in non-invasive samples of 32 seahorses from NW Spain. The number of alleles ranged from 2 to 15 (mean: 6.3) and expected heterozygosity from 0.031 to 0.912 (mean: 0.500). All loci conformed to Hardy—Weinberg expectations and no genotypic disequilibrium was observed between any pair of loci. The theoretical exclusion probabilities for this set of loci, when no parental information exists or when one parent is known, were 0.973 and 0.998, respectively. This study indicates the usefulness of these novel loci for population analysis and kinship studies in Hippocampus guttulatus. Their potential application is extended to the other European seahorse species, since all loci were successfully cross-amplified in H. hippocampus. [ABSTRACT FROM AUTHOR]

Marsh deer (Blastocerus dichotomus) is one of the most exposed large mammals in South America. To aid in the conservation management of the species, nine polymorphic microsatellite loci were isolated and tested on up to 50 animals, showing 3-12 alleles and expected heterozygosity values varying from 0.69 to 0.89. These markers should be of considerable utility in future population and ecological genetics studies of this species. The marsh deer (Blastocerus dichotomus) is the biggest South American species of deer. Originally distributed across a large part of South America, stretching from the south bank of the Amazon river to northern Argentina, significant wild populations are now restricted to the Pantanal, swamplands that cover about 40% of southwest Brazil. The marsh deer is listed as Vulnerable on the Red List of the IUCN. Three populations of the species from three areas in the Paraná River basin (between the states of São Paulo and Mato Grosso do Sul) were recently studied by observing protein polymorphism at 17 loci (Oliveira et al. 2005). Now we are presenting data about isolation of microsatellite markers to improve the results regarding population structure. [ABSTRACT FROM AUTHOR]