Microsatellite markers for Vaccinium from EST and genomic libraries

We present 30 microsatellite loci isolated from expressed sequence tag (EST) and genomic libraries in Vaccinium corymbosum L. Allele number per locus in 11 tetraploid and one diploid V. corymbosum accessions ranged from two to 15 (mean = 8.16) in 24 single‐locus simple sequence repeats (SSRs). Cross‐species amplification in a panel of 12 species representing nine sections ranged from 30 to 100% (mean = 83%).     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

Probability of random sire exclusion using microsatellite markers for parentage verification

Many microsatellite sequences have been described in the bovine genome. Being highly polymorphic these have been suggested as markers for parentage verification and individual identification in cattle. We have evaluated the use of five highly polymorphic microsatellite markers for parentage verification in 14 breeds of cattle in the UK. Three of the microsatellite loci occur within introns in genes: BoLA DRB3 , steroid 21-hydroxylase, and the beta subunit of the follicle-stimulating hormone. The other two are anonymous sites ETH131 and HEL6. Results were analysed by a statistical approach that takes in to account deviations from Hardy-Wienberg equilibrium and linkage disequilibrium for multiple loci. The method of determining the probability of random sire exclusion uses observed genotype frequencies instead of allele frequencies. Independently, the markers used have a probability of between 0.72 and 0.62 of identifying a parentage error, while used together the five markers give, on average across breeds, a probability of 0.99 of excluding an incorrect sire.     

Development of EST‐derived microsatellite markers for Arabidopsis lyrata subspecies petraea (L.)

A set of expressed sequence tag–simple sequence repeat (EST‐SSR) loci has been developed for Arabidopsis lyrata ssp. petraea. From 768 root cDNA clones, 126 microsatellites, including di‐, tri‐, tetra‐ and pentanucleotide repeat motifs were identified and primers were designed to 24 EST‐SSRs. Eleven loci were subsequently screened on 150 individuals sampled from five natural populations, which revealed three to nine alleles per locus (mean 5.36) and expected heterozygosity (H_E) estimates ranging from 0.046 to 0.698. Significant deviations from random mating were observed at 10 EST‐SSR loci, likely due to inbreeding (global F_IS = 0.151) and population structure (global F_ST = 0.246).     

Genetic diversity of Eucalyptus hybrids estimated by genomic and EST microsatellite markers

The knowledge of breeding impacts on the genetic diversity of hybrids of Eucalyptus is crucial to the exploration of genetic resources. We estimated genetic polymorphic parameters of 112 hybrids of Eucalyptus spp. using 10 genomic simple sequence repeats (SSR) markers and 10 expressed sequence tags (EST) microsatellite markers. According to Student’s t-test, there were no significant differences between genomic SSR and EST-SSR markers. Our results also revealed high polymorphism in the hybrids analyzed, indicating that both markers are appropriate for use in genetic breeding programs.     

Six polymorphic microsatellite markers for Plantago maritima

Plantago maritima displays an extraordinary variation in breeding system. In northern Europe, the species occurs as either self‐incompatible or self‐compatible, and as unisexual females or co‐sexual hermaphrodites. The isolation of polymorphic codominant markers will provide the necessary tools to investigate the proportion of self‐fertilized seeds and levels of inbreeding depression in natural populations of this species. Isolation of microsatellite loci was achieved using a membrane‐enrichment method for four loci, and a streptavidin‐coated‐beads method for two loci. Primers were designed in microsatellite flanking sequences and were analysed using fluorescent labels.     

Characterization of nuclear DNA microsatellite markers in the ant Cataglyphis cursor

The ant Cataglyphis cursor is exceptional in that unmated workers are potentially able to lay both male and female eggs. We characterized eight pairs of primers for microsatellite loci, developed from genomic DNA for this species. Variability was tested with DNA from 19 workers and all eight loci were highly polymorphic, displaying 5–10 alleles and a high level of heterozygosity. Cross‐species amplifications indicate that these microsatellites might be useful in genetic studies of other species belonging to the genus Cataglyphis.     

