Insight into the Organization of the B10v3 Cucumber Genome by Integration of Biological and Bioinformatic Data.

The availability of a well-organized and annotated reference genome is essential for genome research and the analysis of re-sequencing approaches. The B10v3 cucumber (Cucumis sativus L.) reference genome has been sequenced and assembled into 8035 contigs, a small fraction of which have been mapped to individual chromosomes. Currently, bioinformatics methods based on comparative homology have made it possible to re-order the sequenced contigs by mapping them to the reference genomes. The B10v3 genome (North-European, Borszczagowski line) was rearranged against the genomes of cucumber 9930 ('Chinese Long' line) and Gy14 (North American line). Furthermore, a better insight into the organization of the B10v3 genome was obtained by integrating the data available in the literature on the assignment of contigs to chromosomes in the B10v3 genome with the results of the bioinformatic analysis. The combination of information on the markers used in the assembly of the B10v3 genome and the results of FISH and DArT-seq experiments confirmed the reliability of the in silico assignment. Approximately 98% of the protein-coding genes within the chromosomes were assigned and a significant proportion of the repetitive fragments in the sequenced B10v3 genome were identified using the RagTag programme. In addition, BLAST analyses provided comparative information between the B10v3 genome and the 9930 and Gy14 data sets. This revealed both similarities and differences in the functional proteins found between the coding sequences region in the genomes. This study contributes to better knowledge and understanding of cucumber genome line B10v3.

miRNA Profiling and Its Role in Multi-Omics Regulatory Networks Connected with Somaclonal Variation in Cucumber (Cucumis sativus L.).

The role of miRNAs in connection with the phenomenon of somaclonal variation, which occurs during plant in vitro culture, remains uncertain. This study aims to investigate the possible role of miRNAs in multi-omics regulatory pathways in cucumber somaclonal lines. For this purpose, we performed sRNA sequencing (sRNA-seq) from cucumber fruit samples identified 8, 10 and 44 miRNAs that are differentially expressed between somaclones (S1, S2, S3 lines) and the reference B10 line of Cucumis sativus. For miRNA identification, we use ShortStack software designed to filter miRNAs from sRNAs according to specific program criteria. The identification of predicted in-silico targets revealed 2,886 mRNAs encoded by 644 genes. The functional annotation of miRNA's target genes and gene ontology classification revealed their association with metabolic processes, response to stress, multicellular organism development, biosynthetic process and catalytic activity. We checked with bioinformatic analyses for possible interactions at the level of target proteins, differentially expressed genes (DEGs) and genes affected by genomic polymorphisms. We assume that miRNAs can indirectly influence molecular networks and play a role in many different regulatory pathways, leading to somaclonal variation. This regulation is supposed to occur through the process of the target gene cleavage or translation inhibition, which in turn affects the proteome, as we have shown in the example of molecular networks. This is a new approach combining levels from DNA-seq through mRNA-seq, sRNA-seq and in silico PPI in the area of plants' somaclonal variation.

Influence of transgenesis on genome variability in cucumber lines with a thaumatin II gene.

The development of new plant varieties by genetic modification aims at improving their features or introducing new qualities. However, concerns about the unintended effects of transgenes and negative environmental impact of genetically modified plants are an obstacle for the use of these plants in crops. To analyze the impact of transgenesis on plant genomes, we analyze three cucumber transgenic lines with an introduced thaumatin II gene. After genomes sequencing, we analyzed the transgene insertion site and performed variant prediction. As a result, we obtained similar number of variants for all analyzed lines (average of 4307 polymorphisms), with high abundance in one region of chromosome 4. According to SnpEff analysis, the presence of genomic variants generally does not influence the genome functionality, as less than 2% of polymorphisms have high impact. Moreover, analysis indicates that these changes were more likely induced by in vitro culture than by the transgenesis itself. The insertion site analysis shows that the region of transgene integration could cause changes in gene expression, by gene disruption or loss of promoter region continuity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00990-8.

Correction to: A high-quality cucumber genome assembly enhances computational comparative genomics.

The authors would like to correct the citation for the North European B10 line.

A high-quality cucumber genome assembly enhances computational comparative genomics.

Genetic variation is expressed by the presence of polymorphisms in compared genomes of individuals that can be transferred to next generations. The aim of this work was to reveal genome dynamics by predicting polymorphisms among the genomes of three individuals of the highly inbred B10 cucumber (Cucumis sativus L.) line. In this study, bioinformatic comparative genomics was used to uncover cucumber genome dynamics (also called real-time evolution). We obtained a new genome draft assembly from long single molecule real-time (SMRT) sequencing reads and used short paired-end read data from three individuals to analyse the polymorphisms. Using this approach, we uncovered differentiation aspects in the genomes of the inbred B10 line. The newly assembled genome sequence (B10v3) has the highest contiguity and quality characteristics among the currently available cucumber genome draft sequences. Standard and newly designed approaches were used to predict single nucleotide and structural variants that were unique among the three individual genomes. Some of the variant predictions spanned protein-coding genes and their promoters, and some were in the neighbourhood of annotated interspersed repetitive elements, indicating that the highly inbred homozygous plants remained genetically dynamic. This is the first bioinformatic comparative genomics study of a single highly inbred plant line. For this project, we developed a polymorphism prediction method with optimized precision parameters, which allowed the effective detection of small nucleotide variants (SNVs). This methodology could significantly improve bioinformatic pipelines for comparative genomics and thus has great practical potential in genomic metadata handling.

Impact of Climate Change on Regulation of Genes Involved in Sex Determination and Fruit Production in Cucumber.

Environmental changes, both natural and anthropogenic, mainly related to rising temperatures and water scarcity, are clearly visible around the world. Climate change is important for crop production and is a major issue for the growth and productivity of cucumbers. Processes such as sex determination, flower morphogenesis and fruit development in cucumbers are highly sensitive to various forms of stress induced by climatic changes. It is noteworthy that many factors, including genetic factors, transcription factors, phytohormones and miRNAs, are crucial in regulating these processes and are themselves affected by climate change. Changes in the expression and activity of these factors have been observed as a consequence of climatic conditions. This review focuses primarily on exploring the effects of climate change and abiotic stresses, such as increasing temperature and drought, on the processes of sex determination, reproduction, and fruit development in cucumbers at the molecular level. In addition, it highlights the existing research gaps that need to be addressed in order to improve our understanding of the complex interactions between climate change and cucumber physiology. This, in turn, may lead to strategies to mitigate the adverse effects and enhance cucumber productivity in a changing climate.

Understanding Transcription Factors and How They Affect Processes in Cucumber Sex Determination.

Plant reproduction is a fundamental process on Earth from the perspective of biodiversity, biomass gain, and crop productivity. It is therefore important to understand the sex determination process, and many researchers are investigating the molecular basis of this phenomenon. However, information on the influence of transcription factors (TFs), genes that encode DNA-binding proteins, on this process is limited, although cucumber is a model plant in this regard. In the present study, based on RNA-seq data for differentially expressed genes (DEGs), we aimed to investigate the regulatory TFs that may influence the metabolic processes in the shoot apex containing the forming flower buds. Therefore, the annotation of the genome of the B10 cucumber line was supplemented with the assigned families of transcription factors. By performing ontology analyses of the DEGs, the processes they participate in were identified, and TFs were located among the results. In addition, TFs that have significantly overrepresented targets among DEGs were detected, and sex-specific interactome network maps were generated, indicating the regulatory TFs based on their effects on DEGs and furthermore, on the processes leading to the formation of different-sex flowers. Among the most overrepresented TF families in the sex comparisons were the NAC, bHLH, MYB, and bZIP families. An interaction network analysis indicated the most abundant families among DEGs' regulatory TFs were MYB, AP2/ERF, NAC, and bZIP, and those with the most significant impact on developmental processes were identified, namely the AP/ERF family, followed by DOF, MYB, MADS, and others. Thus, the networks' central nodes and key regulators were identified with respect to male, female, and hermaphrodite forms. Here, we proposed the first model of the regulatory network of TFs that influences the metabolism of sex development in cucumber. These findings may help us to understand the molecular genetics and functional mechanisms underlying sex determination processes.

Genome-wide discovery of DNA variants in cucumber somaclonal lines.

The emergence of somaclonal variability in in vitro cultures is undesirable during micropropagation, but this phenomenon may be a source of genetic variability sought by breeders. The main factors that affect the appearance of variability are known, but the exact mechanism has not yet been determined. In this paper, we used next-generation sequencing and comparative genomics to study changes in the genomes of cucumber lines resulting from in vitro regeneration and somaclonal mutation in comparison to a reference, the highly inbred B10 line. The total number of obtained polymorphisms differed between the three somaclonal lines S1, S2 and S3, with 8369, 7591 and 44510, respectively. Polymorphisms occurred most frequently in non-coding regions and were mainly SNPs. High-impact changes accounted for 1%-3% of all polymorphisms and most often caused an open reading frame shift. Functional analysis of genes affected by high impact variants showed that they were related to transport, biosynthetic processes, nucleotide-containing compounds and cellular protein modification processes. The obtained results indicated significant factors affecting somaclonal variability and the appearance of changes in the genome, and demonstrated a lack of dependence between phenotype and the number of genomic polymorphisms.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II.

