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.

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.

The Pomegranate Deciduous Trait Is Genetically Controlled by a PgPolyQ-MADS Gene.

The pomegranate (Punica granatum L.) is a deciduous fruit tree that grows worldwide. However, there are variants, which stay green in mild winter conditions and are determined evergreen. The evergreen trait is of commercial and scientific importance as it extends the period of fruit production and provides opportunity to identify genetic functions that are involved in sensing environmental cues. Several different evergreen pomegranate accessions from different genetic sources grow in the Israeli pomegranate collection. The leaves of deciduous pomegranates begin to lose chlorophyll during mid of September, while evergreen accessions continue to generate new buds. When winter temperature decreases 10°C, evergreen variants cease growing, but as soon as temperatures arise budding starts, weeks before the response of the deciduous varieties. In order to understand the genetic components that control the evergreen/deciduous phenotype, several segregating populations were constructed, and high-resolution genetic maps were assembled. Analysis of three segregating populations showed that the evergreen/deciduous trait in pomegranate is controlled by one major gene that mapped to linkage group 3. Fine mapping with advanced F3 and F4 populations and data from the pomegranate genome sequences revealed that a gene encoding for a putative and unique MADS transcription factor (PgPolyQ-MADS) is responsible for the evergreen trait. Ectopic expression of PgPolyQ-MADS in Arabidopsis generated small plants and early flowering. The deduced protein of PgPolyQ-MADS includes eight glutamines (polyQ) at the N-terminus. Three-dimensional protein model suggests that the polyQ domain structure might be involved in DNA binding of PgMADS. Interestingly, all the evergreen pomegranate varieties contain a mutation within the polyQ that cause a stop codon at the N terminal. The polyQ domain of PgPolyQ-MADS resembles that of the ELF3 prion-like domain recently reported to act as a thermo-sensor in Arabidopsis, suggesting that similar function could be attributed to PgPolyQ-MADS protein in control of dormancy. The study of the evergreen trait broadens our understanding of the molecular mechanism related to response to environmental cues. This enables the development of new cultivars that are better adapted to a wide range of climatic conditions.

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.

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

BACKGROUND: 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). RESULTS: 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. CONCLUSIONS: 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).

Revealing the Genetic Components Responsible for the Unique Photosynthetic Stem Capability of the Wild Almond Prunus arabica (Olivier) Meikle.

Almond [Prunus dulcis (Mill.) D. A. Webb] is a major deciduous fruit tree crop worldwide. During dormancy, under warmer temperatures and inadequate chilling hours, the plant metabolic activity increases and may lead to carbohydrate deficiency. Prunus arabica (Olivier) Meikle is a bushy wild almond species known for its green, unbarked stem, which stays green even during the dormancy period. Our study revealed that P. arabica green stems assimilate significantly high rates of CO2 during the winter as compared to P. dulcis cv. Um el Fahem (U.E.F.) and may improve carbohydrate status throughout dormancy. To uncover the genetic inheritance and mechanism behind the P. arabica stem photosynthetic capability (SPC), a segregated F1 population was generated by crossing P. arabica to U.E.F. Both parent's whole genome was sequenced, and SNP calling identified 4,887 informative SNPs for genotyping. A robust genetic map for U.E.F. and P. arabica was constructed (971 and 571 markers, respectively). QTL mapping and association study for the SPC phenotype revealed major QTL [log of odd (LOD) = 20.8] on chromosome 7 and another minor but significant QTL on chromosome 1 (LOD = 3.9). As expected, the P. arabica allele in the current loci significantly increased the SPC phenotype. Finally, a list of 64 candidate genes was generated. This work sets the stage for future research to investigate the mechanism regulating the SPC trait, how it affects the tree's physiology, and its importance for breeding new cultivars better adapted to high winter temperatures.

Fine Mapping of the "black" Peel Color in Pomegranate (Punica granatum L.) Strongly Suggests That a Mutation in the Anthocyanidin Reductase (ANR) Gene Is Responsible for the Trait.

