CuGenDBv2: an updated database for cucurbit genomics.

The Cucurbitaceae (cucurbit) family consists of about 1,000 species in 95 genera, including many economically important and popular fruit and vegetable crops. During the past several years, reference genomes have been generated for >20 cucurbit species, and variome and transcriptome profiling data have been rapidly accumulated for cucurbits. To efficiently mine, analyze and disseminate these large-scale datasets, we have developed an updated version of Cucurbit Genomics Database. The updated database, CuGenDBv2 (http://cucurbitgenomics.org/v2), currently hosts 34 reference genomes from 27 cucurbit species/subspecies belonging to 10 different genera. Protein-coding genes from these genomes have been comprehensively annotated by comparing their protein sequences to various public protein and domain databases. A novel 'Genotype' module has been implemented to facilitate mining and analysis of the functionally annotated variome data including SNPs and small indels from large-scale genome sequencing projects. An updated 'Expression' module has been developed to provide a comprehensive gene expression atlas for cucurbits. Furthermore, synteny blocks between any two and within each of the 34 genomes, representing a total of 595 pair-wise genome comparisons, have been identified and can be explored and visualized in the database.

QTL mapping of resistance to Pseudoperonospora cubensis clade 2, mating type A1, in Cucumis melo and dual-clade marker development.

KEY MESSAGE: This is the first identification of QTLs underlying resistance in Cucumis melo to an isolate of Pseudoperonospora cubensis identified as Clade 2/mating type A1. Pseudoperonospora cubensis, causal organism of cucurbit downy mildew (CDM), causes severe necrosis and defoliation on Cucumis melo (melon). A recombinant inbred line population (N = 169) was screened against an isolate of P. cubensis (Clade 2/mating type A1) in replicated greenhouse and growth chamber experiments. SNPs (n = 5633 bins) identified in the RIL population were used for quantitative trait loci (QTL) mapping. A single major QTL on chromosome 10 (qPcub-10.3-10.4) was consistently associated with resistance across all experiments, while a second major QTL on chromosome 8 (qPcub-8.3) was identified only in greenhouse experiments. These two major QTLs were identified on the same chromosomes (8 and 10) but in different locations as two major QTLs (qPcub-8.2 and qPcub-10.1) previously identified for resistance to P. cubensis Clade 1/mating type A2. Kompetitive allele-specific PCR (KASP) markers were developed for these four major QTLs and validated in the RIL population through QTL mapping. These markers will provide melon breeders a high-throughput genotyping toolkit for development of melon cultivars with broad tolerance to CDM.

Distinct Genomic Loci Underlie Quantitative Resistance to Meloidogyne enterolobii Galling and Reproduction in Citrullus amarus.

Meloidogyne enterolobii is a virulent species of root-knot nematode that threatens watermelon (Citrullus lanatus) production in the southeastern United States. There are no known sources of root-knot nematode resistance in cultivated C. lanatus. Specific genotypes of a wild watermelon relative, C. amarus, are resistant against M. incognita but the genetics that underly this resistance are still unknown and it is not clear that this same resistance will be effective against M. enterolobii. To identify and characterize new sources of resistance to M. enterolobii, we screened 108 diverse C. amarus lines alongside a susceptible C. lanatus cultivar (Charleston Gray) for resistance against M. enterolobii. Different C. amarus genotypes ranged from resistant to susceptible for the three resistance phenotypes measured; mean percent root system galled ranged from 10 to 73%, mean egg mass counts ranged from 0.3 to 64.5, and mean eggs per gram of root ranged from 326 to 146,160. We used each of these three resistance phenotypes combined with whole-genome resequencing data to conduct a genome-wide association scan that identified significant associations between M. enterolobii resistance and 11 single-nucleotide polymorphisms (SNPs) within the C. amarus genome. Interestingly, SNPs associated with reduced galling and egg masses were located within a single quantitative trait locus (QTL) on chromosome Ca03, while reductions in nematode eggs per gram of root were associated with separate QTL on chromosomes Ca04 and Ca08. The results of this study suggest that multiple genes are involved with M. enterolobii resistance in C. amarus and the SNPs identified will assist with efforts to breed for M. enterolobii resistance in watermelon.

Broad Resistance to Post-Harvest Fruit Rot in USVL Watermelon Germplasm Lines to Isolates of Phytophthora capsici Across the United States.

