Complete Genome Sequence of Peanut Mottle Virus Identified from Soybean (Glycine Max L.) Using RNA-Sequencing

Complete genome sequence of Peanut mottle virus identified from soybean (Glycine max L.) using RNA-sequencing

Yeonhwa Jo1, Hoseong Choi2, Soo Yeon Choi3, Sang-Min Kim3, Bong Choon Lee3, and Won Kyong Cho1* 1Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 2Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea 3Crop Foundation Division, National Institute of Crop Science, Rural Development Administration, Wanju 55365, Republic of Korea

RNA-sequencing을 이용하여 콩에서 동정한 Peanut mottle virus의 전체 유전체 서열

조연화1 ･ 최호성2 ･ 최수연3 ･ 김상민3 ･ 이봉춘3 ･ 조원경1* 1서울대학교 농업생명과학연구원, 2서울대학교 농생명공학부, 3농촌진흥청 국립식량과학원

(Received June 26, 2020; Revised July 20, 2020; Accepted July 21, 2020)

To identify viruses infecting soybean (Glycine max L.), we Breuil et al., 2008). PeMoV is transmitted by diverse aphid collected soybean leaf samples showing viral disease symptoms species, seed, and mechanical inoculation. The hosts of PeMoV and subjected them to RNA-sequencing. De novo transcriptome are peanuts (Arachis hypogaea) (de Breuil et al., 2008), soybeans assembly followed by BLAST search identified Peanut mottle (Glycine max L.) (Lim et al., 2014), French beans (Phaseolus virus (PeMoV) belonging to the genus Potyvirus in the family vulgaris), and peas (Pisum sativum) (Soumya et al., 2014). Potyviridae. We obtained the complete genome of PeMoV isolate Daegu with 9,708 nucleotides encoding polypeptide The previous study has reported that soybean plant infected (3,099 aa) and pretty interesting potyviridae ORF (PIPO) (74 aa) by PeMoV showed mosaic, mottle, yellowing, and stunting proteins. The phylogenetic analysis using 10 available complete symptoms (Lim et al., 2014). genomes for PeMoV isolates demonstrated that PeMoV isolate In order to identify viruses infecting soybean, we collected Daegu is grouped with five other PeMoV isolates from Korea, soybean leaf samples showing viral disease symptoms in June China, Kenya, and Brazil. 2016. Of the collected soybean samples, a soybean leaf sample Keywords: genome, peanut mottle virus, RNA-seq, soybean, collected from Daegu in Korea was subjected to virus identi- virus fication. We extracted total RNA using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. A library for RNA-sequencing (RNA-seq) was prepared using Peanut mottle virus (PeMoV) is a single-stranded RNA virus the TruSeq RNA Library Prep Kit (Illumina). The prepared belonging to the genus Potyvirus in the family Potyviridae (de library was paired-end (100 bp × 2) sequenced by the Illumina HiSeq 2000 system (Macrogen). The raw sequence data were *For correspondence. E-mail: [email protected]; used for the de novo transcriptome assembly using Trinity Tel.: +82-2-880-4687; Fax: +82-2-873-2317 Complete genome sequence of Peanut mottle virus ∙ 319

version 2.0.6 with default parameters (Haas et al., 2013). as polyprotein with 3,099 amino acids (aa) (position 123~ Assembled contigs were subjected to BLASTX search against 9,422) and pretty interesting potyviridae ORF (PIPO) with 74 aa NCBI’s non-redundant protein database using standalone BLAST (position 2,924~3,148). The polyprotein showed sequence version 2.1.19 installed in the Linux system. We extracted only similarity to the polyprotein of PeMoV isolate Liaoning virus-associated contigs based on the BLASTX result using (AXK59834.1) from China with 99.65% identity, whereas the E-value 1e-10 as a cutoff. Two contigs 4,858 and 4,876 bp in PIPO showed sequence similarity to the PIPO of PeMoV strain length were associated with PeMoV. We combined the two M (YP_006393468.1) from the US with 91.89% identity. contigs, in which 15 bp nucleotides (nt) overlapped. Finally, We generated the phylogenetic tree using 10 available we obtained the complete genome of PeMoV isolate Daegu, complete genomes of PeMoV including isolate Daegu using which was 9,708 nt in length (Fig. 1A). BLASTN search the MEGA7 program (Kumar et al., 2016) and revealed four showed that PeMoV isolate Daegu showed sequence similarity different groups of PeMoV isolates (Fig. 1B). PeMoV isolate to PeMoV isolate Liaoning (GenBank MH270528.1) identified Daegu was grouped together with three isolates from Korea, an from peanut in China with 100% coverage and 99.44% nt isolate from China, an isolate from Brazil, and an isolate from identity. The genome of PeMoV encodes two proteins referred to Kenya. Interestingly, the hosts in Group A, including soybean,

