Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.

• Klíčová slova
• DNA damage repair, DNA-protein crosslinks, Forward genetics, Genetic mapping, High-throughput sequencing, Mapping-by-sequencing, SNP calling, Zebularine,
• MeSH
• Arabidopsis * genetika   MeSH
• fenotyp MeSH
• genom rostlinný MeSH
• mapování chromozomů * metody   MeSH
• mutace MeSH
• sekvenování celého genomu metody   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * metody   MeSH
• Publikační typ
• časopisecké články MeSH

Micropterigidae is regarded as the sister group of all the other Lepidoptera, providing important insights into the evolution of Lepidoptera. However, the gene and protein profiles of silk from Micropterigidae have not yet been identified. In this study, we investigate the components of silk cocoons of the micropterigid species Neomicropteryx cornuta. Here we show that the protein fibroin heavy chain (FibH) is absent in the silk of N. cornuta and that the putative homolog of fibroin light chain (FibL) is also absent or severely altered. This is confirmed by transcriptome and genome analyses of the conserved regions in this species. The examination of the synteny around the fibH genes in several Lepidoptera and Trichoptera species shows that the genomic region containing this gene is absent in another micropterigid species, Micropterix aruncella. In contrast, we found putative orthologs of fibH and fibL in the representative transcripts of another distinct clade, Eriocraniidae. This study shows that the loss of FibH and the loss or severe divergence of FibL occurrs specifically in the family Micropterigidae and reveals dynamic evolutionary changes in silk composition during the early evolution of Lepidoptera. It also shows that silk proteins without FibH can form a solid cocoon.

• MeSH
• fibroiny * genetika   metabolismus   chemie   MeSH
• fylogeneze MeSH
• hedvábí genetika   chemie   MeSH
• hmyzí proteiny genetika   metabolismus   chemie   MeSH
• molekulární evoluce MeSH
• můry genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibroiny * MeSH
• hedvábí MeSH
• hmyzí proteiny MeSH

Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.

• Klíčová slova
• Limnephilus flavicornis, Plectrocnemia conspersa, Fibroin, Gene duplication, Hydrophobicity, Sericin,
• MeSH
• fibroiny genetika   metabolismus   chemie   MeSH
• hedvábí * metabolismus   chemie   MeSH
• hmyz metabolismus   genetika   MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• proteom MeSH
• proteomika metody   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Genome size variation is a crucial aspect of plant evolution, influenced by a complex interplay of factors. Repetitive elements, which are fundamental components of genomic architecture, often play a role in genome expansion by selectively amplifying specific repeat motifs. This study focuses on Amomum, a genus in the ginger family (Zingiberaceae), known for its 4.4-fold variation in genome size. Using a robust methodology involving PhyloNet reconstruction, RepeatExplorer clustering, and repeat similarity-based phylogenetic network construction, we investigated the repeatome composition, analyzed repeat dynamics, and identified potential hybridization events within the genus. Our analysis confirmed the presence of four major infrageneric clades (A-D) within Amomum, with clades A-C exclusively comprising diploid species (2n = 48) and clade D encompassing both diploid and tetraploid species (2n = 48 and 96). We observed an increase in the repeat content within the genus, ranging from 84% to 89%, compared to outgroup species with 75% of the repeatome. The SIRE lineage of the Ty1-Copia repeat superfamily was prevalent in most analyzed ingroup genomes. We identified significant difference in repeatome structure between the basal Amomum clades (A, B, C) and the most diverged clade D. Our investigation revealed evidence of ancient hybridization events within Amomum, coinciding with a substantial proliferation of multiple repeat groups. This finding supports the hypothesis that ancient hybridization is a driving force in the genomic evolution of Amomum. Furthermore, we contextualize our findings within the broader context of genome size variations and repeatome dynamics observed across major monocot lineages. This study enhances our understanding of evolutionary processes within monocots by highlighting the crucial roles of repetitive elements in shaping genome size and suggesting the mechanisms that drive these changes.

• Klíčová slova
• 5S rDNA, Zingiberaceae, genome evolution, genome size, interspecific hybridization, phylogeny, repeatome, repetitive DNA,
• Publikační typ
• časopisecké články MeSH

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

• MeSH
• alely MeSH
• membránové glykoproteiny genetika   MeSH
• telomery metabolismus   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• Trypanosoma * metabolismus   MeSH
• trypanosomové variantní povrchové glykoproteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové glykoproteiny MeSH
• trypanosomové variantní povrchové glykoproteiny MeSH

