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This article describes a novel method to identify as yet undiscovered telomere sequences, which combines next generation sequencing (NGS) with BAL31 digestion of high molecular weight DNA. The method was applied to two groups of plants: i) dicots, genus Cestrum, and ii) monocots, Allium species (e.g. A. ursinum and A. cepa). Both groups consist of species with large genomes (tens of Gb) and a low number of chromosomes (2n=14-16), full of repeat elements. Both genera lack typical telomeric repeats and multiple studies have attempted to characterize alternative telomeric sequences. However, despite interesting hypotheses and suggestions of alternative candidate telomeres (retrotransposons, rDNA, satellite repeats) these studies have not resolved the question. In a novel approach based on the two most general features of eukaryotic telomeres, their repetitive character and sensitivity to BAL31 nuclease digestion, we have taken advantage of the capacity and current affordability of NGS in combination with the robustness of classical BAL31 nuclease digestion of chromosomal termini. While representative samples of most repeat elements were ensured by low-coverage (less than 5%) genomic shot-gun NGS, candidate telomeres were identified as under-represented sequences in BAL31-treated samples., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Phylogenetic divergence in Asparagales plants is associated with switches in telomere sequences. The last switch occurred with divergence of the genus Allium (Amaryllidaceae) from the other Allioideae (formerly Alliaceae) genera, resulting in uncharacterized telomeres maintained by an unknown mechanism. To characterize the unknown Allium telomeres, we applied a combination of bioinformatic processing of transcriptomic and genomic data with standard approaches in telomere biology such as BAL31 sensitivity tests, terminal restriction fragment analysis, the telomere repeat amplification protocol (TRAP), and fluorescence in situ hybridization (FISH). Using these methods, we characterize the unusual telomeric sequence (CTCGGTTATGGG)n present in Allium species, demonstrate its synthesis by telomerase, and characterize the telomerase reverse transcriptase (TERT) subunit of Allium cepa. Our findings open up the possibility of studying the molecular details of the evolutionary genetic change in Allium telomeres and its possible role in speciation. Experimental studies addressing the implications of this change in terms of the interplay of telomere components may now be designed to shed more light on telomere functions and evolution in general., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)