BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

Background

Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively.

Results

We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.

Conclusion

These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.

     

Local spread of classical swine fever upon virus introduction into The Netherlands: Mapping of areas at high risk

Background

In the recent past, the introduction of Classical Swine Fever Virus (CSFV) followed by between-herd spread has given rise to a number of large epidemics in The Netherlands and Belgium. Both these countries are pork-exporting countries. Particularly important in these epidemics has been the occurrence of substantial "neighborhood transmission" from herd to herd in the presence of base-line control measures prescribed by EU legislation. Here we propose a calculation procedure to map out "high-risk areas" for local between-herd spread of CSFV as a tool to support decision making on prevention and control of CSFV outbreaks. In this procedure the identification of such areas is based on an estimated inter-herd distance dependent probability of neighborhood transmission or "local transmission". Using this distance-dependent probability, we derive a threshold value for the local density of herds. In areas with local herd density above threshold, local transmission alone can already lead to epidemic spread, whereas in below-threshold areas this is not the case. The first type of area is termed 'high-risk' for spread of CSFV, while the latter type is termed 'low-risk'.

Results

As we show for the case of The Netherlands, once the distance-dependent probability of local transmission has been estimated from CSFV outbreak data, it is possible to produce a map of the country in which areas of high-risk herds and of low-risk herds are identified. We made these maps even more informative by estimating border zones between the two types of areas. In these border zones the risk of local transmission of infection to a nearby high-risk area exceeds a certain level.

Conclusion

The risk maps provide an easily understandable visualization of the spatial heterogeneities in transmission risk. They serve as a tool for area-specific designs of control strategies, and possibly also for spatial planning of areas where livestock farming is allowed. Similar risk maps can in principle be constructed for other highly-transmissible livestock infections that spread via neighborhood transmission.

     

Mapping carcass and meat quality QTL on Sus Scrofa chromosome 2 in commercial finishing pigs

Quantitative trait loci (QTL) affecting carcass and meat quality located on SSC2 were identified using variance component methods. A large number of traits involved in meat and carcass quality was detected in a commercial crossbred population: 1855 pigs sired by 17 boars from a synthetic line, which where homozygous (A/A) for IGF2. Using combined linkage and linkage disequilibrium mapping (LDLA), several QTL significantly affecting loin muscle mass, ham weight and ham muscles (outer ham and knuckle ham) and meat quality traits, such as Minolta-L* and -b*, ultimate pH and Japanese colour score were detected. These results agreed well with previous QTL-studies involving SSC2. Since our study is carried out on crossbreds, different QTL may be segregating in the parental lines. To address this question, we compared models with a single QTL-variance component with models allowing for separate sire and dam QTL-variance components. The same QTL were identified using a single QTL variance component model compared to a model allowing for separate variances with minor differences with respect to QTL location. However, the variance component method made it possible to detect QTL segregating in the paternal line (e.g. HAMB), the maternal lines (e.g. Ham) or in both (e.g. pHu). Combining association and linkage information among haplotypes improved slightly the significance of the QTL compared to an analysis using linkage information only.     

Neurotoxic,cytotoxic, apoptotic and antiproliferative effects of some marine algae extracts on the NA2B cell line

Oxidative stress contributes to cancer pathologies and to apoptosis. Marine algae exhibit cytotoxic, antiproliferative and apoptotic effects; their metabolites have been used to treat many types of cancer. We investigated in culture extracts of Petalonia fascia, Jania longifurca and Halimeda tuna to determine their effects on mouse neuroblastoma cell line, NA2B. NA2B cells were treated with algae extracts, and the survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of algae extracts on oxidative stress in NA2B cells also were investigated using nitric oxide synthase (NOS) immunocytochemistry and apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling. We observed significant neurite inhibition with moderate damage by the neurotoxicity-screening test (NST) at IC₅₀ dilutions of the extracts. MTT demonstrated that J. longifurca extracts were more toxic than P. fascia and H. tuna extracts. We found an increase of endothelial and inducible NOS immunostaining for oxidative stress and TUNEL analysis revealed increased apoptosis after application of extract. Our findings suggest that the algae we tested may have potential use for treatment of cancer.     

Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study

Background

Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.

Methods

28 patients (14 patients with mild allergic asthma and 14 healthy non-allergic controls) were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests (Wilcoxon, Mann Whitney and Spearman correlation).

Results

13 patients with mild asthma (6 females, 19-29 y) and 11 healthy controls (10 females, 19-31 y) had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients (t = -1: 706 s vs. t = 6: 498 s; p = 0.02), but not of controls (t = -1: 693 s vs. t = 6: 636 s; p = 0.65). The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection (r = -0.517 and -0.528 resp., both p = 0.01).Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma (26.0 ng/mL vs. 11.5 ng/mL in healthy controls, p = 0.04). Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.

Conclusions

Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.

Trial registration

This trial was registered at the Dutch trial registry (http://www.trialregister.nl): NTR1677.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Breeding value prediction for production traits in layer chickens using pedigree or genomic relationships in a reduced animal model

Background

Genomic selection involves breeding value estimation of selection candidates based on high-density SNP genotypes. To quantify the potential benefit of genomic selection, accuracies of estimated breeding values (EBV) obtained with different methods using pedigree or high-density SNP genotypes were evaluated and compared in a commercial layer chicken breeding line.

Methods

The following traits were analyzed: egg production, egg weight, egg color, shell strength, age at sexual maturity, body weight, albumen height, and yolk weight. Predictions appropriate for early or late selection were compared. A total of 2,708 birds were genotyped for 23,356 segregating SNP, including 1,563 females with records. Phenotypes on relatives without genotypes were incorporated in the analysis (in total 13,049 production records).The data were analyzed with a Reduced Animal Model using a relationship matrix based on pedigree data or on marker genotypes and with a Bayesian method using model averaging. Using a validation set that consisted of individuals from the generation following training, these methods were compared by correlating EBV with phenotypes corrected for fixed effects, selecting the top 30 individuals based on EBV and evaluating their mean phenotype, and by regressing phenotypes on EBV.

Results

Using high-density SNP genotypes increased accuracies of EBV up to two-fold for selection at an early age and by up to 88% for selection at a later age. Accuracy increases at an early age can be mostly attributed to improved estimates of parental EBV for shell quality and egg production, while for other egg quality traits it is mostly due to improved estimates of Mendelian sampling effects. A relatively small number of markers was sufficient to explain most of the genetic variation for egg weight and body weight.     

Enrichment of DNRA bacteria in a continuous culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h⁻¹). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.     

Genomic selection of purebred animals for crossbred performance in the presence of dominant gene action

Background

Genomic selection is an appealing method to select purebreds for crossbred performance. In the case of crossbred records, single nucleotide polymorphism (SNP) effects can be estimated using an additive model or a breed-specific allele model. In most studies, additive gene action is assumed. However, dominance is the likely genetic basis of heterosis. Advantages of incorporating dominance in genomic selection were investigated in a two-way crossbreeding program for a trait with different magnitudes of dominance. Training was carried out only once in the simulation.

Results

When the dominance variance and heterosis were large and overdominance was present, a dominance model including both additive and dominance SNP effects gave substantially greater cumulative response to selection than the additive model. Extra response was the result of an increase in heterosis but at a cost of reduced purebred performance. When the dominance variance and heterosis were realistic but with overdominance, the advantage of the dominance model decreased but was still significant. When overdominance was absent, the dominance model was slightly favored over the additive model, but the difference in response between the models increased as the number of quantitative trait loci increased. This reveals the importance of exploiting dominance even in the absence of overdominance. When there was no dominance, response to selection for the dominance model was as high as for the additive model, indicating robustness of the dominance model. The breed-specific allele model was inferior to the dominance model in all cases and to the additive model except when the dominance variance and heterosis were large and with overdominance. However, the advantage of the dominance model over the breed-specific allele model may decrease as differences in linkage disequilibrium between the breeds increase. Retraining is expected to reduce the advantage of the dominance model over the alternatives, because in general, the advantage becomes important only after five or six generations post-training.

Conclusion

Under dominance and without retraining, genomic selection based on the dominance model is superior to the additive model and the breed-specific allele model to maximize crossbred performance through purebred selection.