(A)

(B)

Fig. 1. Genomic features and phylogenetic relationship of PeMoV isolate Daegu. (A) Genomic features of PeMoV isolate Daegu. Two gray bars indicate the identified contigs associated with the PeMoV isolate Daegu. (B) Phylogenetic relationship of PeMoV isolate Daegu with other PeMoV isolates. Available 10 PeMoV complete genomes including PeMoV isolate Daegu indicated by orange color box were aligned by the ClustalW program with default parameters. The phylogenetic tree was constructed using the MEGA6 program with Maximum likelihood method and 500 bootstrap replicates. Soybean mosaic virus (SMV) was used as an outgroup.

Korean Journal of Microbiology, Vol. 56, No. 3 320 ∙ Jo et al.

peanut, adzuki bean, and pinto peanut, varied. Taken together, Development (No. PJ01498301)” conducted by the Rural we report the complete genome of PeMoV isolate Daegu from Development Administration, Republic of Korea. soybean by RNA-seq.

Nucleotide sequence accession number Conflict of Interest The complete genome sequence of Peanut mottle virus isolate Daegu has been deposited in GenBank under the The authors declare that the research was conducted in the accession number MT603852. absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

적 요 References 콩 감염 바이러스를 동정하기 위하여 바이러스 병증을 보이 는 콩 잎 샘플을 채취하였다. 채취한 잎 샘플은 RNA-sequencing de Breuil S, Nievas MS, Giolitti FJ, Giorda LM, and Lenardon SL. 2008. Occurrence, prevalence, and distribution of viruses infecting 에 이용되었다. 전사체 조립 및 BLAST 검색을 통해 Potyvirus peanut in Argentina. Plant Dis. 92, 1237–1240. 속 및 Potyviridae과에 속하는 Peanut mottle virus (PeMoV)를 Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, 동정하였다. PeMoV 분리주 Daegu의 유전체는 9,708 뉴클레 Bowden J, Couger MB, Eccles D, Li B, Lieber M, et al. 2013. De 오티드로 구성되어 있으며, 폴리펩타이드(3,099 aa)와 PIPO (74 novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. aa) 단백질을 코딩한다. 이용 가능한 PeMoV 10개 유전체를 이 Protoc. 8, 1494–1512. 용하여 계통수 분석을 수행한 결과 PeMoV 분리주 Daegu는 Kumar S, Stecher G, and Tamura K. 2016. MEGA7: molecular 한국, 중국, 케냐 및 브라질에서 동정된 다른 PeMoV 분리주와 evolutionary genetics analysis version 7.0 for bigger datasets. 그룹을 이루었다. Mol. Biol. Evol. 33, 1870–1874. Lim S, Lee YH, Igori D, Zhao F, Yoo RH, Lee SH, Baek IY, and Moon JS. 2014. First report of Peanut mottle virus infecting soybean in South Korea. Plant Dis. 98, 1285. Acknowledgments Soumya K, Yogita M, Prasanthi Y, Anitha K, Kishor PBK, Jain RK, and Mandal B. 2014. Molecular characterization of Indian isolate This work was carried out with the support of the “Cooperative of Peanut mottle virus and immunodiagnosis using bacterial Research Program for Agriculture Science & Technology expressed core capsid protein. Virusdisease 25, 331–337.

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