Interaction between QTLs induces an advance in ethylene biosynthesis during melon fruit ripening

The coexistence of both climacteric and non-climacteric genotypes and the availability of a set of genetic and genomic resources make melon a suitable model for genetic studies of fruit ripening. We have previously described a QTL, ETHQB3.5, which induces climacteric fruit ripening in the near-isogenic line (NIL) SC3-5 that harbors an introgression on linkage group (LG) III from the non-climacteric melon accession PI 161375 in the, also non-climacteric cultivar, “Piel de Sapo” genetic background. In the current study, a new major QTL, ETHQV6.3, on LG VI was detected on an additional introgression in the same NIL. These QTLs are capable, individually, of inducing climacteric ripening in the non-climacteric background, the effects of ETHQV6.3 being greater than that of ETHQB3.5. The QTLs interact epistatically, advancing the timing of ethylene biosynthesis during ripening and, therefore, the climacteric responses. ETHQV6.3 was fine-mapped to a 4.5 Mb physical region of the melon genome, probably in the centromeric region of LG VI. The results presented will be of value in the molecular identification of the gene underlying ETHQV6.3     

Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.     

Development of a genomic library of near isogenic lines (NILs) in melon (Cucumis melo L.) from the exotic accession PI161375

A doubled haploid line (DHL) population of melon derived from a cross between the Korean cultivar “Songwhan Charmi” accession PI161375 (SC), included in the horticultural group conomon, and the Spanish cultivar “Piel de Sapo” (PS), included in the horticultural group inodorus, was used to develop a collection of near isogenic lines (NILs). These parental lines represent very different melon cultivar groups, with important differences at fruit, plant, disease response and molecular level. This cross is one of the most polymorphic ones within melon germplasm. Selected DHLs were backcrossed to PS and further backcrossing and selfing was performed, monitoring introgressions from SC using molecular markers covering the melon genetic map. A final collection of 57 NILs was obtained, containing a unique independent introgression from SC in the PS genetic background. The introgressions within the collection cover at least 85% of the SC genome with an average introgression size of 41 cM, corresponding to 3.4% of the SC genome. The average resolution for mapping genes or quantitative trait loci is 18.90 cM. This set of NILs is a potentially powerful tool for the study of quantitative trait locus involved in melon fruit quality and other important complex traits, and the introduction of new genetic variability in modern cultivars from exotic sources. The NILs can also be used as pre-competitive breeding lines in melon breeding projects.     

Shaping melons: agronomic and genetic characterization of QTLs that modify melon fruit morphology

The consistency of quantitative trait locus (QTL) effects among genetic backgrounds is a key factor for introgressing QTLs from initial mapping experiments into applied breeding programs. We have selected four QTLs (fs6.4, fw4.3, fw4.4 and fw8.1) involved in melon fruit morphology that had previously been detected in a collection of introgression lines derived from the cross between a Spanish cultivar, “Piel de Sapo,” and the Korean accession PI161375 (Songwan Charmi). Introgression lines harboring these QTLs were crossed with an array of melon inbred lines representative of the most important cultivar types. Hybrids of the introgression and inbred lines, with the appropriate controls, were evaluated in replicated agronomic trials. The effects of the QTLs were consistent among the different genetic backgrounds, demonstrating the utility of these QTLs for applied breeding programs in modifying melon fruit morphology. Three QTLs, fw4.4, fs6.4 and fs12.1 were subjected to further study in order to map them more accurately by substitution mapping using a new set of introgression lines with recombination events within the QTL chromosome region. The position of the QTLs was narrowed down to 36–5 cM, depending on the QTL. The results presented in the current study set the basis for the use of these QTLs in applied breeding programs and for the molecular characterization of the genes underlying them.     

Bin mapping of genomic and EST-derived SSRs in melon (Cucumis melo L.)