Anthocyanins are important dietary and health-promoting substances present in high quantities in the peel and arils of the pomegranate (Punica granatum L.) fruit. Yet, there is a high variation in the content of anthocyanin among different pomegranate varieties. The 'Black' pomegranate variety (P.G.127-28) found in Israel contains exceptionally high levels of anthocyanins in its fruit peel which can reach up to two orders of magnitude higher content as compared to that of other pomegranate varieties' peel anthocyanins. Biochemical analysis reveals that delphinidin is highly abundant in the peel of 'Black' variety. The pattern of anthocyanin accumulation in the fruit peel during fruit development of 'Black' variety differs from that of other pomegranates. High anthocyanin levels are maintained during all developmental stages. Moreover, the accumulation of anthocyanin in the fruit peel of 'Black' variety is not dependent on light. Genetic analysis of an F2 population segregating for the "black" phenotype reveals that it is determined by a single recessive gene. Genetic mapping of the F2 population using single nucleotide polymorphism (SNP) markers identified few markers tightly linked to the "black" phenotype. Recombination analysis of the F2 population and F3 populations narrowed the "black" trait to an area of 178.5 kb on the draft genome sequence of pomegranate cv. 'Dabenzi.' A putative anthocyanidin reductase (ANR) gene is located in this area. Only pomegranate varieties displaying the "black" trait carry a base pair deletion toward the end of the gene, causing a frame shift resulting in a shorter protein. We propose that this mutation in the ANR gene is responsible for the different anthocyanin composition and high anthocyanin levels of the "black" trait in pomegranate.

High-throughput marker discovery in melon using a self-designed oligo microarray.

BACKGROUND: Genetic maps constitute the basis of breeding programs for many agricultural organisms. The creation of these maps is dependent on marker discovery. Melon, among other crops, is still lagging in genomic resources, limiting the ability to discover new markers in a high-throughput fashion. One of the methods used to search for molecular markers is DNA hybridization to microarrays. Microarray hybridization of DNA from different accessions can reveal differences between them--single-feature polymorphisms (SFPs). These SFPs can be used as markers for breeding purposes, or they can be converted to conventional markers by sequencing. This method has been utilized in a few different plants to discover genetic variation, using Affymetrix arrays that exist for only a few organisms. We applied this approach with some modifications for marker discovery in melon. RESULTS: Using a custom-designed oligonucleotide microarray based on a partial EST collection of melon, we discovered 6184 putative SFPs between the parents of our mapping population. Validation by sequencing of 245 SFPs from the two parents showed a sensitivity of around 79%. Most SFPs (81%) contained single-nucleotide polymorphisms. Testing the SFPs on another mapping population of melon confirmed that many of them are conserved. CONCLUSION: Thousands of new SFPs that can be used for genetic mapping and molecular-assisted breeding in melon were discovered using a custom-designed oligo microarray. A portion of these SFPs are conserved and can be used in different breeding populations. Although improvement of the discovery rate is still needed, this approach is applicable to many agricultural systems with limited genomic resources.

A "White" Anthocyanin-less Pomegranate (Punica granatum L.) Caused by an Insertion in the Coding Region of the Leucoanthocyanidin Dioxygenase (LDOX; ANS) Gene.

Color is an important determinant of pomegranate fruit quality and commercial value. To understand the genetic factors controlling color in pomegranate, chemical, molecular and genetic characterization of a "white" pomegranate was performed. This unique accession is lacking the typical pomegranate color rendered by anthocyanins in all tissues of the plant, including flowers, fruit (skin and arils) and leaves. Steady-state gene-expression analysis indicated that none of the analyzed "white" pomegranate tissues are able to synthesize mRNA corresponding to the PgLDOX gene (leucoanthocyanidin dioxygenase, also called ANS, anthocyanidin synthase), which is one of the central structural genes in the anthocyanin-biosynthesis pathway. HPLC analysis revealed that none of the "white" pomegranate tissues accumulate anthocyanins, whereas other flavonoids, corresponding to biochemical reactions upstream of LDOX, were present. Molecular analysis of the "white" pomegranate revealed the presence of an insertion and an SNP within the coding region of PgLDOX. It was found that the SNP does not change amino acid sequence and is not fully linked with the "white" phenotype in all pomegranate accessions from the collection. On the other hand, genotyping of pomegranate accessions from the collection and segregating populations for the "white" phenotype demonstrated its complete linkage with the insertion, inherited as a recessive single-gene trait. Taken together, the results indicate that the insertion in PgLDOX is responsible for the "white" anthocyanin-less phenotype. These data provide the first direct molecular, genetic and chemical evidence for the effect of a natural modification in the LDOX gene on color accumulation in a fruit-bearing woody perennial deciduous tree. This modification can be further utilized to elucidate the physiological role of anthocyanins in protecting the tree organs from harmful environmental conditions, such as temperature and UV radiation.

