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Background

The cucumber, Cucumis sativus, is one of the most widely consumed fruit vegetables the world over. The history of its dispersal to the Occident from its centre of origin, the Indian subcontinent, has been incorrectly understood for some time, due to the confusion of cucumbers with vegetable melons. Iconographic and literary evidence has shown that cucumber was absent in Roman times, up to 500 CE, but present in Europe by late medieval times, 1300. The objective of the present investigation was to determine more accurately when the cucumber arrived in Europe and by what route.

Findings and Conclusions

The evidence for the movement of C. sativus westward is entirely lexicographical until the 10th century. Syriac, Persian and Byzantine Greek sources suggest the presence of cucumbers, to the east and north-east of the Mediterranean Sea (modern Iran, Iraq and Turkey), by the 6th or 7th century. Arabic medical writings suggest the presence of cucumbers in Spain as early as the mid-9th century and in Tunisia by the early 10th century. Descriptive evidence in Arabic establishes the presence of cucumbers in Andalusia by the second half of the 10th century. Latin translations from Arabic sources indicate the presence of cucumbers in southern Italy by the second half of the 11th century. These writings, together with lexicographical discrepancies in names of cucurbits in late medieval Latin writings, suggest that cucumber was introduced to Europe by two independent diffusions. One diffusion appears to have been overland from Persia into eastern and northern Europe and preceded the Islamic conquests. The other, subsequent diffusion into western and southern Europe, was probably by a mostly maritime route from Persia or the Indian subcontinent into Andalusia.

Background

Cucumber, Cucumis sativus L. (2n = 2 × = 14) and melon, C. melo L. (2n = 2 × = 24) are two important vegetable species in the genus Cucumis (family Cucurbitaceae). Both species have an Asian origin that diverged approximately nine million years ago. Cucumber is believed to have evolved from melon through chromosome fusion, but the details of this process are largely unknown. In this study, comparative genetic mapping between cucumber and melon was conducted to examine syntenic relationships of their chromosomes.

Results

Using two melon mapping populations, 154 and 127 cucumber SSR markers were added onto previously reported F2- and RIL-based genetic maps, respectively. A consensus melon linkage map was developed through map integration, which contained 401 co-dominant markers in 12 linkage groups including 199 markers derived from the cucumber genome. Syntenic relationships between melon and cucumber chromosomes were inferred based on associations between markers on the consensus melon map and cucumber draft genome scaffolds. It was determined that cucumber Chromosome 7 was syntenic to melon Chromosome I. Cucumber Chromosomes 2 and 6 each contained genomic regions that were syntenic with melon chromosomes III+V+XI and III+VIII+XI, respectively. Likewise, cucumber Chromosomes 1, 3, 4, and 5 each was syntenic with genomic regions of two melon chromosomes previously designated as II+XII, IV+VI, VII+VIII, and IX+X, respectively. However, the marker orders in several syntenic blocks on these consensus linkage maps were not co-linear suggesting that more complicated structural changes beyond simple chromosome fusion events have occurred during the evolution of cucumber.

Conclusions

Comparative mapping conducted herein supported the hypothesis that cucumber chromosomes may be the result of chromosome fusion from a 24-chromosome progenitor species. Except for a possible inversion, cucumber Chromosome 7 has largely remained intact in the past nine million years since its divergence from melon. Meanwhile, many structural changes may have occurred during the evolution of the remaining six cucumber chromosomes. Further characterization of the genomic nature of Cucumis species closely related to cucumber and melon might provide a better understanding of the evolutionary history leading to modern cucumber.

Background

Melon, Cucumis melo, and cucumber, C. sativus, are among the most widely cultivated crops worldwide. Cucumis, as traditionally conceived, is geographically centered in Africa, with C. sativus and C. hystrix thought to be the only Cucumis species in Asia. This taxonomy forms the basis for all ongoing Cucumis breeding and genomics efforts. We tested relationships among Cucumis and related genera based on DNA sequences from chloroplast gene, intron, and spacer regions (rbcL, matK, rpl20-rps12, trnL, and trnL-F), adding nuclear internal transcribed spacer sequences to resolve relationships within Cucumis.

