Biology subjects

Vrana, J.

Publications and source records attributed to Vrana, J..

7 recordsLinked to original sources

bioRxiv2022

Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop

Computational tools addressing various components of design-build-test-learn loops (DBTL) for the construction of synthetic genetic networks exist, but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called DART (Design Assemble Round Trip). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection, and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions.

synthetic biology↗

bioRxiv2020

Anatomy and transcription dynamics of wheat ribosomal RNA loci revealed by optical mapping and RNA sequencing

Background and AimsThree out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly identical units spanning over several megabases of sequence. Due to this peculiar structure, the number of rRNA genes, their sequence composition and expression status remain unclear, especially in complex polyploid genomes harbouring multiple loci. Here we conducted a complex study to decipher structure and activity of both major and minor rRNA loci in hexaploid bread wheat (Triticum aestivum). MethodsWe employed an original, multi-omics approach, combining chromosome flow sorting and optical mapping with transcriptome and methylome sequencing. Key ResultsThe former two techniques enabled unbiased quantification of rDNA units in particular loci of the wheat genome. Total number of rRNA genes organized in tandem arrays was 4388, with 64.1, 31.4, 3.9 and 0.7% located in short arms of chromosomes 6B, 1B, 5D and 1A, respectively. At the expression level, only 1B and 6B loci contributed to transcription at roughly 2:1 ratio. The 1B:6B ratio varied among five analysed tissues (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG contexts in the silenced 5D locus compared to the active 1B and 6B loci. ConclusionsA fine genomic organization and tissue-specific expression of rRNA loci were deciphered, for the first time, in a complex polyploid species. We documented various mechanisms of rRNA dosage control, including gene elimination and stable inactivation related to nucleolar subdominance of A and D-genome loci, and a subtle, developmentally regulated silencing of one of the major loci. The results are discussed in the context of wheat evolution and transcription regulation.

genomics↗

bioRxiv2020

DNA replication and chromosome positioning throughout the interphase in three-dimensional space of plant nuclei

Despite the recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains limited. In this study, DNA replication timing and interphase chromosome positioning was analyzed in seven Poaceae species differing in genome size. A multidisciplinary approach combining newly replicated DNA labelling by EdU, nuclei sorting by flow cytometry, three-dimensional immuno-FISH, and confocal microscopy revealed similar replication timing order for telomeres and centromeres as well as for euchromatin and heterochromatin in all seven species. The Rabl configuration of chromosomes that lay parallel to each other and their centromeres and telomeres are localized at opposite nuclear poles, was observed in wheat, oat, rye and barley with large genomes, as well as in Brachypodium with a small genome. On the other hand, chromosomes of rice with a small genome and maize with relatively large genome did not assume proper Rabl configuration. In all species, the interphase chromosome positioning inferred from the location of centromeres and telomeres was stable throughout the interphase. These observations extend earlier studies indicating a more complex relation between genome size and interphase chromosome positioning, which is controlled by factors currently not known. HighlightTelomere and centromere replication timing and interphase chromosome positioning in seven grass species differing in genome size indicates a more complex relation between genome size and the chromosome positioning.

plant biology↗

bioRxiv2019

Chromosome-scale genome assembly provides insights into rye biology, evolution, and agronomic potential

We present a chromosome-scale annotated assembly of the rye (Secale cereale L. inbred line Lo7) genome, which we use to explore Triticeae genomic evolution, and ryes superior disease and stress tolerance. The rye genome shares chromosome-level organization with other Triticeae cereals, but exhibits unique retrotransposon dynamics and structural features. Crop improvement in rye, as well as in wheat and triticale, will profit from investigations of rye gene families implicated in pathogen resistance, low temperature tolerance, and fertility control systems for hybrid breeding. We show that rye introgressions in wheat breeding panels can be characterised in high-throughput to predict the yield effects and trade-offs of rye chromatin.

genomics↗

bioRxiv2019

The genome of cowpea (Vigna unguiculata Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. A high-quality assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single molecule real-time sequencing, optical and genetic mapping, and a novel assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared to other warm-season legumes that have been sequenced. A surprising outcome of this study is the identification of a chromosomal inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence also facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A new numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris).

genomics↗

bioRxiv2017

Large-scale structural variation detection in subterranean clover subtypes using optical mapping validated at nucleotide level

Whole genome sequencing has been widely used to detect structural variations (SVs). However, the limited single molecule size makes it difficult to characterize large-scale SVs in a genome because they cannot fully cover such vast and complex regions. Recently, optical mapping in nanochannels has provided novel resolution to detect large-scale SVs by comparing the physical location of the nickase recognition sequence in genomes. Other than in humans, SVs discovered in plants by optical mapping have not been validated. To assess the accuracy of SV calling in plants by optical mapping, we selected two genetically diverse subspecies of the Trifolium model species, subterranean clover cvs. Daliak and Yarloop. The SVs discovered by BioNano optical mapping (BOM) were validated using Illumina short reads. In the analysis, BOM identified 12 large-scale regions containing deletions and 19 containing insertions in Yarloop. The 12 large-scale regions contained 71 small deletions when validated by Illumina short reads. The results suggest that BOM could detect the total size of deletions and insertions, but it could not precisely report the location and actual quantity of SVs in the genome. Nucleotide-level validation is crucial to confirm and characterize SVs reported by optical mapping. The accuracy of SV detection by BOM is highly dependent on the quality of reference genomes and the density of selected nickases.

bioinformatics↗

bioRxiv2017

Maternal X chromosome upregulation in both sexes initiates dosage compensation evolution

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100044).\n\nSex chromosomes have repeatedly evolved from a pair of autosomes1. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced Y gene expression and eventual Y gene loss. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown and dosage compensation is poorly understood in plants5. Here we show a novel dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno for the evolution of X inactivation in mammals.

evolutionary biology↗