Single-nucleotide polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity.

Pomegranate is a valuable crop that is grown commercially in many parts of the world. Wild species have been reported from India, Turkmenistan and Socotra. Pomegranate fruit has a variety of health-beneficial qualities. However, despite this crop's importance, only moderate effort has been invested in studying its biochemical or physiological properties or in establishing genomic and genetic infrastructures. In this study, we reconstructed a transcriptome from two phenotypically different accessions using 454-GS-FLX Titanium technology. These data were used to explore the functional annotation of 45,187 fully annotated contigs. We further compiled a genetic-variation resource of 7,155 simple-sequence repeats (SSRs) and 6,500 single-nucleotide polymorphisms (SNPs). A subset of 480 SNPs was sampled to investigate the genetic structure of the broad pomegranate germplasm collection at the Agricultural Research Organization (ARO), which includes accessions from different geographical areas worldwide. This subset of SNPs was found to be polymorphic, with 10.7% loci with minor allele frequencies of (MAF<0.05). These SNPs were successfully used to classify the ARO pomegranate collection into two major groups of accessions: one from India, China and Iran, composed of mainly unknown country origin and which was more of an admixture than the other major group, composed of accessions mainly from the Mediterranean basin, Central Asia and California. This study establishes a high-throughput transcriptome and genetic-marker infrastructure. Moreover, it sheds new light on the genetic interrelations between pomegranate species worldwide and more accurately defines their genetic nature.

The molecular and biochemical basis for varietal variation in sesquiterpene content in melon (Cucumis melo L.) rinds.

A combined chemical, biochemical and molecular study was conducted to understand the differential accumulation of volatile sesquiterpenes in melon fruits. Sesquiterpenes were present mainly in the rinds of climacteric varieties, and a great diversity in their composition was found among varieties. Sesquiterpenes were generally absent in non-climacteric varieties. Two climacteric melon varieties, the green-fleshed 'Noy Yizre'el', and the orange-fleshed 'Dulce' were further examined. In 'Noy Yizre'el' the main sesquiterpenes accumulated are delta-cadinene, gamma-cadinene and alpha-copaene, while alpha-farnesene is the main sesquiterpene in 'Dulce'. Sesquiterpene synthase activities, mainly restricted to rinds of mature fruits, were shown to generate different sesquiterpenes in each variety according to the compositions found in rinds. EST melon database mining yielded two novel cDNAs coding for members of the Tps gene family termed CmTpsNY and CmTpsDul respectively, that are 43.2% similar. Heterologous expression in E. coli of CmTpsNY produced mainly delta-copaene, alpha-copaene, beta-caryophyllene, germacrene D, alpha-muurolene, gamma-cadinene, delta-cadinene, and alpha-cadinene, while CmTpsDul produced alpha-farnesene only. CmTpsNY was mostly expressed in 'Noy Yizre'el' rind while CmTpsDul expression was specific to 'Dulce' rind. None of these genes was expressed in rinds of the non-climacteric 'Tam Dew' cultivar. Our results indicate that different sesquiterpene synthases encoded by different members of the Tps gene family are active in melon varieties and this specificity modulates the accumulation of sesquiterpenes. The genes are differentially transcriptionally regulated during fruit development and according to variety and are likely to be associated with chemical differences responsible for the unique aromas of melon varieties.