Results

Analyses of combined chloroplast sequences (4,375 aligned nucleotides) for 123 of the 130 genera of Cucurbitaceae indicate that the genera Cucumella, Dicaelospermum, Mukia, Myrmecosicyos, and Oreosyce are embedded within Cucumis. Phylogenetic trees from nuclear sequences for these taxa are congruent, and the combined data yield a well-supported phylogeny. The nesting of the five genera in Cucumis greatly changes the natural geographic range of the genus, extending it throughout the Malesian region and into Australia. The closest relative of Cucumis is Muellerargia, with one species in Australia and Indonesia, the other in Madagascar. Cucumber and its sister species, C. hystrix, are nested among Australian, Malaysian, and Western Indian species placed in Mukia or Dicaelospermum and in one case not yet formally described. Cucumis melo is sister to this Australian/Asian clade, rather than being close to African species as previously thought. Molecular clocks indicate that the deepest divergences in Cucumis, including the split between C. melo and its Australian/Asian sister clade, go back to the mid-Eocene.

Conclusion

Based on congruent nuclear and chloroplast phylogenies we conclude that Cucumis comprises an old Australian/Asian component that was heretofore unsuspected. Cucumis sativus evolved within this Australian/Asian clade and is phylogenetically far more distant from C. melo than implied by the current morphological classification.

The prominent satellites of the Cucurbitaceae Cucumis melo (melon) and Cucumis sativus (cucumber) have been characterized, in actinomycin/CsCl gradients where the satellite sequences can be separated from ribosomal, organelle, and main band DNA the location of the satellites is different indicating a different GC content. The purified satellite of C. melo is cut by HindIII into a repeat unit of 380 bp; AluI digestion gives rise to two bands (about 80 and 220 bp in size). The HindIII repeat unit if cloned into pBR325 exhibits new recognition sites for HpaII leaving two bands with 150 and 80 bp suggesting methylation of the C/CGG cutting site in the uncloned material. The restriction pattern indicates an internal sequence repeat within the 380 bp HindIII fragment. The C. sativus satellite is cut by AluI to a repeat unit of 180 bp showing no other recognition site for the restriction enzymes tested so far. About 10% sequence homology has been determined between the C. melo and C. sativus satellites by cross hybridization studies. A high methylation degree of cytosines has been measured for both satellites and the ribosomal DNA of C. sativus (about 30%). No transcription products of the C. melo satellite were found during seedling development.

Images

Background

Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited.

Result

We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences.

Conclusion

The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.

• Background The gorgeous frescoes organized by the master Renaissance painter Raphael Sanzio (1483–1520) and illustrating the heavenly adventures of Cupid and Psyche were painted between 1515 and 1518 to decorate the Roman villa (now known as the Villa Farnesina) of the wealthy Sienese banker Agostino Chigi (1466–1520). Surrounding these paintings are festoons of fruits, vegetables and flowers painted by Giovanni Martini da Udine (1487–1564), which include over 170 species of plants. A deconstruction and collation of the cucurbit images in the festoons makes it possible to evaluate the genetic diversity of cucurbits in Renaissance Italy 500 years ago.

• Findings The festoons contain six species of Old World cucurbits, Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis sativus (cucumber), Ecballium elaterium (squirting cucumber), Lagenaria siceraria (bottle gourd) and Momordica balsamina (balsam apple), and two or three species of New World cucurbits, Cucurbita maxima, C. pepo and, perhaps, C. moschata (pumpkin, squash, gourd). The images of C. maxima are the first illustrations of this species in Europe.

