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Six putative α-galactosidase genes (α-Gals), three acid forms (CsGAL1, CsGAL2, CsGAL3) and three alkaline forms (CsAGA1, CsAGA2, CsAGAL3), were found in the cucumber genome. It is interesting to know the expression pattern and possible function of these α-Gals in the cucumber plant since it is a stachyose-translocating species. In this study, full-length cDNAs of six α-Gals were cloned and heterologously expressed. The result showed that all recombinant proteins revealed acid or alkaline α-Gal activities with different substrate specificities and pH or temperature responding curves, indicating their distinct roles in cucumber plants. Phylogenetic analysis of collected α-Gal amino acid sequences from different plants indicated that the ancestor of both acid and alkaline α-Gals existed before monocots and dicots separated. Generally, six α-Gal genes are universally expressed in different cucumber organs. CsGAL2 highly expressed in fasting-growing leaves, fruits and germinating seeds; CsGAL3 mainly distributes in vacuoles and significantly expressed in cucumber fruits, senescent leaves and seeds during late stage germination; The expression of CsAGA1 increased from leaf 1 to leaf 3 (sink leaves) and then declined from leaf 4 to leaf 7 (source leaves), and this isoform also highly expressed in male flowers and germinating seeds at early stage; CsAGA2 significantly expressed in cucumber leaves and female flowers; CsAGA3 is localized in plastids and also actively expressed in senescent leaves and germinating seeds; The role of CsGAL1 in cucumber plants is now unclear since its expression was relatively low in all organs. According to their expression patterns, subcellular localizations and previously reported functions of these isoforms in other plants, combining the data of soluble sugars contents in different tissues, the possible functions of these α-Gals were discussed., Competing Interests: The authors have declared that no competing interests exist.

Domesticated crops with high yield and quality are frequently susceptible to pathogen attack, whereas enhancement of disease resistance generally compromises crop yield. The underlying mechanisms of how plant development and disease resistance are coordinately programed remain elusive. Here, we showed that the basic Helix-Loop-Helix (bHLH) transcription factor Cucumis sativus Irregular Vasculature Patterning (CsIVP) was highly expressed in cucumber vascular tissues. Knockdown of CsIVP caused severe vasculature disorganization and abnormal organ morphogenesis. CsIVP directly binds to vascular-related regulators YABBY5 (CsYAB5), BREVIPEDICELLUS (CsBP), and AUXIN/INDOLEACETIC ACIDS4 (CsAUX4) and promotes their expression. Knockdown of CsYAB5 resulted in similar phenotypes as CsIVP-RNA interference (RNAi) plants, including disturbed vascular configuration and abnormal organ morphology. Meanwhile, CsIVP-RNAi plants were more resistant to downy mildew and accumulated more salicylic acid (SA). CsIVP physically interacts with NIM1-INTERACTING1 (CsNIMIN1), a negative regulator in the SA signaling pathway. Thus, CsIVP is a novel vasculature regulator functioning in CsYAB5-mediated organ morphogenesis and SA-mediated downy mildew resistance in cucumber., Competing Interests: The authors have declared that no competing interests exist.

The commercial yield of cucurbit crops infected with Cucumber mosaic virus (CMV) severely decreases. Chemical treatments against CMV are not effective; therefore, genetic resistance is considered the primary line of defense. Here, we studied resistance to CMV in cucumber inbred line '02245' using a recombinant inbred line (RIL) population generated from a cross between '65G' and '02245' as susceptible and resistant parents, respectively. Genetic analysis revealed that CMV resistance in cucumber is quantitatively inherited. Analysis of the RIL population revealed that a quantitative trait locus (QTL) was found on chromosome 6; named cmv6.1, this QTL was delimited by SSR9-56 and SSR11-177 and explained 31.7% of the phenotypic variation in 2016 and 28.2% in 2017. The marker SSR11-1, which is close to the locus, was tested on 78 different cucumber accessions and found to have an accuracy of 94% in resistant and moderately resistant lines but only 67% in susceptible lines. The mapped QTL was delimited within a region of 1,624.0 kb, and nine genes related to disease resistance were identified. Cloning and alignment of the genomic sequences of these nine genes between '65G' and '02245' revealed that Csa6M133680 had four single-base substitutions within the coding sequences (CDSs) and two single-base substitutions in its 3'-untranslated region, and the other eight genes showed 100% nucleotide sequence identity in their exons. Expression pattern analyses of Csa6M133680 in '65G' and '02245' revealed that the expression levels of Csa6M133680 significantly differed between '65G' and '02245' at 80 h after inoculation with CMV and that the expression in '02245' was 4.4 times greater than that in '65G'. The above results provide insights into the fine mapping and marker-assisted selection in cucumber breeding for CMV resistance., Competing Interests: The authors have declared that no competing interests exist.

