Analysis of the structure and dynamics of human serum albumin

A computational approach to designing a peptide-based ligand for the purification of human serum albumin (HSA) was undertaken using molecular docking and molecular dynamics (MD) simulation. A three-step procedure was performed to design a specific ligand for HSA. Based on the candidate pocket structure of HSA (warfarin binding site), a peptide library was built. These peptides were then docked into the pocket of HSA using the GOLD program. The GOLDscore values were used to determine the affinity of peptides for HSA. Consequently, the dipeptide Trp–Trp, which shows a high GOLDscore value, was selected and linked to a spacer arm of Lys[CO(CH₂)₅NH] on the surface of ECH-lysine sepharose 4 gel. For further evaluation, the Autodock Vina program was used to dock the linked compound into the pocket of HSA. The docking simulation was performed to obtain a first guess of the binding structure of the spacer–Trp–Trp–HSA complex and subsequently analyzed by MD simulations to assess the reliability of the docking results. These MD simulations indicated that the ligand–HSA complex remains stable, and water molecules can bridge between the ligand and the protein by hydrogen bonds. Finally, absorption spectroscopic studies were performed to illustrate the appropriateness of the binding affinity of the designed ligand toward HSA. These studies demonstrate that the designed dipeptide can bind preferentially to the warfarin binding site. Graphical Abstract

Three-step computational approach to the design of a dipeptide ligand for human serum albumin purification exploiting structure-based docking and molecular dynamics simulation     

Influence of CdCl2 and CdSO4 supplementation on Cd distribution and ligand environment in leaves of the Cd hyperaccumulator Noccaea (Thlaspi) praecox

Background and aims

The aim was to investigate whether different Cd salts in the nutrient solution of the Cd/Zn hyperaccumulator Noccaea (Thlaspi) praecox alter leaf Cd distribution and Cd ligand environment, and plant fitness.

Methods

Plants were grown for 8 weeks with 100/300 μM CdCl₂ or CdSO₄. Leaf biomass, and total chlorophyll, anthocyanin, Cd, Cl, S and P concentrations were monitored. Cd localisation and ligand environment in leaves were analysed using quantitative synchrotron-based micro-X-ray fluorescence imaging, and Cd K-edge X-ray absorption fine structure and Cd L₃-edge micro-X-ray absorption near-edge structure measurements.

Results

Cd uptake and plant fitness were comparable for CdCl₂ and CdSO₄ treatments, and depended on applied Cd concentration. In all treatments, Cd preferentially accumulated with high concentrations of Cl in vacuoles of large vacuolarised epidermal cells, bound mainly to oxygen-based (O)-ligands. In the mesophyll of CdCl₂? treated plants, Cd was preferentially sequestered in vacuoles, while for CdSO₄, Cd accumulated preferentially in the apoplast. In the symplast, O-ligands increased with increasing Cd concentrations; in the apoplast, sulphur-based (S)-ligands prevailed.

Conclusions

Cd partitioning between leaf mesophyll apoplast and symplast and the Cd ligand environment in N. praecox depend on the Cd salt type and concentration added to the nutrient solution.     

The significance of D-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps

Background

D-amino acids are far less abundant in nature than L-amino acids. Both L- and D-amino acids enter soil from different sources including plant, animal and microbial biomass, antibiotics, faeces and synthetic insecticides. Moreover, D-amino acids appear in soil due to abiotic or biotic racemization of L-amino acids. Both L- and D-amino acids occur as bound in soil organic matter and as “free“ amino acids dissolved in soil solution or exchangeably bound to soil colloids. D-amino acids are mineralized at slower rates compared to the corresponding L-enantiomers. Plants have a capacity to directly take up “free“ D-amino acids by their roots but their ability to utilize them is low and thus D-amino acids inhibit plant growth.

Scope

The aim of this work is to review current knowledge on D-amino acids in soil and their utilization by soil microorganisms and plants, and to identify critical knowledge gaps and directions for future research.

Conclusion

Assessment of “free“ D-amino acids in soils is currently complicated due to the lack of appropriate extraction procedures. This information is necessary for consequent experimental determination of their significance for crop production and growth of plants in different types of managed and unmanaged ecosystems. Hypotheses on occurrence of “free“ D-amino acids in soil are presented in this review.     

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Background

The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated.

Results

The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples.

Conclusions

This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.     

QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber

Key message

Next-generation sequencing enabled a fast discovery of a major QTL controlling early flowering in cucumber, corresponding to the FT gene conditioning flowering time in Arabidopsis.