Background

A critical analysis was made of cucurbit descriptions in Dioscorides' De Materia Medica, Columella's De Re Rustica and Pliny's Historia Naturalis, works on medicine, agriculture and natural science of the 1st century ce, as well as the Mishna and Tosefta, compilations of rabbinic law derived from the same time period together with cucurbit images dating from antiquity including paintings, mosaics and sculpture. The goal was to identify taxonomically the Mediterranean cucurbits at the time of the Roman Empire.

Findings

By ancient times, long-fruited forms of Cucumis melo (melon) and Lagenaria siceraria (bottle gourd) were selected, cultivated and used as vegetables around the Mediterranean and, in addition, bottle-shaped fruits of L. siceraria were employed as vessels. Citrullus lanatus (watermelons) and round-fruited forms of Cucumis melo (melons) were also consumed, but less commonly. A number of cucurbit species, including Bryonia alba, B. dioica, Citrullus colocynthis and Ecballium elaterium, were employed for medicinal purposes. No unequivocal evidence was found to suggest the presence of Cucumis sativus (cucumber) in the Mediterranean area during this era. The cucumis of Columella and Pliny was not cucumber, as commonly translated, but Cucumis melo subsp. melo Flexuosus Group (snake melon or vegetable melon).

Background

Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has high intra-specific genetic variation, morphologic diversity and a small genome size (450 Mb), which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. A number of genetic and genomic resources have already been developed, such as several genetic maps and BAC genomic libraries. These tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. However, no physical map of any Cucurbitaceae has yet been developed. A project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. A BAC-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced.

Results

A melon physical map was constructed using a 5.7 × BAC library and a genetic map previously developed in our laboratories. High-information-content fingerprinting (HICF) was carried out on 23,040 BAC clones, digesting with five restriction enzymes and SNaPshot labeling, followed by contig assembly with FPC software. The physical map has 1,355 contigs and 441 singletons, with an estimated physical length of 407 Mb (0.9 × coverage of the genome) and the longest contig being 3.2 Mb. The anchoring of 845 BAC clones to 178 genetic markers (100 RFLPs, 76 SNPs and 2 SSRs) also allowed the genetic positioning of 183 physical map contigs/singletons, representing 55 Mb (12%) of the melon genome, to individual chromosomal loci. The melon FPC database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.

Conclusions

Here we report the construction of the first physical map of a Cucurbitaceae species described so far. The physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. The data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in Spain, adopting a whole genome shotgun approach based on 454 sequencing data.

Background

Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods.

Results

In order to gain insight into the structure of a significant portion of the melon genome we set out to perform massive sequencing of pools of BAC clones. For this, a set of 57 BAC clones from a double haploid line was sequenced in two pools with the 454 system using both shotgun and paired-end approaches. The final assembly consists of an estimated 95% of the actual size of the melon BAC clones, with most likely complete sequences for 50 of the BACs, and a total sequence coverage of 39x. The accuracy of the assembly was assessed by comparing the previously available Sanger sequence of one of the BACs against its 454 sequence, and the polymorphisms found involved only 1.7 differences every 10,000 bp that were localized in 15 homopolymeric regions and two dinucleotide tandem repeats. Overall, the study provides approximately 6.7 Mb or 1.5% of the melon genome. The analysis of this new data has allowed us to gain further insight into characteristics of the melon genome such as gene density, average protein length, or microsatellite and transposon content. The annotation of the BAC sequences revealed a high degree of collinearity and protein sequence identity between melon and its close relative Cucumis sativus (cucumber). Transposon content analysis of the syntenic regions suggests that transposition activity after the split of both cucurbit species has been low in cucumber but very high in melon.

Conclusions

The results presented here show that the strategy followed, which combines shotgun and BAC-end sequencing together with anchored marker information, is an excellent method for sequencing specific genomic regions, especially from relatively compact genomes such as that of melon. However, in agreement with other results, this map-based, BAC approach is confirmed to be an expensive way of sequencing a whole plant genome. Our results also provide a partial description of the melon genome's structure. Namely, our analysis shows that the melon genome is highly collinear with the smaller one of cucumber, the size difference being mainly due to the expansion of intergenic regions and proliferation of transposable elements.