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.

Determinate cucumber is a type of short vines, fewer nodes, and terminal flowers, it is suitable for high-density planting and available harvesting in field cultivation, whereas the indeterminate cucumber is preferred to cultivate in greenhouses. However, many biotic or abiotic stresses could lead indeterminate cucumber to be determinate in greenhouse cultivation, which may decrease yield and fruit quality. Therefore, it is urgent and essential to investigate the key factors forming determinate and terminal flowering in cucumber. In this study, two close background inbred lines were selected and conducted the miRNA and mRNA high throughput sequencing. Interestingly, ethylene-associated miRNAs and mRNAs were intensively obtained, indicating that the plant hormone ethylene is a key factor impacting determinate and terminal flowering in cucumber. The ethylene metabolites analysis showed that significant higher ethylene was observed in determinate line than that in the indeterminate line. The RT-qPCR validation of ethylene related miRNAs Cas-miR172, Cas-miR396, and Cas-miR414 and their target mRNAs showed a significant negative correlation. These data suggested that ethylene-associated miRNAs might affect determinate and terminal flower phenotypes by regulating their target genes expression. This study not only provides a potential molecular mechanism for determinate formation in cucumber but also establishes a method to demonstrate important physiological processes through the comprehensive association of miRNA and mRNA high-throughput sequencing.

Mechanized harvesting of cucumbers offers significant advantages compared to manual labor as both shortages and costs of labor increase. However the efficient use of machines depends on breeding plants with longer peduncles, but the genetic and molecular basis of fruit peduncle development in cucumber is not well understood. In this study, F2 populations were developed from a cross between two inbred lines, 1101 with a long peduncle and 1694 with a short peduncle. These were grown at two field sites, Hainan, with a tropical marine climate, in December 2014, and Beijing, with a warm temperate climate, in May 2015. Electron microscope examination of the pith cells in the peduncles of the two parental lines showed that line 1101 had significantly greater numbers of smaller cells compared to line 1694. The inheritance of cucumber fruit peduncle length (FPL) was investigated by the mixed major gene and polygene inheritance model. Genetic analysis indicated that FPL in cucumber is quantitatively inherited and controlled by one additive major gene and additive-dominant polygenes (D-2 model). A total of 1460 pairs of SSR (simple sequence repeat) primers were analyzed to identify quantitative trait loci (QTLs). Two similar genetic maps with 78 SSR markers which covered 720.6 cM in seven linkage groups were constructed based on two F2 populations. QTL analysis from the data collected at the two field sites showed that there are two minor QTLs on chromosome 1, named qfpl1.1 and qfpl1.2, and one major QTL on chromosome 6, named qfpl6.1. The marker UW021226, which was the closest one to qfpl6.1, had an accuracy rate of 79.0% when tested against plants selected from populations of the two parents. The results from this study provide insights into the inheritance and molecular mechanism of the variation of FPL in cucumber, and further research will be carried out to fine map qfpl6.1 to develop more accurate markers for MAS breeding., Competing Interests: The authors have declared that no competing interests exist.