Abstract

Next-generation sequencing technologies are making it faster and more efficient to establish the association of agronomic traits with molecular markers or candidate genes, which is the requirement for marker-assisted selection in molecular breeding. Early flowering is an important agronomic trait in cucumber (Cucumis sativus L.), but the underlying genetic mechanism is unknown. In this study, we identified a candidate gene for early flowering QTL, Ef1.1 through QTL-seq. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Muromskij” (early flowering) and “9930” (late flowering) suggested quantitative nature of flowering time in cucumber. Genome-wide comparison of SNP profiles between the early and late-flowering bulks constructed from F₂ plants identified a major QTL, designated Ef1.1 on cucumber chromosome 1 for early flowering in Muromskij, which was confirmed by microsatellite marker-based classical QTL mapping in the F₂ population. Joint QTL-seq and traditional QTL analysis delimited Ef1.1 to an 890 kb genomic region. A cucumber gene, Csa1G651710, was identified in this region, which is a homolog of the FLOWERING LOCUS T (FT), the main flowering switch gene in Arabidopsis. Quantitative RT-PCR study of the expression level of Csa1G651710 revealed significantly higher expression in early flowering genotypes. Data presented here provide support for Csa1G651710 as a possible candidate gene for early flowering in the cucumber line Muromskij.     

Mycophenolate mofetil inhibits the development of Coxsackie B3-virus-induced myocarditis in mice

Background

Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results

Observation of survival percentages over time and detection of LD₅₀s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions

These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.     

Mapping and identification of a Cicer arietinum NSP2 gene involved in nodulation pathway

Key message

For the first time the putative NSP2 gene in chickpea has been identified using pairs of NILs differing for the Rn1 / rn1 nodulation gene that was located in LG5 of chickpea genetic map.

Abstract

An intraspecific cross between the mutant non-nodulating genotype PM233, carrying the recessive gene rn1, and the wild-type CA2139 was used to develop two pairs of near-isogenic lines (NILs) for nodulation in chickpea. These pairs of NILs were characterized using sequence tagged microsatellite site (STMS) markers distributed across different linkage groups (LGs) of the chickpea genetic map leading to the detection of polymorphic markers located in LG5. Using this information, together with the genome annotation in Medicago truncatula, a candidate gene (NSP2) known to be involved in nodulation pathway was selected for mapping in chickpea. The full length sequence obtained in chickpea wild-type (CaNSP2) was 1,503 bp. Linkage analysis in an F₃ population of 118 plants derived from the cross between the pair of NILS NIL7-2A (nod) × NIL7-2B (non-nod) revealed a co-localization between CaNSP2 and Rn1 gene. These data implicate the CaNSP2 gene as a candidate for identity to Rn1, and suggest that it could act in the nodulation signaling transduction pathway similarly to that in other legumes species.     

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages

Background

HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain.

Methods

Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD).

Results

Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC₅₀s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N -Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those displayed by fungal molecules from Fusarium sp., shown in the 1990s to inhibit HIV-1 integration.

Conclusion

The 3D model presented here supports the idea that INSTIs dock at the putative acceptor DNA-binding site in a IN/viral DNA complex. This mechanism of enzyme inhibition, likely to be exploited by some natural products, might disclose future strategies for inhibition of nucleic acid-manipulating enzymes.     

Association mapping, patterns of linkage disequilibrium and selection in the vicinity of the PHYTOCHROME C gene in pearl millet

Key message

Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate.

Abstract

Major efforts are currently underway to dissect the phenotype–genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F₂ families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.     

Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

Background

Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding.

Results

In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces.

Conclusions

NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.     

Molecular dissection of the human antibody response to the structural repeat epitope of Plasmodium falciparum sporozoite from a protected donor

Background

The circumsporozoite surface protein is the primary target of human antibodies against Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)_n, (NPNA)_n. In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity.

Methods

The antibody repertoire from a protected individual immunized by the bites of irradiated P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)₃ antibody fragments that recognized the PfCSP repeat epitope were rescued.

Results

Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of V_H3 and V_κI families for heavy and light chain respectively with moderate affinity for the ligand.

Conclusion

The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single V_H/V_L pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)_n will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum.     

A genome-wide association study using international breeding-evaluation data identifies major loci affecting production traits and stature in the Brown Swiss cattle breed

Background

Fusarium graminearum sensu stricto (s.s.) is an ubiquitous pathogen of cereals. The economic impact of Fusarium head blight (FHB) is characterized by crop losses and mycotoxin contamination. Our objective was to associate SNP diversity within candidate genes with phenotypic traits. A total of 77 F. graminearum s.s. isolates was tested for severity of fungal infection (= aggressiveness) and deoxynivalenol (DON) production in an inoculated field experiment at two locations in each of two years. For seven genes known to control fungal growth (MetAP1, Erf2) or DON production (TRI1, TRI5, TRI6 TRI10 and TRI14) single nucleotides polymorphic sites (SNPs) were determined and evaluated for the extent of linkage disequilibrium (LD). Associations of SNPs with both phenotypic traits were tested using linear mixed models.