Downy mildew, caused by the Oomycete pathogen Pseudoperonospora cubensis, is one of the most destructive diseases of cucumber (Cucumis sativus L.) and muskmelon (C. melo L.). Although the process of pathogenesis is well understood, there are few disease control options available. The development and deployment of resistant cultivars is generally considered to be the best approach to control downy mildew. The recently completed sequencing of the cucumber genome provides a great opportunity for reliable and thorough study of the sequence and function of resistance genes in the Cucurbitaceae, which will help us to understand the resistance mechanisms and metabolic pathways activated by these genes. It can be anticipated that, in the near future, we will have more information about the genetic bases of resistance to downy mildew in Cucumis, which will facilitate efforts to breed for resistance to this pathogen.

Background

Melon (Cucumis melo) is a horticultural specie of significant nutritional value, which belongs to the Cucurbitaceae family, whose economic importance is second only to the Solanaceae. Its small genome of approx. 450 Mb coupled to the high genetic diversity has prompted the development of genetic tools in the last decade. However, the unprecedented existence of a transcriptomic approaches in melon, highlight the importance of designing new tools for high-throughput analysis of gene expression.

Results

We report the construction of an oligo-based microarray using a total of 17,510 unigenes derived from 33,418 high-quality melon ESTs. This chip is particularly enriched with genes that are expressed in fruit and during interaction with pathogens. Hybridizations for three independent experiments allowed the characterization of global gene expression profiles during fruit ripening, as well as in response to viral and fungal infections in plant cotyledons and roots, respectively. Microarray construction, statistical analyses and validation together with functional-enrichment analysis are presented in this study.

Conclusion

The platform validation and enrichment analyses shown in our study indicate that this oligo-based microarray is amenable for future genetic and functional genomic studies of a wide range of experimental conditions in melon.

Background

The melon belongs to the Cucurbitaceae family, whose economic importance among vegetable crops is second only to Solanaceae. The melon has a small genome size (454 Mb), which makes it suitable for molecular and genetic studies. Despite similar nuclear and chloroplast genome sizes, cucurbits show great variation when their mitochondrial genomes are compared. The melon possesses the largest plant mitochondrial genome, as much as eight times larger than that of other cucurbits.

Results

The nucleotide sequences of the melon chloroplast and mitochondrial genomes were determined. The chloroplast genome (156,017 bp) included 132 genes, with 98 single-copy genes dispersed between the small (SSC) and large (LSC) single-copy regions and 17 duplicated genes in the inverted repeat regions (IRa and IRb). A comparison of the cucumber and melon chloroplast genomes showed differences in only approximately 5% of nucleotides, mainly due to short indels and SNPs. Additionally, 2.74 Mb of mitochondrial sequence, accounting for 95% of the estimated mitochondrial genome size, were assembled into five scaffolds and four additional unscaffolded contigs. An 84% of the mitochondrial genome is contained in a single scaffold. The gene-coding region accounted for 1.7% (45,926 bp) of the total sequence, including 51 protein-coding genes, 4 conserved ORFs, 3 rRNA genes and 24 tRNA genes. Despite the differences observed in the mitochondrial genome sizes of cucurbit species, Citrullus lanatus (379 kb), Cucurbita pepo (983 kb) and Cucumis melo (2,740 kb) share 120 kb of sequence, including the predicted protein-coding regions. Nevertheless, melon contained a high number of repetitive sequences and a high content of DNA of nuclear origin, which represented 42% and 47% of the total sequence, respectively.

Conclusions

Whereas the size and gene organisation of chloroplast genomes are similar among the cucurbit species, mitochondrial genomes show a wide variety of sizes, with a non-conserved structure both in gene number and organisation, as well as in the features of the noncoding DNA. The transfer of nuclear DNA to the melon mitochondrial genome and the high proportion of repetitive DNA appear to explain the size of the largest mitochondrial genome reported so far.

