Xiuyan Zhou

Development of novel chloroplast microsatellite markers for Cucumis from sequence database

The development of chloroplast microsatellite (cpSSR) markers in Cucumis species and analysis of their polymorphism and transferability were reported. Fifteen microsatellite markers, represented by mononucleotide repeats, were developed from the complete sequence of Cucumis sativus chloroplast genome. Intraspecific variation was successfully detected in C. sativus and C. melo and revealed mean 1.6 and 1.9 alleles per cpSSR locus, respectively. With the exception of two exon region-located cpSSR markers being monomorphic, each of the others amplified polymorphic fragments in C. sativus or C. melo. A total of 34 polymorphic loci were detected with these cpSSR markers in the two species. Transferability of the newly developed cpSSR markers was checked on an additional set of 41 Cucurbitaceae accessions (belonging to 12 different species), and except for two markers with no amplification in Cucurbita maxima, the others could be transferable to all the accessions tested. Of the 15 cpSSR markers, 14 markers generated fragments with expected band sizes and 13 markers detected interspecific polymorphism among the accessions. Intraspecific polymorphism was also observed within four Cucurbitaceae species excluding C. sativus and C. melo.

Analysis of cytoplasmic variation in a cucumber germplasm collection using chloroplast microsatellite markers

Nine PCR-based markers were developed from the microsatellites in non-coding regions of chloroplast genome of Cucumis sativus and used to detect chloroplast DNA variation. These markers successfully detected intraspecific polymorphism among 37 cucumber accessions containing Chinese native germplasms (CNGs) and non-Chinese germplasms (NCGs). Each marker detected between two and four alleles and the diversity value of the makers ranged from 0.105 to 0.528. Based on the data from allele size variation, a total of 17 distinct haplotypes were identified from the 35 accessions (excluding the two accessions possessing null genes). Three haplotypes were prevalent among CNGs but most NCGs had unique haplotype. No identical haplotype was found between CNGs and NCGs, reflecting lack of exchange of CNGs with others in the 60–80s of last century. A wild species (C. hystrix Chakr.) tested herein shared a haplotype with some CNGs, suggesting that it could be the ancestry of C. sativus or at least had a common ancestral lineage. The genetic relationship among the 37 cucumber accessions was further analyzed through construction of dendrogram based on Jaccard coefficient of similarity obtained from the allele sizes. All the CNGs were clustered into a group (containing the wild accession) that distinctly differed from the other four groups containing NCGs. This result agreed with the findings above obtained from haplotype analysis. Our research documented here will offer useful information for cucumber breeding.

Expression and functional analysis of the transcription factor-encoding Gene CsERF004 in cucumber during Pseudoperonospora cubensis and Corynespora cassiicola infection

BackgroundCucumber downy mildew, caused by P. cubensis, is an important leaf disease that can severely affect cucumber production. In recent years, cucumber target spot, caused by C. cassiicola, has been reported in both Asia and Europe and is now considered as a major disease disrupting cucumber production. Single-disease-resistant cucumber varieties have been unable to satisfy production needs.To explore the molecular mechanisms of cucumber resistance to these two diseases, cucumber cultivars D9320 (resistant to downy mildew and target spot) and D0401 (susceptible to downy mildew and target spot) were used as experimental materials in this study. We used transcriptome sequencing technology to identify genes related to disease resistance and verified using transgenic technology.ResultsWe screened out the cucumber resistance-related gene CsERF004 using transcriptome sequencing technology. Induction by pathogens, salicylic acid (SA), and ethylene (ET) resulted in the up-regulation of CsERF004. Three treatments, namely, inoculation with C. cassiicola alone, inoculation with P. cubensis alone, and simultaneous inoculation with both pathogens, all resulted in the significant and sustained up-regulation of CsERF004 in the resistant cultivar D9320, during the early stage of infection. In the susceptible cultivar D0401, CsERF004 expression was also significantly up-regulated at the later stage of infection but to a lesser extent and for a shorter duration than in the resistant cultivar D9320. The CsERF004 gene encodes a protein localizes to the nucleus. The over-expression of CsERF004 in the susceptible cultivar D0401 resulted in the significant up-regulation of the CsPR1 and CsPR4 genes and increased the levels of SA and ET, which enhanced the resistance of cucumber to downy mildew and target spot.ConclusionsAnalyses of the CsERF004 expression pattern in disease-resistant and susceptible cucumber cultivars and transgenic validation indicate that CsERF004 confers resistance to P. cubensis and C. cassiicola. The findings of this study can help to better understanding of mechanisms of response to pathogens and in establishment the genetic basis for the development of cucumber broad-spectrum resistant cultivars.

Transcriptome profiling of cucumber genome expression in response to long-term low nitrogen stress

The present study aimed to delineate the genes mediating nitrogen metabolism in cucumber (Cucumis sativus L.) and elucidate the mechanisms underlying the response to long-term nitrogen limitation. As an economically important crop, cucumber is strongly nitrogen dependent. The mechanisms underlying nitrogen metabolism in cucumber are not fully known. This study found that cucumber developed to a 3.5-leaf stage with reduced plant size and biomass under chronic low nitrogen stress condition. Gene expression profiling and analysis of cucumber roots and leaves under nitrogen-starved condition identified a total of 2991 unigenes as reliable differentially expressed genes (DEGs). A comprehensive analysis of the transcriptome revealed that the mechanisms underlying the response of cucumber roots to nitrogen-deficient stress were considerably different from those of its leaves. Importantly, the DEGs involved in the photosynthesis were almost downregulated, suggesting that the photosystem was sensitive to nitrogen starvation. Otherwise, the nitrate metabolic pathway of cucumber was suppressed by nitrogen deficiency, which was further confirmed by quantitative reverse transcription–polymerase chain reaction. This study represents a comparative analysis of the transcriptome levels of roots and leaves of cucumber, which possibly provides a valuable resource for further investigating the mechanism underlying plant response to long-term nitrogen limitation stress along with the candidate genes controlling the nitrogen metabolism.

Differential proteomic analysis of dwarf characteristics in cucumber (Cucumis sativus Linn.) stems

AbstractDwarfism was one of the mo st important agronomic traits in cucumber breeding. The current study was conducted to identify dwarf-related proteins using two-dimensional electrophoresis. Twenty-two differentially expressed protein spots were detected between dwarf and vine genotypes while 20 of them were successfully identified by MALDI-TOF/TOF MS. Out of 20 identified proteins in dwarf genotype, 14 were up-regulated and six were down-regulated. The classification of differential proteins showed that the identified proteins were functionally involved in photosynthesis, energy metabolism, cytoskeletal functions, transduction and signal regulation, detoxification and redox regulation. Five differentially regulated proteins were analyzed using the technique quantitative real-time PCR (qRT-PCR). The results showed that four proteins, including histone deacetylase, the EIN2-like protein, chlorophyll A/B binding protein, and ubiquitin-conjugating enzyme Spm2 (E2) were up-regulated and one F-box family protein was down-regulated in the dwarf genotype. Further western blot analysis revealed that only E2 protein was detected in the dwarf genotype, indicating its important role in the cucumber dwarf trait.

Expression and functional analysis of the Propamocarb-related gene CsDIR16 in cucumbers

BackgroundCucumber downy mildew is among the most important diseases that can disrupt cucumber production. Propamocarb, also known as propyl-[3-(dimethylamino)propyl]carbamate (PM), is a systemic carbamate fungicide pesticide that is widely applied in agricultural production because of its high efficiency of pathogens control, especially cucumber downy mildew. However, residual PM can remain in cucumbers after the disease has been controlled. To explore the molecular mechanisms of PM retention, cucumber cultivars ‘D9320’ (with the highest residual PM content) and ‘D0351’ (lowest residual PM content) were studied. High-throughput tag-sequencing (Tag-Seq) results showed that the CsDIR16 gene was related to PM residue, which was verified using transgenic technology.ResultsWe investigated the activity of a dirigent cucumber protein encoded by the CsDIR16 in gene response to stress induced by PM treatment. Gene-expression levels of CsDIR16 were up-regulated in the fruits, leaves, and stems of ‘D0351’ plants in response to PM treatment. However, in cultivar ‘D9320’, CsDIR16 levels were down-regulated in the leaves and stems after PM treatment, with no statistically significant differences observed in the fruits. Induction by jasmonic acid, abscisic acid, polyethylene glycol 4000, NaCl, and Corynespora cassiicola Wei (Cor) resulted in CsDIR16 up-regulation in ‘D0351’ and ‘D9320’. Expression after salicylic acid treatment was up-regulated in ‘D0351’, but was down-regulated in ‘D9320’. CsDIR16 overexpression lowered PM residues, and these were more rapidly reduced in CsDIR16(+) transgenic ‘D9320’ plants than in wild-type ‘D9320’ and CsDIR16(−) transgenic plants.ConclusionsAnalyses of the CsDIR16-expression patterns in the cucumber cultivars with the highest and lowest levels of PM residue, and transgenic validation indicated that CsDIR16 plays a positive role in reducing PM residues. The findings of this study help understand the regulatory mechanisms occurring in response to PM stress in cucumbers and in establishing the genetic basis for developing low-pesticide residue cucumber cultivars.

The novel ethylene-responsive factor CsERF025 affects the development of fruit bending in cucumber

Key messageOverexpression of CsERF025 induces fruit bending by promoting the production of ethylene.AbstractCucumber fruit bending critically affects cucumber quality, but the mechanism that causes fruit bending remains unclear. To better understand this mechanism, we performed transcriptome analyses on tissues from the convex (C1) and concave (C2) sides of bending and straight (S) fruit at 2 days post anthesis (DPA). We identified a total of 281 differentially expressed genes (DEGs) from both the convex and concave sides of bent fruit that showed significantly different expression profiles relative to straight fruits. Of these 281 DEGs, 196 were up-regulated (C1/S_C2/S) and 85 were down-regulated (C1/S_C2/S). Among the 196 up-regulated DEGs, the transcriptional levels of genes related to ethylene biosynthesis and signaling pathways were significantly higher in bending fruit compared with straight fruit. CsERF025 showed the largest difference in expression between bending and straight fruit. CsERF025 is an AP2/ERF gene encoding a protein that localizes to the nucleus. Overexpression of this gene increased the bending rate of cucumber fruits and increased the angle of bending. CsERF025 increased both the expression of ethylene biosynthesis-related genes and the production of ethylene. The application of exogenous 1-aminocyclopropane-l-carboxylic acid (ACC) to straight fruits from control plants promoted fruit bending. Thus, CsERF025 enhances the production of ethylene and thereby promotes fruit bending in cucumber.