Watermelon is an important cucurbit vegetable crop grown in most of the United States. Phytophthora fruit rot of watermelon caused by Phytophthora capsici has been a major factor, limiting production for the past 15 years in the southeastern United States. The U.S. Department of Agriculture, Agricultural Research Service released five Phytophthora fruit rot-resistant germplasm lines for use in breeding programs. These lines were developed by phenotyping using a local isolate of P. capsici from South Carolina. The present study was undertaken to determine if these resistant lines had broad resistance to diverse P. capsici isolates collected from different states and crops. Five resistant germplasm lines (USVL020-PFR, USVL203-PFR, USVL782-PFR, USVL489-PFR, and USVL531-MDR) and two susceptible cultivars, Sugar Baby and Mickey Lee, used as checks were grown in a field in 2014 and 2015 to produce fruit for evaluation. Mature fruit were harvested and placed in a walk-in growth chamber and inoculated with 20 different P. capsici isolates. The chamber was maintained at 26 ± 2°C and high relative humidity (>95%) using a humidifier. All five resistant germplasm lines were significantly more resistant than the two susceptible checks to all 20 P. capsici isolates. Among the five resistant germplasm lines, USVL020-PFR, USVL782-PFR, and USVL531-MDR had broad resistance. Some P. capsici isolates induced minor lesions and rot on USVL489-PFR compared with the other resistant lines. Variation in virulence and genetic diversity among the 20 P. capsici isolates was also observed. The five watermelon germplasm lines will be useful for developing commercial watermelon cultivars with broad resistance to P. capsici.

Genome-Wide Association Analysis of Resistance to Pseudoperonospora cubensis in Citron Watermelon.

Cultivated sweet watermelon (Citrullus lanatus) is an important vegetable crop for millions of people around the world. There are limited sources of resistance to economically important diseases within C. lanatus, whereas C. amarus has a reservoir of traits that can be exploited to improve C. lanatus for resistance to biotic and abiotic stresses. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is an emerging threat to watermelon production. We screened 122 C. amarus accessions for resistance to CDM over two tests (environments). The accessions were genotyped by whole-genome resequencing to generate 2,126,759 single nucleotide polymorphic (SNP) markers. A genome-wide association study was deployed to uncover marker-trait associations and identify candidate genes underlying resistance to CDM. Our results indicate the presence of wide phenotypic variability (1.1 to 57.8%) for leaf area infection, representing a 50.7-fold variation for CDM resistance across the C. amarus germplasm collection. Broad-sense heritability estimate was 0.55, implying the presence of moderate genetic effects for resistance to CDM. The peak SNP markers associated with resistance to P. cubensis were located on chromosomes Ca03, Ca05, Ca07, and Ca11. The significant SNP markers accounted for up to 30% of the phenotypic variation and were associated with promising candidate genes encoding leucine-rich repeat receptor-like protein kinase and the WRKY transcription factor. This information will be useful in understanding the genetic architecture of the P. cubensis-Citrullus spp. patho-system as well as development of resources for genomics-assisted breeding for resistance to CDM in watermelon.

Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops.

The Cucurbitaceae family (cucurbit) includes several economically important crops, such as melon, cucumber, watermelon, pumpkin, squash and gourds. During the past several years, genomic and genetic data have been rapidly accumulated for cucurbits. To store, mine, analyze, integrate and disseminate these large-scale datasets and to provide a central portal for the cucurbit research and breeding community, we have developed the Cucurbit Genomics Database (CuGenDB; http://cucurbitgenomics.org) using the Tripal toolkit. The database currently contains all available genome and expressed sequence tag (EST) sequences, genetic maps, and transcriptome profiles for cucurbit species, as well as sequence annotations, biochemical pathways and comparative genomic analysis results such as synteny blocks and homologous gene pairs between different cucurbit species. A set of analysis and visualization tools and user-friendly query interfaces have been implemented in the database to facilitate the usage of these large-scale data by the community. In particular, two new tools have been developed in the database, a 'SyntenyViewer' to view genome synteny between different cucurbit species and an 'RNA-Seq' module to analyze and visualize gene expression profiles. Both tools have been packed as Tripal extension modules that can be adopted in other genomics databases developed using the Tripal system.

Quantitative Trait Loci Mapping of Resistance to Powdery Mildew Race 1 in a Recombinant Inbred Line Population of Melon.

