A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.)

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance

Diversidad genética de la colección núcleo de silvestres de fríjol común (Phaseolus vulgaris L.) analizados mediante microsatélites fluorescentes

Las colecciones núcleo desarrollan un papel importante en la utilización de los recursos genéticos, ya que poseen el material prioritario para la mejora. El desarrollo y la aplicación de SSRs facilitan la adquisición de una gran cantidad de información genética para la caracterización de colecciones de germoplasma. El objetivo de este estudio fue Caracterizar la diversidad genética de 104 accesiones silvestres de la Colección Núcleo de fríjol común (Phaseolus vulgaris L.) procedentes de la Unidad de Recursos Genéticos del CIAT (URG-CIAT), mediante el uso de 36 marcadores moleculares tipo microsatélites analizados con una técnica de fluorescencia. Se identificaron un total de 492 alelos con un promedio de 13.30 alelos por marcador, y un promedio de Contenido de Información Polimórfica de 0.64. Usando los programas NTSyS y STRUCTURE, se identificaron cinco grupos bien definidos correspondiendo a los dos principales acervos: Mesoamérica y Andino, y los centros menores de Colombia, Ecuador-Norte del Perú y Guatemala. Accesiones provenientes de Guatemala y el sur de México presentaron diferenciación genética y estructural con los otros acervos y centros menores. Se presenta una alta diversidad genética en el acervo Mesoamérica que en el Andino, presentándose en este ultimo asociaciones discretas en ciertas regiones, posiblemente reflejando el aislamiento geográfico y genético. Un alto valor de FST confirmó la congruente separación de los grupos en cuanto a suestructura genética

Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes

Carmina Gallardo, Evans A. Taracha,Edward Okoth & Richard P. Bishop are ILRI authorsComplete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach

Data from: Resequencing of common bean identifies regions of inter-gene pool introgression and provides comprehensive resources for molecular breeding

Common bean is the most important grain legume for human consumption and a main nutrition source in the tropics. Because bean production is reduced by both abiotic and biotic constraints, current breeding efforts are focused on the development of improved varieties with tolerance to these stresses. We characterized materials from different breeding programs spanning three continents to understand their sequence diversity and advance the development of molecular breeding tools. For this, 37 varieties belonging to P. vulgaris, P. acutifolius, and P. coccineus were sequenced by whole-genome sequencing (WGS), identifying more than 40 million genomic variants. Evaluation of nuclear DNA content and analysis of copy number variation revealed important differences in genomic content not only between P. vulgaris and two other domesticated Phaseolus species, but also within P. vulgaris, affecting hundreds of protein-coding genomic regions. A large number of inter-gene-pool introgressions were identified. Furthermore, interspecific introgressions for disease resistance in breeding lines were mapped. Evaluation of newly developed SNP markers within previously discovered QTL for common bacterial blight and angular leaf spot provide improved specificity to tag sources of resistance to these diseases. We expect that this dataset will provide a deeper molecular understanding of breeding germplasm and deliver molecular tools for germplasm development, aiming to increase the efficiency of bean breeding programs

Data from: Resequencing of common bean identifies regions of inter-gene pool introgression and provides comprehensive resources for molecular breeding

Common bean is the most important grain legume for human consumption and a main nutrition source in the tropics. Because bean production is reduced by both abiotic and biotic constraints, current breeding efforts are focused on the development of improved varieties with tolerance to these stresses. We characterized materials from different breeding programs spanning three continents to understand their sequence diversity and advance the development of molecular breeding tools. For this, 37 varieties belonging to P. vulgaris, P. acutifolius, and P. coccineus were sequenced by whole-genome sequencing (WGS), identifying more than 40 million genomic variants. Evaluation of nuclear DNA content and analysis of copy number variation revealed important differences in genomic content not only between P. vulgaris and two other domesticated Phaseolus species, but also within P. vulgaris, affecting hundreds of protein-coding genomic regions. A large number of inter-gene-pool introgressions were identified. Furthermore, interspecific introgressions for disease resistance in breeding lines were mapped. Evaluation of newly developed SNP markers within previously discovered QTL for common bacterial blight and angular leaf spot provide improved specificity to tag sources of resistance to these diseases. We expect that this dataset will provide a deeper molecular understanding of breeding germplasm and deliver molecular tools for germplasm development, aiming to increase the efficiency of bean breeding programs

African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site

The role of the ancestral sylvatic cycle of the African swine fever virus (ASFV) is not well understood in the endemic areas of eastern Africa. We therefore analysed the ASF infection status on samples collected from 51 free-ranging warthogs (Phacocherus africanus) and 1576 Ornithodorus porcinus ticks from 26 independent warthog burrows at a single ranch in Kenya. Abattoir samples from 83 domestic pigs without clinical symptoms, originating from specific locations with no recent reported ASF outbreaks were included in this study. All samples were derived from areas of central Kenya, where ASF outbreaks have been reported in the past. Infection with ASFV was confirmed in 22% of O. porcinus pools, 3.22% of adult warthog serum samples and 49% of domestic pig serum samples by using p72-based PCR. All of the warthog sera were positive for anti-ASFV antibodies, investigated by using ELISA, but none of the domestic pig sera were positive. Twenty O. porcinus-, 12 domestic pig- and three warthogderived viruses were genotyped at four polymorphic loci. The ASFV isolates from ticks and domestic pigs clustered within p72 genotype X. By contrast, ASF viruses genotyped directly from warthog sera, at same locality as the tick isolates, were within p72 genotype IX and genetically similar to viruses causing recent ASF outbreaks in Kenya and Uganda. This represents the first report of the co-existence of different ASFV genotypes in warthog burrow-associated ticks and adult wild warthogs. The data from this and earlier studies suggest transfer of viruses of at least two different p72 genotypes, from wild to domestic pigs in East Africa

Molecular Diagnosis of African Swine Fever by a New Real-Time PCR Using Universal Probe Library

A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n=46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n=260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous ?-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF

Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness, makes antibody detection techniques essential in control programmes. Recent reports appears to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1,000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs

Surveillance for African Swine fever in Nigeria, 2006-2009

African swine fever (ASF) has had significant economic and social impact in Nigeria since 1997. However, there has been no effective national response to bring it under control. In this report, we confirm that ASF is still prevalent and widespread in Nigeria. Results from both serosurveillance and virological analyses indicated that ASF is present in most of the agro-ecological zones of the country. Nine per cent (9%) of serum samples and 48% of tissue samples were positive for ASF virus antibody and genome, respectively. Areas with high pig-related activities (marketing, consumption and farming) have higher prevalences compared with areas with less pig activities. Farm-gate buyers, marketing systems and transport of untested pigs within the country assist with the circulation of the virus. Only by putting in place a comprehensive routine surveillance and testing system, reorganizing the market and transportation systems for pigs, implementing on-farm bio-security protocols and considering the option of compensation will it be possible to achieve a significant reduction in ASF prevalence in Nigeria