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The Detection of Plant Viruses in Korean Ginseng (Panax ginseng) through RNA Sequencing

Korean ginseng (Panax ginseng) is a dicotyledonous, medicinal, perennial plant belonging to the genus Panax of the family Araliaceae. We investigated the occurrence and incidence of plant viruses in Panax ginseng in Korea. A total of 656 leaf samples were combined into one and total RNA was extracted from the polled sample, using RNA sequencing (RNA-Seq), a metatranscriptome analysis of the plant virome was conducted. The virus present in Panax ginseng was confirmed by reverse transcription polymerase chain reaction (RT-PCR) assay using virus-specific primers. In RNA-Seq data analysis, the multiplication protein of four viral contigs including Aristotelia chilensis virus 1 (AcV1), Turnip mosaic virus (TuMV), Watermelon mosaic virus (WMV), and Tobamovirus multiplication protein were discovered. From our metatranscriptome analysis and RT-PCR assay, TuMV and WMV were detected, whereas the three viruses reported in China such as tomato yellow leaf curl China virus; panax notoginseng virus A; and panax virus Y were not found in this study. The distribution of domestic ginseng viruses seems different from that recorded in China. Overall, this is the first plant virome analysis of Panax ginseng in Korea.

Korean ginseng, traditionally referred to as "Insam" (Panax ginseng) is dicotyledonous, medicinal, perennial plant belonging to the genus Panax of the family Araliaceae; there are six species in the genus Panax (P. ginseng, P. japonicum [Japanese ginseng], P. notoginseng [Chinese ginseng], P. pseudoginseng [Himalayan ginseng], P. quinquefolius [American ginseng], and P. trifolius [Dwarf ginseng]). In previous reports, six viruses were reported in the genus Panax: tomato yellow leaf curl china virus (TYLCCNV) (Li et al., 2014), panax notoginseng virus A (PnVA) (Guo et al., 2016), panax notoginseng virus B (reported only in the NCBI GenBank, MF614102), panax notoginseng virus Y (reported only in the NCBI GenBank no., FJ816101), panax virus Y (PanVY) (Yan et al., 2010), and watermelon mosaic virus (WMV) (Jung et al., 2013). TYLCCNV belongs to a member of the genus Begomovirus and was reported in China. TYLCCNV-infected P. notoginseng shows foliar yellowing, shrinking, and blistering symptoms. Bemisia tabasi is known as a potential insect vector of TYLCCNV. PnVA belongs to a member of the genus putative Totivirus and has been reported in China. PnVAinfected P. notoginseng shows mild mosaic symptom. PanVY, as a member of the genus Potyvirus, has also been reported in China. PanVY-infected P. notoginseng shows typical distortion and mosaic symptoms. The pathogenicity of panax notoginseng virus B and panax notoginseng virus Y is uncertain; only their nucleotide sequences have Lee et al. 644 been reported in the GenBank database. In Korea, only the WMV has been reported in P. ginseng. The WMV is a member of the Potyvirus, which is the second largest genus of plant viruses based on the International Committee on Taxonomy of Viruses. The WMV strain Insam (WMV-Insam) infecting P. ginseng was first reported in Korea using large-scale oligonucleotide chip and reverse transcrip-tion polymerase chain reaction (RT-PCR) assay (Jung et al., 2013). Unlike other WMV isolates that cause economic damage to the Cucurbitaceae, including cucumber, pumpkin, and watermelon, it has been known that the WMV-Insam strain is not transmitted to the Cucurbitaceae plants by mechanical inoculation (Choi et al., 2014).
Due to the detection limit of conventional diagnostic   (Min et al., 2017, Oh et al., 2018, Yang et al., 2019. This study reports the first metatranscriptomic analysis of the P. ginseng virome using RNA-Seq and RT-PCR assay for identifying the occurrence and incidence of P. ginseng viruses in Korea.

