[Show abstract][Hide abstract] ABSTRACT: Melon (Cucumis melo) fruits exhibit phenotypic diversity in several key quality determinants such as taste, color and aroma. Sucrose, carotenoids and volatiles are recognized as the key compounds shaping the above corresponding traits yet the full network of biochemical events underlying their synthesis have not been comprehensively described. To delineate the cellular processes shaping fruit quality phenotypes, a population of recombinant inbred lines (RIL) was used as a source of phenotypic and genotypic variations. In parallel, ripe fruits were analyzed for both the quantified level of 77 metabolic traits directly associated with fruit quality and for RNA-seq based expression profiles generated for 27,000 unigenes. First, we explored inter-metabolite association patterns; then, we described metabolites versus gene association patterns; finally, we used the correlation-based associations for predicting uncharacterized synthesis pathways.
Based on metabolite versus metabolite and metabolite versus gene association patterns, we divided metabolites into two key groups: a group including ethylene and aroma determining volatiles whose accumulation patterns are correlated with the expression of genes involved in the glycolysis and TCA cycle pathways; and a group including sucrose and color determining carotenoids whose accumulation levels are correlated with the expression of genes associated with plastid formation.
The study integrates multiple processes into a genome scale perspective of cellular activity. This lays a foundation for deciphering the role of gene markers associated with the determination of fruit quality traits.
[Show abstract][Hide abstract] ABSTRACT: Background
Melon fruit flesh color is primarily controlled by the “golden” single nucleotide polymorhism of the “Orange” gene, CmOr, which dominantly triggers the accumulation of the pro-vitamin A molecule, β-carotene, in the fruit mesocarp. The mechanism by which CmOr operates is not fully understood. To identify cellular and metabolic processes associated with CmOr allelic variation, we compared the transcriptome of bulks of developing fruit of homozygous orange and green fruited F3 families derived from a cross between orange and green fruited parental lines.
Pooling together F3 families that share same fruit flesh color and thus the same CmOr allelic variation, normalized traits unrelated to CmOr allelic variation. RNA sequencing analysis of these bulks enabled the identification of differentially expressed genes. These genes were clustered into functional groups. The relatively enriched functional groups were those involved in photosynthesis, RNA and protein regulation, and response to stress.
The differentially expressed genes and the enriched processes identified here by bulk segregant RNA sequencing analysis are likely part of the regulatory network of CmOr. Our study demonstrates the resolution power of bulk segregant RNA sequencing in identifying genes related to commercially important traits and provides a useful tool for better understanding the mode of action of CmOr gene in the mediation of carotenoid accumulation.
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The online version of this article (doi:10.1186/s12870-015-0661-8) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Melon (Cucumis melo L.) genetic maps were compiled by the International Cucurbit Genomics Initiative (ICuGI) before the release of the melon genome. However, due to the use of different marker sets, the position of ICuGI markers in the genome remained unknown, complicating the integration of previous genetic mapping studies in the genome. We looked for the genome position of 870 simple sequence repeat and single nucleotide polymorphism (SNP) markers from the ICuGI map, locating 836 of them in the melon pseudochromosomes v3.5.1, and integrating them with previously available SNPs to reach a total of 1850 markers mapped in the genome sequence. The number of markers per scaffold ranged from 1 to 105, with an average of 13, thus improving on the previous studies in melon. Twenty-three of the markers mapped on virtual chromosome “0”, twelve of them being included in the ICuGI map, which could assist in the anchoring of some unanchored contigs and scaffolds. Genetic and physical distance comparison showed a good collinearity between them, confirming the quality of the ICuGI map. A higher recombination rate was also usually found at the ends of the chromosomes, whereas a drastic reduction was observed in the putative pericentromeric regions. Quantitative trait loci (QTL) previously located in the ICuGI map were also anchored in the genome. This work offers the opportunity to supplement the genetic maps available up to now with the genomic resources resulting from the melon genome sequencing to facilitate comparative mapping in melon between past and new studies, and to overcome some of the current limitations in QTL gene identification.
