Chromosomal assignment of microsatellite loci in cotton

Microsatellite markers or simple sequence repeats (SSRs) represent a new class of genetic markers for cotton (Gossypium sp.). Sixty-five SSR primer pairs were used to amplify 71 marker loci and genotype 13 monosomic and 27 monotelodisomic cotton cytogenetic stocks. Forty-two SSR loci were assigned to cotton chromosomes or chromosome arms. Thirty SSRs were not located to specific chromosomes in this study. Nineteen marker loci were shown to occur on the A subgenome and 11 on the D subgenome by screening accessions of G. herbaceum (2n = 2x = 26 = 2A1) and G. raimondii (2n = 2x = 26 = 2D5). The aneuploid stocks proved to be very powerful tools for localizing SSR markers to individual cotton chromosomes. Multiplex PCR bins of the SSR primers and semiautomated detection of the amplified products were optimized in this experiment. Thirteen multiplex PCR bins were optimized to contain an average of 4 SSR primer pairs per bin. This provides a protocol for high-throughput genotyping of cotton SSRs that improves the efficiency of genetic mapping and marker-assisted programs utilizing SSR markers.     

Temporal-method estimates of Ne from highly polymorphic loci

The temporal method is used widely to estimategenetic effective population size (N _e), a parameter of fundamental interestto studies of evolutionary and conservationbiology. The statistical properties oftemporal-method estimates have not beenexplored for highly polymorphic DNA markersthat often contain many alleles occurring invery low frequencies. We used a Monte Carlosimulation approach to assess accuracy andprecision of the temporal method whenimplemented with haplotypic/allelic data atmitochondrial (mt)DNA and nuclear-encodedmicrosatellite DNA loci. Estimates of N _e were between 2%–106% greater thantheir true values in 48 simulationsparameterized using different demographicscenarios, models of mutation, and samplesizes. Overestimation of N _e resultsfrom a bias in the approximation used by Waples(1989) to derive the relationship between theexpected temporal variance (F) and N _e when allele frequencies are very closeto 0 or 1. Our results show that one commonlyapplied solution to this problem, binning oflow-frequency alleles, results in a trade-offof accuracy and precision in some cases. Weshow that both chi-square and normalapproximations are appropriate for estimating95% confidence intervals of N _e andwe develop a power analysis based on thechi-square distribution to estimate samplesizes and allelic diversity required toevaluate specific hypotheses. For highlypolymorphic loci like mtDNA andmicrosatellites, the increased precisionafforded by the presence of rare allelesoutweighs the upward bias in temporal-methodestimates of N _e.     

RFLP analysis of highly polymorphic loci in barley

Summary Barley middle-repeat sequences were screened for their ability to discriminate 51 barley commercial varieties. Two hordein clones, a clone encoding a leaf-specific thionin, a desiccation induced cDNA clone, a clone coding for 5S-rRNA and one corresponding to ubiquitin genes were tested. A very sensitive RFLP technique including four cutter restriction enzymes and denaturing 4% polyacrylamide gels were used to evidence the highest level of polymorphism.The RFLP data were analyzed by computer. Some probe/enzyme combinations were able to differentiate a large number of the cultivars tested, whereas three probe/enzyme combinations succeeded in identifying all the varieties. The use of this RFLP method can thus be suggested for cultivar identification in barley.     

Chromosomal assignment and genomic structure of Il15

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the β and γ chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.     

Eight highly polymorphic microsatellite loci for the Australian gecko Heteronotia binoei

Eight highly polymorphic dinucleotide microsatellite loci were developed for the Bynoe's gecko, Heteronotia binoei. Across the species as a whole, expected heterozygosities for the loci range from 0.59 to 0.92, with observed numbers of alleles ranging from 13 to 27. All eight loci successfully amplify in each of the three most widespread sexual chromosome races of Heteronotia binoei, and with the exception of one locus in one race all are polymorphic. All eight loci also amplify in hybrid parthenogenetic Bynoe's geckos, in several other sexual chromosome races, and in related Heteronotia species.     

