Long-Read cDNA Sequencing Revealed Novel Expressed Genes and Dynamic Transcriptome Landscape of Triticale (x Triticosecale Wittmack) Seed at Different Developing Stages

Developing seed is a unique stage of plant development with highly dynamic changes in transcriptome. Here, we aimed to detect the novel previously unannotated, genes of the triticale (x Triticosecale Wittmack, AABBRR genome constitution) genome that are expressed during different stages and at different parts of the developing seed. For this, we carried out the Oxford Nanopore sequencing of cDNA obtained for middle (15 days post-anthesis, dpa) and late (20 dpa) stages of seed development. The obtained data together with our previous direct RNA sequencing of early stage (10 dpa) of seed development revealed 39,914 expressed genes including 7128 (17.6%) genes that were not previously annotated in A, B, and R genomes. The bioinformatic analysis showed that the identified genes belonged to long non-coding RNAs (lncRNAs), protein-coding RNAs, and TE-derived RNAs. The gene set analysis revealed the transcriptome dynamics during seed development with distinct patterns of over-represented gene functions in early and middle/late stages. We performed analysis of the lncRNA genes polymorphism and showed that the genes of some of the tested lncRNAs are indeed polymorphic in the triticale collection. Altogether, our results provide information on thousands of novel loci expressed during seed development that can be used as new targets for GWAS analysis, the marker-assisted breeding of triticale, and functional elucidation

Long-Read cDNA Sequencing Revealed Novel Expressed Genes and Dynamic Transcriptome Landscape of Triticale (x Triticosecale Wittmack) Seed at Different Developing Stages

Developing seed is a unique stage of plant development with highly dynamic changes in transcriptome. Here, we aimed to detect the novel previously unannotated, genes of the triticale (x Triticosecale Wittmack, AABBRR genome constitution) genome that are expressed during different stages and at different parts of the developing seed. For this, we carried out the Oxford Nanopore sequencing of cDNA obtained for middle (15 days post-anthesis, dpa) and late (20 dpa) stages of seed development. The obtained data together with our previous direct RNA sequencing of early stage (10 dpa) of seed development revealed 39,914 expressed genes including 7128 (17.6%) genes that were not previously annotated in A, B, and R genomes. The bioinformatic analysis showed that the identified genes belonged to long non-coding RNAs (lncRNAs), protein-coding RNAs, and TE-derived RNAs. The gene set analysis revealed the transcriptome dynamics during seed development with distinct patterns of over-represented gene functions in early and middle/late stages. We performed analysis of the lncRNA genes polymorphism and showed that the genes of some of the tested lncRNAs are indeed polymorphic in the triticale collection. Altogether, our results provide information on thousands of novel loci expressed during seed development that can be used as new targets for GWAS analysis, the marker-assisted breeding of triticale, and functional elucidation

Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome

Sequencing and epigenetic profiling of target genes in plants are important tasks with various applications ranging from marker design for plant breeding to the study of gene expression regulation. This is particularly interesting for plants with big genome size for which whole-genome sequencing can be time-consuming and costly. In this study, we asked whether recently proposed Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing for plant species with big genome size. We applied nCATS to sequence the full-length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that while the target gene enrichment per se was quite high for the three glutenin genes (up to 645×), the sequencing depth that was achieved from two MinION flowcells was relatively low (5–17×). However, this sequencing depth was sufficient for various tasks including detection of InDels and single-nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS, we uncovered SNP and InDel variation of full-length glutenin genes providing useful information for marker design and deciphering of variation of individual Glu-1By alleles. Moreover, we demonstrated that glutenin genes possess a ‘gene-body’ methylation epigenetic profile with hypermethylated CDS part and hypomethylated promoter region. The obtained information raised an interesting question on the role of gene-body methylation in glutenin gene expression regulation. Taken together, our work disclosures the potential of the nCATS approach for sequencing of target genes in plants with big genome size

A Pipeline NanoTRF as a New Tool for De Novo Satellite DNA Identification in the Raw Nanopore Sequencing Reads of Plant Genomes