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most important diseases of melon. Although there are several pathogenic races of P. xanthii, race 1 is the predominant race in South Carolina and in other parts of the United States. We used a densely genotyped recombinant inbred line melon population for traditional quantitative trait loci (QTL) mapping, to identify two major (qPx1-5 and qPx1-12) and two minor (qPx1-4 and qPx1-10) QTLs (named according to race - chromosome number) associated with resistance to P. xanthii race 1. QTL mapping of disease severity in multiple tissues (hypocotyl, cotyledons, true leaves, and stems) identified the same genetic basis of resistance in all tissue types. Whole-genome resequencing of the parents was used for marker development across the major QTLs and functional annotation of single nucleotide polymorphisms (SNPs) for candidate gene analysis. Kompetitive allele-specific PCR (KASP) markers were tightly linked to the QTL peaks of qPx1-5 (pm1-5_25329892, pm1-5_25461503 and pm1-5_25625375) and qPx1-12 (pm1-12_22848920 and pm1-12_22904659) in the population and will enable efficient marker-assisted introgression of powdery mildew resistance into improved germplasm. Candidate genes were identified in both major QTL intervals that encode putative R genes with missense mutations between the parents. The candidate genes provide targets for future breeding efforts and a fundamental examination of resistance to powdery mildew in melon.

Identifying Races of Fusarium oxysporum f. sp. niveum in South Carolina Recovered From Watermelon Seedlings, Plants, and Field Soil.

Fusarium wilt of watermelon (Citrullus lanatus), caused by the soilborne fungus Fusarium oxysporum f. sp. niveum, is the most serious disease of watermelon in South Carolina and other southeastern U.S. states. Isolates of F. oxysporum collected from field-grown plants, greenhouse-grown seedlings, and field soil between 1999 and 2018 were inoculated onto three differential watermelon cultivars to identify races. Of 197 isolates obtained from plants, 12% were nonpathogenic, 2% were race 0, 23% were race 1, and 63% were race 2. One collection of isolates from greenhouse seedlings was exclusively race 1 and the other was exclusively race 2. Seventeen of 81 soil isolates were pathogenic: five were race 1 and 12 were race 2. Reactions of C. amarus PI 296341-FR, Carolina Strongback, and SP-6, cultigens with resistance to race 2, did not differ significantly among five highly virulent race 2 isolates and a standard race 2 isolate, indicating a lack of a race 3 phenotype. Forma specialis-specific primers matched phenotypic race identification for 74% of the isolates. Race-specific primers based on a secreted-in-xylem elicitor present in race 0 and 1 isolates matched phenotypic race identification for 66% of the isolates. Because a majority of the F. oxysporum f. sp. niveum isolates from South Carolina were race 2, integrated management practices should be used until commercial cultivars with resistance to race 2 are available.

QTL mapping of resistance to bacterial fruit blotch in Citrullus amarus.

KEY MESSAGE: Six QTLs were associated with affected leaf area in response to inoculation with Acidovorax citrulli in a recombinant inbred line population of Citrullus amarus. Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, has the potential to devastate production of watermelon and other cucurbits. Despite decades of research on host-plant resistance to A. citrulli, no germplasm has been found with immunity and only a few sources with various levels of BFB resistance have been identified, but the genetic basis of resistance in these watermelon sources are not known. Most sources of resistance are plant introductions of Citrullus amarus (citron melon), a closely related species that crosses readily with cultivated watermelon (Citrullus lanatus L.). In this study, we evaluated a recombinant inbred line population (N = 200), derived from a cross between BFB-resistant (USVL246-FR2) and BFB-susceptible (USVL114) C. amarus lines, for foliar resistance to A. citrulli in three replicated greenhouse trials. We found the genetics of BFB resistance to be complicated by strong environmental influence, low heritability and significant genotype-by-environment interactions. QTL mapping of affected leaf area identified six QTL that each explained between 5 and 15% of the variation in BFB resistance in the population. This study represents the first identification of QTL associated with resistance to A. citrulli in any cucurbit.

Genome-Wide Association Study of Resistance to Xanthomonas gardneri in the USDA Pepper (Capsicum) Collection.

Bacterial spot Xanthomonas species cause significant disease outbreaks on tomato and pepper in tropical and subtropical regions throughout the world. Host resistance has been one of the key components of integrated disease management approaches to mitigate plant pathogens. Although a number of resistance genes have been identified in pepper against bacterial spot xanthomonads, emergence of bacterial spot pathogen variants capable of overcoming these sources and changing pathogen distributions reinforce the importance of identifying novel candidates to incorporate into breeding programs. We conducted a genome-wide association study (GWAS) on a diverse U.S. Department of Agriculture collection of pepper germplasm including different species of Capsicum to identify novel sources of disease resistance against a highly virulent X. gardneri strain isolated from a recent outbreak. GWAS identified highly significant single nucleotide polymorphisms associated with defoliation in response to infection with X. gardneri. Functionally relevant candidate genes encoded products involved in disease resistance/susceptibility, hormone signaling, and basal resistance against multiple pathogens in various host-pathogen systems. The X. gardneri-resistant genotypes and quantitative trait loci identified in this study provide alleles that could be used for a resistance gene pyramiding effort against different species of bacterial spot xanthomonads in pepper.

