Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity.

Genetic diversity of the cestode Echinococcus multilocularis in red foxes at a continental scale in Europe

BACKGROUND: Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level. METHODOLOGY/PRINCIPAL FINDINGS: The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas. CONCLUSIONS/SIGNIFICANCE: The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a "mainland-island" system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.

Genetic diversity among alfalfa genotypes (Medicago sativa L.) of non-dormant cultivars using SSR markers and agronomic traits

The aim of this study was to assess genetic diversity among 40 alfalfa (Medicago sativa L.) genotypes of different non-dormant (FD=8) cultivars. Biomass yield, regrowth speed and reaction to spring black stem, lepto leaf spot, and rust were evaluated. Analyses of variances were performed using a mixed model to examine the agronomic variation among individuals. A principal component analysis on standardized agronomic data was performed. Agronomic data were also used to calculate Gower's distance and UPGMA algorithm. For the molecular analysis, six SSR markers were evaluated and 84 alleles were identified. The genetic distance was estimated using standard Nei's distance. Average standard genetic diversity was 0.843, indicating a high degree of variability among genotypes. Finally, a generalized procrustes analysis was performed to calculate the correlation between molecular and agronomic distance, indicating a 65.4% of consensus. This value is likely related to the low number of individuals included in the study, which might have underestimated the real phenotypic variability among genotypes. Despite the low number of individuals and SSR markers analyzed, this study provides a baseline for future diversity studies to identify genetically distant alfalfa individuals or cultivars.

Caracterización genética de germoplasma de maní cultivado (Arachis hypogaea L.) mediante el empleo de marcadores microsatélites

El maní cultivado (Arachis hypogaea L.), es una especie de gran importancia económica, nativo de América del Sur. Se divide en dos subespecies y seis variedades botánicas. Genéticamente es alotetraploide, constituido por dos juegos genómicos duplicados. El empleo de marcadores microsatélites resulta más apropiado para realizar la caracterización genética de esta especie, puesto que permiten detectar un elevado nivel de polimorfismo. El objetivo del presente trabajo fue caracterizar la diversidad genética existente en las entradas de germoplasma de maní cultivado pertenecientes al Banco Activo de Germoplasma de Maní del Instituto Nacional de Tecnología Agropecuaria (INTA). Veinticinco entradas fueron genotipificadas con 23 marcadores microsatélites, de los cuales, 17 resultaron polimórficos. Se observaron 75 fragmentos polimórficos amplificados, con un promedio de 4,41 alelos por locus y un rango de 1 a 9 alelos. El contenido de información polimórfica osciló entre 0,15 y 0,58. El valor de la diversidad genética promedio fue de 0,165. Tanto el análisis de conglomerados como el de coordenadas principales evidenciaron dos grupos, uno formado por los materiales representantes de la subespecie fastigiata y otro por los de la subespecie hypogaea. Los resultados del análisis molecular de la varianza mostraron varianza tanto dentro como entre, las subespecies analizadas.

Detection of growth-related QTLs in turbot (Scophtalmus maximux)

Background The turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor. Results Eight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection. Conclusions The use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21–23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21–33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores ≥1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

ANX7, a candidate tumor suppressor gene for prostate cancer

The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P = 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Genetic analysis along an invasion pathway reveals endemic cryptic taxa, but a single species with little population structure in the introduced range

The invasion pathways of pest arthropods can be traced using genetic tools to develop an understanding of the processes that have shaped successful invasions and to inform both pest management and conservation strategies in their non-native and native ranges, respectively. The redlegged earth mite, Halotydeus destructor, is a major economic pest in Australia, successfully establishing and spreading after arrival from South Africa more than 100 years ago. Halotydeus destructor has recently expanded its range and evolved resistance to numerous pesticides in Australia, raising questions around its origin and spread. Location: South Africa and Australia. Methods: We sampled H. destructor populations in South Africa and Australia and developed a microsatellite marker library. We then examined genetic variation using mtDNA and microsatellite markers across both native and invasive ranges to determine endemic genetic diversity within South Africa, identify the likely origin of invasive populations and test genetic divergence across Australia. Results: The data show that H. destructor comprises a cryptic species complex in South Africa, with putative climatic/host plant associations that may correspond to regional variation. A lineage similar to that found near Cape Town has spread throughout Western and eastern Australia, where populations remain genetically similar. Main conclusions: Tracing the invasion pathway of this economically important pest revealed cryptic lineages in South Africa which points to the need for a taxonomic revision. The absence of significant genetic structure across the wide invasive range of H. destructor within Australia has implications for the development (and spread) of pesticide resistance and also points to recent local adaptation in physiological traits.

