The morphological response of Emiliania huxleyi to seawater carbonate chemistry changes: an inter-strain comparison

Four strains of the coccolithophore Emiliania huxleyi (RCC1212, RCC1216, RCC1238, RCC1256) were grown in dilute batch culture at four CO2 levels ranging from ~200 µatm to ~1200 µatm. Coccolith morphology was analyzed based on scanning electron micrographs. Three of the four strains did not exhibit a change in morphology over the CO2 range tested. One strain (RCC1256) displayed an increase in the percentage of malformed coccoliths with increasing CO2 concentration. We conclude that the sensitivity of the coccolith-shaping machinery to carbonate chemistry changes is strain-specific. Although it has been shown before that carbonate chemistry related changes in growth- and calcification rate are strain-specific, there seems to be no consistent correlation between coccolith morphology and growth or calcification rate. We did not observe an increase in the percentage of incomplete coccoliths in RCC1256, indicating that the coccolith-shaping machinery per se is affected by acidification and not the signalling pathway that produces the stop-signal for coccolith growth.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli

RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognised adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.

The Clinical Impact and Molecular Biology of del(17p) in Multiple Myeloma Treated with Conventional or Thalidomide-Based Therapy

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P <0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group. (C) 2011 Wiley-Liss, Inc.

Building a 'Repository of Science': the importance of integrating Biobanks within molecular pathology programmes

Repositories containing high quality human biospecimens linked with robust and relevant clinical and pathological information are required for the discovery and validation of biomarkers for disease diagnosis, progression and response to treatment. Current molecular based discovery projects using either low or high throughput technologies rely heavily on ready access to such sample collections. It is imperative that modern biobanks align with molecular diagnostic pathology practices not only to provide the type of samples needed for discovery projects but also to ensure requirements for ongoing sample collections and the future needs of researchers are adequately addressed. Biobanks within comprehensive molecular pathology programmes are perfectly positioned to offer more than just tumour derived biospecimens; for example, they have the ability to facilitate researchers gaining access to sample metadata such as digitised scans of tissue samples annotated prior to macrodissection for molecular diagnostics or pseudoanonymised clinical outcome data or research results retrieved from other users utilising the same or overlapping cohorts of samples. Furthermore, biobanks can work with molecular diagnostic laboratories to develop standardized methodologies for the acquisition and storage of samples required for new approaches to research such as ‘liquid biopsies’ which will ultimately feed into the test validations required in large prospective clinical studies in order to implement liquid biopsy approaches for routine clinical practice. We draw on our experience in Northern Ireland to discuss how this harmonised approach of biobanks working synergistically with molecular pathology programmes is key for the future success of precision medicine.

Challenges in molecular testing in non-small-cell lung cancer patients with advanced disease

Lung cancer diagnostics have progressed greatly in the previous decade. Development of molecular testing to identify an increasing number of potentially clinically actionable genetic variants, using smaller samples obtained via minimally invasive techniques, is a huge challenge. Tumour heterogeneity and cancer evolution in response to therapy means that repeat biopsies or circulating biomarkers are likely to be increasingly useful to adapt treatment as resistance develops. We highlight some of the current challenges faced in clinical practice for molecular testing of EGFR, ALK, and new biomarkers such as PDL1. Implementation of next generation sequencing platforms for molecular diagnostics in non-small-cell lung cancer is increasingly common, allowing testing of multiple genetic variants from a single sample. The use of next generation sequencing to recruit for molecularly stratified clinical trials is discussed in the context of the UK Stratified Medicine Programme and The UK National Lung Matrix Trial.

Determining day length and temperature regulation of flowering: a molecular and modelling approach

Thesis (Ph.D.)--University of Washington, 2016-08

Arabidopsis MYC transcription factors are the target of hormonal salicylic acid/jasmonic acid cross talk in response to Pieris brassicae egg extract.

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated.