QTL Mapping Identifies Novel Source of Resistance to Fusarium Wilt Race 1 in Citrullus amarus.

Fusarium wilt race 1, caused by the soilborne fungus Fusarium oxysporum Schlechtend.: Fr. f. sp. niveum (E.F. Sm.) W.C. Snyder & H.N. Hans (Fon), is a major disease of watermelon (Citrullus lanatus) in the United States and throughout the world. Although Fusarium wilt race 1 resistance has been incorporated into several watermelon cultivars, identification of additional genetic sources of resistance is crucial if a durable and sustainable level of resistance is to be continued over the years. We conducted a genetic mapping study to identify quantitative trait loci (QTLs) associated with resistance to Fon race 1 in segregating populations (F2:3 and recombinant inbred lines) of Citrullus amarus (citron melon) derived from the Fon race 1 resistant and susceptible parents USVL246-FR2 and USVL114, respectively. A major QTL (qFon1-9) associated with resistance to Fon race 1 was identified on chromosome 9 of USVL246-FR2. This discovery provides a novel genetic source of resistance to Fusarium wilt race 1 in watermelon and, thus, an additional host-resistance option for watermelon breeders to further the effort to mitigate this serious phytopathogen.

Cucurbit Rootstocks Resistant to Fusarium oxysporum f. sp. niveum Remain Resistant When Coinfected by Meloidogyne incognita in the Field.

Interspecific hybrid squash (Cucurbita maxima × Cucurbita moschata) rootstocks used to graft watermelon (Citrullus lanatus var. lanatus) are resistant to Fusarium oxysporum f. sp. niveum, the fungus that causes Fusarium wilt of watermelon, but they are susceptible to Meloidogyne incognita, the southern root knot nematode. A new citron (Citrullus amarus) rootstock cultivar Carolina Strongback is resistant to F. oxysporum f. sp. niveum and M. incognita. The objective of this study was to determine if an interaction between M. incognita and F. oxysporum f. sp. niveum race 2 occurred on grafted or nongrafted triploid watermelon susceptible to F. oxysporum f. sp. niveum race 2. In 2016 and 2018, plants of nongrafted cultivar Fascination and Fascination grafted onto Carolina Strongback and interspecific hybrid squash cultivar Carnivor were inoculated or not inoculated with M. incognita before transplanting into field plots infested or not infested with F. oxysporum f. sp. niveum race 2. Incidence of Fusarium wilt and area under the disease progress curve did not differ when hosts were inoculated with F. oxysporum f. sp. niveum alone or F. oxysporum f. sp. niveum and M. incognita together. Fusarium wilt was greater on nongrafted watermelon (78% mean incidence) than on both grafted rootstocks and lower on Carnivor (1% incidence) than on Carolina Strongback (12% incidence; P ≤ 0.01). Plants not inoculated with F. oxysporum f. sp. niveum did not wilt. At the end of the season, Carnivor had a greater percentage of the root system galled than the other two hosts, whereas galling did not differ on Fascination and Carolina Strongback. F. oxysporum f. sp. niveum reduced marketable weight of nongrafted Fascination with and without coinoculation with M. incognita. M. incognita reduced marketable weight of Fascination grafted onto Carnivor compared with noninoculated, nongrafted Fascination. In conclusion, cucurbit rootstocks that are susceptible and resistant to M. incognita retain resistance to F. oxysporum f. sp. niveum when they are coinfected with M. incognita.

Construction of a genome-anchored, high-density genetic map for melon (Cucumis melo L.) and identification of Fusarium oxysporum f. sp. melonis race 1 resistance QTL.