Denaturing high performance liquid chromatography for the detection of microsatellite instability using Bethesda and pentaplex marker panels

Microsatellite instability (MSI) is a characteristic molecular phenotype of tumors from the hereditary nonpolyposis colorectal cancer (Lynch) syndrome. Routine MSI screening of tumors in patients is an efficient prescreening tool for the population-based detection of Lynch syndrome in the absence of family cancer history. We describe here the optimization of a denaturing high performance liquid chromatography (DHPLC) assay for MSI analysis with the

The genomic Echinococcus microsatellite EmsB sequences: from a molecular marker to the epidemiological tool

In the field of molecular and epidemiological parasitology, characterization of fast evolving genetic markers appears as an important challenge to consider the diversity and genetic structure of parasites. The study of respective populations can help us to understand their adaptive strategies to survive and perpetuate the species within different host populations, all trying to resist infection. In the past, the relative monomorphic features of Echinococcus multilocularis, the causative agent of alveolar echinococcosis and a severe human parasitic disease, did not stimulate studies dealing with the genetic variability of Echinococcus species or respective populations. A recently developed, characterized and validated original multilocus microsatellite, named EmsB, tandemly repeated in the genome, offered an additional opportunity for this line of investigation. We have compiled in this review new insights brought by this molecular tracker on the transmission activity of Echinococcus among different hosts and at different geographical scales.

Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii)

Habitat fragmentation as a result of urbanisation is a growing problem for native lizard species. The Eastern Water Dragon (Physignathus lesueurii) is a social arboreal agamid lizard, native to Australia. This species represents an ideal model species to investigate the effect of urbanisation because of their prominent abundance in the urban landscape. Here we describe the isolation and characterisation of a novel set of 74 di-, tri-, and tetramicrosatellites from which 18 were selected and optimised into two multiplexes. The 18 microsatellites generated a total 148 alleles across the two populations. The number of alleles per locus varied from 2 to 18 alleles and measures of Ho and He varied from 0.395 to 0.877 and from 0.441 to 0.880, respectively. We also present primers for four novel mitochondrial DNA (mtDNA) markers. The combined length of the four mtDNA marker pairs was 2,528 bp which included 15 nucleotides changes. In comparison to threatened species, which are generally characterised by small population sizes, the Eastern Water Dragon represents an ideal model species to investigate the effect of urbanisation on their behavioural ecology and connectivity patterns among populations.

Development and characterisation of tri- and tetra-nucleotide polymorphic microsatellite markers for skipjack tuna (Katsuwonus pelamis)

Skipjack tuna (katsuwonus pelamis) (SJT) is the largest tuna fishery in all the major oceans around the world, and the largest marine fishery in Sri Lanka. Knowledge of genetic population structure and effective population size of SJT in the Indian Ocean and other major oceans, however, is still lacking for better management practices and conservation strategies. We developed microsatellite genetic markers using SJT around Sri Lanka in the Indian Ocean, and characterise one tri- and seven tetra-nucleotide microsatellite loci isolated from enriched genomic libraries from SJT, to provide tools for addressing both conservation and fisheries management questions. An analysis of these eight microsatellite markers in two populations of SJT from eastern Sri Lanka (n = 44) and the Maldives Islands (n = 53) showed that all eight microsatellites were polymorphic with an average number of alleles per locus of 11.80 (range 5-27). Expected heterozygosities at marker loci ranged from 0.450 to 0.961. These markers are being used currently to characterise population structure and extent of natural gene flow in SJT populations from the eastern and western Indian Ocean. No significant linkage disequilibrium was detected among any loci pairs.

Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 Pyrosequencing

Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10–5) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

Development and characterization of microsatellite markers in the sea squirt, Halocynthia roretzi

We characterized nine new microsatellite markers isolated from (GT) n and (CT) n microsatellite- enriched genomic libraries of the sea squirt ( Halocynthia roretzi ). All markers were polymorphic in 92 individuals from a single natural population with 2–21 (mean 9.22) alleles per locus. The observed and expected heterozy-gosity of these markers were 0.086–0.886 and 0.102–0.870, respectively.One marker (Hr2004) significantly deviated from Hardy–Weinberg equilibrium. These new microsat-ellite markers should be useful for assessing the genetic diversity and population structure in H. roretzi.