Comparative Metabolomics and Molecular Phylogenetics of Melon (Cucumis melo, Cucurbitaceae) Biodiversity.

The broad variability of Cucumis melo (melon, Cucurbitaceae) presents a challenge to conventional classification and organization within the species. To shed further light on the infraspecific relationships within C. melo, we compared genotypic and metabolomic similarities among 44 accessions representative of most of the cultivar-groups. Genotyping-by-sequencing (GBS) provided over 20,000 single-nucleotide polymorphisms (SNPs). Metabolomics data of the mature fruit flesh and rind provided over 80,000 metabolomic and elemental features via an orchestra of six complementary metabolomic platforms. These technologies probed polar, semi-polar, and non-polar metabolite fractions as well as a set of mineral elements and included both flavor- and taste-relevant volatile and non-volatile metabolites. Together these results enabled an estimate of "metabolomic/elemental distance" and its correlation with the genetic GBS distance of melon accessions. This study indicates that extensive and non-targeted metabolomics/elemental characterization produced classifications that strongly, but not completely, reflect the current and extensive genetic classification. Certain melon Groups, such as Inodorous, clustered in parallel with the genetic classifications while other genome to metabolome/element associations proved less clear. We suggest that the combined genomic, metabolic, and element data reflect the extensive sexual compatibility among melon accessions and the breeding history that has, for example, targeted metabolic quality traits, such as taste and flavor.

Deciphering genetic factors that determine melon fruit-quality traits using RNA-Seq-based high-resolution QTL and eQTL mapping.

Combined quantitative trait loci (QTL) and expression-QTL (eQTL) mapping analysis was performed to identify genetic factors affecting melon (Cucumis melo) fruit quality, by linking genotypic, metabolic and transcriptomic data from a melon recombinant inbred line (RIL) population. RNA sequencing (RNA-Seq) of fruit from 96 RILs yielded a highly saturated collection of > 58 000 single-nucleotide polymorphisms, identifying 6636 recombination events that separated the genome into 3663 genomic bins. Bin-based QTL analysis of 79 RILs and 129 fruit-quality traits affecting taste, aroma and color resulted in the mapping of 241 QTL. Thiol acyltransferase (CmThAT1) gene was identified within the QTL interval of its product, S-methyl-thioacetate, a key component of melon fruit aroma. Metabolic activity of CmThAT1-encoded protein was validated in bacteria and in vitro. QTL analysis of flesh color intensity identified a candidate white-flesh gene (CmPPR1), one of two major loci determining fruit flesh color in melon. CmPPR1 encodes a member of the pentatricopeptide protein family, involved in processing of RNA in plastids, where carotenoid and chlorophyll pigments accumulate. Network analysis of > 12 000 eQTL mapped for > 8000 differentially expressed fruit genes supported the role of CmPPR1 in determining the expression level of plastid targeted genes. We highlight the potential of RNA-Seq-based QTL analysis of small to moderate size, advanced RIL populations for precise marker-assisted breeding and gene discovery. We provide the following resources: a RIL population genotyped with a unique set of SNP markers, confined genomic segments that harbor QTL governing 129 traits and a saturated set of melon eQTLs.

The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii.

The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway.

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).

Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence 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 the 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.

The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

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.

Metabolism of soluble sugars in developing melon fruit: a global transcriptional view of the metabolic transition to sucrose accumulation.

The sweet melon fruit is characterized by a metabolic transition during its development that leads to extensive accumulation of the disaccharide sucrose in the mature fruit. While the biochemistry of the sugar metabolism pathway of the cucurbits has been well studied, a comprehensive analysis of the pathway at the transcriptional level allows for a global genomic view of sugar metabolism during fruit sink development. We identified 42 genes encoding the enzymatic reactions of the sugar metabolism pathway in melon. The expression pattern of the 42 genes during fruit development of the sweet melon cv Dulce was determined from a deep sequencing analysis performed by 454 pyrosequencing technology, comprising over 350,000 transcripts from four stages of developing melon fruit flesh, allowing for digital expression of the complete metabolic pathway. The results shed light on the transcriptional control of sugar metabolism in the developing sweet melon fruit, particularly the metabolic transition to sucrose accumulation, and point to a concerted metabolic transition that occurs during fruit development.

Trifluralin herbicide-induced resistance of melon to fusarium wilt involves expression of stress- and defence-related genes.

SUMMARY To identify genes involved in trifluralin herbicide-induced resistance of melon to Fusarium oxysporum f. sp. melonis, suppression subtractive hybridization (SSH) and cDNA-amplified fragment-length polymorphism (cDNA-AFLP) were used. A total of 123 clones-60 of which have never been isolated from melon-were isolated, sequenced and annotated. A significant proportion (35%) of the total 123 clones exhibited similarity to genes that have been formerly described as stress- or defence-related. Thirty-two selected clones were subjected to a detailed expression analysis, one-third of which were found to be up-regulated in response to trifluralin treatment and/or fusarium inoculation. The putative roles of seven of these clones in stress are discussed. Furthermore, the expression of four stress-related and up-regulated genes was enhanced when the plants were subjected to salinity stress, suggesting that trifluralin induces a general stress response which protects the plant against fusarium wilt.