Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo).

Linking genotype with phenotype is a fundamental goal in biology and requires robust data for both. Recent advances in plant-genome sequencing have expedited comparisons among multiple-related individuals. The abundance of structural genomic within-species variation that has been discovered indicates that a single reference genome cannot represent the complete sequence diversity of a species, leading to the expansion of the pan-genome concept. For high-resolution forward genetics, this unprecedented access to genomic variation should be paralleled and integrated with phenotypic characterization of genetic diversity. We developed a multi-parental framework for trait dissection in melon (Cucumis melo), leveraging a novel pan-genome constructed for this highly variable cucurbit crop. A core subset of 25 diverse founders (MelonCore25), consisting of 24 accessions from the two widely cultivated subspecies of C. melo, encompassing 12 horticultural groups, and 1 feral accession was sequenced using a combination of short- and long-read technologies, and their genomes were assembled de novo. The construction of this melon pan-genome exposed substantial variation in genome size and structure, including detection of ~300 000 structural variants and ~9 million SNPs. A half-diallel derived set of 300 F2 populations, representing all possible MelonCore25 parental combinations, was constructed as a framework for trait dissection through integration with the pan-genome. We demonstrate the potential of this unified framework for genetic analysis of various melon traits, including rind color intensity and pattern, fruit sugar content, and resistance to fungal diseases. We anticipate that utilization of this integrated resource will enhance genetic dissection of important traits and accelerate melon breeding.

Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux.

ß-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, ß-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-lowß) that lowered fruit ß-carotene levels with impaired chromoplast biogenesis. Cmor-lowß 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 ß-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of ß-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-lowß 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 ß-carotene to dramatically affect both carotenoid content and plastid fate.

A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with Orange allelic variation and fruit ß-carotene accumulation in melon fruit.

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, ß-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. RESULTS: 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. CONCLUSIONS: 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.

The Role of Carotenogenic Metabolic Flux in Carotenoid Accumulation and Chromoplast Differentiation: Lessons From the Melon Fruit.

Carotenoids have various roles in plant physiology. Plant carotenoids are synthesized in plastids and are highly abundant in the chromoplasts of ripening fleshy fruits. Considerable research efforts have been devoted to elucidating mechanisms that regulate carotenoid biosynthesis, yet, little is known about the mechanism that triggers storage capacity, mainly through chromoplast differentiation. The Orange gene (OR) product stabilizes phytoene synthase protein (PSY) and triggers chromoplast differentiation. OR underlies carotenoid accumulation in orange cauliflower and melon. The OR's 'golden SNP', found in melon, alters the highly evolutionary conserved Arginine108 to Histidine and controls ß-carotene accumulation in melon fruit, in a mechanism yet to be elucidated. We have recently shown that similar carotenogenic metabolic flux is active in non-orange and orange melon fruit. This flux probably leads to carotenoid turnover but known carotenoid turnover products are not detected in non-orange fruit. Arrest of this metabolic flux, using chemical inhibitors or mutations, induces carotenoid accumulation and biogenesis of chromoplasts, regardless of the allelic state of OR. We suggest that the 'golden SNP' induces ß-carotene accumulation probably by negatively affecting the capacity to synthesize downstream compounds. The accumulation of carotenoids induces chromoplast biogenesis through a metabolite-induced mechanism. Carotenogenic turnover flux can occur in non-photosynthetic tissues, which do not accumulate carotenoids. Arrest of this flux by the 'golden SNP' or other flux-arrest mutations is a potential tool for the biofortification of agricultural products with carotenoids.

Environmental effects on molecular and phenotypic variation in populations of Eruca sativa across a steep climatic gradient.

In Israel Eruca sativa has a geographically narrow distribution across a steep climatic gradient that ranges from mesic Mediterranean to hot desert environments. These conditions offer an opportunity to study the influence of the environment on intraspecific genetic variation. For this, we combined an analysis of neutral genetic markers with a phenotypic evaluation in common-garden experiments, and environmental characterization of populations that included climatic and edaphic parameters, as well as geographic distribution. A Bayesian clustering of individuals from nine representative populations based on amplified fragment length polymorphism (AFLP) divided the populations into a southern and a northern geographic cluster, with one admixed population at the geographic border between them. Linear mixed models, with cluster added as a grouping factor, revealed no clear effects of environment or geography on genetic distances, but this may be due to a strong association of geography and environment with genetic clusters. However, environmental factors accounted for part of the phenotypic variation observed in the common-garden experiments. In addition, candidate loci for selection were identified by association with environmental parameters and by two outlier methods. One locus, identified by all three methods, also showed an association with trichome density and herbivore damage, in net-house and field experiments, respectively. Accordingly, we propose that because trichomes are directly linked to defense against both herbivores and excess radiation, they could potentially be related to adaptive variation in these populations. These results demonstrate the value of combining environmental and phenotypic data with a detailed genetic survey when studying adaptation in plant populations. This article describes the use of several types of data to estimate the influence of the environment on intraspecific genetic variation in populations originating from a steep climatic gradient. In addition to molecular marker data, we made use of phenotypic evaluation from common garden experiments, and a broad GIS based environmental data with edaphic information gathered in the field. This study, among others, lead to the identification of an outlier locus with an association to trichome formation and herbivore defense, and its ecological adaptive value is discussed.