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