The flesh color of Cucumis melo (melon) 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 being associated 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 single nucleotide polymorphisms (SNPs) between CmOr alleles in orange versus green/white‐flesh fruit, a single SNP causes a change of an evolutionarily highly conserved 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. The 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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
β-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) golden ...single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowβ) that lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP. Moreover, Cmor-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of β-carotene to dramatically affect both carotenoid content and plastid fate.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Summary
Fruit ripening is divided into climacteric and non‐climacteric types depending on the presence or absence of a transient rise in respiration rate and the production of autocatalytic ethylene. ...Melon is ideal for the study of fruit ripening, as both climacteric and non‐climacteric varieties exist. Two introgressions of the non‐climacteric accession PI 161375, encompassed in the QTLs ETHQB3.5 and ETHQV6.3, into the non‐climacteric ‘Piel de Sapo’ background are able to induce climacteric ripening independently. We report that the gene underlying ETHQV6.3 is MELO3C016540 (CmNAC‐NOR), encoding a NAC (NAM, ATAF1,2, CUC2) transcription factor that is closely related to the tomato NOR (non‐ripening) gene. CmNAC‐NOR was functionally validated through the identification of two TILLING lines carrying non‐synonymous mutations in the conserved NAC domain region. In an otherwise highly climacteric genetic background, both mutations provoked a significant delay in the onset of fruit ripening and in the biosynthesis of ethylene. The PI 161375 allele of ETHQV6.3 is similar to that of climacteric lines of the cantalupensis type and, when introgressed into the non‐climacteric ‘Piel de Sapo’, partially restores its climacteric ripening capacity. CmNAC‐NOR is expressed in fruit flesh of both climacteric and non‐climacteric lines, suggesting that the causal mutation may not be acting at the transcriptional level. The use of a comparative genetic approach in a species with both climacteric and non‐climacteric ripening is a powerful strategy to dissect the complex mechanisms regulating the onset of fruit ripening.
Significance Statement
Regulatory mechanisms common to climacteric and non‐climacteric fruit ripening are not fully understood. Melon is a unique model species presenting both climacteric and non‐climacteric types. ETHQV6.3 QTL allele introgressed into a non‐climacteric background partially restores climacteric ripening. We show that ETHQV6.3 is encoded by the NAC‐domain transcription factor MELO3C016540 (CmNAC‐NOR). Mutations in CmNAC‐NOR in a climacteric genetic background delay fruit ripening and biosynthesis of ethylene, confirming its role in this process.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Melon is an important crop that exhibits broad variation for fruit morphology traits that are the substrate for genetic mapping efforts. In the post-genomic era, the link between genetic maps and ...physical genome assemblies is key for leveraging QTL mapping results for gene cloning and breeding purposes. Here, using a population of 164 melon recombinant inbred lines (RILs) that were subjected to genotyping-by-sequencing, we constructed and compared high-density sequence- and linkage-based recombination maps that were aligned to the reference melon genome. These analyses reveal the genome-wide variation in recombination frequency and highlight regions of disrupted collinearity between our population and the reference genome. The population was phenotyped over 3 years for fruit size and shape as well as rind netting. Four QTLs were detected for fruit size, and they act in an additive manner, while significant epistatic interaction was found between two neutral loci for this trait. Fruit shape displayed transgressive segregation that was explained by the action of four QTLs, contributed by alleles from both parents. The complexity of rind netting was demonstrated on a collection of 177 diverse accessions. Further dissection of netting in our RILs population, which is derived from a cross of smooth and densely netted parents, confirmed the intricacy of this trait and the involvement of major locus and several other interacting QTLs. A major netting QTL on chromosome 2 co-localized with results from two additional populations, paving the way for future study toward identification of a causative gene for this trait.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the ...regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression ofORfrom Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression ofAtORHis
(R90H) orSbORHis
(R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found thatAtORAla
(R90A) functioned similarly toAtORHis
to promote carotenoid overproduction. Neither AtOR nor AtORHisgreatly affected carotenogenic gene expression. AtORHisexhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHistriggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability ofAtORHis
in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstratesORHis/Ala
as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlyingORHis
-regulated carotenoid accumulation.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Dear Editor, Potyviruses such as Papaya ring-spot virus （PRSV） cause important yield losses in cucurbits. Two distinct resistant alleles were identified in the Cucumis melo germplasm. Accession PI ...414723 （Supplemental Table 1） possesses mono- genic resistance, controlled by the Prv2 allele, and reacts to PRSV by systemic necrotic lesions; plants with the PryI allele, described in cultivar WMR-29, remain symptomless （Pitrat and Lecoq, 1983）. Fusarium oxysporum f. sp. melonis （FUS） exclusively attacks melon, causing severe wilt. Monogenic dominant resistance was described against races O, 1, and 2. The Fore-2 gene, controlling resistance to races 0 and 1, was cloned by Joobeur et al. （2004）, and encodes a nucleotide bindina domain （NB）-Ieucine rich repeat （LRR） protein.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and ...other volatiles. l–methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with 13C‐ and 2H‐labeled l–methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l‐methionine aminotransferase and preserves the main carbon skeleton of l‐methionine. The second route apparently involves the action of an l‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l–isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the ...bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.
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).
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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