Ten polymorphic microsatellite loci identified from a small insert genomic library for Peronospora tabacina

Ten polymorphic microsatellite loci for the obligate biotrophic, oomycete pathogen of tobacco, Peronospora tabacina, were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the 162 loci identified were composed of dinucleotide repeats, whereas only 4% and 11% were tri-and tetra-nucleotide repeats respectively. About 82% of all the microsatellites were perfect and within the library; only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic and 34 either failed to amplify or produced ambiguous/inconsistent results. The 10 polymorphic loci were characterized with 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East and North and South America. The number of alleles per locus was either three or four with a mean of 3.2, and the mean number of genotypes per locus was 3.6. Observed heterozygosity was 0.32-0.95, whereas expected heterozygosity was 0.44-0.69 for these loci. All loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci was 0.35-0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species of Peronospora.     

Cultivar identification in T. aestivum using highly polymorphic RFLP probes

Two probes, specific for HMW-glutenins and -gliadins have been used to identify 50 common wheat Italian cultivars, most of which are closely related, and four common wheat cultivars originating outside Italy. The probes revealed complex polymorphic patterns; three probe/enzyme combinations had the necessary sensitivity for the identification of all 54 cultivars. As already shown for potato and barley, the use of four-cutter restriction enzymes and polyacrylamide gels proved particularly useful for detecting polymorphism.     

Microsatellite‐enriched genomic libraries as a source of polymorphic loci for Schistosoma mansoni

Microsatellite markers for Schistosoma mansoni were developed using four genomic microsatellite‐enriched libraries. Microsatellites were observed in 65.4% of all sequences. Primer pairs were designed and tested for 23 loci. Eighteen loci produced amplification products, out of which 11 were polymorphic and were further characterized on 100 individuals of S. mansoni. Two to 19 alleles per locus were detected. The average values of expected and observed heterozygosities among the 11 loci were 0.79 and 0.59, respectively.     

Chromosomal assignment of 11 loci in the rat by mouse-rat somatic hybrids and linkage

Eleven rat genes have been assigned to rat chromosomes by use of mouse × rat somatic hybrids and/or use of linkage to known chromosome markers. Among them, the genes for the inducible nitric oxide synthase (Nos2) and for a vasoactive intestinal peptide receptor (Vipr) are potential candidates for genetic regulation of blood pressure and were localized to rat Chromosomes (Chrs) 10 and 8 respectively. Genes for gastric H,K-ATPase alpha subunit (Atp4a). Class I alcohol dehydrogenase (Adh), and aldolase C (Aldoc) were localized to Chrs 1, 2, and 10 respectively, and thus provide more DNA markers for genetic mapping of quantitative trait loci for blood pressure on those chromosomes. Genes for alkaline phosphatase (Alp1) and cardiac AE-3 Cl^-/HCO₃ ^- exchanger (Ae3) were both localized to Chr 9. Genes for glutamate dehydrogenase (Glud) and gastric H,K-ATPase beta subunit (Atp4b) were localized to Chr 16. The ornithine decarboxylase (Odc) gene and ornithine decarboxylase pseudogene (Odcp) were localized to Chrs 6 and 11 respectively.     

Characterization of highly polymorphic nuclear microsatellite loci in Juniperus communis L.

We developed five nuclear microsatellite markers in Juniperus communis L. using an enriched library method. Samples from 28 juniper individuals were collected in Spain, Germany and Slovakia and were analysed at the five loci. A high level of allelic diversity with values ranging from nine to 23 alleles was found. These highly polymorphic markers will be used in ongoing population genetic studies to evaluate the genetic resources and to contribute to the maintenance of genetic diversity of juniper in Middle Europe.     

Isolation of highly polymorphic microsatellite loci from the temperate damselfish Parma microlepis

Microsatellites were isolated from the damselfish Parma microlepis (Günther 1862) (Pomacentridae) and screened for 100 individuals. Seven of the eight loci tested were highly polymorphic, having 14–43 alleles with average heterozygosities between 0.86 and 0.97. These loci should be informative for studies on population genetics of this species.     