Polymorphic microsatellite markers isolated from partially enriched genomic library of Chitala chitala

A total eight polymorphic microsatellite loci were obtained from genomic library of Indian feather back, Chitala chitala (order Osteoglossiformes, family Notopteridae) and the 46 samples were analysed to determine genetic variation. The mean number of alleles per locus ranged from 4.50 to 5.25, and expected heterozygosity ranged from 0.124 to 0.852. Deviation from Hardy–Weinberg equilibrium expectations (P < 0.002) was observed at loci Cch2, Cch9 (Bhaghirathi) and Cch9 (Brahmaputra). The identified microsatellite loci were found promising for population genetics studies of C. chitala and related species Notopterus notopterus (family Notopteridae).     

Breeding system of a plesiomorphic floral type: an investigation of small flowered Streptocarpus (Gesneriaceae) species

Six different floral morphologies can be found among the ca. 146 species of Streptocarpus occurring in Africa and Madagascar. One of these, a simple, small pouch type, was found to be plesiomorphic for the genus after mapping these floral types onto a molecular phylogeny. The breeding systems and population structure of three species possessing the plesiomorphic small floral morphology, S. micranthus, S. ibityensis and S. lanatus, have been investigated using nuclear microsatellite markers. Significant deviations from panmixia were found both at the between and within population level (S. micranthus θ = 0.708, f = 0.786; S. ibityensis, θ = 0.173, f = 0.138; S. lanatus, θ=0.539, f = 0.646). As a florally diverse genus, Streptocarpus is unusual in having a predominantly selfing, mixed mating breeding system as a plesiomorphic condition.     

New microsatellite markers in chicken optimized for automated fluorescent genotyping

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4·0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).     

Isolation and characterization of polymorphic microsatellite loci from an EST library of turbot (Scophthalmus maximus) and cross‐species amplification

In the present study, we report 12 polymorphic microsatellite loci developed from a cDNA library from the turbot, Scophthalmus maximus. Observed and expected heterozygosities varied from 0.20 to 1.00 and from 0.18 to 0.78, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. Cross‐species amplifications of these microsatellites in five additional fish species revealed between five and 11 positive amplifications and between zero and four polymorphic loci per species.     

Development of microsatellite markers in polyploid persimmon (Diospyros kaki Lf) from an enriched genomic library

The oriental persimmon (Diospyros kaki Lf) is believed to have originated in China with subsequent introduction into Japan and Korea in ancient times. The species was then brought to Europe, Brazil and the USA from Japan in the 19th century. Recent studies highlighted the poor state of identification of cultivars in these countries due to incorrect labelling and presence of synonyms among local varieties. Thus, molecular marker characterization of germplasm resources is of great value for genetic resource preservation and plant breeding of persimmon. Therefore, to identify accessions for further plant breeding and germplasm management, 37 microsatellite loci were developed from a CT/AG‐enriched persimmon genomic library.     

Interspecific microsatellite markers for the study of pinniped populations

Microsatellites have rapidly become the marker of choice for a wide variety of population genetic studies. Here we describe 20 pinniped microsatellite markers which have been tested across 18 pinniped species. The majority of these markers have broad utility in all pinnipeds and provide a strong base for detailed population genetic studies in the Pinnipedia.     

Developing EPIC markers for chalcidoid Hymenoptera from EST and genomic data

Increasing numbers of phylogeographic studies make comparative inferences about the histories of co-distributed species. Although the aims of such studies are best achieved by jointly analysing sequences from multiple loci in a model-based framework, such data currently exist for few nonmodel systems. We used existing genomic data and expressed sequence tags (ESTs) for Hymenoptera and other insects to design intron-crossing primers for 40 loci, mainly ribosomal proteins, for chalcidoid parasitoids. Amplification success was scored on a range of taxa associated with two natural communities; oak galls and figs. Taxa were chosen at increasing distance from Nasonia, which was used for primer design, (i) within Pteromalids, (ii) within Chalcidoidea (Eupelmidae, Eulophidae, Eurytomidae, Ormyridae, Torymidae) and (iii) for a selection of distantly related gall and fig wasps (Cynipidae, Agaonidae). To assess the utility of these loci for phylogeographic and population genetic studies, we compared genetic diversity between Western Palaearctic refugia for two species. Our results show that it is feasible to design a large number of exon-primed-intron-crossing (EPIC) loci that may be informative about phylogeographic history within species but amplify across a large taxonomic range.     