Total RNA extraction and RNA library construction.
To construct the RNA library of P. ginseng viruses, all collected samples were combined into one sample and total RNA was extracted from the polled sample using a Quant-IT RiboGreen (Invitrogen, Carlsbad, CA, USA). To assess the integrity of the total RNA, samples were subjected to the TapeStation RNA screentape (Agilent Technologies, Santa Clara, CA, USA). Only high-quality RNA preparations with RNA integrity number greater than 7.0 were used for RNA library construction. Total RNA quality and quantity were verified using a NanoDrop 1000 spectrometer (Thermo Scientific, Welmington, DE, USA) and Bioanalyzer 2100 (Agilent Technologies). A library was independently prepared with 1 μg of total RNA using TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The rRNA in total RNA was depleted by TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Following the depletion of the rRNA, the remaining purified, fragmented, and primed RNA was used for cDNA synthesis. The cleaved RNA fragments were copied into the first-strand cDNA using reverse transcriptase and random hexamer (5'-NNNNNN-3').
De novo transcriptome assembly. One hundred bp pairedend (2 × 100 bp) RNA-Seq based on next-generation se- quencing (NGS) was performed using Illumina Hiseq 4000 sequencer by Macrogen Inc. (Seoul, Korea). The quality of the sequence was verified using FastQC v0.11.7. Before the commencement of the analysis, raw reads, adapter sequences, and low-quality read sequences were filtered using Trimmomatic 0.38. Trimmed reads were assembled using a Trinity program with default parameters. The Trinity program was used for de novo transcriptome assembly, and was then assembled into transcript contigs. These contigs were further processed for read alignment and abundance estimation using Bowtie 1.

Confirmation of RNA-Seq result using RT-PCR assay.
To confirm the RNA-Seq result, the total RNA of the pooled leaf sample was extracted using the easy-spin Total RNA Extraction Kit (iNtRON, Seongnam, Korea) and following the manufacturer's instruction RT-PCR assay using SR-8000 (GeNetBio, Daejeon, Korea) was conducted. The viral gene-specific primer, based on GenBank reference sequences and nucleotide sequences of the viral contigs from RNA-Seq, was designed for RT-PCR assay (Table 1). To verify the presence of reported viruses in P. notoginseng (TYLCCNV, PnVA, and PanVY) RT-PCR assay was performed using gene-specific primers (Table 1).  Fig. 3, Supplementary Tables 1-3). Most WMV contigs identified were similar to other reported WMV-Insam strains with a nucleotide identity of 99-100%. Among the identified viral contigs, small contigs that corresponds to the WMV with a length less than 1,000 nt were also found, but mostly covered complete genome of WMV-Insam strains (Fig. 3C).
Thus, it appears to be the same WMV isolate previously

RT-PCR assay for RNA-Seq data verification.
In RNA-Seq analysis large contigs (>8,000 nt) corresponding to WMV and TuMV, were mainly identified (Fig. 3). To verify the RNA-Seq data in individual samples, RT-PCR assay was conducted using gene-specific primers to detect TuMV (R.O.K. Patent No. 1006250190000) (Lee et al., 2006) and WMV (Jung et al., 2013). In RT-PCR assays for all collected samples (656 leaf samples), 3 (0.46%) samples were TuMV-positive (two samples of Yeoju and one sample of Hoengseong) and 139 (21.2%) samples were WMV-positive (Table 3). Interestingly, the infection rate of WMV showed noticeable regional differences, and this is probably related to the regional distribution of plant viral insect vectors such as aphids (Table 3). Tobamovirus was not detected using RT-PCR assay for the single pooled sample with a degenerated primer set for detecting Tobamovirus (Letschert et al., 2002). Therefore, the existence of the Tobamovirus is uncertain; therefore, the most commonly found virus of P. ginseng is the WMV. TuMV, on the other hand, is considered the minor virus of P. ginseng, given its low infection rate (0.61%). In RT-PCR assay using a polled samples, AcV1 was detected by RT-PCR assay using sequence-specific primers, designed based on the AcV1-like contigs from our RNA-Seq data. However, it is unclear whether AcV1 infects P. ginseng because only two small contigs (214 nt and 299 nt), corresponding to the partial regions of the genome of AcV1, were found in the RNA-Seq data and the pathogenicity of AcV1 was not yet been determined (Villareses et al., 2015). Viruses in the genus Panax that were reported in China, such as TY- LCCNV, PnVA, and PanVY were not found in the RNA-Seq data from P. ginseng. TYLCCNV, PnVA, and PanVY were also not detected by RT-PCR assay. From these results, the distribution of domestic ginseng virus species seems different from those recorded in China; only two viruses (WMV and TuMV) were detected in domestically grown P. ginseng by RNA-Seq metatranscriptome analysis and RT-PCR assay (Table 4). This is the first metatranscriptome analysis of plant virome from P. ginseng using NGS-based RNA-Seq and RT-PCR based analyses for virus identification and detection and its distribution, which will provide important basic data  for the development of virus-free plants. Further research and continuous field monitoring are therefore needed to clarify potential pathogenicity and economic impact of viruses on P. ginseng.

Conflicts of Interest
No potential conflict of interest relevant to this article was reported.