Long et al. J of Biol Res-Thessaloniki (2016) 23:6 DOI 10.1186/s40709-016-0048-5 Journal of Biological Research-Thessaloniki RESEARCH Open Access Sequencing and characterization of leaf transcriptomes of six diploid Nicotiana species Ni Long1, Xueliang Ren2, Zhidan Xiang3, Wenting Wan1 and Yang Dong1,4* Abstract Background: Nicotiana belongs to the Solanaceae family that includes important crops such as tomato, potato, eggplant, and pepper. Nicotiana species are of worldwide economic importance and are important model plants for scientific research. Here we present the comparative analysis of the transcriptomes of six wild diploid Nicotiana spe- cies. Wild relatives provide an excellent study system for the analysis of the genetic basis for various traits, especially disease resistance. Results: Whole transcriptome sequencing (RNA-seq) was performed for leaves of six diploid Nicotiana species, i.e. Nicotiana glauca, Nicotiana noctiflora, Nicotiana cordifolia, Nicotiana knightiana, Nicotiana setchellii and Nicotiana tomentosiformis. For each species, 9.0–22.3 Gb high-quality clean data were generated, and 67,073–182,046 tran- scripts were assembled with lengths greater than 100 bp. Over 90 % of the ORFs in each species had significant similarity with proteins in the NCBI non-redundant protein sequence (NR) database. A total of 2491 homologs were identified and used to construct a phylogenetic tree from the respective transcriptomes in Nicotiana. Bioinformatic analysis identified resistance gene analogs, major transcription factor families, and alkaloid transporter genes linked to plant defense. Conclusions: This is the first report on the leaf transcriptomes of six wild Nicotiana species by Illumina paired-end sequencing and de novo assembly without a reference genome. These sequence resources hopefully will provide an opportunity for identifying genes involved in plant defense and several important quality traits in wild Nicotiana and will accelerate functional genomic studies and genetic improvement efforts of Nicotiana or other important Solan- aceae crops in the future. Keywords: Nicotiana, Transcriptome, De novo assembly, Phylogenetic relationship, Nicotiana setchellii, Nicotiana cordifolia, Nicotiana knightiana, Nicotiana tomentosiformis, Nicotiana noctiflora, Nicotiana glauca Background includes over 75 naturally occurring species, almost The genus Nicotiana is a member of the Solanaceae or half of which are allopolyploid [3]. The genus Nicotiana nightshade family, which includes many economically contains species of scientific and economic importance, important crop plants such as tomato, potato, eggplant, with different evolutionary histories resulting to highly and pepper. According to Goodspeed [1] and Good- complex genomes [5]. Of all species, only Nicotiana speed & Thompson [2], Nicotiana was initially divided tabacum (common tobacco) and Nicotiana rustica are into three subgenera and 14 sections. Recently, this genus cultivated worldwide, whereas the others are wild spe- was reclassified into 13 sections based on morphologi- cies. Moreover, Nicotiana benthamiana is used exten- cal, cytological, and DNA sequence data [3, 4]. Nicotiana sively as a model to study plant-pathogen interactions. Several other species, such as Nicotiana alata and Nico- tiana sylvestris, are grown as ornamentals. In N. tabacum *Correspondence: dongyang@dongyang‑lab.org breeding programs, wild Nicotiana species are valuable 1 Faculty of Life Science and Technology, Kunming University of Science and Technology, South Jingming Road No.727, Kunming 650500, Yunnan, sources for identifying genes involved in disease and pest China resistance, important quality traits, and phytochemicals, Full list of author information is available at the end of the article which are not present in cultivated varieties [6]. © 2016 Long et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Long et al. J of Biol Res-Thessaloniki (2016) 23:6 Page 2 of 12 Plants are constantly under the attack of bacteria, for several crops including tobacco. This is mainly due to fungi, viruses, nematodes and insect pests. Some of the lack of genomic resources hampering the develop- them have successfully invaded crop plants, causing dis- ment of genetic markers for investigating species diver- eases and reducing crop quality and yield. To protect gence, adaptation and demographic processes in natural against pathogens, plants have evolved various defense populations. mechanisms. Plant disease resistance (R) genes play a In the present study, we selected six wild Nicotiana spe- key role in defending plants from a range of pathogens. cies for analyses, which included N. glauca, N. noctiflora, For instance, N genes from tobacco confer resistance to N. cordifolia, N. knightiana, N. setchellii, and N. tomen- tobacco mosaic virus (TMV) [7]. In recent years, a set of tosiformis. These diploid Nicotiana species (all with chro- 112 known and 104,310 putative R genes fighting against mosome numbers of 2n = 24) were chosen because they 122 different pathogens have been identified in 233 plant are repositories of pathogen resistant genes (Table 1). species [8]. Most of the characterized R genes share a few The six wild Nicotiana species belong to three sections: highly conserved domains, including nucleotide binding Noctiflorae, Paniculatae and Tomentosae. Trait introgres- site (NBS), leucine-rich repeat (LRR), Toll/Interleukin-1 sion from wild relatives has been used to improve crop receptor (TIR) and coiled-coil (CC) domains [9–11]. species. For example, characters from at least 13 differ- These conservative domains provide convenient and reli- ent species have been transferred into tobacco [4]. With able means for rapidly identifying and cloning R genes or advances in next-generation sequencing (NGS) technolo- resistance gene analogs (RGAs). gies, genomic data for several Nicotiana species have Identification of Nicotiana R genes and RGAs can- become available [21–25]. These data revealed that some not only help elucidate the molecular mechanisms of Nicotiana genomes are large compared with other Solan- host-pathogen interaction, but also benefit breeding aceae species such as the tomato [5]. For most wild Nico- programs for disease resistance in Nicotiana and other tiana species, very few genomic sequences are currently important Solanaceae crops. Transcriptomic sequences available. In this study, we performed transcriptome can be useful substitutes for gene discovery in species sequencing using the Illumina paired-end sequencing without sequenced genomes. In the past, a large RGA technique with the aim of identifying expressed RGAs, pool has been mined from transcriptomic sequences and transcription factors important in plant defense, and expressed sequence tags (ESTs) of coffee [12], Phaseolus alkaloid transporter genes by data mining. Our results vulgaris [13], Curcuma longa [14] and Cocos nucifera will provide a useful basis for future identification and [15]. Wild Nicotiana species are known to resist a vari- cloning of interest genes in wild Nicotiana and contrib- ety of pathogens. For example, N. glauca has attractive ute to the improvement of cultivated tobacco and other potentials to resist black root rot (BRR), potato virus Y important Solanaceae crops. (PVY), tobacco etch virus (TEV), anthracnose (An), pow- dery mildew (PM), rattle virus (RV) and tobacco streak Results and discussion virus (TS) [16–18]. Nicotiana noctiflora is resistant to Assembly of RNA‑seq reads and evaluation PM and PVY. Nicotiana cordifolia shows resistance to The Illumina paired-end sequencing yielded 100 bp TS. Nicotiana knightiana manifests high resistance to paired-end independent reads from each insert of cDNA. An, PM, root knot nematodes (RK), PVY and TEV. Nico- After stringent quality assessment and data filtering, tiana setchellii shows resistance to RV and TEV. Nicoti- reads with Q20 bases (those with a base quality greater ana tomentosiformis is resistant to cyst nematodes (CN), RK, RV and TEV [16, 17]. These observations suggest that wild Nicotiana species are excellent depositories of Table 1 Summary of the six wild Nicotiana species investi- R genes and RGAs, but relevant analyses of these genes gated in this study have been lacking. Species Sections Subgenus Resistance to diseases In Nicotiana species, alkaloids (e.g. nicotine) are believed to function as a chemical defense mechanism N. glauca Noctiflorae Petunioides BRR, An, PM, RV, TEV, TS, against pathogens and herbivores. Nicotine and related PVY pyridine alkaloids are synthesized in the tobacco root and N. noctiflora Noctiflorae Petunioides PM, PVY then translocated to the aerial parts of the plant [19, 20]. N. cordifolia Paniculatae Rustica TS Thus the translocation of nicotine from the root to the N. knightiana Paniculatae Rustica An, PM, RK, TEV, PVY leaves is very important in tobacco defenses. N. setchellii Tomentosae Tabacum RV, TEV Comparative studies of closely related species can N. tomentosiformis Tomentosae