Epigenetic Stress and Long-Read cDNA Sequencing of Sunflower (Helianthus annuus L.) Revealed the Origin of the Plant Retrotranscriptome.

Transposable elements (TEs) contribute not only to genome diversity but also to transcriptome diversity in plants. To unravel the sources of LTR retrotransposon (RTE) transcripts in sunflower, we exploited a recently developed transposon activation method ('TEgenesis') along with long-read cDNA Nanopore sequencing. This approach allows for the identification of 56 RTE transcripts from different genomic loci including full-length and non-autonomous RTEs. Using the mobilome analysis, we provided a new set of expressed and transpositional active sunflower RTEs for future studies. Among them, a Ty3/Gypsy RTE called SUNTY3 exhibited ongoing transposition activity, as detected by eccDNA analysis. We showed that the sunflower genome contains a diverse set of non-autonomous RTEs encoding a single RTE protein, including the previously described TR-GAG (terminal repeat with the GAG domain) as well as new categories, TR-RT-RH, TR-RH, and TR-INT-RT. Our results demonstrate that 40% of the loci for RTE-related transcripts (nonLTR-RTEs) lack their LTR sequences and resemble conventional eucaryotic genes encoding RTE-related proteins with unknown functions. It was evident based on phylogenetic analysis that three nonLTR-RTEs encode GAG (HadGAG1-3) fused to a host protein. These HadGAG proteins have homologs found in other plant species, potentially indicating GAG domestication. Ultimately, we found that the sunflower retrotranscriptome originated from the transcription of active RTEs, non-autonomous RTEs, and gene-like RTE transcripts, including those encoding domesticated proteins.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans.

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Pipeline for the Rapid Development of Cytogenetic Markers Using Genomic Data of Related Species.

Repetitive DNA including tandem repeats (TRs) is a significant part of most eukaryotic genomes. TRs include rapidly evolving satellite DNA (satDNA) that can be shared by closely related species, their abundance may be associated with evolutionary divergence, and they have been widely used for chromosome karyotyping using fluorescence in situ hybridization (FISH). The recent progress in the development of whole-genome sequencing and bioinformatics tools enables rapid and cost-effective searches for TRs including satDNA that can be converted into molecular cytogenetic markers. In the case of closely related taxa, the genome sequence of one species (donor) can be used as a base for the development of chromosome markers for related species or genomes (target). Here, we present a pipeline for rapid and high-throughput screening for new satDNA TRs in whole-genome sequencing of the donor genome and the development of chromosome markers based on them that can be applied in the target genome. One of the main peculiarities of the developed pipeline is that preliminary estimation of TR abundance using qPCR and ranking found TRs according to their copy number in the target genome; it facilitates the selection of the most prospective (most abundant) TRs that can be converted into cytogenetic markers. Another feature of our pipeline is the probe preparation for FISH using PCR with primers designed on the aligned TR unit sequences and the genomic DNA of a target species as a template that enables amplification of a whole pool of monomers inherent in the chromosomes of the target species. We demonstrate the efficiency of the developed pipeline by the example of FISH probes developed for A, B, and R subgenome chromosomes of hexaploid triticale (BBAARR) based on a bioinformatics analysis of the D genome of Aegilops tauschii (DD) whole-genome sequence. Our pipeline can be used to develop chromosome markers in closely related species for comparative cytogenetics in evolutionary and breeding studies.