[Show abstract][Hide abstract] ABSTRACT: The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba melons (Cucumis melo Inodorous Group) accumulate naringenin chalcone, a yellow flavonoid pigment. RNA-Seq analysis of bulksbulked segregants, representing the tails of a population segregating for naringenin chalcone accumulation, followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding gene (CmKFB) that, when expressed, negatively regulates naringenin chalcone accumulation. Further metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, while CmKFB expression diverts the biochemical flux towards coumarins and general phenylpropanoids. These results demonstrate that CmKFB functions as a post-transcriptional regulator which diverts flavonoid metabolic flux.
[Show abstract][Hide abstract] ABSTRACT: Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequencerelated amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ∼2 Kb and have expected genotype calls in parents/F 1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a '9930' 'Gy14 ' recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.
PLoS ONE 04/2015; 10(4):e0124101. DOI:10.1371/journal.pone.0124101 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Melon (Cucumis melo) flesh color is genetically determined and can be white, light green or orange with β-carotene being the predominant pigment. We associated carotenoid accumulation in melon fruit flesh with polymorphism within CmOr, a homolog of the cauliflower BoOr gene, and identified CmOr as the previously described gf locus in melon. CmOr was found to co-segregate with fruit flesh color and presented two haplotypes (alleles) in a broad germplasm collection, one being associated with orange flesh and the second with either white or green flesh. Allelic variation of CmOr does not affect its transcription or protein level. The variation also does not affect its plastid subcellular localization. Among the identified SNPs between CmOr alleles in orange versus green/white-flesh fruit, a single SNP causes a change of an evolutionarily highlyconserved arginine to histidine in the CmOr protein. Functional analysis of CmOr haplotypes in an Arabidopsis callus system confirmed the ability of the CmOr orange haplotype to induce β-carotene accumulation. Site-directed mutagenesis of the CmOr green/white haplotype to change the CmOR arginine to histidine triggered β-carotene accumulation. Identification of the "golden" SNP in CmOr, which is responsible for the non-orange and orange melon fruit phenotypes, provides new tools for studying the Or mechanism of action and suggests genome editing of the Or gene for nutritional biofortification of crops. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The Plant Journal 03/2015; 82(2). DOI:10.1111/tpj.12814 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cucurbitacins are a group of bitter tasting oxygenated tetracyclic triterpenes that are produced in the Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defense against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter tasting watermelon Citrullus lanatus var. citroides as well as in their wild ancestor C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1) all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity while CcCDS1, did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1, isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.
[Show abstract][Hide abstract] ABSTRACT: In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates β-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'(. HPLC analysis revealed that 'yofI accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A to T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.
Archives of Biochemistry and Biophysics 08/2013; 539(2). DOI:10.1016/j.abb.2013.08.006 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Success in grafting depends on the identification of a compatible rootstock (RS) that promotes rapid formation of vascular connections between the RS and the scion, and rapid resumption of root and shoot growth. In this study, we used compatible and incompatible Cucurbita rootstocks, with a melon (Cucumis melo L. 'Arava') scion, to identify physiological and biochemical factors in the scion-RS interface that could be associated with graft compatibility. Anatomical characterisation of the grafting interface showed that the regeneration and differentiation of vascular elements was similar, at first, in both compatible and incompatible grafted seedlings although, in the latter, a protective layer was formed, but did not interfere with communication between the RS and the scion. Water uptake and sugar distribution between the plant canopy and the roots, measured 14 d after grafting (DAG) were not correlated with compatibility or incompatibility. At 24 DAG, both water uptake and root sugar concentrations decreased significantly in the incompatible RS, reflecting deterioration of the RS. At 24 DAG, part of the RS collapsed, but only in incompatible grafted seedlings. Histochemical staining revealed that superoxide, H 2 O 2 , peroxidase (POX) activity, and lignin deposits at the scion-rootstock interface were similar in both compatible and incompatible grafting combinations at 14 DAG; but, at 24 DAG, H 2 O 2 and superoxide levels were higher in the incompatible grafted transplants. In addition, cell-wall POX and superoxide dismutase (SOD) activities were lower in the incompatible RS-scion interface at 14 and 24 DAG. These results suggest that a physical barrier is unlikely to be formed between the incompatible partners early after grafting, but that lower anti-oxidant enzyme activities and higher levels of reactive oxygen species (ROS) in the incompatible RS-scion interface may be responsible for degradation of the grafting zone.
