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: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: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:Background. Ovary culture has been a useful way to generate double haploid (DH) plant in cucumber (Cucumis sativus L.). However, the rate of embryo induction and the ability for induced embryo to grow into normal embryo are quite low. Moreover, the s mechanism of cucumber embryogenesis remains ambiguous. In this study, the molecular basis for cucumber embryogenesis was explored to set up basis for a more efficient ovary culture method. Differentially expressed genes during embryogenesis process, including the early stages of embryo formation, embryo maturation and shoot formation, were investigated using transcriptomic sequencing. Methods. Based on the cytological observation of cucumber ovary culture, the ovary culture can be divided into three stages:early embryo development, embryo maturation (from pre-embryos to cotyledon embryos) and the shoot formation stage. six key time points were selected for transcriptome sequencing and analysis. Results. We firstly conducted cytological observations which suggest that cell enlargement is the symbol for gametophytes to switch to sporophyte development pathway during early embryogenesis stage. In this stage, RNA-seq revealed 3468 up-regulated genes, including hormone signal transduction genes, hormone response genes and stress-induced genes. The reported embryogenesis-related genes BBM, HSP90 and AGL were also actively expressed during this stage. The total 480 genes that function in protein complex binding, microtubule binding, tetrapyrrole binding, tubulin binding and other microtubule activities were continuously up-regulated during the embryo maturation stage, indicating that the cytoskeleton structure was continuously being built and maintained by the action of microtubule-binding proteins and enzyme modification during embryo development. In shoot formation stage, 1383 genes were up-regulated, which were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, phenylalanine metabolism, and starch and sucrose metabolism. The shoot formation stage might be regulated by 6 transcription factors that contained a B3 domain, 9 genes in the AP2/ERF family and 2 genes encoded WUS homologous domain proteins. Conclusions. These findings offer a valuable framework for explaining the transcriptional regulatory mechanism underlying embryogenesis during cucumber ovary culture.
Project description:Trichomes are the hair-like structures that are widely present on the surface of aerial organs and function in plant defense against biotic and abiotic stresses. Previous studies focus on the single cell trichomes in Arabidopsis and cotton, or multicellular glandular trichomes in tomato, but the developmental process and molecular mechanisms controlling multicellular non-glandular trichome development are largely neglected. Here, we extensively characterized the fruit trichome (spine) development in wild type cucumber and in a tiny branched hair (tbh) mutant that contains a spontaneous mutation and has hairless foliage and smooth fruit surface. Our data indicated that cucumber trichome was multicellular and non-glandular, with no branches or endoreduplication. Further, the major feature of cucumber trichome development was spine base expansion. Transcriptome profiling through Digital Gene Expression indicated that meristem-related genes and transcription factors were implicated in the fruit spine development, and polarity regulators were upregulated during spine base expansion. qRT-PCR verified the reliability of our RNA-SEQ data, and in situ hybridization confirmed the enriched expression of meristem regulators CUP-SHAPED COTYLEDON3 (CUC3) and STM (SHOOT MERISTEMLESS) , as well as the abaxial identity gene KANADI (KAN) in cucumber fruit spine. Together, our results suggest a distinct regulatory pathway involving meristem genes and polarity regulators in multicellular trichome development in cucumber. Using Digital Gene Expression technology to compare the genome-wide gene expression profiles in the fruit spines of wild type cucumber and the tbh mutant, as well as the fruit spines on fruits of 0.5cm and 1.6cm long, repectively. Two biological repelicates were generated for each tissue.
Project description:Trichomes are the hair-like structures that are widely present on the surface of aerial organs and function in plant defense against biotic and abiotic stresses. Previous studies focus on the single cell trichomes in Arabidopsis and cotton, or multicellular glandular trichomes in tomato, but the developmental process and molecular mechanisms controlling multicellular non-glandular trichome development are largely neglected. Here, we extensively characterized the fruit trichome (spine) development in wild type cucumber and in a tiny branched hair (tbh) mutant that contains a spontaneous mutation and has hairless foliage and smooth fruit surface. Our data indicated that cucumber trichome was multicellular and non-glandular, with no branches or endoreduplication. Further, the major feature of cucumber trichome development was spine base expansion. Transcriptome profiling through Digital Gene Expression indicated that meristem-related genes and transcription factors were implicated in the fruit spine development, and polarity regulators were upregulated during spine base expansion. qRT-PCR verified the reliability of our RNA-SEQ data, and in situ hybridization confirmed the enriched expression of meristem regulators CUP-SHAPED COTYLEDON3 (CUC3) and STM (SHOOT MERISTEMLESS) , as well as the abaxial identity gene KANADI (KAN) in cucumber fruit spine. Together, our results suggest a distinct regulatory pathway involving meristem genes and polarity regulators in multicellular trichome development in cucumber.
Project description:Phloem is essential for higher plant development and survival by transporting photosynthetic products and systemic signals from source to sink organs. To date, most phloem studies were performed with the phloem exudate for physiological, transcriptomic or proteomic analyses. However, as yet, no transcriptomic profiling was performed to the phloem tissue itself, probably due to the technique difficulties to access the phloem tissue inside the plant body. In this study, laser microdissection combined with RNA-Seq technology was used to gain phloem-specific transcript profiling in three different organs (pedicle, stalk and fruit) of cucumber, a model plant species for phloem research. We found that transcription factors and biotic/abiotic stress related genes are highly enriched in the sink organs, while cell growth via calcium ion, hormone actions and cell cycle control was largely restricted in the pedicle and stalk, and genes implicated in transporting amino acids and sugars are mostly up-regulated in the pedicle. Further, we found excellent corroboration between phloem-specific gene expression, and physiological characterization with phloem function. In addition, we identified 432 cucumber-unique genes and several phloem-specific markers for future functional studies. This study provides new insights into the molecular genetics of the function of phloem tissue in cucumber, rather than the conventional phloem sap analyses.
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.
Project description:Colletotrichum orbiculare Whi2, yeast stress response Whi2 homolog, is involved in switch from biotrophic to necrotrophic stage. To elucidate downstream genes regulated by Co Whi2, we have conducted DNA microarray. About 3100 genes were up or down regulated in the Co whi2Î mutant compared with the wild-type. In particularly, 44 genes among up-regulated 58 genes in the Co whi2Î mutant are ribosomal protein related gene. Eukaryote is widely conserved TOR (Target Of Rapamycin) which is known to regulator of ribosomal gene expression. To elucidate whether up-regulated ribosomal genes in the Co whi2Î mutant are regulated by TOR activity, we have conducted DNA microarray in the Co whi2Î mutant treated with rapamycin inhibiting TOR activity. The enormous ribosomal gene expression in the Co whi2Î mutant treated with the rapamycin is lower than that without rapamycin treatment. In gene expression of the Co whi2Î mutant, the wild-type and the Co whi2Î mutant infecting on cucumber cotyledons were assessed at 4 hours post-inoculation. In gene expression of the Co whi2Î mutant with rapamycin treatment, Co whi2Î mutant treated with 100nM rapamycin and Co whi2Î mutant without rapamycin treatment infecting on cucumber cotyledons were assesed at 4 hours post-inoculation. Four replication were performed for each experiments.
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.