EST‐SSR markers from the Pacific oyster,Crassostrea gigas

Ten polymorphic microsatellite repeat markers were identified from Crassostrea gigas, expressed sequence tags (EST) deposited in public sequence database. Number of alleles per locus ranged from three to 18, expected and observed heterozygosities ranged from 0.071 to 0.738 and from 0.306 to 0.913, respectively. Marker transferability was tested on other two Crassostrea species and polymorphic products were detected at nine loci. EST‐derived simple sequence repeats provide robust, informative and potentially transferable polymorphic markers suitable for population genetic, parentage, and mapping studies of C. gigas.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Development of EST‐SSRs in Cucumis sativus from sequence database

Simple sequence repeat markers derived from expressed sequence tags (EST‐SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. We have identified 20 polymorphic SSR markers from cucumber ESTs deposited in public sequence database. The average allele number was 3.3 per locus, ranging from two to six alleles during screening 20 cucumber genotypes with the mean expected heterozygosity of 0.477. Amplification products were also detected by 13 pairs of primer in Cucumis melo. These informative EST‐SSR markers can be used in cucumber genetic improvement projects.     

Predicting polymorphic EST‐SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST‐SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co‐segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.     

Di‐ and tetranucleotide microsatellite markers for the Alpine newt (Triturus alpestris): characterization and cross‐priming in five congeners

We developed a set of di‐ and tetranucleotide microsatellite markers for the Alpine newt, Triturus alpestris. Polymorphism as detected in 39 individuals ranged from 3 to 32 alleles at a locus. Cross‐priming with samples of five other Triturus species showed extremely poor levels of cross‐species utility. Still, these markers are suitable for studies of inter‐ and intrapopulation genetic diversity in the focal species.     

Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

Nine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.     

When North and South don’t mix: genetic connectivity of a recently endangered oceanic cycad,Cycas micronesica,in Guam using EST‐microsatellites

Subject to environmental changes and recurrent isolation in the last ca. 250 Ma, cycads are often described as relicts of a previously common lineage, with populations characterized by low genetic variation and restricted gene flow. We found that on the island of Guam, the endemic Cycas micronesica has most of the genetic variation of 14 EST‐microsatellites distributed within each of 18 genetic populations, from 24 original sampling sites. There were high levels of genetic variation in terms of total number of alleles and private alleles, and moderate levels of inbreeding. Restricted but ongoing gene flow among populations within Guam reveals a genetic mosaic, probably more typical of cycads than previously assumed. Contiguous cycad populations in the north of Guam had higher self‐recruitment rates compared to fragmented populations in the south, with no substantial connection between them except for one population. Guam’s genetic mosaic may be explained by the influence of forest continuity, seed size, edaphic differences, and human transport of cycads. Also important are the extent of synchrony among flushes of reproductive female seed‐bearing sporophylls and restricted pollen movement by an obligate mutualist and generalist insects. An NADH EST‐locus under positive selection may reflect pressure from edaphic differences across Guam. This and three other loci are ideal candidates for ecological genomic studies. Given this species’ vulnerability due to the recent introduction of the cycad aulacaspis scale, we also identify priority populations for ex situ conservation, and provide a genetic baseline for understanding the effects of invasive species on cycads in the Western Pacific, and islands in general.     

Isolation and cross‐species amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus,F. serratus and Ascophyllum nodosum (Heterokontophyta,Fucaceae)

The Fucaceae is a family of brown seaweeds that dominate and frequently co‐occur on North Atlantic rocky shores. We developed nine polymorphic microsatellite markers for the fucoid seaweeds Fucus vesiculosus, F. serratus and Ascophyllum nodosum using a combined, enriched library. Six of these loci were polymorphic in at least two species, showing from two to eight alleles with heterozygosities ranging from 0.41 to 0.85. Loci were also tested on F. spiralis, revealing five polymorphic microsatellite loci in this species.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Isolation and characterization of 15 microsatellite loci in the poplar rust fungus,Melampsora larici‐populina,and cross‐amplification in related species

We developed 15 microsatellite loci in the poplar rust fungus, Melampsora larici‐populina, using two enrichment protocols. Polymorphism of each locus was assessed on a panel of 30 isolates, comprising three subpanels (world, regional and local scales). Thirteen loci were polymorphic with three to eight alleles detected. The 15 loci were also tested on five related Melampsora species, M. allii‐populina, M. medusae f. sp. deltoidae, M. larici‐tremulae, M. rostrupii and M. pinitorqua, and partial or global cross‐amplification events were detected.     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Development and characterization of microsatellite markers for Prosopis chilensis and Prosopis flexuosa and cross‐species amplification

Prosopis chilensis and Prosopis flexuosa (Fabaceae) are closely related hardwood arboreal species that are widely distributed in the arid regions of Argentina. The development of highly polymorphic markers, such as microsatellites, is desirable for genetic studies of these species. Here, we present the development and characterization of six polymorphic microsatellite markers in P. chilensis and P. flexuosa. These markers showed a polymorphism information content between 0.14 and 0.85 and the number of alleles varied from two to 13 considering both species. All markers revealed a broad cross‐species affinity when tested in seven other Prosopis species. All primers amplified in at least five species.     

