Mega-sized pericentromeric blocks of simple telomeric repeats and their variants reveal patterns of chromosome evolution in ancient Cycadales genomes

Simple telomeric repeats composed of six to seven iterating nucleotide units are important sequences typically found at the ends of chromosomes. Here we analyzed their abundance and homogeneity in 42 gymnosperm (29 newly sequenced), 29 angiosperm (one newly sequenced), and eight bryophytes using bioinformatics, conventional cytogenetic and molecular biology approaches to explore their diversity across land plants. We found more than 10 000-fold variation in the amounts of telomeric repeats among the investigated taxa. Repeat abundance was positively correlated with increasing intragenomic sequence heterogeneity and occurrence at non-telomeric positions, but there was no correlation with genome size. The highest abundance/heterogeneity was found in the gymnosperm genus Cycas (Cycadaceae), in which megabase-sized blocks of telomeric repeats (i.e., billions of copies) were identified. Fluorescent in situ hybridization experiments using variant-specific probes revealed canonical Arabidopsis-type telomeric TTTAGGG repeats at chromosome ends, while pericentromeric blocks comprised at least four major telomeric variants with decreasing abundance: TTTAGGG>TTCAGGG >TTTAAGG>TTCAAGG. Such a diversity of repeats was not found in the sister cycad family Zamiaceae or in any other species analyzed. Using immunocytochemistry, we showed that the pericentromeric blocks of telomeric repeats overlapped with histone H3 serine 10 phosphorylation signals. We show that species of Cycas have amplified their telomeric repeats in centromeric and telomeric positions on telocentric chromosomes to extraordinary high levels. The ancestral chromosome number reconstruction suggests their occurrence is unlikely to be the product of ancient Robertsonian chromosome fusions. We speculate as to how the observed chromosome dynamics may be associated with the diversification of cycads.This project was supported by the Czech Academy of Science, Czech Science Foundation (22-16826S), Czech National Infrastructure for Biological data (ELIXIR CZ, LM2018131), NERC and China Scholarship Council (CSC). JP benefited from a Ramón y Cajal grant Ref: RYC-2017-2274 funded by MCIN/AEI/INTRODUCTION RESULTS Identification and quantification of telomeric repeats in high-throughput reads In silico identification of telomeric repeat variants Southern blot hybridization analysis of telomeric variants Identification of cycad centromeres by immunostaining of chromatin FISH analysis of telomeric variants Evolution of chromosome numbers and genome sizes across cycads DISCUSSION Variable abundance of telomeric repeats in plant genomes Origin of telomeric repeat variants in cycad genomes Epigenetic modification of telomeric repeats Chromosome evolution in cycads CONCLUSION EXPERIMENTAL PROCEDURES Plant material DNA isolation and Illumina sequencing Estimation of telomeric repeats abundance and diversity from high-throughput sequencing data Ancestral chromosome and genome size reconstruction Southern blot hybridization and DNA methylation analysis DNA probe preparation for FISH and southern blotting FISH Immunohistochemical staining of chromosomes ACKNOWLEDGEMENTS Author CONTRIBUTION

INFLUENCE OF INACTIVATION METHODS ON PATHOGEN DIAGNOSTICS BY MEANS OF INSTRUMENTAL METHODS

Mass spectrometry as an identification method for microorganisms is rapidly developing in the last years. However, this method is not suitable for detection of agents in complex matrices and it has to be preceded by clean-up procedures and particular agent’s concentration. In the case of high-risk pathogens, such a separation method may pose a hazard for the laboratory staff. Therefore, various methods for pathogen inactivation were studied. Their influence on pre-concentration and separation of microorganisms by means of preparative and capillary isoelectric formation was studied. Most of the disinfectant agents disrupted the cells integrity and made their following separation impossible. The most promising was freeze-dry samples inactivation using hydrogen peroxide vapor at 300 ppm concentration. Inactivation of the lyophilized bacterial agents caused an only inconclusive shift of mass spectra in MALDI-TOF MS analysis, but it caused a quite significant change of isoelectric point. Inactivation of bacterial spores required at least 2 hours of exposure. Increased vapor concentration caused damage to the cells. Heating up the samples up to 60 °C enabled to increase the vapor concentration and made the inactivation faster without influencing the mass spectra. Moreover, the influence of thermal inactivation of bacterial agents at 60 °C for 16 hours was studied. There was no significant change in mass spectral in MALDI-TOF MS analysis, but it did not work for sporulating bacterias. Simultaneously, the longtime of inactivation was a significant drawback. The possibilities of the detection and the identification of inactivated pathogens are an object of ongoing research