Cucumber is an economically important vegetable crop worldwide. Powdery mildew (PM) is one of the most severe diseases that can affect cucumber crops. There have been several research efforts to isolate PM resistance genes for breeding PM-resistant cucumber. In the present study, we used a chromosome segment substitution line, SSL508-28, which carried PM resistance genes from the donor parent, JIN5-508, through twelve generations of backcrossing with a PM-susceptible inbred line, D8. We performed whole-genome resequencing of SSL508-28 and D8 to identify single nucleotide polymorphisms (SNPs), and insertions and deletions (indels). When compared against the reference genome of the inbred cucumber line 9930, a total of 468,616 SNPs and 67,259 indels were identified in SSL508-28, and 537,352 SNPs and 91,698 indels were identified in D8. Of these, 3,014 non-synonymous SNPs and 226 frameshift indels in SSL508-28, and 3,104 non-synonymous SNPs and 251 frameshift indels in D8, were identified. Bioinformatics analysis of these variations revealed a total of 15,682 SNPs and 6,262 indels between SSL508-28 and D8, among which 120 non-synonymous SNPs and 30 frameshift indels in 94 genes were detected between SSL508-28 and D8. Finally, out of these 94 genes, five resistance genes with nucleotide-binding sites and leucine-rich repeat domains were selected for qRT-PCR analysis. This revealed an upregulation of two transcripts, Csa2M435460.1 and Csa5M579560.1, in SSL508-28. Furthermore, the results of qRT-PCR analysis of these two genes in ten PM resistant and ten PM susceptible cucumber lines showed that when exposed to PM, Csa2M435460.1 and Csa5M579560.1 exhibited a higher expression level of resistant lines than susceptible lines. This indicates that Csa2M435460.1 and Csa5M579560.1 are candidate genes for PM resistance in cucumber. In addition, the non-synonymous SNPs in Csa2M435460.1 and Csa5M579560.1, identified in SSL508-28 and D8, might be the key to high PM-resistance in SSL508-28., Competing Interests: The authors have declared that no competing interests exist.

Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses., Competing Interests: The authors have declared that no competing interests exist.

Suboptimal temperature stress often causes heavy yield losses of vegetables by suppressing plant growth during winter and early spring. Gibberellin acid (GA) has been reported to be involved in plant growth and acquisition of mineral nutrients. However, no studies have evaluated the role of GA in the regulation of growth and nutrient acquisition by vegetables under conditions of suboptimal temperatures in greenhouse. Here, we investigated the roles of GA in the regulation of growth and nitrate acquisition of cucumber (Cucumis sativus L.) plants under conditions of short-term suboptimal root-zone temperatures (Tr). Exposure of cucumber seedlings to a Tr of 16°C led to a significant reduction in root growth, and this inhibitory effect was reversed by exogenous application of GA. Expression patterns of several genes encoding key enzymes in GA metabolism were altered by suboptimal Tr treatment, and endogenous GA concentrations in cucumber roots were significantly reduced by exposure of cucumber plants to 16°C Tr, suggesting that inhibition of root growth by suboptimal Tr may result from disruption of endogenous GA homeostasis. To further explore the mechanism underlying the GA-dependent cucumber growth under suboptimal Tr, we studied the effect of suboptimal Tr and GA on nitrate uptake, and found that exposure of cucumber seedlings to 16°C Tr led to a significant reduction in nitrate uptake rate, and exogenous application GA can alleviate the down-regulation by up regulating the expression of genes associated with nitrate uptake. Finally, we demonstrated that N accumulation in cucumber seedlings under suboptimal Tr conditions was improved by exogenous application of GA due probably to both enhanced root growth and nitrate absorption activity. These results indicate that a reduction in endogenous GA concentrations in roots due to down-regulation of GA biosynthesis at transcriptional level may be a key event to underpin the suboptimal Tr-induced inhibition of root growth and nitrate uptake. These findings may have important practical implications in effective mitigation of suboptimal temperature-induced vegetable loss under greenhouse conditions.

Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera.

Recent, rapid radiations make species-level phylogenetics difficult to resolve. We used a multiplexed, high-throughput sequencing approach to identify informative genomic regions to resolve phylogenetic relationships at low taxonomic levels in Begonia from a survey of sixteen species. A long-range PCR method was used to generate draft plastid genomes to provide a strong phylogenetic backbone, identify fast evolving regions and provide informative molecular markers for species-level phylogenetic studies in Begonia.

Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber.

Spines or trichomes on the fruit of cucumbers enhance their commercial value in China. In addition, glabrous mutants exhibit resistance to aphids and therefore their use by growers can reduce pesticide residues. Previous studies have reported two glabrous mutant plants containing the genes, csgl1 and csgl2. In the present study, a new glabrous mutant, NCG157, was identified showing a gene interaction effect with csgl1 and csgl2. This mutant showed the glabrous character on stems, leaves, tendrils, receptacles and ovaries, and there were no spines or tumors on the fruit surface. Inheritance analysis showed that a single recessive gene, named csgl3, determined the glabrous trait. An F2 population derived from the cross of two inbred lines 9930 (a fresh market type from Northern China that exhibits trichomes) and NCG157 (an American processing type with glabrous surfaces) was used for genetic mapping of the csgl3 gene. By combining bulked segregant analysis (BAS) with molecular markers, 18 markers, including two simple sequence repeats (SSR), nine insertion deletions (InDel) and seven derived cleaved amplified polymorphism sequences (dCAPs), were identified to link to the csgl3 gene. All of the linked markers were used as anchor loci to locate the csgl3 gene on cucumber chromosome 6. The csgl3 gene was mapped between the dCAPs markers dCAPs-21 and dCAPs-19, at genetic distances of 0.05 cM and 0.15 cM, respectively. The physical distance of this region was 19.6 kb. Three markers, InDel-19, dCAPs-2 and dCAPs-11, co-segregated with csgl3. There were two candidate genes in the region, Csa6M514860 and Csa6M514870. Quantitative real-time PCR showed that the expression of Csa6M514870 was higher in the tissues of 9930 than that of NCG157, and this was consistent with their phenotypic characters. Csa6M514870 is therefore postulated to be the candidate gene for the development of trichomes in cucumber. This study will facilitate marker-assisted selection (MAS) of the smooth plant trait in cucumber breeding and provide for future cloning of csgl3.

Cucumber gray mold caused by Botrytis cinerea is considered one of the most serious cucumber diseases. With the advent of Hi-seq technology, it is possible to study the plant-pathogen interaction at the transcriptome level. To the best of our knowledge, this is the first application of RNA-seq to identify cucumber and B. cinerea differentially expressed genes (DEGs) before and after the plant-pathogen interaction. In total, 248,908,688 raw reads were generated; after removing low-quality reads and those containing adapter and poly-N, 238,341,648 clean reads remained to map the reference genome. There were 3,512 cucumber DEGs and 1,735 B. cinerea DEGs. GO enrichment and KEGG enrichment analysis were performed on these DEGs to study the interaction between cucumber and B. cinerea. To verify the reliability and accuracy of our transcriptome data, 5 cucumber DEGs and 5 B. cinerea DEGs were chosen for RT-PCR verification. This is the first systematic transcriptome analysis of components related to the B. cinerea-cucumber interaction. Functional genes and putative pathways identified herein will increase our understanding of the mechanism of the pathogen-host interaction.

Very young cucumber (Cucumis sativus) fruit are highly susceptible to infection by the oomycete pathogen, Phytophthora capsici. As the fruit complete exponential growth, at approximately 10-12 days post pollination (dpp), they transition to resistance. The development of age-related resistance (ARR) is increasingly recognized as an important defense against pathogens, however, underlying mechanisms are largely unknown. Peel sections from cucumber fruit harvested at 8 dpp (susceptible) and 16 dpp (resistant) showed equivalent responses to inoculation as did whole fruit, indicating that the fruit surface plays an important role in defense against P. capsici. Exocarp from 16 dpp fruit had thicker cuticles, and methanolic extracts of peel tissue inhibited growth of P. capsici in vitro, suggesting physical or chemical components to the ARR. Transcripts specifically expressed in the peel vs. pericarp showed functional differentiation. Transcripts predominantly expressed in the peel were consistent with fruit surface associated functions including photosynthesis, cuticle production, response to the environment, and defense. Peel-specific transcripts that exhibited increased expression in 16 dpp fruit relative to 8 dpp fruit, were highly enriched (P<0.0001) for response to stress, signal transduction, and extracellular and transport functions. Specific transcripts included genes associated with potential physical barriers (i.e., cuticle), chemical defenses (flavonoid biosynthesis), oxidative stress, penetration defense, and molecular pattern (MAMP)-triggered or effector-triggered (R-gene mediated) pathways. The developmentally regulated changes in gene expression between peels from susceptible- and resistant- age fruits suggest programming for increased defense as the organ reaches full size.