Background

Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method.

Results

A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV.

Conclusion

This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.

Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed.

Background

Cucumber, Cucumis sativus L., is an economically and nutritionally important crop of the Cucurbitaceae family and has long served as a primary model system for sex determination studies. Recently, the sequencing of its whole genome has been completed. However, transcriptome information of this species is still scarce, with a total of around 8,000 Expressed Sequence Tag (EST) and mRNA sequences currently available in GenBank. In order to gain more insights into molecular mechanisms of plant sex determination and provide the community a functional genomics resource that will facilitate cucurbit research and breeding, we performed transcriptome sequencing of cucumber flower buds of two near-isogenic lines, WI1983G, a gynoecious plant which bears only pistillate flowers, and WI1983H, a hermaphroditic plant which bears only bisexual flowers.

Result

Using Roche-454 massive parallel pyrosequencing technology, we generated a total of 353,941 high quality EST sequences with an average length of 175bp, among which 188,255 were from gynoecious flowers and 165,686 from hermaphroditic flowers. These EST sequences, together with ~5,600 high quality cucumber EST and mRNA sequences available in GenBank, were clustered and assembled into 81,401 unigenes, of which 28,452 were contigs and 52,949 were singletons. The unigenes and ESTs were further mapped to the cucumber genome and more than 500 alternative splicing events were identified in 443 cucumber genes. The unigenes were further functionally annotated by comparing their sequences to different protein and functional domain databases and assigned with Gene Ontology (GO) terms. A biochemical pathway database containing 343 predicted pathways was also created based on the annotations of the unigenes. Digital expression analysis identified ~200 differentially expressed genes between flowers of WI1983G and WI1983H and provided novel insights into molecular mechanisms of plant sex determination process. Furthermore, a set of SSR motifs and high confidence SNPs between WI1983G and WI1983H were identified from the ESTs, which provided the material basis for future genetic linkage and QTL analysis.

Conclusion

A large set of EST sequences were generated from cucumber flower buds of two different sex types. Differentially expressed genes between these two different sex-type flowers, as well as putative SSR and SNP markers, were identified. These EST sequences provide valuable information to further understand molecular mechanisms of plant sex determination process and forms a rich resource for future functional genomics analysis, marker development and cucumber breeding.

Melon, Cucumis melo L. is an important vegetable crop worldwide. At present, there are phenomena of homonyms and synonyms present in the melon seed markets of China, which could cause variety authenticity issues influencing the process of melon breeding, production, marketing and other aspects. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for cultivar identification. The aim of this study was to construct a DNA fingerprinting database of major melon cultivars, which could provide a possibility for the establishment of a technical standard system for purity and authenticity identification of melon seeds. In this study, to develop the core set SSR markers, 470 polymorphic SSRs were selected as the candidate markers from 1219 SSRs using 20 representative melon varieties (lines). Eighteen SSR markers, evenly distributed across the genome and with the highest contents of polymorphism information (PIC) were identified as the core marker set for melon DNA fingerprinting analysis. Fingerprint codes for 471 melon varieties (lines) were established. There were 51 materials which were classified into17 groups based on sharing the same fingerprint code, while field traits survey results showed that these plants in the same group were synonyms because of the same or similar field characters. Furthermore, DNA fingerprinting quick response (QR) codes of 471 melon varieties (lines) were constructed. Due to its fast readability and large storage capacity, QR coding melon DNA fingerprinting is in favor of read convenience and commercial applications.

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.

