A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.

Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci of P. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289-296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types

Familial Paget's disease of bone: Nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. Susceptibility loci for Paget's disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q121.1-q22 (PDB2) in different pedigrees, We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Paget's disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log,, of the odds ratio (LOD) scores < -2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < -2.0) centered on D18S42, These data confirm the genetic heterogeneity of Paget's disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.

Genetic dependence of the specific T-cell cytokine response to Porphyromonas gingivalis in mice

Background: Susceptibility to periodontal infections may, in part, be genetically determined. Porphyromonas gingivalis is a major periodontopathogen, and the immune response to this organism requires T-cell help. The aim of the present study was to examine the specific T-cell cytokine responses to P gingivalis outer membrane antigens in a mouse model and their relationship with H-2 haplotype. Methods: BALB/c and DBA/2J (H-2(d)), CBACaH (H-2(k)), and C57BL6 (H-2(b)) mice were immunized with P gingivalis outer membrane antigens weekly for 3 weeks. One week after the final injection, the spleens were removed, and 6 T-cell lines specific for P gingivalis were established for each mouse strain. The percentage of CD4 and CD8 cells in the P gingivalis-specific T-cell lines staining positive for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10 was determined by 2-color flow cytometry. Results: The cytokine profiles of T-cell lines from BALB/c and DBA/2J mice showed no significant differences. Significantly fewer IL4+, IFN-gamma+, and IL-10+ CD4 cells than IL-4+, IFN-gamma+, and IL-10+ CD8 cells, respectively, were demonstrated for both strains. P gingivalis-specific T-cell lines generated from CBACaH mice were similar to those generated from BALB/c and DBA/2J mice; however, the mean percentage of IL4+ CD4 cells in CBACaH mice was lower than the percentage of IFN-gamma+ CD4 cells. Also, the mean percentage of IFN-gamma+ CD4 cells in CBACaH mice was significantly increased compared to DBA/2J mice. Unlike the other 3 strains, T-cell lines established from C57BL6 mice contained similar percentages of cytokine-positive cells, although the percentage of IL-4+ CD4 cells was reduced in comparison to the percentage of CD8 cells. However, comparisons with the other 3 strains demonstrated a higher percentage of IL-4+ CD4 cells than in lines established from the spleens of DBA/2J mice, IFN-gamma+ CD4 cells than in lines established from BALB/c and CBACaH mice, and IL-10+ CD4 cells than in lines established from all 3 other strains. No significant differences in the percentage of positive CD8 cells were demonstrated between lines in the 4 strains of mice. Conclusion: The specific T-cell response to P gingivalis in mice may, in the case of the CD4 response, depend on MHC genes. These findings are consistent with the concept that patient susceptibility is important to the outcome of periodontal infection and may, in part, be genetically determined.

The limits of environmental responsibility - A stormwater case study

The motivation for concern about the environment beyond one's neighborhood is still relatively poorly understood. This article examines the determinants of feelings of responsibility at a regional watershed level. Using demographic, attitudinal, self-reported behavior and neighborhood mapping measures from four cities in Australia, five hypotheses were derived. These were that wider environmental concerns would depend on (a) the physical and social characteristics of the respondents' neighborhoods, (b) the size of their perceived neighborhoods, (c) the length of residence at their localities, (d) educational level and attitudes toward environmental moral responsibility (and the interaction between them), and (e) the level of reported environmentally friendly behavior. Support was gained for all hypotheses except length of residence and the role of general moral attitudes toward the environment. It is concluded that to explain community action at the regional level, it is important to include both spatial and psychological insights and methodologies in research.

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKTI1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athktl/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Genetic consequences of sequential founder events by an island-colonizing bird

The importance of founder events in promoting evolutionary changes on islands has been a subject of long-running controversy. Resolution of this debate has been hindered by a lack of empirical evidence from naturally founded island populations. Here we undertake a genetic analysis of a series of historically documented, natural colonization events by the silvereye species-complex (Zosterops lateralis), a group used to illustrate the process of island colonization in the original founder effect model. Our results indicate that single founder events do not affect levels of heterozygosity or allelic diversity, nor do they result in immediate genetic differentiation between populations. Instead, four to five successive founder events are required before indices of diversity and divergence approach that seen in evolutionarily old forms. A Bayesian analysis based on computer simulation allows inferences to be made on the number of effective founders and indicates that founder effects are weak because island populations are established from relatively large flocks. Indeed, statistical support for a founder event model was not significantly higher than for a gradual-drift model for all recently colonized islands. Taken together, these results suggest that single colonization events in this species complex are rarely accompanied by severe founder effects, and multiple founder events and/or long-term genetic drift have been of greater consequence for neutral genetic diversity.

Identity and genetic diversity of the sorghum ergot pathogen in Australia

Sorghum ergot was first discovered in Australia in 1996. It affects seed production and grain usage in stock feed due to concerns of animal toxicity. Three species of Claviceps are known to cause ergot of sorghum with different epidemiological, animal toxicity, and management implications. Claviceps africana was identified as the causal agent but morphological differences between isolates raised the possibility of more than one species being involved. The major aim of this study was to identify the Claviceps species causing sorghum ergot and to determine the genetic diversity among isolates of the ergot pathogen from Australia and overseas. Symptom development, sequencing of the ITS1 region, and radiolabelled DNA amplification fingerprints (RAF) were used to confirm that ergot of sorghum in Australia is caused by C. africana. The morphology of sphacelia, microconidia, macroconidia, and secondary conidia of all 36 Australian isolates studied matched the description for C. africana and the DNA sequence of the ITS1 region of 2 selected Australian isolates was identical to that of C. africana. Based on RAF analysis of 110 Australian and overseas isolates of Claviceps spp., C. africana isolates could be clearly distinguished (

Combining the analyses of introgressive hybridisation and linkage mapping to investigate the genetic architecture of population divergence in the lake whitefish (Coregonus clupeaformis, Mitchill)

Adaptation and reproductive isolation, the engines of biological diversity, are still elusive when discussing the genetic bases of speciation. Namely, the number of genes and magnitude of selection acting positively or negatively on genomic traits implicated in speciation is contentious. Here, we describe the first steps of an ongoing research program aimed at understanding the genetic bases of population divergence and reproductive isolation in the lake whitefish (Coregonus clupeaformis). A preliminary linkage map originating from a hybrid cross between dwarf and normal ecotypes is presented, whereby some of the segregating AFLP markers were found to be conserved among natural populations. Maximum-likelihood was used to estimate hybrid indices from non-diagnostic markers at 998 AFLP loci. This allowed identification of the most likely candidate loci that have been under the influence of selection during the natural hybridisation of whitefish originating from different glacial races. As some of these loci could be identified on the linkage map, the possibility that selection of traits in natural populations may eventually be correlated to specific chromosomal regions was demonstrated. The future prospects and potential of these approaches to elucidate the genetic bases of adaptation and reproductive isolation among sympatric ecotypes of lake whitefish is discussed.