Seedlings of Cucumis sativus L. (cv. 'Zhongnong 16') were artificially inoculated with Cucumber green mottle mosaic virus (CGMMV) at the three-true-leaf stage. Leaf and flower samples were collected at different time points post-inoculation (10, 30 and 50 d), and processed by high throughput sequencing analysis to identify candidate miRNA sequences. Bioinformatic analysis using screening criteria, and secondary structure prediction, indicated that 8 novel and 23 known miRNAs (including 15 miRNAs described for the first time in vivo) were produced by cucumber plants in response to CGMMV infection. Moreover, gene expression profiles (p-value <0.01) validated the expression of 3 of the novel miRNAs and 3 of the putative candidate miRNAs and identified a further 82 conserved miRNAs in CGMMV-infected cucumbers. Gene ontology (GO) analysis revealed that the predicted target genes of these 88 miRNAs, which were screened using the psRNATarget and miRanda algorithms, were involved in three functional categories: 2265 in molecular function, 1362 as cellular components and 276 in biological process. The subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the predicted target genes were frequently involved in metabolic processes (166 pathways) and genetic information processes (40 pathways) and to a lesser degree the biosynthesis of secondary metabolites (12 pathways). These results could provide useful clues to help elucidate host-pathogen interactions in CGMMV and cucumber, as well as for the screening of resistance genes.

Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a '9930' × 'Gy14' recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed.

Long intergenic noncoding RNAs (lincRNAs) are intergenic transcripts with a length of at least 200 nt that lack coding potential. Emerging evidence suggests that lincRNAs from animals participate in many fundamental biological processes. However, the systemic identification of lincRNAs has been undertaken in only a few plants. We chose to use cucumber (Cucumis sativus) as a model to analyze lincRNAs due to its importance as a model plant for studying sex differentiation and fruit development and the rich genomic and transcriptome data available. The application of a bioinformatics pipeline to multiple types of gene expression data resulted in the identification and characterization of 3,274 lincRNAs. Next, 10 lincRNAs targeted by 17 miRNAs were also explored. Based on co-expression analysis between lincRNAs and mRNAs, 94 lincRNAs were annotated, which may be involved in response to stimuli, multi-organism processes, reproduction, reproductive processes, and growth. Finally, examination of the evolution of lincRNAs showed that most lincRNAs are under purifying selection, while 16 lincRNAs are under natural selection. Our results provide a rich resource for further validation of cucumber lincRNAs and their function. The identification of lincRNAs targeted by miRNAs offers new clues for investigations into the role of lincRNAs in regulating gene expression. Finally, evaluation of the lincRNAs suggested that some lincRNAs are under positive and balancing selection.

Background: Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics., Principal Findings: A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation., Conclusions: We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