Background

Cucumber, Cucumis sativus L. is an important vegetable crop worldwide. Until very recently, cucumber genetic and genomic resources, especially molecular markers, have been very limited, impeding progress of cucumber breeding efforts. Microsatellites are short tandemly repeated DNA sequences, which are frequently favored as genetic markers due to their high level of polymorphism and codominant inheritance. Data from previously characterized genomes has shown that these repeats vary in frequency, motif sequence, and genomic location across taxa. During the last year, the genomes of two cucumber genotypes were sequenced including the Chinese fresh market type inbred line '9930' and the North American pickling type inbred line 'Gy14'. These sequences provide a powerful tool for developing markers in a large scale. In this study, we surveyed and characterized the distribution and frequency of perfect microsatellites in 203 Mbp assembled Gy14 DNA sequences, representing 55% of its nuclear genome, and in cucumber EST sequences. Similar analyses were performed in genomic and EST data from seven other plant species, and the results were compared with those of cucumber.

Results

A total of 112,073 perfect repeats were detected in the Gy14 cucumber genome sequence, accounting for 0.9% of the assembled Gy14 genome, with an overall density of 551.9 SSRs/Mbp. While tetranucleotides were the most frequent microsatellites in genomic DNA sequence, dinucleotide repeats, which had more repeat units than any other SSR type, had the highest cumulative sequence length. Coding regions (ESTs) of the cucumber genome had fewer microsatellites compared to its genomic sequence, with trinucleotides predominating in EST sequences. AAG was the most frequent repeat in cucumber ESTs. Overall, AT-rich motifs prevailed in both genomic and EST data. Compared to the other species examined, cucumber genomic sequence had the highest density of SSRs (although comparable to the density of poplar, grapevine and rice), and was richest in AT dinucleotides. Using an electronic PCR strategy, we investigated the polymorphism between 9930 and Gy14 at 1,006 SSR loci, and found unexpectedly high degree of polymorphism (48.3%) between the two genotypes. The level of polymorphism seems to be positively associated with the number of repeat units in the microsatellite. The in silico PCR results were validated empirically in 660 of the 1,006 SSR loci. In addition, primer sequences for more than 83,000 newly-discovered cucumber microsatellites, and their exact positions in the Gy14 genome assembly were made publicly available.

Conclusions

The cucumber genome is rich in microsatellites; AT and AAG are the most abundant repeat motifs in genomic and EST sequences of cucumber, respectively. Considering all the species investigated, some commonalities were noted, especially within the monocot and dicot groups, although the distribution of motifs and the frequency of certain repeats were characteristic of the species examined. The large number of SSR markers developed from this study should be a significant contribution to the cucurbit research community.

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar – Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

Knowing the extent and structure of genetic variation in germplasm collections is essential for the conservation and utilization of biodiversity in cultivated plants. Cucumber is the fourth most important vegetable crop worldwide and is a model system for other Cucurbitaceae, a family that also includes melon, watermelon, pumpkin and squash. Previous isozyme studies revealed a low genetic diversity in cucumber, but detailed insights into the crop's genetic structure and diversity are largely missing. We have fingerprinted 3,342 accessions from the Chinese, Dutch and U.S. cucumber collections with 23 highly polymorphic Simple Sequence Repeat (SSR) markers evenly distributed in the genome. The data reveal three distinct populations, largely corresponding to three geographic regions. Population 1 corresponds to germplasm from China, except for the unique semi-wild landraces found in Xishuangbanna in Southwest China and East Asia; population 2 to Europe, America, and Central and West Asia; and population 3 to India and Xishuangbanna. Admixtures were also detected, reflecting hybridization and migration events between the populations. The genetic background of the Indian germplasm is heterogeneous, indicating that the Indian cucumbers maintain a large proportion of the genetic diversity and that only a small fraction was introduced to other parts of the world. Subsequently, we defined a core collection consisting of 115 accessions and capturing over 77% of the SSR alleles. Insight into the genetic structure of cucumber will help developing appropriate conservation strategies and provides a basis for population-level genome sequencing in cucumber.

Background

Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.

Results

Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups.

Conclusions

Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.

Background

Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon.

Results

We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs.

Conclusion

We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.

The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and α-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[13C6]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective α-keto acids, utilizing α-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants.

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.

Background

Although melon (Cucumis melo L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library.

Results

We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes.

Conclusion

The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes.