Agnieszka Skarzyńska

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.

The construction of genomic libraries in BAC and its practical application and bioinformatic usage

Libraries constructed in bacterial artificial chromosome (BAC) vectors are essential in modern genomics for all organisms such as plants, animals, insects, algae and microbes. Cloning using a bacterial artificial chromosome (BAC) allows to obtain high quality genomic libraries that are used for physical mapping, cytogenetic research, identification and isolation of genes, as well as for gene sequencing and genomic assembly. Here we describe the methodology of BAC library construction, characterization and the newest application and bioinformatics usage in the post-genomic era.

Comparison of bioinformatics programs for analysis of single nucleotide variants

Changes in genomic sequence might influence the gene expression, protein function and, what is related to phenotype of the organism. The Next Generation Sequencing provides a big amount of data that could be used in predicting the single nucleotide variants between analyzed and reference genome. Herein we compare three tools for predicting the structural variants: Freebayes, GATK toolkit and DeepVariant. Predictions with usage of each program were made on cucumber lines and the results were compared. Our analysis indicates that in order to obtain more precise and reliable variant predictions it is worth to use more than one program for detecting polymorphisms and cross-check the results.

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.

Biological significance, computational analysis, and applications of plant microRNAs

AbstractmicroRNA molecules belong to a class of small non-coding RNAs composed of 21–24 nucleotides and have been identified in most eukaryotes. These small RNA molecules can either transcriptionally or post-transcriptionally regulate expression of their target messenger RNAs. Access to the latest RNA-profiling technologies (e.g. high-throughput sequencing) in combination with computational analysis has contributed to rapid development in the field of miRNA research. Species-specific and highly conserved miRNAs’ control in plants biological processes. Nevertheless, regulatory functions of plant miRNAs have not been still fully understood. Hence, one of the major challenges in plant miRNA research is to find out their regulatory activities that may create an opportunity to develop new strategies for improving crops. This paper provides an overview of the current knowledge concerning the mechanisms related to plant gene regulation via miRNAs. Moreover, it includes an updated overview on the bioinformatic approaches that are available for identification of new miRNAs and their targets. It also includes some specific data on key functions of plant miRNAs to show potential impact of such small RNA molecules on diverse biological processes and their biotechnological significance. Current challenges and future perspectives have also been highlighted.

Assembly of cucumber (Cucumis sativus L.) somaclones

The development of next generation sequencing opens the possibility of using sequencing in various plant studies, such as finding structural changes and small polymorphisms between species and within them. Most analyzes rely on genomic sequences and it is crucial to use well-assembled genomes of high quality and completeness. Herein we compare commonly available programs for genomic assembling and newly developed software - dnaasm. Assemblies were tested on cucumber (Cucumis sativus L.) lines obtained by in vitro regeneration (somaclones), showing different phenotypes. Obtained results shows that dnaasm assembler is a good tool for short read assembly, which allows obtaining genomes of high quality and completeness.

Bioinformatics and expressional analysis of cDNA clones from floral buds

The application of genomic approaches may serve as an initial step in understanding the complexity of biochemical network and cellular processes responsible for regulation and execution of many developmental tasks. The molecular mechanism of sex expression in cucumber is still not elucidated. A study of differential expression was conducted to identify genes involved in sex determination and floral organ morphogenesis. Herein, we present generation of expression sequence tags (EST) obtained by differential hybridization (DH) and subtraction technique (cDNA-DSC) and their characteristic features such as molecular function, involvement in biology processes, expression and mapping position on the genome.

Laser capture microdissection to study flower morphogenesis

Laser Capture Microdissection (LCM) is a sample preparation microscopic method that enables isolation of an interesting cell or cells population from human, animal or plant tissue. This technique allows for obtaining pure sample from heterogeneous mixture. From isolated cells, it is possible to obtain the appropriate quality material used for genomic research in transcriptomics, proteomics and metabolomics. We used LCM method to study flower morphogenesis and specific bud’s organ organization and development. The genes expression level in developing flower buds of male (B10) and female (2gg) lines were analyzed with qPCR. The expression was checked for stamen and carpel primordia obtained with LCM and for whole flower buds at successive stages of growth.

The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

Bioinformatics pipeline for functional identification and characterization of proteins

The new sequencing methods, called Next Generation Sequencing gives an opportunity to possess a vast amount of data in short time. This data requires structural and functional annotation. Functional identification and characterization of predicted proteins could be done by in silico approches, thanks to a numerous computational tools available nowadays. However, there is a need to confirm the results of proteins function prediction using different programs and comparing the results or confirm experimentally. Here we present a bioinformatics pipeline for structural and functional annotation of proteins.