Eight PCR primers to amplify EST‐linked microsatellites in the Eastern oyster,Crassostrea virginica genome

Twenty‐four microsatellite repeat sequences were identified by screening a total of 4446 eastern oyster, Crassostrea virginica, expressed sequence tags. Polymerase chain reaction primers were designed to amplify 12 of these loci. After optimizing reaction parameters, eight loci showed high variability with consistent amplification that could be scored unambiguously. Ninety two C. virginica from the James River, VA, were genotyped at these loci. Number of alleles per locus ranged from 11 to 53, expected and observed heterozygosities ranged from 0.69 to 0.97, and from 0.30 to 0.99, respectively. Discrepancies between expected and observed heterozygosities were common and likely caused by null alleles.     

Species‐diagnostic microsatellite loci for the fig wasp genus Pegoscapus

To obtain tools for the estimation of inbreeding and assignment of offspring to matrilines, we developed 13 microsatellite loci from the fig wasps that pollinate Ficus obtusifolia. Based on morphological studies, it was thought that a single species (Pegoscapus hoffmeyeri) pollinated this fig. However, our data revealed the presence of two coexisting cryptic species. Several diagnostic microsatellite markers may be used to distinguish these two cryptic species. The new microsatellites can be used across a wide range of fig‐pollinating wasp species for both evolutionary and population genetic studies.     

Ten new microsatellite loci for the yellowhammer (Emberiza citrinella) and their cross‐species applicability among related taxa

We describe isolation and characterization of the first microsatellite loci specifically developed for a very common European passerine bird, the yellowhammer Emberiza citrinella. Nine out of our 10 loci are polymorphic within the species E. citrinella. Number of alleles ranged from two to 21 per locus and observed heterozygosity between 0.20 and 0.91. Four primer pairs also yielded reproducible results in other species of Emberizidae. These loci comprise a set of molecular markers for various applications, from moderately polymorphic loci suitable for population studies to highly polymorphic loci for pedigree analysis in Emberizidae.     

Isolation and characterization of polymorphic microsatellite loci from an EST‐library of red sea bream (Chrysophrys major) and cross‐species amplification

Microsatellite markers have been developed from a complementary DNA (cDNA) library of red sea bream, Chrysophrys major. Twenty‐eight microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. Observed and expected heterozygosities varied from 0.33 to 1.00 and from 0.38 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and six positive amplifications and between 0 and 6 polymorphic loci per species.     

Microsatellite loci for the Siberian flying squirrel,Pteromys volans

We report the isolation and characterization of polymorphic microsatellite loci for the Siberian flying squirrel Pteromys volans. The seven most useful loci had between six and 11 alleles and expected heterozygosities ranging from 0.477 to 0.866. We also tested the utility of these loci in other squirrel species, northern flying squirrels (Glaucomys sabrinus and G. volans) and the common red squirrel (Sciurus vulgaris). Three of the Siberian flying squirrel loci were polymorphic in other squirrel species, suggesting a limited potential for cross‐species use.     

Additional microsatellites for Sparus aurata and cross‐species amplification within the Sparidae family

Six new microsatellite loci were isolated and characterized in 32 individuals from a farm population of gilthead seabream (Sparus aurata). Expected heterozygosity at all loci was high, ranging from 0.835 to 0.958 with between 10 and 27 alleles per locus. A multiplex polymerase chain reaction protocol was developed using four of the loci. Cross‐species amplification of the loci was tested in six species of the Sparidae family and four loci were successfully amplified in two or more related species.     

AFLP-based genetic linkage maps of the blue mussel (Mytilus edulis)

We report the construction of the first genetic linkage map in the blue mussel, Mytilus edulis. AFLP markers were used in 86 full-sib progeny from a controlled pair mating, applying a double pseudo-test cross strategy. Thirty-six primer pairs generated 2354 peaks, of which 791 (33.6%) were polymorphic in the mapping family. Among those, 341 segregated through the female parent, 296 through the male parent (type 1:1) and 154 through both parents (type 3:1). Chi-square goodness-of-fit tests revealed that 71% and 73% of type 1:1 and 3:1 markers respectively segregated according to Mendelian inheritance. Sex-specific linkage maps were built with mapmaker 3.0 software. The female framework map consisted of 121 markers ordered into 14 linkage groups, spanning 862.8 cM, with an average marker spacing of 8.0 cM. The male framework map consisted of 116 markers ordered into 14 linkage groups, spanning 825.2 cM, with an average marker spacing of 8.09 cM. Genome coverage was estimated to be 76.7% and 75.9% for the female and male framework maps respectively, rising to 85.8% (female) and 86.2% (male) when associated markers were included. Twelve probable homologous linkage group pairs were identified and a consensus map was built for nine of these homologous pairs based on multiple and parallel linkages of 3:1 markers, spanning 816 cM, with joinmap 4.0 software.