We report the development of 158 primer pairs flanking SSR motifs in genomic (gSSR) and EST (EST-SSR) melon sequences, all yielding polymorphic bands in melon germplasm, except one that was polymorphic only in Cucurbita species. A similar polymorphism level was found among EST-SSRs and gSSRs, between dimeric and trimeric EST-SSRs, and between EST-SSRs placed in the open reading frame or any of the 5′- or 3′-untranslated regions. Correlation between SSR length and polymorphism was only found for dinucleotide EST-SSRs located within the untranslated regions, but not for trinucleotide EST-SSRs. Transferability of EST-SSRs to Cucurbita species was assayed and 12.7% of the primer pairs amplified at least in one species, although only 5.4% were polymorphic. A set of 14 double haploid lines from the cross between the cultivar “Piel de Sapo” and the accession PI161375 were selected for the bin mapping approach in melon. One hundred and twenty-one SSR markers were newly mapped. The position of 46 SSR loci was also verified by genotyping the complete population. A final bin-map was constructed including 80 RFLPs, 212 SSRs, 3 SNPs and the Nsv locus, distributed in 122 bins with an average bin length of 10.2 cM and a maximum bin length of 33 cM. Map density was 4.2 cM/marker or 5.9 cM/SSR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Non‐invasive quantification of ethylene in attached fruit headspace at 1 p.p.b. by gas chromatography–mass spectrometry

Ethylene is a gaseous plant hormone involved in defense, adaptations to environmental stress and fruit ripening. Its relevance to the latter makes its detection highly useful for physiologists interested in the onset of ripening. Produced as a sharp peak during the respiratory burst, ethylene is biologically active at tens of nl L^?1. Reliable quantification at such concentrations generally requires specialized instrumentation. Here we present a rapid, high‐sensitivity method for detecting ethylene in attached fruit using a conventional gas chromatography–mass spectrometry (GC‐MS) system and in situ headspace collection chambers. We apply this method to melon (Cucumis melo L.), a unique species consisting of climacteric and non‐climacteric varieties, with a high variation in the climacteric phenotype among climacteric types. Using a population of recombinant inbred lines (RILs) derived from highly climacteric (‘Védrantais’, cantalupensis type) and non‐climacteric (‘Piel de Sapo’, inodorus type) parental lines, we observed a significant variation for the intensity, onset and duration of the ethylene burst during fruit ripening. Our method does not require concentration, sampling times over 1 h or fruit harvest. We achieved a limit of detection of 0.41 ± 0.04 nl L^?1 and a limit of quantification of 1.37 ± 0.13 nl L^?1 with an analysis time per sample of 2.6 min. Validation of the analytical method indicated that linearity (>98%), precision (coefficient of variation ≤2%) and sensitivity compared favorably with dedicated optical sensors. This study adds to evidence of the characteristic climacteric ethylene burst as a complex trait whose intensity in our RIL population lies along a continuum in addition to two extremes.     

Ethylene and fruit ripening

The latest advances in our understanding of the relationship between ethylene and fruit ripening are reviewed. Considerable progress has been made in the characterisation of genes encoding the key ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) and in the isolation of genes involved in the ethylene signal transduction pathway, particularly those encoding ethylene receptors ( ETR ). These have allowed the generation of transgenic fruit with reduced ethylene production and the identification of the Nr tomato ripening mutant as an ethylene receptor mutant. Through these tools, a clearer picture of the role of ethylene in fruit ripening is now emerging. In climacteric fruit, the transition to autocatalytic ethylene production appears to result from a series of events where developmentally regulated ACO and ACS gene expression initiates a rise in ethylene production, setting in motion the activation of autocatalytic ethylene production. Differential expression of ACS and ACO gene family members is probably involved in such a transition. Finally, we discuss evidence suggesting that the NR ethylene perception and transduction pathway is specific to a defined set of genes expressed in ripening climacteric fruit and that a distinct ETR pathway regulates other ethylene-regulated genes in both immature and ripening climacteric fruit as well as in non-climacteric fruit. The emerging picture is one where both ethylene-dependent and -independent pathways coexist in both climacteric and non-climacteric fruits. Further work is needed in order to dissect the molecular events involved in individual ripening processes and to understand the regulation of the expression of both ethylene-dependent and -independent genes.     

On the genetic control of heterosis for fruit shape in melon (Cucumis melo L.)

The objective of the present work is to study the genetic basis of heterosis for fruit shape (FS) in melon observed in a cross between the Spanish cultivar "Piel de Sapo" (PS) and the Korean accession PI 161375 (Songwang Charmi [SC]) using a set of near-isogenic lines (NILs) with contrasting phenotypes for FS, each carrying a single chromosomal introgression from SC within the genetic background of PS. We investigated the FS of homozygous NILs, hybrids NIL x PS, and all 2-way crosses between NILs to test the main heterosis hypotheses (dominance, overdominance, and epistatic interactions). Gene action of alleles of quantitative trait loci inducing fruit enlargement was dominance, whereas those inducing rounder fruit were additive or recessive. Only minor epistatic interactions were found. Therefore, the most plausible explanation for FS heterosis in this cross is in agreement with the dominance complementation hypothesis. Over 70% of the hybrid heterosis could be achieved by combining just 2 loci, indicating that the genetic control of FS heterosis in this cross is relatively simple. FS is proposed as a reproductive trait in melon because of the high correlation to the number of seeds produced along the fruit longitudinal axis.     

Identification of quantitative trait loci involved in fruit quality traits in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Two populations [an F₂ and a set of 77 double haploid lines (DHLs)] developed from a cross between a Piel de Sapo cultivar (PS) and the exotic Korean accession PI 161375 were used to detect QTLs involved in melon fruit quality traits: earliness (EA), fruit shape (FS), fruit weight (FW) and sugar content (SSC); and loci involved in the colour traits: external colour (ECOL) and flesh colour (FC). High variation was found, showing transgressive segregations for all traits. The highest correlation among experiments was observed for FS and the lowest for FW and SSC. Correlations among traits within experiments were, in general, not significant. QTL analysis, performed by Composite Interval Mapping, allowed the detection of nine QTLs for EA, eight for FS, six for FW and five for SSC. Major QTLs (R ²>25%) were detected for all traits. QTLs for different traits were no clearly co-localised, suggesting low pleiotropic effects at QTLs. Sixty-one per cent of them were detected in two or more experiments. QTLs for FS were detected in more trials than QTLs for FW and SSC, confirming that FS is under highly hereditable polygenic control. ECOL segregated as yellow:green in both experimental populations. The genetic control of ECOL was found to be complex, probably involving more than two loci with epistatic interactions. One of these loci was mapped on linkage group 9, but the other loci could not be clearly resolved. FC segregated as white:green:orange. The locus responsible for the green FC was mapped on linkage group 1, and it was proposed to correspond to the previously described locus gf. The genetic control of orange FC was complex: two loci in linkage groups 2 and 12 were associated with orange flesh, but larger population sizes would be necessary to elucidate completely the genetic control of orange flesh in this cross. Exotic alleles from PI161375 showed beneficial effects on EA, FW and SSC, indicating the usefulness of PI 161375 as a new source of genetic variability to improve European and American cultivars.Communicated by H.F. Linskens     

Abscisic acid perception and signaling transduction in strawberry: A model for non-climacteric fruit ripening

On basis of fruit differential respiration and ethylene effects, climacteric and non-climacteric fruits have been classically defined. Over the past decades, the molecular mechanisms of climacteric fruit ripening were abundantly described and found to focus on ethylene perception and signaling transduction. In contrast, until our most recent breakthroughs, much progress has been made toward understanding the signaling perception and transduction mechanisms for abscisic acid (ABA) in strawberry, a model for non-climacteric fruit ripening. Our reports not only have provided several lines of strong evidences for ABA-regulated ripening of strawberry fruit, but also have demonstrated that homology proteins of Arabidopsis ABA receptors, including PYR/PYL/RCAR and ABAR/CHLH, act as positive regulators of ripening in response to ABA. These receptors also trigger a set of ABA downstream signaling components, and determine significant changes in the expression levels of both sugar and pigment metabolism-related genes that are closely associated with ripening. Soluble sugars, especially sucrose, may act as a signal molecular to trigger ABA accumulation through an enzymatic action of 9-cis-epoxycarotenoid dioxygenase 1 (FaNCED1). This mini-review offers an overview of these processes and also outlines the possible, molecular mechanisms for ABA in the regulation of strawberry fruit ripening through the ABA receptors.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Genetic control of fruit shape acts prior to anthesis in melon (Cucumis melo L.)

Genetic control of fruit shape in Cucumis melo was studied using QTL analysis in two Recombinant Inbred (RI) populations consisting of 163 and 63 individuals, respectively, obtained by crossing the same round-fruited parent with two different elongated-fruit lines. Fruit shape is mainly explained by fruit length in these two populations. Most QTLs for fruit shape and ovary shape detected were found to co-segregate, thus demonstrating early control of fruit shape during ovary development. A high level of correlation between fruit shape and ovary shape was also found in 14 unrelated genetic lines, a finding which suggests that control of fruit shape by gene(s) active early in the ovary is a general feature in C. melo. Two major flower genes, a ( monoecious) and p ( pentamerous), were shown to have major effects on fruit shape. Major tightly linked QTLs for fruit and ovary shape were found close to the a and p genes, probably reflecting their pleiotropic effect on fruit shape. Moreover, one of the two QTLs detected in the Védrantais x PI 414723 population was also found in the Védrantais x PI 161375 population. Variation of fruit shape in melon could be due to variations having quantitative effects on a large set of genes that are probably involved in ovary development.     

A set of EST-SNPs for map saturation and cultivar identification in melon

Background

There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs.

Results

EST-based SNPs were discovered after resequencing ESTs between the parental lines of the PI 161375 (SC) × 'Piel de sapo' (PS) genetic map or using in silico SNP information from EST databases. In total 200 EST-based SNPs were mapped in the melon genetic map using a bin-mapping strategy, increasing the map density to 2.35 cM/marker. A subset of 45 SNPs was used to study variation in a panel of 48 melon accessions covering a wide range of the genetic diversity of the species. SNP analysis correctly reflected the genetic relationships compared with other marker systems, being able to distinguish all the accessions and cultivars.

Conclusion

This is the first example of a genetic map in a cucurbit species that includes a major set of SNP markers discovered using ESTs. The PI 161375 × 'Piel de sapo' melon genetic map has around 700 markers, of which more than 500 are gene-based markers (SNP, RFLP and SSR). This genetic map will be a central tool for the construction of the melon physical map, the step prior to sequencing the complete genome. Using the set of SNP markers, it was possible to define the genetic relationships within a collection of forty-eight melon accessions as efficiently as with SSR markers, and these markers may also be useful for cultivar identification in Occidental melon varieties.     

The respiratory climacteric is present in Charentais (Cucumis melo cv. Reticulatus F1 Alpha) melons ripened on or off the plant

Ripening of climacteric fruit is accompanied by an increasein respiration and autocatalytic ethylene synthesis. In harvestedmelons, there is variation in the magnitude and duration ofthe respiratory climacteric depending on the cultivar. It hasrecently been reported that, while the ripening-associated increasein ethylene production is present, the respiratory climactericis absent in ripening melon fruit attached to the plant, leadingto the suggestion that climacteric respiration is an artifactof harvest. To address the universality of this phenomenon,ripening behaviour in the melon cultivar Charentais (Cucumismelo cv. Reticulatus F1 Alpha), was investigated and the resultsshow that the respiratory climacteric occurs in fruit ripenedboth on and off the plant. Key words: Cucumis melo, ethylene, respiratory climacteric     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

The complex resistance to cucumber mosaic cucumovirus (CMV) in the melon accession PI161375 is governed by one gene and at least two quantitative trait loci

The complex resistance to cucumber mosaic virus (CMV) present in the exotic melon accession Sonwang Charmi PI161375 (SC) has been studied using two populations, a near-isogenic line (NIL) collection and a doubled haploid line (DHL) collection, both generated from a cross between SC and the cultivar Piel de Sapo as resistant and susceptible parents, respectively. The NIL collection had previously allowed us to describe a single recessive gene, cmv1, which conferred full resistance to CMV strains P9 and P104.82. Screening of the whole DHL population followed by quantitative trait locus (QTL) analysis revealed that resistance to the strains M6 and TL, both non-responsive to cmv1, was quantitative and governed by at least three QTLs. One of them, cmvqw12.1, co-located with cmv1 in linkage group (LG) XII. The QTL analysis mapped another two QTLs in LGIII (cmvqw3.1) and LGX (cmvqw10.1) and showed interaction between cmvqw12.1 and cmvqw3.1. Progeny of crosses between resistant DHLs carrying the three main QTLs showed complete resistance to the strain M6, validating the accuracy of the QTL analysis. However, in our screening, there were resistant DHLs carrying only two QTLs, suggesting that there are other regions involved in resistance to M6 and required when one of the main QTLs is missing. Therefore, resistance to CMV in melon SC is qualitative for some strains and quantitative for the rest. For this late resistance, cmv1 is necessary and explains most of the phenotypic variance, but it is not sufficient, and needs the interaction with other loci.