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.

Comparative transcriptome analysis reveals new molecular pathways for cucumber genes related to sex determination.

KEY MESSAGE: Transcriptome data and qPCR analysis revealed new insight into genes regulatory mechanism related to cucumber sex determination. Cucumber (Cucumis sativus L.) is an economically important crop cultivated worldwide. Enhancing the genomic resources for cucumber may enable the regulation of traits relevant to crop productivity and quality. Sequencing technologies and bioinformatics tools provide opportunities for the development of such resources. The aims of this study were to identify and characterize the genes involved in sex determination and flower morphogenesis in cucumber isogenic lines that differed regarding flower sex type. We obtained transcripts for 933 genes related to shoot apex development, among which 310 were differentially expressed genes (DEGs) among the male, female, and hermaphroditic lines. We performed gene ontology and molecular network analyses and explored the DEGs related to already known processes like: hormone synthesis and signaling, lipid and sugar metabolism; and also newly discovered processes related to cell wall, membrane, and cytoskeleton modifications; ion homeostasis which appears to be important for ethylene perception and signaling, and genes expression mediated by transcription factors related to floral organ identities. We proposed a new model of regulatory mechanism network of sex development in cucumber. Our results may be useful for clarifying the molecular genetics and the functional mechanisms underlying the sex determination processes.

Transcriptome Analyses of Mosaic (MSC) Mitochondrial Mutants of Cucumber in a Highly Inbred Nuclear Background.

Cucumber (Cucumis sativus L.) has a large, paternally transmitted mitochondrial genome. Cucumber plants regenerated from cell cultures occasionally show paternally transmitted mosaic (MSC) phenotypes, characterized by slower growth, chlorotic patterns on the leaves and fruit, lower fertility, and rearrangements in their mitochondrial DNAs (mtDNAs). MSC lines 3, 12, and 16 originated from different cell cultures all established using the highly inbred, wild-type line B. These MSC lines possess different rearrangements and under-represented regions in their mtDNAs. We completed RNA-seq on normalized and non-normalized cDNA libraries from MSC3, MSC12, and MSC16 to study their nuclear gene-expression profiles relative to inbred B. Results from both libraries indicated that gene expression in MSC12 and MSC16 were more similar to each other than MSC3. Forty-one differentially expressed genes (DEGs) were upregulated and one downregulated in the MSC lines relative to B. Gene functional classifications revealed that more than half of these DEGs are associated with stress-response pathways. Consistent with this observation, we detected elevated levels of hydrogen peroxide throughout leaf tissue in all MSC lines compared to wild-type line B. These results demonstrate that independently produced MSC lines with different mitochondrial polymorphisms show unique and shared nuclear responses. This study revealed genes associated with stress response that could become selection targets to develop cucumber cultivars with increased stress tolerance, and further support of cucumber as a model plant to study nuclear-mitochondrial interactions.

Next generation sequencing and omics in cucumber (Cucumis sativus L.) breeding directed research.

In the post-genomic era the availability of genomic tools and resources is leading us to novel generation methods in plant breeding, as they facilitate the study of the genotype and its relationship with the phenotype, in particular for complex traits. In this study we have mainly concentrated on the Cucumis sativus and (but much less) Cucurbitaceae family several important vegetable crops. There are many reports on research conducted in Cucurbitaceae plant breeding programs on the ripening process, phloem transport, disease resistance, cold tolerance and fruit quality traits. This paper presents the role played by new omic technologies in the creation of knowledge on the mechanisms of the formation of the breeding features. The analysis of NGS (NGS-next generation sequencing) data allows the discovery of new genes and regulatory sequences, their positions, and makes available large collections of molecular markers. Genome-wide expression studies provide breeders with an understanding of the molecular basis of complex traits. Firstly a high density map should be created for the reference genome, then each re-sequencing data could be mapped and new markers brought out into breeding populations. The paper also presents methods that could be used in the future for the creation of variability and genomic modification of the species in question. It has been shown also the state and usefulness in breeding the chloroplastomic and mitochondriomic study.