Development and mapping of ten porcine microsatellite markers

Thirty (TG)_n microsatellite clones were isolated from a pig genomic library, sequenced, and tested for their suitability to detect polymorphism on a panel of animals by means of the polymerase chain reaction. Ten of these clones were developed into suitable markers and subsequently segregation of these markers was determined in the five PiGMaP reference pedigrees. A linkage analysis was performed on these 10 microsatellites together with 365 other loci that have been typed on these reference families. Eight of the microsatellites have been mapped to eight different linkage groups that have been previously assigned to different chromosomes (chromosomes 1, 6, 7, 9, 14, 15, 17 and 18). Of the remaining two markers, one is X-linked and the other shows no linkage. The number of alleles detected by these microsatellites, in the reference pedigrees, varied from six to sixteen and the heterozygosity varied from 42 to 85% in the 26 unrelated founder animals of these reference pedigrees.     

Development of nine microsatellite markers for Pomacentrus amboinensis

The relatively long pelagic larval duration of Pomacentrus amboinensis, a tropical fish, suggests the potential for long-distance dispersal; however, several nongenetic studies have found substantial self-recruitment at one location. To analyse patterns of connectivity of this species, primers for nine independent microsatellite loci were developed for P. amboinensis using a magnetic bead enrichment protocol. Twenty individuals from one location were analysed and observed heterozygosities ranged from 0.7 to 0.95. Eight of nine loci were in Hardy-Weinberg equilibrium and no evidence of linkage or null alleles were found.     

Extensive genomic conservation of cattle microsatellite heterozygosity in sheep

We report the evaluation of 1036 bovine microsatellite primer pairs for their suitability as linkage markers in sheep. Approximately 58% (605/1036) of bovine primer pairs amplified a locus in sheep. Sixty-seven per cent (409/605) of amplified loci were detected as polymorphic. Marker heterozygosity, allele number and range of allele sizes were significantly lower in sheep than cattle sampled in this study. However, median fragment size was similar. These data suggest that high-resolution comparative linkage maps between closely related species can be constructed relatively efficiently.     

Chromosomal localization of six bovine microsatellite markers

Six lambda genomic clones containing polymorphic microsatellite (MS) markers were assigned to bovine chromosomes 1, 3, 5, 7, 13 and 24 by fluorescence in situ hybridization (FISH). Linkage data for four MS markers were presented earlier and linkage data for the remaining two on chromosome 7 and 24 are presented here. All assignments either orient or confirm the orientation of linkage groups relative to the centromere. A comparison of physical assignments and linkage intervals was possible on chromosome 5 (three loci, 38 cM) and 13 (two loci, 6 cM).     

Characterization of 42 highly polymorphic bovine microsatellite markers

We have isolated 42 highly polymorphic microsatellite (GT/CA)_n markers from Japanese black cattle Wagyu ( Bos taurus ). Forty-one of the markers were assigned to bovine autosomes with lod scores > 6, through linkage analyses performed on the International Bovine Reference Family Panel (IBRP). The remaining marker showed X-linked inheritance. These markers exhibited an average heterozygosity value of 0.67 with between four and 17 alleles on the IBRP.     

Development and polymorphisms of microsatellite markers for hinoki (Chamaecyparis obtusa)

Fifteen microsatellite markers were developed from a microsatellite‐enriched library and characterized using 32 Chamaecyparis obtusa individuals. The number of alleles ranged from two to 27 per locus, with observed heterozygosity ranging from 0.281 to 0.906. The polymorphism information content (PIC) was also calculated for each marker and the average was 0.796 ± 0.024. These microsatellite markers will be useful for investigating population genetics, reproductive ecology, tree improvement and constructing linkage maps of the species.