Characterization of highly polymorphic nuclear microsatellite loci from the ascidian Ciona intestinalis

Eight nuclear polymorphic microsatellite markers were characterized from the ascidian Ciona intestinalis whole genome sequence. The behaviour of these loci was investigated against two geographically distinct populations: one from Plymouth, UK the other from the Fusaro Lagoon, Italy, both belonging to the type A Ciona cryptic species. The markers exhibited six to 29 alleles and average observed heterozygosity ranging from 0.06 to 0.73. These new microsatellite loci demonstrated to be valuable tools for both population genetic analysis at different scales and genetic identification of mutant phenotypes frequently encountered in Mediterranean populations of C. intestinalis.     

Ten new highly polymorphic microsatellite loci in the blood clam Scapharca broughtonii

Ten novel microsatellite loci were isolated from blood clam Scapharca broughtonii, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. The number of alleles ranged from 11 to 23, and the observed and expected heterozygosities ranged from 0.63 to 0.93 and 0.66 to 0.95, respectively. Four loci showed significant Hardy–Weinberg disequilibrium at P < 0.05 level. The high variabilities revealed in this study suggest that microsatellites should prove useful for various genetic investigations.     

Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism

Microsatellite loci are regions of DNA containing tandem repeats of a short sequence motif; they occur abundantly in all eukaryotic genomes and have been shown to be a rich source of highly polymorphic genetic markers in humans and other mammals. These loci are particularly suitable for population studies because they can be relatively easily scored using a combination of polymerase chain reaction (PCR) amplification of each locus followed by electrophoresis to separate alleles. This paper details a method for finding these loci in any species. This method demonstrates that trinucleotide microsatellite loci are abundant and highly polymorphic in the social wasp Polistes annularis , whereas allozyme electrophoresis reveals very little polymorphism. The first six loci examined were all polymorphic with a mean observed heterozygosity of 0.62; in comparison average heterozygosity of 33 allozymes was 0.035. We suggest that this method can be used to detect variation where other methods have failed, making it an ideal tool for population and conservation geneticists who must deal with populations lacking other types of genetic variability.     

New polymorphic microsatellite loci for Theobroma cacao: isolation and characterization of microsatellites from enriched genomic libraries

Seventeen polymorphic microsatellite markers were isolated from enriched genomic libraries for Theobroma cacao, providing additional tools for studying the genetic diversity and map saturation of this species. These markers were characterized in 32 accessions of the T. cacao germplasm collection from the Centro de Pesquisas do Cacau. The number of alleles at each locus varied from 2 to 8, with an average of 4.41 alleles per locus. The polymorphism information content varied from 0.060 to 0.695, with an average of 0.333. The markers characterized in this study will be employed in map saturation studies and diversity assessments of cacao genotypes.     

Development and characterization of highly polymorphic microsatellite loci for the Western Spadefoot toad, Pelobates cultripes

Nine highly polymorphic microsatellite markers were isolated and characterized for the Western Spadefoot, Pelobates cultripes. Remarkably, for this amphibian species high numbers of microsatellites were found as part of larger repeat containing regions, making primer design difficult. For nine loci, primers were designed successfully and genotyping of individuals was reliable and consistent. Number of alleles and heterozygosity for these loci ranged from 9 to 34 and from 0.72 to 0.94, respectively. The high levels of polymorphism revealed by our developed loci should provide insight into population genetic structure and levels of dispersal for this typical Mediterranean temporary pond-breeding amphibian.     

Eleven new highly polymorphic microsatellite loci in the yellow croaker,Pseudosciaena crocea

We developed 11 new microsatellite markers in Pseudosciaena crocea by screening an enriched genomic library using nonradioactive polymerase chain reaction (PCR) techniques. All loci were found to be polymorphic with an average of 14.9 alleles per locus (range four to 30). The mean observed and expected heterozygosities were 0.86 (range 0.57–1.00) and 0.90 (range 0.62–0.98), respectively. Four loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for population genetic studies of P. crocea.