Nicotiana glauca whole-genome investigation for cT-DNA study
BMC Research Notes volume 11, Article number: 18 (2018)
Nicotiana glauca (tree tobacco) is a naturally transgenic plant, containing sequences acquired from Agrobacterium rhizogenes by horizontal gene transfer. Besides, N. glauca contains a wide profile of alkaloids of medical interest.
We report a high-depth sequencing and de novo assembly of N. glauca full genome and analysis of genome elements with bacterial origin. The draft genome assembly is 3.2 Gb, with N50 size of 31.1 kbp. Comparative analysis confirmed the presence of single, previously described gT insertion. No evidence was acquired to support idea of multiple T-DNA insertions in the N. glauca genome. Our data is the first comprehensive de novo assembly of tree tobacco and provide valuable information for researches in pharmacological and in phylogenetic fields.
Nicotiana glauca (tree tobacco) is a member of the Solanaceae family, which includes important crops (potato, tomato, eggplant, pepper) and many medicinal plants . This diploid plant is native to South America and is one of the first Nicotiana species with Agrobacterium cellular T-DNA (cT-DNA) . Its cT-DNA is a partial, inverted repeat, called gT . Tree tobacco belongs to the section Noctiflorae. Sequencing of the genomes of N. tomentosiformis and N. otophora (section Tomentosae) and N. tabacum (section Nicotiana) allowed the detection of previously unknown multiple cT-DNAs , raising the question whether there are other T-DNA insertions in the N. glauca. NGS data can help answer this question. Besides, N. glauca contains a profile of alkaloids different from N. tabacum . The plant is used for medicinal purposes. Comparative analysis of genomic data of phylogenetically distant tobacco species will provide valuable information on the genetic basis for various traits, especially secondary metabolism. Our data complement the list of species for the comparative genomics of Nicotiana, which opens up new opportunities for pharmacological and phylogenetic studies.
One plant isolate was sequenced on Illumina HiSeq machine, yielding in total 210 Gb of raw sequence data. De novo assembly resulted in 385116 scaffolds, with N50 and L50 of 31.1 kbp and 27293 respectively. Genome size suggested by K-mer analysis is 2 Gb, while the final size of the assembled genome equaled 3.2 Gb. Comparative analyses of N. glauca scaffolds against genome assembly of N. tabacum TN90 cultivar strain resulted in 3.2 Gbp of aligned sequences median identity of 88%. T-DNA analysis revealed sequences homologous to agrobacterial genes orf13a, orf13, orf14, rolC, rolB and mis. The fragment of T-DNA obtained in the assembly is organized in an imperfect inverted repeat. The similarity of the nucleotide sequences, that we found, and sequence of gT, previously described by Suzuki  was 99%, while its similarity to Agrobacterium T-DNA is 77–89%. Sequences of PCR fragments, amplified from T-DNA/plantDNA junction areas, coincide with known ones (Acs. AB071335, AB071334).
Leaf tissue of aseptic plants N. glauca was used for DNA extraction, with a modified version of Doyle and Doyle protocol , yielding 30 ng/μl of high molecular weight DNA.
Purified genomic DNA from N. glauca was used to construct both pair-end and mate pair libraries in order to generate a high coverage de novo assembly. A pair-end library with an insert size of 350 bp was constructed using the TruSeq® Nano DNA Library Prep Reference Guide. To improve resolution of repeats during the assembly stage and scaffolding process, one mate pair library with an insert size of 4 kbp was constructed, according to the Nextera® Mate Pair Library Prep Reference Guide.
Read sequencing, quality analysis and filtering
Pair-end and mate pair libraries were sequenced on four and two lanes using Illumina HiSeq. Quality of raw reads was analyzed with the FastQC  program, followed by filtering and trimming raw PE reads with Trimgalore . Mate pair raw reads were processed and splitted with Nextclip  and additionally filtered with Trimgalore .
The genome was assembled with the MaSuRCA-3.2.2 genome assembler , [config in data file 1].
Whole genome alignment of Nicotiana glauca and Nicotiana tabacum
To identify the location of the N. glauca cT-DNA insertion relative to the N. tabacum genome, we mapped all N. glauca scaffolds to N. tabacum scaffolds downloaded from the Sol Genomics Network . To increase accuracy of alignment we masked all known plant repeat classes and their homologs in the N. glauca genome. For repeat identification, we used the RepeatMasker software  and the latest Repbase Update library from 09.27.2017. For whole genome alignment, we used the Last software .
The Last software  was used to carry out the alignment of the database, containing all known T-DNA-like sequences, that were detected as part of cT-DNA [data file 2], to the N. glauca genome. To reaffirm T-DNA/plantDNA junction areas Long PCR was carried out using “LONG PCR enzyme Mix” (Thermo scientific) according to the instructions for the kit (Table 1).
85% of the mate pair library proved to be PCR duplicates, which we filtered before assembling. Low coverage of MP reads resulted in low N50 and big number of contigs and scaffolds. A better quality or/and a bigger number of MP libraries should be used in future to improve the assembly.
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TM developed the overall project design. GK, PD and TM wrote the paper. GK collected the N. glauca sample and extracted DNA from the sample. GK and DP constructed libraries. DP sequenced the genome of N. glauca. PD assembled the N. glauca genome and analyzed whole genome alignments. GK, PD and TM performed cT-DNA analysis. All authors read and approved the final manuscript.
The authors thank Professor Otten (Institut de Biologie Moléculaire des Plantes, Strasbourg) and Professor Lutova (Saint Petersburg University, St. Petersburg) for useful discussion and critical reading of the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The assembly sequences are available at DDBJ/ENA/GenBank as WGS project under the Accession PGPE00000000. The SRA for whole-genome sequencing can be accessed at NCBI SRA via Reference Numbers: SRX3419913, SRX3419914, SRX3419915, SRX3419916, SRX3419917, SRX3419918.
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1. Khafizova G, Dobrynin P, Polev D. FigShare https://doi.org/10.6084/m9.figshare.5732427.v1 (2017).
2. Khafizova G, Dobrynin P. FigShare https://doi.org/10.6084/m9.figshare.5645854.v1 (2017).
3. Khafizova G, Matveeva T. FigShare https://doi.org/10.6084/m9.figshare.5754120.v1 (2018).
Ethics approval and consent to participate
This paper was supported by a Grant to Tatiana Matveeva from the Russian Science Foundation 16-16-10010 and a Grant from Saint Petersburg State University №1.52.1647.2016.
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Khafizova, G., Dobrynin, P., Polev, D. et al. Nicotiana glauca whole-genome investigation for cT-DNA study. BMC Res Notes 11, 18 (2018). https://doi.org/10.1186/s13104-018-3127-x