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.

Extreme sugar accumulation in late fig ripening is accompanied by global changes in sugar metabolism and transporter gene expression.

Female fig (Ficus carica L.) fruit are characterized by a major increase in volume and sugar content during the final week of development. A detailed developmental analysis of water and dry matter accumulation during these final days indicated a temporal separation between the increase in volume due to increasing water content and a subsequent sharp increase in sugar content during a few days. The results present fig as an extreme example of sugar import and accumulation, with calculated import rates that are one order of magnitude higher than those of other sugar-accumulating sweet fruit species. To shed light on the metabolic changes occurring during this period, we followed the expression pattern of 80 genes encoding sugar metabolism enzymes and sugar transporter proteins identified in fig fruit. A parallel comparison with male fig fruits, which do not accumulate sugar during ripening, highlighted the genes specifically related to sugar accumulation. Tissue-specific analysis indicated that the expression of genes involved in sugar metabolism and transport undergoes a global transition.

Underground heterosis for yield improvement in melon.

Heterosis, the superiority of hybrids over their parents, is a major genetic force associated with plant fitness and crop yield enhancement. We investigated root-mediated yield heterosis in melons (Cucumis melo) by characterizing a common variety grafted onto 190 hybrid rootstocks, resulting from crossing 20 diverse inbreds in a diallel-mating scheme. Hybrid rootstocks improved yield by more than 40% compared with their parents, and the best hybrid yield outperformed the reference commercial variety by 65% under both optimal and minimal irrigation treatments. To characterize the genetics of underground heterosis we conducted whole genome re-sequencing of the 20 founder lines, and showed that parental genetic distance was no predictor for the level of heterosis. Through inference of the 190 hybrid genotypes from their parental genomes, followed by genome-wide association analysis, we mapped multiple quantitative trait loci for root-mediated yield. Yield enhancement of the four best-performing hybrid rootstocks was validated in multiple experiments with four different scion varieties. Our grafting approach is complementary to the common roots genetic approach that focuses mainly on variation in root system architecture, and is a step towards discovery of candidate genes involved in root function and yield enhancement.

Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops.

The Cucurbitaceae family (cucurbit) includes several economically important crops, such as melon, cucumber, watermelon, pumpkin, squash and gourds. During the past several years, genomic and genetic data have been rapidly accumulated for cucurbits. To store, mine, analyze, integrate and disseminate these large-scale datasets and to provide a central portal for the cucurbit research and breeding community, we have developed the Cucurbit Genomics Database (CuGenDB; http://cucurbitgenomics.org) using the Tripal toolkit. The database currently contains all available genome and expressed sequence tag (EST) sequences, genetic maps, and transcriptome profiles for cucurbit species, as well as sequence annotations, biochemical pathways and comparative genomic analysis results such as synteny blocks and homologous gene pairs between different cucurbit species. A set of analysis and visualization tools and user-friendly query interfaces have been implemented in the database to facilitate the usage of these large-scale data by the community. In particular, two new tools have been developed in the database, a 'SyntenyViewer' to view genome synteny between different cucurbit species and an 'RNA-Seq' module to analyze and visualize gene expression profiles. Both tools have been packed as Tripal extension modules that can be adopted in other genomics databases developed using the Tripal system.

Sugar-regulated susceptibility of tomato fruit to Colletotrichum and Penicillium requires differential mechanisms of pathogenicity and fruit responses.

Colletotrichum gloeosporioides and Penicillium expansum cause postharvest diseases in tropical and deciduous fruit. During colonization, C. gloeosporioides and P. expansum secrete ammonia in hosts with low sugar content (LowSC) and gluconic acid in hosts with high sugar content (HighSC), respectively, as a mechanism to modulate enhanced pathogenicity. We studied the pathogens interactions with tomato lines of similar genetic background but differing in their sugar content. Colletotrichum gloeosporioides showed enhanced colonization of the LowSC line with differential expression response of 15% of its genes including enhanced relative expression of glycosyl hydrolases, glucanase and MFS-transporter genes. Enhanced colonization of P. expansum occurred in the HighSC line, accompanied by an increase in carbohydrate metabolic processes mainly phosphoenolpyruvate carboxykinase, and only 4% of differentially expressed genes. Gene response of the two host lines strongly differed depending on the sugar level. Limited colonization of HighSC line by C. gloeosporioides was accompanied by a marked alteration of gene expression compared the LowSC response to the same pathogen; while colonization by P. expansum resulted in a similar response of the two different hosts. We suggest that this differential pattern of fungal/host responses may be the basis for the differential of host range of both pathogens in nature.

High-density NGS-based map construction and genetic dissection of fruit shape and rind netting in Cucumis melo.

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.

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 multi-allelic APRR2 gene is associated with fruit pigment accumulation in melon and watermelon.

Color and pigment contents are important aspects of fruit quality and consumer acceptance of cucurbit crops. Here, we describe the independent mapping and cloning of a common causative APRR2 gene regulating pigment accumulation in melon and watermelon. We initially show that the APRR2 transcription factor is causative for the qualitative difference between dark and light green rind in both crops. Further analyses establish the link between sequence or expression level variations in the CmAPRR2 gene and pigment content in the rind and flesh of mature melon fruits. A genome-wide association study (GWAS) of young fruit rind color in a panel composed of 177 diverse melon accessions did not result in any significant association, leading to an earlier assumption that multiple genes are involved in shaping the overall phenotypic variation in this trait. Through resequencing of 25 representative accessions and allelism tests between light rind accessions, we show that multiple independent single nucleotide polymorphisms in the CmAPRR2 gene are causative of the light rind phenotype. The multi-haplotypic nature of this gene explains the lack of detection power obtained through genotyping by sequencing-based GWAS and confirms the pivotal role of this gene in shaping fruit color variation in melon. This study demonstrates the power of combining bi- and multi-allelic designs with deep sequencing, to resolve lack of power due to high haplotypic diversity and low allele frequencies. Due to its central role and broad effect on pigment accumulation in fruits, the APRR2 gene is an attractive target for carotenoid bio-fortification of cucurbit crops.

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential.

Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration.

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.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

BACKGROUND: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. RESULTS: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. CONCLUSIONS: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

A 'golden' SNP in CmOr governs the fruit flesh color of melon (Cucumis melo).

The flesh color of Cucumis melo (melon) is genetically determined, and can be white, light green or orange, with ß-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 ß-carotene accumulation. Site-directed mutagenesis of the CmOr green/white haplotype to change the CmOR arginine to histidine triggered ß-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.

A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon.

The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba muskmelons (Cucumis melo 'Inodorous Group') accumulate naringenin chalcone, a yellow flavonoid pigment. With RNA-sequencing analysis of bulked segregants representing the tails of a population segregating for naringenin chalcone accumulation followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding (CmKFB) gene that, when expressed, negatively regulates naringenin chalcone accumulation. Additional metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, whereas CmKFB expression diverts the biochemical flux toward coumarins and general phenylpropanoids. These results show that CmKFB functions as a posttranscriptional regulator that diverts flavonoid metabolic flux.

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.

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.

Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

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.

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function.

The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor <7 million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with the wax coverage of wild species exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a Solanum habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene.

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates ß-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'). HPLC analysis revealed that 'yofI' accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of ß-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to ß-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A-T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.

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.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa.

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.