Zbigniew Przybecki

The Genome Sequence of the North-European Cucumber (Cucumis sativus L.) Unravels Evolutionary Adaptation Mechanisms in Plants

Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar – Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.

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.

Identification of genes up-regulated during somatic embryogenesis of cucumber

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.

The construction and characteristics of a BAC library for Cucumis sativus L. 'B10'.

Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.

Molecular Cytogenetic Analysis of Cucumis Wild Species Distributed in Southern Africa: Physical Mapping of 5S and 45S rDNA with DAPI

Wild Cucumis species have been divided into Australian/Asian and African groups using morphological and phylogenetic characteristics, and new species have been described recently. No molecular cytogenetic information is available for most of these species. The crossability between 5 southern African Cucumis species (C. africanus, C. anguria, C. myriocarpus, C. zeyheri, and C. heptadactylus) has been reported; however, the evolutionary relationship among them is still unclear. Here, a molecular cytogenetic analysis using FISH with 5S and 45S ribosomal DNA (rDNA) was used to investigate these Cucumis species based on sets of rDNA-bearing chromosomes (rch) types I, II and III. The molecular cytogenetic and phylogenetic results suggested that at least 2 steps of chromosomal rearrangements may have occurred during the evolution of tetraploid C. heptadactylus. In step 1, an additional 45S rDNA site was observed in the chromosome (type III). In particular, C. myriocarpus had a variety of rch sets. Our results suggest that chromosomal rearrangements may have occurred in the 45S rDNA sites. We propose that polyploid evolution occurred in step 2. This study provides insights into the chromosomal characteristics of African Cucumis species and contributes to the understanding of chromosomal evolution in this genus.

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.

In Situ Reverse Transcription PCR on Plant Tissues

In situ detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, in situ detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. The advantage of in situ methods over the conventional techniques (e.g., Northern blot, reverse transcription polymerase chain reaction [RT-PCR], or real-time PCR) is that they allow the investigation of the putative spatial distribution of nucleic acid products activity in a heterogeneous cell population. In this chapter, we describe a protocol for in situ RT-PCR detection of specific messenger RNA in cucumber (Cucumis sativus), although this protocol can be used for any plant species, floral buds, and somatic embryo tissue sections on glass microscope slides. A successful in situ RT-PCR procedure requires the optimization of many conditions related to the tissue types used, for example, a cells age, size, and composition, which may influence the detection of RT-PCR products, as well as specific transcript availability. Moreover, parameters, such as the fixation time, thermal cycling set-up, and the time of detection of RT-PCR products, also should be optimized. The importance of the other factors also is estimated in the protocol. In addition several types of controls that are necessary for a trustworthy in situ RT-PCR method are being discussed.

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.

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.