Soybean Viromes in the Republic of Korea Revealed by RT-PCR and Next-Generation Sequencing

Abstract

Soybean (Glycine max L.) is one of the most important crop plants in the Republic of Korea. Here, we conducted a soybean virome study. We harvested a total of 172 soybean leaf samples showing disease symptoms from major soybean-growing regions in the Republic of Korea. Individual samples were examined for virus infection by RT-PCR. Moreover, we generated eight libraries representing eight provinces by pooling samples and four libraries from single samples. RNA-seq followed by bioinformatics analyses revealed 10 different RNA viruses infecting soybean. The proportion of viral reads in each transcriptome ranged from 0.2 to 31.7%. Coinfection of different viruses in soybean plants was very common. There was a single dominant virus in each province, and this geographical difference might be related to the soybean seeds that transmit viruses. In this study, 32 viral genome sequences were assembled and successfully used to analyze the phylogenetic relationships and quasispecies nature of the identified RNA viruses. Moreover, RT-PCR with newly developed primers confirmed infection of the identified viruses in each library. Taken together, our soybean virome study provides a comprehensive overview of viruses infecting soybean in eight geographical regions in the Republic of Korea and four single soybean plants in detail.

Keywords: Korea; RNA-seq; mutation; soybean; viral genome; virome; virus.

Figure 1

Soybean viral disease symptoms and eight major provinces in the Republic of Korea for soybean virome study. Soybean leaf sample showing viral disease symptoms such as leaf mottling collected from Gyeongbuk (A) and Gangwon (B). Leaf sample showing viral disease symptoms indicated by yellow-colored circle used for virome study. (C) Map displaying eight major provinces in the Republic of Korea from which soybean leaf samples were collected.

Figure 2

Identification of viruses and viral populations from 12 different libraries. Pie charts displaying the proportion of identified viruses based on the number of contigs (A) and number of viral reads (B). (C) Number of libraries in which individual viruses were identified. (D) Number of identified viruses in each library. Proportion of identified viruses based on viral reads (E) and fragments per kilobase of transcript per million mapped reads (FPKM) values (F) in each library. (G) Proportion of viral reads in each library. Abbreviations: Gangwon (GAWO); Gyeonggi (GYGI); Chungbuk (CHBU); Chungnam (CHNA); Jeonbuk (JEBU); Jeonnam (JENA); Gyeongbuk (GYBU); Gyeongnam (GYNA); number (No.); pooled (p).

Figure 3

Phylogenetic relationship of WVMV, PeMoV, SYMMV, and SYCMV. Phylogenetic trees of WVMV (A), PeMoV (B), SYMMV (C), and SYCMV (D) constructed using complete genome sequences for individual virus species. Nucleotide sequences were aligned by MAFFT, and phylogenetic trees were constructed using IQ-TREE with maximum likelihood method and bootstrap with 1000 iterations. Virus genomes derived from this study are indicated by red color. Best-fit models for each virus species are as follows: GTR+F+I+G4 (WVMV), TN+F+I (PeMoV), TIM2e+I+G4 (SYMMV), and TIM2e+I+G4 (SYCMV). The generated phylogenetic trees were visualized by the FigTree (version 1.4.4).

Figure 4

Phylogenetic relationship of PSV RNA1, PSV RNA2, PSV RNA3, and SMV. Phylogenetic trees of PSV RNA1 (A), PSV RNA2 (B), PSV RNA3 (C), and SMV (D) constructed using complete genome sequences for individual virus species. Nucleotide sequences were aligned by MAFFT, and phylogenetic trees were constructed using IQ-TREE with maximum likelihood method and bootstrap with 1000 iterations. Virus genomes derived from this study are indicated by red color. Best-fit models for each virus species are as follows: TIM2+F+I+G4 (PSV_RNA1), TN+F+G4 (PSV_RNA2), TIM2+F+G4 (PSV_RNA3), and GTR+F+I+G4 (SMV). The generated phylogenetic trees were visualized by the FigTree (version 1.4.4).

Figure 5

Identification of SNPs for viruses from four single plants. (A) Genome structure of PSV and positions of identified SNPs. (B) Genome structure of PeMoV and positions of identified SNPs. (C) Genome structure of SYMMV and positions of identified SNPs. (D) Genome structure of SMV and positions of identified SNPs. We used a reference genome for the genome structure of individual virus species. Information of the complete viral genome sequences used for SNP analysis, including isolate name, accession number, and genome size, is indicated. The SNPs were visualized by the Tablet program.

Figure 6

Confirmation of eight major viruses infecting soybean by RT-PCR. (A) The individual viral genome structure displays the target virus sequence position amplified by newly designed primer pairs indicated by gray-colored bar. The detailed information of primer pairs can be found in Table S8. (B) Amplified PCR products by RT-PCR were visualized by agarose gel electrophoresis. The Actin11 gene of soybean was used as a positive control. We used the same total RNA for both RNA-seq and RT-PCR.