[Show abstract][Hide abstract] ABSTRACT: The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.
[Show abstract][Hide abstract] ABSTRACT: Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.
[Show abstract][Hide abstract] ABSTRACT: Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.
Proceedings of the Royal Society B: Biological Sciences 11/2011; 279(1734):1791-6. DOI:10.1098/rspb.2011.2095 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A number of molecular marker linkage maps have been developed for melon (Cucumis melo L.) over the last two decades. However, these maps were constructed using different marker sets, thus, making comparative analysis among maps difficult. In order to solve this problem, a consensus genetic map in melon was constructed using primarily highly transferable anchor markers that have broad potential use for mapping, synteny, and comparative quantitative trait loci (QTL) analysis, increasing breeding effectiveness and efficiency via marker-assisted selection (MAS).
Under the framework of the International Cucurbit Genomics Initiative (ICuGI, http://www.icugi.org), an integrated genetic map has been constructed by merging data from eight independent mapping experiments using a genetically diverse array of parental lines. The consensus map spans 1150 cM across the 12 melon linkage groups and is composed of 1592 markers (640 SSRs, 330 SNPs, 252 AFLPs, 239 RFLPs, 89 RAPDs, 15 IMAs, 16 indels and 11 morphological traits) with a mean marker density of 0.72 cM/marker. One hundred and ninety-six of these markers (157 SSRs, 32 SNPs, 6 indels and 1 RAPD) were newly developed, mapped or provided by industry representatives as released markers, including 27 SNPs and 5 indels from genes involved in the organic acid metabolism and transport, and 58 EST-SSRs. Additionally, 85 of 822 SSR markers contributed by Syngenta Seeds were included in the integrated map. In addition, 370 QTL controlling 62 traits from 18 previously reported mapping experiments using genetically diverse parental genotypes were also integrated into the consensus map. Some QTL associated with economically important traits detected in separate studies mapped to similar genomic positions. For example, independently identified QTL controlling fruit shape were mapped on similar genomic positions, suggesting that such QTL are possibly responsible for the phenotypic variability observed for this trait in a broad array of melon germplasm.
Even though relatively unsaturated genetic maps in a diverse set of melon market types have been published, the integrated saturated map presented herein should be considered the initial reference map for melon. Most of the mapped markers contained in the reference map are polymorphic in diverse collection of germplasm, and thus are potentially transferrable to a broad array of genetic experimentation (e.g., integration of physical and genetic maps, colinearity analysis, map-based gene cloning, epistasis dissection, and marker-assisted selection).
[Show abstract][Hide abstract] ABSTRACT: Production of vegetables starting with grafted seedlings is common in many countries with documented advantages over non-grafted seedlings, such as improved disease resistance and enhanced abiotic stress tolerance/resistance. However, the involvement of hormonal effects in the responses of grafted seedlings to abiotic stresses is not clear yet. The aim of the present study was to investigate the effect of ABA and of anti ethylene agents STS and AV G and ascorbic acid (as an anti-oxidant) on the performance of melon/pumpkin grafted transplants under salinity condition. Non-grafted melon transplants were compared to melon grafted on itself or melon grafted on pumpkin rootstock. The transplants were grown in hydroponic solutions containing either normal nutrient concentrations or nutrient solutions containing NaCl at a concentration of 100 mM. The results show that transplant growth was inhibited under salinity and that there were differences in the response of the three transplant groups to salinity stress with the self grafted melons being the most tolerant to salinity. Application of either ascorbic acid or ABA, Silver thiosulphate (STS) (ethylene action blocker) or aminoethoxyvinylglycine (AVG) (ethylene biosynthesis blocker) to the root medium, protected against the growth inhibition caused by high salinity and also protected against growth inhibition caused by application of high ethephon concentration to the roots. Salinity increased oxidative stress in the roots but ABA, STS and ascorbic acid treatments reduced oxidative stress associated with salinity stress. Finally, we demonstrate that melon self grafting (melon/melon) was superior to non grafted melon as well as to melon grafted on pumpkin rootstock under control as well as under salinity conditions. The possible mechanisms of ethylene and ABA effects are discussed.