Project description:Genotyping arrays are tools for high throughput genotyping, which is required in genome-wide association studies (GWAS). Since the first cucumber genome draft was reported, genetic maps were constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and other sequence-related amplified polymorphism (SRAP). In this study we developed the first cucumber genotyping array which consisted of 32,864 single nucleotide polymorphisms (SNPs). These markers cover the cucumber genome every 2.1Kb and have parents/F1 hybridizations as a training set. The training set was validated with Fludigm technology and had 98% concordance. The application of the genotyping array was illustrated by constructed a genetic map of 600 cM in length based on recombinant inbred lines (RIL) population of a 9930XGy14 cross of which compromise of 11564 SNPs. The markers collinearity between the genetic map and genome references of the two parents estimated as R2=0.97. Moreover, this comparison supports a translocation in the beginning of chromosome 5 that occurred in the lineage of 9930 and Gy14 as well as local variation in the recombination rate. We also used the array to investigate the local allele frequencies along the cucumber genome and found specific region with segregation distortions. We believe that the genotyping array together with the training set would be a powerful tool in applications such as quantitative-trait loci (QTL) analysis and GWAS.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene expression profiles of cucumber under short-term chilling stress. The goals of this study are to transcriptome analysis of cucumber leaves under chilling stress. Methods: mRNA profiles of seedlings exposed to an air temperature of 6°C in the absence of light at 0, 2, 6, and 12 h were generated by deep sequencing, in triplicate, using Illumina Hiseq platform. The reference genome and gene model annotation files were downloaded from the genome website (http://cucurbitgenomics.org/). An index of the reference genome was built using Bowtie v.2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v.2.0.12. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 55.7 million clean reads was generated. Based on the threshold values of absolute value of log2 ratio ≥ 1 and FDR ≤ 0.05, a total of 2113 DEGs was identified at three time points (2, 6, and 12 h). A total of 30 genes was detected at all time points. The number of DEGs increased with time. In total, 100 TFs from 22 families in three subsets were detected. And 19 kinase families were identified in three subsets. The DEGs identified by RNA sequencing were confirmed by qRT-PCR analysis, indicating that the data were reliable. These findings provide information that can be useful for investigating the molecular mechanisms underlying the response to chilling stress in cucumber and other plants. Conclusions: The results presented here reveal changes in the transcriptome profile of cucumber in response to chilling stress. Exposure to a low temperature induced genes involved in hormone regulation, lipid metabolism, and photosynthesis, including NAC, WRKY, AP2/ERF, ERD, MYB as well as zinc finger TFs and protein kinases such as receptor-like protein kinase, MAPK, and CDK. Most TFs were upregulated whereas CDKs were downregulated. These findings provide information that can be useful for investigating the molecular mechanisms underlying the response to chilling stress in cucumber and other plants.
Project description:Plant viruses are a major threat for a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant immunity is associated with changes in the gene expression and consequently, cellular proteome to ensure virus resistance. Proteomics proved to be an extremely valuable tool for discovering multiple targets for the rational design of plant protection strategies. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant ´Heliana´ and susceptible ´Vanda´. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Upon protein extraction and filter-aided sample preparations, peptides were profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1,516 reproducibly quantified proteins using label-free approach, 133 were differentially abundant among genotypes or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Advanced bioinformatics revealed a modular network of affected host proteins. Direct comparison of both genotypes in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection showed worse preservation of energy processes and less robust translation in susceptible cultivar versus resistant genotype. Contrary, susceptible cultivar had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines a step toward durable virus resistance in cucurbits and other crops.
Project description:The plant vascular system is essential for the enlarged plant stature and successful colonizzation the land by delivering resources throughout the plants and providing mechanical support. Despite several regulators of vascular patterning have been reported, how vascular system mediates stress resistance remain largely unknown. Here we identified a CsIND transcription factor that is specifically expressed in the xylem and phloem tissues in cucumber. Knock down of CsIND by RNAi lead to dwarf plants with enlarged or disorganized vascular systems in all aerial organs. The content of both auxin and jasmonic acid were increased in the CsIND-RNAi lines. Transcriptome profiling by RNA-Seq hints CsIND-regulated gene networks for defense response and vascular development. Biochemical analyses verified that CsIND directly binds to well-known vascular regulators including CsCCR1, CsMYB116, CsYAB5, CsBP and CsAUX, and physically interacts with dorsiventral patterning genes CsKAN2 and CsYAB5. Further, CsIND-RNAi plants displayed significantly enhanced tolerance to nitrogen dificency and resistance to cucumber downy mildew. Therefore, CsIND regulates vascular formation and resistance to biotic and abiotic stresses in cucumber, through the combinarory interactions with well-known vascular regulaors and hormone metabolism and signaling pathways.
Project description:The carpel number (CN) is an important fruit trait affecting fruit shape, size, and internal quality in cucumber. CsCLAVATA3 (CsCLV3) was previously showed to be the simply inherited gene responsible for carpel number variation in cucumber, but the molecular mechanism of CsCLV3 regulating carpel number remains elusive. Here, we found that the expression of CsCLV3 was negatively correlated with carpel number variation in different cucumber lines. Knock down of CsCLV3 by RNAi led to increased number of petals and carpels, suggesting that CsCLV3 functions as a negative regulator for floral organ number in cucumber. WUSCHEL (WUS) has been well characterized to promote CLV3-expressing stem cell activity in a non-cell autonomous manner to regulate meristem maintenance and floral organ number. However, here we found the expression region of CsCLV3 overlaps with CsWUS in the basal domain of meristem, and CsCLV3 interact with CsWUS at the protein level through binding to the WUS-box motif. Overexpression of CsFUL1, a FRUITFULL-like MADS-box gene involved in fruit length regulation, resulted in increased number of floral organs in cucumber. Biochemical analyses indicated that CsFUL1 can directly bind to CsWUS promoter to stimulate its expression. Further, we found that auxin participates in carpel number variation in cucumber through physical interaction of AUXIN RESPONSE FACTOR 14 (CsARF14) and CsWUS. Therefore, CsFUL1 and CsARF14 are two new players in the WUS-CLV pathway in determining carpel number variation in cucumber.