KEY MESSAGE: Four QTLs and an epistatic interaction were associated with disease severity in response to inoculation with Fusarium oxysporum f. sp. melonis race 1 in a recombinant inbred line population of melon. The USDA Cucumis melo inbred line, MR-1, harbors a wealth of alleles associated with resistance to several major diseases of melon, including powdery mildew, downy mildew, Alternaria leaf blight, and Fusarium wilt. MR-1 was crossed to an Israeli cultivar, Ananas Yok'neam, which is susceptible to all of these diseases, to generate a recombinant inbred line (RIL) population of 172 lines. In this study, the RIL population was genotyped to construct an ultra-dense genetic linkage map with 5663 binned SNPs anchored to the C. melo genome and exhibits the overall high quality of the assembly. The utility of the densely genotyped population was demonstrated through QTL mapping of a well-studied trait, resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) race 1. A major QTL co-located with the previously validated resistance gene Fom-2. In addition, three minor QTLs and an epistatic interaction contributing to Fom race 1 resistance were identified. The MR-1 × AY RIL population provides a valuable resource for future QTL mapping studies and marker-assisted selection of disease resistance in melon.

Suppression of Bacterial Blight on Mustard Greens with Host Plant Resistance and Acibenzolar-S-Methyl.

Bacterial blight, caused by Pseudomonas cannabina pv. alisalensis, attacks the leaves of most brassica vegetables, including mustard greens (Brassica juncea). 'Carolina Broadleaf,' a new mustard cultivar, is resistant to bacterial blight, whereas 'Florida Broadleaf,' a commonly grown cultivar, is susceptible. Acibenzolar-S-methyl (trade name Actigard) has been used to manage bacterial diseases caused by P. syringae on a variety of crops. The objective of this study was to evaluate host plant resistance and acibenzolar-S-methyl alone and in combination to manage bacterial blight. Three field experiments were done in spring and fall 2011 and fall 2014. In each experiment, acibenzolar-S-methyl was applied twice as a foliar spray, once before and once after plants were inoculated. Severity of bacterial blight was 81% less on nontreated Carolina Broadleaf than on nontreated Florida Broadleaf (P ≤ 0.0003). Acibenzolar-S-methyl applications reduced severity of bacterial blight by 55% compared with the water control treatment on susceptible Florida Broadleaf. Mean weight of diseased leaves, averaged across acibenzolar-S-methyl treatments, was 53% less with Carolina Broadleaf than with Florida Broadleaf (P < 0.0001). However, acibenzolar-S-methyl applied at the recommended rate (14.2 g/ha) significantly injured leaves of Carolina Broadleaf in two experiments and injured leaves of Florida Broadleaf in one experiment. Overall, host plant resistance was more effective than acibenzolar-S-methyl for managing bacterial blight on mustard greens.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

QTL Mapping and Marker Development for Tolerance to Sulfur Phytotoxicity in Melon (Cucumis melo).

Elemental sulfur is an effective, inexpensive fungicide for many foliar pathogens, but severe phytotoxicity prohibits its use on many melon varieties. Sulfur phytotoxicity causes chlorosis and necrosis of leaf tissue, leading to plant death in the most sensitive lines, while other varieties have little to no damage. A high-density, genotyping-by-sequencing (GBS)-based genetic map of a recombinant inbred line (RIL) population segregating for sulfur tolerance was used for a quantitative trait loci (QTL) mapping study of sulfur phytotoxicity in melon. One major (qSulf-1) and two minor (qSulf-8 and qSulf-12) QTL were associated with sulfur tolerance in the population. The development of Kompetitive Allele-Specific PCR (KASP) markers developed across qSulf-1 decreased the QTL interval from 239 kb (cotyledons) and 157 kb (leaves) to 97 kb (both tissues). The markers were validated for linkage to sulfur tolerance in a set of melon cultivars. These KASP markers can be incorporated into melon breeding programs for introgression of sulfur tolerance into elite melon germplasm.

Gene expression in developing watermelon fruit.

BACKGROUND: Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. RESULTS: High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis. CONCLUSION: This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.

Development of a one-step immunocapture real-time TaqMan RT-PCR assay for the broad spectrum detection of Pepino mosaic virus.

A real-time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse Pepino mosaic virus (PepMV) isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single conserved TaqMan probe to broaden its reaction to cover all available PepMV strains. This duo-primer real-time RT-PCR assay was evaluated against US1, US2, Ch1, Ch2 and 25 field isolates collected from six major commercial tomato greenhouse facilities in U.S. and Canada in 2006. Under optimum reaction conditions, sensitivity of the detection was as low as 100 fg of purified viral RNA. This assay was also evaluated for its efficiency in detecting PepMV in various levels of contaminated seed samples. Using immuno-capture sample preparation, real-time RT-PCR was able to detect PepMV in one infested seed in 1000. This level of sensitivity indicated that the one-step immuno-capture duo-primer TaqMan real-time RT-PCR developed in the present study could be used for routine seed health assays.