Shahar Cohen

A consensus linkage map for molecular markers and quantitative trait loci associated with economically important traits in melon (Cucumis melo L.).

BackgroundA 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).ResultsUnder 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.ConclusionsEven 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).

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.

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.

Oligogenic inheritance of resistance to zucchini yellow mosaic virus in melons

The melon (Cucumis melo) plant introduction PI 414723 was observed to segregate for resistance after inoculation with a highly virulent non-aphid-transmissible strain of zucchini yellow mosaic potyvirus (ZYMV-NAT). By self-pollination of selected resistant plants, the percentage of resistant plants increased in successive generations until nearly 100% in the S7 progeny. A reciprocal cross was made between a resistant S5 plant of PI 414723 and a plant of the susceptible cultivar Dulce. Parental-, filial-, and backcross- generation plants were inoculated with ZYMV-NAT. One-half of the F1 plants, regardless of the direction of the cross, were resistant, indicating that the PI 414723 individual used for crossing was heterozygous. The F2 generation obtained by self-pollination of resistant F1 plants segregated to resistant and susceptible in accordance with a 27:37 ratio, indicating that, in this cross, three complementary dominant genes are needed for resistance to be expressed. The resistance to ZYMV carried by PI 414723 is oligogenic with the number of genes observed to segregate in crossing depending on the genotype of the susceptible parent.

Single-gene resistance to zucchini yellow mosaic virus in Cucurbita moschata

SummaryThe mode of inheritance for resistance to zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata was determined from F1, F2, and backcross progenies of the cross between the susceptible cultivar Waltham Butternut from the U.S.A. and a resistant inbred line of the Menina cultivar from Portugal. Resistance to ZYMV in Menina was conferred by a single dominant gene designated Zym.

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

Significance We identified the biosynthetic pathway for the nonsugar sweetener mogroside V, a noncaloric with a sweetening strength 250-fold that of sucrose. This compound is produced by the fruit of the endemic Chinese cucurbit Siraitia grosvenoriii, also known as monk fruit and luo-han-guo. The metabolic pathway was identified using a combination of genomic and transcriptomic databases of the Siraitia plant, together with a large-scale functional expression of candidate genes. The novelty of the pathway could be attributed to a highly coordinated gene expression pattern responsible for the unique epoxidations, hydroxylations, and glucosylations leading to the sweet mogrosides. These discoveries will facilitate the development of alternative natural sweeteners. 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. Copyright

Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

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.

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon.

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.

Genomic Aspects of Melon Fruit Quality

Fruit quality in melon (Cucumis melo) and in other cucurbit species is primarily determined by sweetness, acidity, aroma, color and shelf-life. During ripening, the mesocarp (fruit flesh), the consumed tissue, generally softens due to degradation of cell walls, and accumulates soluble sugars, organic acids, volatiles and additional secondary metabolites. Flesh and rind color undergo developmental changes, the most noticeable of which are changes in pigmentation. This chapter reviews the current knowledge of genes that regulate, or participate in, the major metabolic pathways affecting sugar and acid metabolism (sweetness), volatile organic compounds (aroma) and pigments (color) of the melon fruit.