Michał Wojcieszek

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.

Karyotype analysis and chromosomal distribution of repetitive DNA sequences of cucumis metuliferus using fluorescence in situ hybridization.

Cucumis metuliferus (2n = 24) is a cultivated species of the Cucumis genus which is a potential genetic resource for Cucumis crops. Although some cytogenetic research has been reported, there is no study of karyotyping in this species. Here, we used 4′,6-diamidino-2-phenylindole and chromomycin A3 staining to identify 12 pairs of chromosomes in early-metaphase cells. Fluorescence in situ hybridization revealed the chromosomal distribution patterns of the 5S and 45S ribosomal DNA (rDNA) genes, telomeres, and 3 different satellite repeats. The 2 major signals of the 45S rDNA were located on the satellite of chromosome 11, and the 2 signals of the 5S rDNA and 2 minor signals of the 45S rDNA were located on chromosome 12. The telomere probes hybridized to the ends of all chromosomes. The 3 satellite DNAs were localized at the ends of chromosomes 1, 2, 4-10, and at the end of the short arm of chromosome 3. In summary, we reported the identification of all chromosomes of C. metuliferus. We also depicted the location of 5S and 45S rDNA, the telomere motif sequence, CmetSat1, CmetSatT2, and CmetmSat1 in an ideogram.

Genomes correction and assembling: present methods and tools

Recent rapid development of next generation sequencing (NGS) technologies provided significant impact into genomics field of study enabling implementation of many de novo sequencing projects of new species which was previously confined by technological costs. Along with advancement of NGS there was need for adjustment in assembly programs. New algorithms must cope with massive amounts of data computation in reasonable time limits and processing power and hardware is also an important factor. In this paper, we address the issue of assembly pipeline for de novo genome assembly provided by programs presently available for scientist both as commercial and as open – source software. The implementation of four different approaches – Greedy, Overlap – Layout – Consensus (OLC), De Bruijn and Integrated resulting in variation of performance is the main focus of our discussion with additional insight into issue of short and long reads correction.

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.

Comparison of de novo assembly statistics of Cucumis sativus L.

Genome sequencing is the core of genomic research. With the development of NGS and lowering the cost of procedure there is another tight gap - genome assembly. Developing the proper tool for this task is essential as quality of genome has important impact on further research. Here we present comparison of several de Bruijn assemblers tested on C. sativus genomic reads. The assessment shows that newly developed software - dnaasm provides better results in terms of quantity and quality. The number of generated sequences is lower by 5 - 33% with even two fold higher N50. Quality check showed reliable results were generated by dnaasm. This provides us with very strong base for future genomic analysis.

Detection of genomic rearrangements in cucumber using genomecmp software

Comparative genomic by increasing information about the genomes sequences available in the databases is a rapidly evolving science. A simple comparison of the general features of genomes such as genome size, number of genes, and chromosome number presents an entry point into comparative genomic analysis. Here we present the utility of the new tool genomecmp for finding rearrangements across the compared sequences and applications in plant comparative genomics.

Identification and bioinformatics comparison of two novel phosphatases in monoecious and gynoecious cucumber lines

Two Arabidopsis thaliana genes from the PP2C family of protein phosphatases (AtABI1 and AtABI2) were used to find orthologous genes in the Cucumis sativus L. cv. Borszczagowski (cucumber) genome. Cucumber has been used as a model plant for sex expression studies because although it has been defined as a monoecious species, numerous genotypes are known to produce only female, only male, or hermaphroditic flowers. We identified two new orthologous genes of AtABI1 and AtABI2 in the cucumber genome and named them CsABI1 and CsABI2. To determine the relationships between the regulation of CsABI1 and CsABI2 and flower morphogenesis in cucumber, we performed various computational analyses to define the structure of the genes, and to predict regulatory elements and protein motifs in their sequences. We also performed an expression analysis to identify differences in the expression levels of CsABI1 and CsABI2 in vegetative and generative tissues (leaf, shoot apex, and flower buds) of monoecious (B10) and gynoecious (2gg) cucumber lines. We found that the expressions of CsABI1 and CsABI2 differed in male and female floral buds, and correlated these findings with the abscisic acid signaling pathways in male and female flowers.

Bioinformatic investigation of the role of ubiquitins in cucumber flower morphogenesis

Three cDNA clones were used to screen cucumber genome in order to find genes and proteins. Functional annotation reveals that they are correlated with ubiquitination pathways. Various bioinformatics tools were used to screen and check protein sequences features such as: the presence of specific domains, transmembrane regions, cleavage site and cellular placement. The computational analysis for promotor region shows many binding sites for transcription factors, which could regulate the expression of genes. In order to check gene expression levels in developing flower buds of monoecious (B10) and gynoecious (2gg) cucumber lines, the real – time PCR technique was applied. The expression was checked for the whole buds and only for the 3rd and 4th whorls of bud when generative organ are form which were obtained by Laser Capture Microdissection (LCM) technique.

The discrepancies in the results of bioinformatics tools for genomic structural annotation

A major focus of sequencing project is to identify genes in genomes. However it is necessary to define the variety of genes and the criteria for identifying them. In this work we present discrepancies and dependencies from the application of different bioinformatic programs for structural annotation performed on the cucumber data set from Polish Consortium of Cucumber Genome Sequencing. We use Fgenesh, GenScan and GeneMark to automated structural annotation, the results have been compared to reference annotation.

Development of genome viewer (Web Omics Viewer) for managing databases of cucumber genome

Cucumber is an important plant in horticulture and science world. Sequencing projects of C. sativus genome enable new methodological aproaches in further investigation of this species. Accessibility is crucial to fully exploit obtained information about detail structure of genes, markers and other characteristic features such contigs, scaffolds and chromosomes. Genome viewer is one of tools providing plain and easy way for presenting genome data for users and for databases administration. Gbrowse - the main viewer has several very useful features but lacks in managing simplicity. Our group developed new genome browser Web Omics Viewer (WOV), keeping functionality but improving utilization and accessibility to cucumber genome data.