Cimex lectularius, known as the common bed bug, is a widespread hematophagous human ectoparasite and urban pest that is not known to be a vector of any human infectious disease agents. However, few studies in the era of molecular biology have profiled the microorganisms harbored by field populations of bed bugs. The objective of this study was to examine the viruses present in a large sampling of common bed bugs and related bat bugs (Cimex pipistrelle). RNA sequencing was undertaken on an international sampling of > 500 field-collected bugs, and multiple workflows were used to assemble contigs and query these against reference nucleotide databases to identify viral genomes. Shuangao bed bug virus 2, an uncharacterized rhabdovirus previously discovered in Cimex hemipterus from China, was found in several bed bug pools from the USA and Europe, as well as in C. pipistrelle, suggesting that this virus is common among bed bug populations. In addition, Shuangao bed bug virus 1 was detected in a bed bug pool from China, and sequences matching Enterobacteria phage P7 were found in all bed bug pools, indicating the ubiquitous presence of phage-derived elements in the genome of the bed bug or its enterobacterial symbiont. However, viral diversity was low in bed bugs in our study, as no other viral genomes were detected with significant coverage. These results provide evidence against frequent virus infection in bed bugs. Nonetheless, our investigation had several important limitations, and additional studies should be conducted to better understand the prevalence and composition of viruses in bed bugs. Most notably, our study largely focused on insects from urban areas in industrialized nations, thus likely missing infrequent virus infections and those that could occur in rural or tropical environments or developing nations.

• Klíčová slova
• Arbovirus, Bed bug, Cimex lectularius, Cimex pipistrelle, Metatranscriptomics, RNAseq, Virus,
• MeSH
• infestace ektoparazity * MeSH
• lidé MeSH
• štěnice * genetika   MeSH
• viry * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Čína MeSH
• Evropa MeSH

Epigenetics deals with changes in gene expression that are not caused by modifications in the primary sequence of nucleic acids. These changes beyond primary structures of nucleic acids not only include DNA/RNA methylation, but also other reversible conversions, together with histone modifications or RNA interference. In addition, under particular conditions (such as specific ion concentrations or protein-induced stabilization), the right-handed double-stranded DNA helix (B-DNA) can form noncanonical structures commonly described as "non-B DNA" structures. These structures comprise, for example, cruciforms, i-motifs, triplexes, and G-quadruplexes. Their formation often leads to significant differences in replication and transcription rates. Noncanonical RNA structures have also been documented to play important roles in translation regulation and the biology of noncoding RNAs. In human and animal studies, the frequency and dynamics of noncanonical DNA and RNA structures are intensively investigated, especially in the field of cancer research and neurodegenerative diseases. In contrast, noncanonical DNA and RNA structures in plants have been on the fringes of interest for a long time and only a few studies deal with their formation, regulation, and physiological importance for plant stress responses. Herein, we present a review focused on the main fields of epigenetics in plants and their possible roles in stress responses and signaling, with special attention dedicated to noncanonical DNA and RNA structures.

• Klíčová slova
• Acetylation, Chromatin, Epigenetics, G-quadruplex, Gene expression, Histone, Methylation, Non-B DNA, Stress signaling,
• MeSH
• DNA genetika   chemie   MeSH
• epigeneze genetická MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nukleové kyseliny * MeSH
• RNA genetika   chemie   MeSH
• rostliny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nukleové kyseliny * MeSH
• RNA MeSH

Human ribosomal DNA is represented by hundreds of repeats in each cell. Every repeat consists of two parts: a 13 kb long 47S DNA with genes encoding 18S, 5.8S, and 28S RNAs of ribosomal particles, and a 30 kb long intergenic spacer (IGS). Remarkably, transcription does not take place in all the repeats. The transcriptionally silent genes are characterized by the epigenetic marks of the inactive chromatin, including DNA hypermethylation of the promoter and adjacent areas. However, it is still unknown what causes the differentiation of the genes into active and silent. In this study, we examine whether this differentiation is related to the nucleotide sequence of IGS. We isolated ribosomal DNA from the nucleoli of human-derived HT1080 cells, and separated methylated and non-methylated DNA by chromatin immunoprecipitation. Then, we used PCR to amplify a 2 kb long region upstream of the transcription start and sequenced the product. We found that six SNVs and a series of short deletions in a region of simple repeats correlated with the DNA methylation status. These data indicate that variability of IGS sequence may initiate silencing of the ribosomal genes. Our study also suggests a number of pathways to this silencing that involve micro-RNAs and/or non-canonical DNA structures.

• Klíčová slova
• micro-RNAs, non-canonical DNA structures, rDNA, sequence variability, transcription,
• MeSH
• intergenová DNA MeSH
• lidé MeSH
• mezerníky ribozomální DNA genetika   MeSH
• ribozomální DNA genetika   MeSH
• ribozomy * MeSH
• RNA ribozomální 28S genetika   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• intergenová DNA MeSH
• mezerníky ribozomální DNA MeSH
• ribozomální DNA MeSH
• RNA ribozomální 28S MeSH

Psyllids are phloem-feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host-symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure.

• MeSH
• Enterobacteriaceae genetika   MeSH
• Hemiptera * mikrobiologie   MeSH
• hmyz MeSH
• lidé MeSH
• mikrobiota * genetika   MeSH
• Pyrus * MeSH
• RNA ribozomální 16S genetika   MeSH
• symbióza MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.

• Klíčová slova
• crambidae, mediterranean flour moth, mucin, pyralidae, synteny, wax moth,
• Publikační typ
• časopisecké články MeSH