bZIP proteins are one of the largest transcriptional regulators playing crucial roles in plant development, physiological processes, and biotic/abiotic stress responses. Despite the availability of recently published draft genome sequence of Cucumis sativus, no comprehensive investigation of these family members has been presented for cucumber. We have identified 64 bZIP transcription factor-encoding genes in the cucumber genome. Based on structural features of their encoded proteins, CsbZIP genes could be classified into 6 groups. Cucumber bZIP genes were expanded mainly by segmental duplication rather than tandem duplication. Although segmental duplication rate of the CsbZIP genes was lower than that of Arabidopsis, rice and sorghum, it was observed as a common expansion mechanism. Some orthologous relationships and chromosomal rearrangements were observed according to comparative mapping analysis with other species. Genome-wide expression analysis of bZIP genes indicated that 64 CsbZIP genes were differentially expressed in at least one of the ten sampled tissues. A total of 4 CsbZIP genes displayed higher expression values in leaf, flowers and root tissues. The in silico micro-RNA (miRNA) and target transcript analyses identified that a total of 21 CsbZIP genes were targeted by 38 plant miRNAs. CsbZIP20 and CsbZIP22 are the most targeted by miR165 and miR166 family members, respectively. We also analyzed the expression of ten CsbZIP genes in the root and leaf tissues of drought-stressed cucumber using quantitative RT-PCR. All of the selected CsbZIP genes were measured as increased in root tissue at 24th h upon PEG treatment. Contrarily, the down-regulation was observed in leaf tissues of all analyzed CsbZIP genes. CsbZIP12 and CsbZIP44 genes showed gradual induction of expression in root tissues during time points. This genome-wide identification and expression profiling provides new opportunities for cloning and functional analyses, which may be used in further studies for improving stress tolerance in plants.

In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.

The Arabidopsis SUPERMAN (SUP) gene encodes a C2H2 type zinc finger protein that is required for maintaining the boundaries between stamens and carpels, and for regulating development of ovule outer integument. Orthologs of SUP have been characterized in bisexual flowers as well as dioecious species, but it remains elusive in monoecious plants with unisexual flowers on the same individual. Here we isolate the SUP ortholog in Cucumis sativus L (CsSUP), a monoecious vegetable. CsSUP is predominantly expressed in female specific organs: the female flower buds and ovules. Ectopic expression of CsSUP in Arabidopsis can partially complement the fruit development in sup-5 mutant, and its over-expression in wide-type leads to reduced silique length, suppressed stamen development and distorted petal patterning. Our data suggest that CsSUP plays conserved as well as distinct roles during flower and fruit development, and it may function in the boundaries and ovules to balance petal patterning, stamen and ovule development in Arabidopsis.

In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.

Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H(+)/NO₃(-) antiport in cucumber roots in response to N starvation and NO₃(-) induction. The time course of nitrate availability strongly influenced H(+)/NO₃(-) antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H(+)-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H(+)/NO₃(-) antiport in cucumber was significantly stimulated in NO₃(-)-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO₃(-)-induced plants significantly stimulated H(+)/NO₃(-) antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H(+)/NO₃(-) antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase.

Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE) from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2(nd) hour and the 9(th) day, respectively. The ratio of (A+Z)/(V+A+Z) and non-photochemical quenching (NPQ) is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII) in transgenic Arabidopsis under high light stress.

Cucumber is an important model crop and the first species sequenced in Cucurbitaceae family. Compared to the fast increasing genetic and genomics resources, the molecular cytogenetic researches in cucumber are still very limited, which results in directly the shortage of relation between plenty of physical sequences or genetic data and chromosome structure. We mapped twenty-three fosmids anchored by SSR markers from LG-3, the longest linkage group, and LG-4, the shortest linkage group on pachytene chromosomes 3 and 4, using uorescence in situ hybridization (FISH). Integrated molecular cytogenetic maps of chromosomes 3 and 4 were constructed. Except for three SSR markers located on heterochromatin region, the cytological order of markers was concordant with those on the linkage maps. Distinct structural differences between chromosomes 3 and 4 were revealed by the high resolution pachytene chromosomes. The extreme difference of genetic length between LG-3 and LG-4 was mainly attributed to the difference of overall recombination frequency. The significant differentiation of heterochromatin contents in chromosomes 3 and 4 might have a direct correlation with recombination frequency. Meanwhile, the uneven distribution of recombination frequency along chromosome 4 was observed, and recombination frequency of the long arm was nearly 3.5 times higher than that of the short arm. The severe suppression of recombination was exhibited in centromeric and heterochromatin domains of chromosome 4. Whereas a close correlation between the gene density and recombination frequency was observed in chromosome 4, no significant correlation was observed between them along chromosome 3. The comparison between cytogenetic and sequence maps revealed a large gap on the pericentromeric heterochromatin region of sequence map of chromosome 4. These results showed that integrated molecular cytogenetic maps can provide important information for the study of genetic and genomics in cucumber.

Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity.

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.

Background: The R2R3MYB proteins comprise one of the largest families of transcription factors in plants. Although genome-wide analysis of this family has been carried out in some species, little is known about R2R3MYB genes in cucumber (Cucumis sativus L.)., Principal Findings: This study has identified 55 R2R3MYB genes in the latest cucumber genome and the CsR2R3MYB family contained the smallest number of identified genes compared to other species that have been studied due to the absence of recent gene duplication events. These results were also supported by genome distribution and gene duplication analysis. Phylogenetic analysis showed that they could be classified into 11 subgroups. The evolutionary relationships and the intron-exon organizations that showed similarities with Arabidopsis, Vitis and Glycine R2R3MYB proteins were also analyzed and suggested strong gene conservation but also the expansions of particular functional genes during the evolution of the plant species. In addition, we found that 8 out of 55 (∼14.54%) cucumber R2R3MYB genes underwent alternative splicing events, producing a variety of transcripts from a single gene, which illustrated the extremely high complexity of transcriptome regulation. Tissue-specific expression profiles showed that 50 cucumber R2R3MYB genes were expressed in at least one of the tissues and the other 5 genes showed very low expression in all tissues tested, which suggested that cucumber R2R3MYB genes took part in many cellular processes. The transcript abundance level analysis during abiotic conditions (NaCl, ABA and low temperature treatments) identified a group of R2R3MYB genes that responded to one or more treatments., Conclusions: This study has produced a comparative genomics analysis of the cucumber R2R3MYB gene family and has provided the first steps towards the selection of CsR2R3MYB genes for cloning and functional dissection that can be used in further studies to uncover their roles in cucumber growth and development.

The oomycete pathogen, Pseudoperonospora cubensis, is the causal agent of downy mildew on cucurbits, and at present, no effective resistance to this pathogen is available in cultivated cucumber (Cucumis sativus). To better understand the host response to a virulent pathogen, we performed expression profiling throughout a time course of a compatible interaction using whole transcriptome sequencing. As described herein, we were able to detect the expression of 15,286 cucumber genes, of which 14,476 were expressed throughout the infection process from 1 day post-inoculation (dpi) to 8 dpi. A large number of genes, 1,612 to 3,286, were differentially expressed in pair-wise comparisons between time points. We observed the rapid induction of key defense related genes, including catalases, chitinases, lipoxygenases, peroxidases, and protease inhibitors within 1 dpi, suggesting detection of the pathogen by the host. Co-expression network analyses revealed transcriptional networks with distinct patterns of expression including down-regulation at 2 dpi of known defense response genes suggesting coordinated suppression of host responses by the pathogen. Comparative analyses of cucumber gene expression patterns with that of orthologous Arabidopsis thaliana genes following challenge with Hyaloperonospora arabidopsidis revealed correlated expression patterns of single copy orthologs suggesting that these two dicot hosts have similar transcriptional responses to related pathogens. In total, the work described herein presents an in-depth analysis of the interplay between host susceptibility and pathogen virulence in an agriculturally important pathosystem.

MicroRNAs (miRNAs) are endogenous small RNAs playing an important regulatory function in plant development and stress responses. Among them, some are evolutionally conserved in plant and others are only expressed in certain species, tissue or developmental stages. Cucumber is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, two independent small RNA libraries from cucumber leaves and roots were constructed, respectively, and sequenced with the high-throughput Illumina Solexa system. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified. QRT-PCR analysis revealed that some of the cucumber miRNAs were preferentially expressed in certain tissues. With the recently developed 'high throughput degradome sequencing' approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. Our study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

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.

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.

Micro RNAS (miRNAs) are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus) plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand) not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome.In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

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 approximately 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.