Altered regulatory response of Rab7a and Rab9 in MM1 and VV2 subtype of Creutzfeldt-Jakob disease

The present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. We performed a target search for lysosomal network protein, Rab7a and Rab9, in frontal cortex and cerebellum of human brain from patients with sCJD-MM1 and sCJD-VV2. The intracellular level of Rab7a was increased significantly, when compared with healthy age-matched control. Interactions of PrPC and Rab7a/Rab9 were further investigated by using confocal laser scanning microscopy. Immunofluorescence results suggested potential interactions of Rab7a and PrPC. siRNA against the Rab7a gene was used to knockdown the expression of Rab7a protein in primary cell culture of cortical neurons from wild type mice. This depleted Rab7a resulted an impairment of PrPC trafficking leading to an accumulation of PrPC in the endocytosis pathway. Furthermore, interactions of Tau and Rab7a were investigated by using western blot analysis and confocal laser scanning microscopy. Cell cultures of cortex of wildtype mice were treated with siRNA-Tau, siRNA-Rab7 and control siRNA followed by immunofluorescence. The results of immunofluorescence suggested potential interaction of Tau and Rab7a. Cells lines treated with siRNA-Tau, the intracellular levels of Rab7a and Rab9 significantly increases and their localization is also modified. When we transfected this cells lines with siRNA-rab7a the accumulation of Tau decreases in cytosolic region and their localization was also modified when compared with control cells. In conclusion, this study may help to understand and characterize the subtype specific disease progression in CJD cases. Furthermore, it could be a step ahead to development of new treatment strategies for diseases subtype specific manner.

Ecological stoichiometry controls the transformation and retention of plant-derived organic matter to humus in response to nitrogen fertilisation

Carbon (C) sequestration in soils is a means for increasing soil organic carbon (SOC) stocks and is a potential tool for climate change mitigation. One recommended management practice to increase SOC stocks is nitrogen (N) fertilisation, however examples of positive, negative or null SOC effects in response to N addition exist. We evaluated the relative importance of plant molecular structure, soil physical properties and soil ecological stoichiometry in explaining the retention of SOC with and without N addition. We tracked the transformation of 13C pulse-labelled buffel grass (Cenchrus ciliaris L.), wheat (Triticum aestivum L.) and lucerne (Medicago sativa L.) material to the <53 μm silt + clay soil organic C fraction, hereafter named “humus”, over 365-days of incubation in four contrasting agricultural soils, with and without urea-N addition. We hypothesised that: a) humus retention would be soil and litter dependent; b) humus retention would be litter independent once litter C:N ratios were standardised with urea-N addition; and c) humus retention would be improved by urea-N addition. Two and three-way factorial analysis of variance indicated that 13C humus was consistently soil and litter dependent, even when litter C:N ratios were standardised, and that the effect of urea-N addition on 13C humus was also soil and litter dependent. A boosted regression analysis of the effect of 44 plant and soil explanatory variables demonstrated that soil biological and chemical properties had the greatest relative influence on 13C humus. Regression tree analyses demonstrated that the greatest gains in 13C humus occurred in soils of relatively low total organic C, dissolved organic C and microbial biomass C (MBC), or with a combination of relatively high MBC and low C:N ratio. The greatest losses in 13C humus occurred in soils with a combination of relatively high MBC and low total N or increasing C:N ratio. We conclude that soil variables involved in soil ecological stoichiometry exert a greater relative influence on incorporating organic matter as humus compared to plant molecular structure and soil physical properties. Furthermore, we conclude that the effect of N fertilisation on humus retention is dependent upon soil ecological stoichiometry.

Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.

Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

OsHV-1 countermeasures to the Pacific oyster’s anti-viral response

The host-pathogen interactions between the Pacific oyster (Crassostrea gigas) and Ostreid herpesvirus type 1 (OsHV-1) are poorly characterised. Herpesviruses are a group of large, DNA viruses that are known to encode gene products that subvert their host’s antiviral response. It is likely that OsHV-1 has also evolved similar strategies as its genome encodes genes with high homology to C. gigas inhibitors of apoptosis (IAPs) and an interferon-stimulated gene (termed CH25H). The first objective of this study was to simultaneously investigate the expression of C. gigas and OsHV-1 genes that share high sequence homology during an acute infection. Comparison of apoptosis-related genes revealed that components of the extrinsic apoptosis pathway (TNF) were induced in response to OsHV-1 infection, but we failed to observe evidence of apoptosis using a combination of biochemical and molecular assays. IAPs encoded by OsHV-1 were highly expressed during the acute stage of infection and may explain why we didn’t observe evidence of apoptosis. However, C. gigas must have an alternative mechanism to apoptosis for clearing OsHV-1 from infected gill cells as we observed a reduction in viral DNA between 27 and 54 h post-infection. The reduction of viral DNA in C. gigas gill cells occurred after the up-regulation of interferon-stimulated genes (viperin, PKR, ADAR). In a second objective, we manipulated the host’s anti-viral response by injecting C. gigas with a small dose of poly I:C at the time of OsHV-1 infection. This small dose of poly I:C was unable to induce transcription of known antiviral effectors (ISGs), but these oysters were still capable of inhibiting OsHV-1 replication. This result suggests dsRNA induces an anti-viral response that is additional to the IFN-like pathway.

Caracterização fisio-molecular de cultivares de feijão (Phaseolus vulgaris L.) em resposta à deficiência hídrica

The identification of genotypes for drought tolerance has a great importance in breeding programs. The aim of this study was to characterize genotypes of beans in response to drought tolerance in different reproductive stages through physiologic, agronomic and molecular analysis. The experiment was conducted in greenhouse, using a randomized block design with four replicates; 10 cultivars: ANFC 9, ANFP 110, BRS Esplendor, BRSMG Realce, IPR Siriri, IPR Tangará, IPR Tuiuiu, IPR Uirapuru, IAC Imperador and IAC Milênio under two conditions of irrigation: plants irrigated during their entire life cycle, and plants under irrigation suppression in the reproductive stage (R7) until 16% of field capacity, when the irrigation was restored. In the last four days of stress, the gas exchanges were analyzed, and in the last day of stress was analyzed the percentage of closed stomata in the abaxial surface of the leaves, collected in different times of the day (9h, 12h, 15h and 18h). Additionally, plant samples were collected for the following analysis: fresh and dry mass of leaves, stems and legumes, and proline content in leaves and roots. The plants were harvested at the physiological maturity and the yield components and grain yield were determined. In addition, in order to identify polymorphisms in the sequences of promoters and genes related to drought, seven pairs of primers were tested on the group of genotypes. The drought susceptibility indexes (ISS) ranged from 0.65 to 1.10 in the group of genotypes, which the lowest values observed were for IAC Imperador (0.65) and BRS Esplendor (0.87), indicating the ability of these two genotypes to maintain grain yield under water stress condition. All genotypes showed reduction in yield components under water stress. IAC Imperador (43.4%) and BRS Esplendor (60.6%) had the lowest reductions in productivity and kept about 50% of the stomata closed during all the different times evaluated at last day of irrigation suppression. IAC Imperador showed greater water use efficiency and CO2 assimilation rate under drought stress. IPR Tuiuiú, IPR Tangará and IAC Imperador had the highest proline concentrations in the roots. Under water stress condition, there was a strong positive correlation (0.696) between the percentage of stomata closed with the number of grains per plant (0.696) and the fresh mass of leaves (0.731), the maximum percentage of stomata closed 73.71% in water stress. The accumulation of proline in the root was the character that most contributed to the divergence between the genotypes under water deficit, but not always the genotypes that have accumulated more proline were the most tolerant. The polymorphisms in DNA of coding and promoting sequences of transcription factors studied in this experiment did not discriminate tolerant genotypes from the sensitive ones to water stress.

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Background Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms. Methodology/Principal Findings We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR. Conclusion/Significance DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.