Results

Decay of LD was in most instances fast. Two neighboring SNPs in MetAP1 and one SNP in Erf2 were significantly (P < 0.05) associated with aggressiveness explaining proportions of genotypic variance (p _G ) of 25.6%, 0.5%, and 13.1%, respectively. One SNP in TRI1 was significantly associated with DON production (p _G = 4.4).

Conclusions

We argue that using the published sequence information of Fusarium graminearum as a template to amplify comparative sequence parts of candidate genes is an effective method to detect quantitative trait loci. Our findings underline the potential of candidate gene association mapping approaches to identify functional SNPs underlying aggressiveness and DON production for F. graminearum s.s populations.     

A truncated FatB resulting from a single nucleotide insertion is responsible for reducing saturated fatty acids in maize seed oil

Key message

We identified a G-nucleotide insertion in a maize FatB responsible for reducing saturated fatty acids through QTL mapping and map-based cloning and developed an allele-specific DNA marker for molecular breeding.

Abstract

Vegetable oils with reduced saturated fatty acids have signficant health benefits. SRS72NE, a Dow AgroSciences proprietory maize inbred line, was found to contain signficantly reduced levels of palmitic acid and total saturated fatty acids in seed oil when compared to other common inbreds. Using F₂ and F₃ populations derived from a cross between SRS72NE and a normal inbred SLN74, we have demonstrated that the reduced saturated fatty acid phenotype in SRS72NE is controlled by a single QTL on chromosome 9 that explains 79.1 % of palmitic acid and 79.6 % total saturated fatty acid variations. The QTL was mapped to an interval of 105 kb that contains one single gene, a type B fatty acyl-ACP thioesterase (ZmFatB; GRMZM5G829544). ZmFatB alleles from SRS72NE and common inbreds were cloned and sequenced. SRS72NE fatb allele contains a single nucleotide (G) insertion in the 6th exon, which creates a premature stop codon 22 base pairs down stream. As a result, ZmFatB protein from SRS72NE is predicted to contain eight altered and 90 deleted amino acids at its C-terminus. Because the affected region is part of the conserved acyl-ACP thioesterase catalytic domain, the truncated ZmFatB in SRS72NE is likely non-functional. We also show that fatb RNA level in SRS72NE is reduced by 4.4-fold when compared to the normal allele SNL74. A high throughput DNA assay capable of differentiating the normal and reduced saturate fatty acid alleles has been developed and can be used for accelerated molecular breeding.     

Genetically-modified <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">ω</Emphasis>-transaminase: purification and self-assembly facilitating interaction with substrate droplets

Objectives

An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase.

Results

R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %.

Conclusions

The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.

     

Genetic analyses of bolting in bulb onion (Allium cepa L.)

Key message

We present the first evidence for a QTL conditioning an adaptive trait in bulb onion, and the first linkage and population genetics analyses of candidate genes involved in photoperiod and vernalization physiology.

Abstract

Economic production of bulb onion (Allium cepa L.) requires adaptation to photoperiod and temperature such that a bulb is formed in the first year and a flowering umbel in the second. ‘Bolting’, or premature flowering before bulb maturation, is an undesirable trait strongly selected against by breeders during adaptation of germplasm. To identify genome regions associated with adaptive traits we conducted linkage mapping and population genetic analyses of candidate genes, and QTL analysis of bolting using a low-density linkage map. We performed tagged amplicon sequencing of ten candidate genes, including the FT-like gene family, in eight diverse populations to identify polymorphisms and seek evidence of differentiation. Low nucleotide diversity and negative estimates of Tajima’s D were observed for most genes, consistent with purifying selection. Significant population differentiation was observed only in AcFT2 and AcSOC1. Selective genotyping in a large ‘Nasik Red × CUDH2150’ F₂ family revealed genome regions on chromosomes 1, 3 and 6 associated (LOD > 3) with bolting. Validation genotyping of two F₂ families grown in two environments confirmed that a QTL on chromosome 1, which we designate AcBlt1, consistently conditions bolting susceptibility in this cross. The chromosome 3 region, which coincides with a functionally characterised acid invertase, was not associated with bolting in other environments, but showed significant association with bulb sucrose content in this and other mapping pedigrees. These putative QTL and candidate genes were placed on the onion map, enabling future comparative studies of adaptive traits.     

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

Key Message

The critical level for SO ₂ susceptibility of Populus × canescens is approximately 1.2 μL L ^?1 SO ₂ . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO ₂ detoxification.

Abstract

In the present study, uptake, susceptibility and metabolism of SO₂ were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO₂ detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO₂ (0.65, 0.8, 1.0, 1.2 μL L^?1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO₂ assimilation, SO₂ uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO₂ that confers injury to the leaves was 1.2 μL L^?1 SO₂. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO₂ uptake of the plant shoot, whereas SO₂ uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO₂ revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.     

Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Key message

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated.

Abstract

Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F₂ population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10³/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F₁ commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.