High-copy tandemly organized repeats (TRs), or satellite DNA, is an important but still enigmatic component of eukaryotic genomes. TRs comprise arrays of multi-copy and highly similar tandem repeats, which makes the elucidation of TRs a very challenging task. Oxford Nanopore sequencing data provide a valuable source of information on TR organization at the single molecule level. However, bioinformatics tools for de novo identification of TRs in raw Nanopore data have not been reported so far. We developed NanoTRF, a new python pipeline for TR repeat identification, characterization and consensus monomer sequence assembly. This new pipeline requires only a raw Nanopore read file from low-depth (<1×) genome sequencing. The program generates an informative html report and figures on TR genome abundance, monomer sequence and monomer length. In addition, NanoTRF performs annotation of transposable elements (TEs) sequences within or near satDNA arrays, and the information can be used to elucidate how TR–TE co-evolve in the genome. Moreover, we validated by FISH that the NanoTRF report is useful for the evaluation of TR chromosome organization—clustered or dispersed. Our findings showed that NanoTRF is a robust method for the de novo identification of satellite repeats in raw Nanopore data without prior read assembly. The obtained sequences can be used in many downstream analyses including genome assembly assistance and gap estimation, chromosome mapping and cytogenetic marker development

Genomic and Transcriptomic Survey Provides New Insight into the Organization and Transposition Activity of Highly Expressed LTR Retrotransposons of Sunflower (Helianthus annuus L.)

LTR retrotransposons (RTEs) play a crucial role in plant genome evolution and adaptation. Although RTEs are generally silenced in somatic plant tissues under non-stressed conditions, some expressed RTEs (exRTEs) escape genome defense mechanisms. As our understanding of exRTE organization in plants is rudimentary, we systematically surveyed the genomic and transcriptomic organization and mobilome (transposition) activity of sunflower (Helianthus annuus L.) exRTEs. We identified 44 transcribed RTEs in the sunflower genome and demonstrated their distinct genomic features: more recent insertion time, longer open reading frame (ORF) length, and smaller distance to neighboring genes. We showed that GAG-encoding ORFs are present at significantly higher frequencies in exRTEs, compared with non-expressed RTEs. Most exRTEs exhibit variation in copy number among sunflower cultivars and one exRTE Gagarin produces extrachromosomal circular DNA in seedling, demonstrating recent and ongoing transposition activity. Nanopore direct RNA sequencing of full-length RTE RNA revealed complex patterns of alternative splicing in RTE RNAs, resulting in isoforms that carry ORFs for distinct RTE proteins. Together, our study demonstrates that tens of expressed sunflower RTEs with specific genomic organization shape the hidden layer of the transcriptome, pointing to the evolution of specific strategies that circumvent existing genome defense mechanisms

Anisotropic thermal expansion and electronic transitions in the Co3BO5 ludwigite

The investigations of the crystal structure, magnetic and electronic properties of Co3BO5 at high temperatures were carried out using powder X-ray diffraction, magnetic susceptibility, electrical resistivity, and thermopower measurements. The orthorhombic symmetry (Sp.gr. Pbam) was observed at 300 K and no evidence of structural phase transitions was found up to 1000 K. The compound shows a strong anisotropy of the thermal expansion. A large negative thermal expansion along the a-axis is observed over a wide temperature range (T = 300–600 K) with αa = −35 M K−1 at T = 500 K with simultaneous expansion along the b- and c-axes with αb = 70 M K−1 and αc = 110 M K−1, respectively. The mechanisms of thermal expansion are explored by structural analysis. The activation energy of the conductivity decreases significantly above 700 K. Electronic transport was found to be a dominant conduction mechanism in the entire temperature range. The correlations between the thermal expansion, electrical resistivity, and effective magnetic moment were revealed and attributed to the evolution of the spin state of Co3+ ions towards the spin crossover and gradual charge-ordering transition.We are grateful to the Russian Foundation for Basic Research (project no. 20-02-00559 and 21-52-12033) for supporting this paper. This work was performed within the framework of the budget project no. 0287-2021-0013 for the Institute of Chemistry and Chemical Technology SB RAS. We acknowledge the financial support from the Spanish Ministry of Economy, Industry and Competitiviness (MINECO), (Grant No. MAT2017-83468-R) and from the regional Government of Aragón (E12-20R RASMIA project).Peer reviewe

Epigenetic Stress and Long-Read cDNA Sequencing of Sunflower (<i>Helianthus annuus</i> 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

Transposons Hidden in <i>Arabidopsis thaliana</i> Genome Assembly Gaps and Mobilization of Non-Autonomous LTR Retrotransposons Unravelled by Nanotei Pipeline

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing