Sweet potato viromes in eight different geographical regions in Korea and two different cultivars

Abstract

The sweet potato in the family Convolvulaceae is a dicotyledonous perennial plant. Here, we conducted a comprehensive sweet potato virome study using 10 different libraries from eight regions in Korea and two different sweet potato cultivars by RNA-Sequencing. Comprehensive bioinformatics analyses revealed 10 different virus species infecting sweet potato. Moreover, we identified two novel viruses infecting sweet potato referred to as Sweet potato virus E (SPVE) in the genus Potyvirus and Sweet potato virus F (SPVF) in the genus Carlavirus. Of the identified viruses, Sweet potato feathery mottle virus (SPFMV) was the dominant virus followed by Sweet potato virus C (SPVC) and SPVE in Korea. We obtained a total of 30 viral genomes for eight viruses. Our phylogenetic analyses showed many potyvirus isolates are highly correlated with geographical regions. However, two isolates of SPFMV and a single isolate of Sweet potato virus G (SPVG) were genetically distant from other known isolates. The mutation rate was the highest in SPFMV followed by SPVC and SPVG. Two different sweet potato cultivars, Beni Haruka and Hogammi, were infected by seven and five viruses, respectively. Taken together, we provide a complete list of viruses infecting sweet potato in Korea and diagnostic methods.

Figure 1

Geographical regions of collected sweet potato samples in Korea and viral disease symptoms in sweet potato plants. (a) A map displaying eight different geographical regions in Korea in which sweet potato samples were collected. Each region is indicated by a different color with the abbreviated region name. Full names of geographical regions can be found in Table 1. (b) Sweet potato plants grown in field covered by poly mulch sheets to protect soil. (c) Sweet potato plants displaying leaf malformation and purpling. (d) Leaf spots on sweet potato leaf.

Figure 2

Identification of viruses infecting sweet potato and proportion of identified viruses in each library. Pie chart illustrating proportion of identified viruses from all 10 libraries based on virus-associated contigs (a) and virus-associated reads (b). Bar graphs illustrating proportion of identified viruses in each library based on virus-associated contigs (c) and virus-associated reads (d). (e) Number of identified libraries for individual virus. (f) Number of identified viruses in each library. (g) Proportion of virus-associated reads as compared to total sequence reads in each library.

Figure 3

Genome organization and phylogenetic analyses of two novel viruses, SPVE and SPVF. (a) Genome organization of SPVE isolate GS. Polyprotein cleavage sites were also indicated with respective protein names. (b) BLASTN results using complete genome of SPVE against NCBI’s NT database. (c) Maximum likelihood phylogenetic tree of polyprotein amino acid sequences for SPVC, SPVE, SPFMV, SPVG, SPV2, and SPLV. (d) Maximum likelihood phylogenetic tree of genome sequences for SPVC, SPFMV, SPVE, and SPFMV. Two genome sequences for SPVE isolates GS and YJ-B were included in the phylogenetic construction. (e) Genome organization of SPVF isolate GS. (f) BLASTN results using complete genome of SPVF against NCBI’s NT database. (g) Maximum likelihood phylogenetic tree of RdRp amino acid sequences for SPCFV, SPYMV, SPVF, Melon yellowing-associated virus (MYaV), Elderberry carlavirus B (EBCVB), and Poplar mosaic virus (PopMV). (h) Maximum likelihood phylogenetic tree of CP amino acid sequences for SPCFV, SPVF, MYaV, and Garlic latent virus (GLV). (i) Maximum likelihood phylogenetic tree of genome sequences for SPCFV, SPYMV, and SPVF. EBCVA was used as an outgroup. Two genome sequences for SPVF isolates GS and IC were included in the phylogenetic construction. For the phylogenetic tree construction, all available protein or genome sequences homologous to SPVE or SPVF were retrieved from GenBank based on BLASTP and BLASTN searches, respectively. Accession number, isolate name, and virus name were described. Orange color indicates SPVE or SPVF. We used bootstrap replication values of 1,000, and bootstrap values over 70% are shown.

Figure 4

Phylogenetic analyses of SPFMV, SPVC, SPVG, SPLV, SPV2, and SPLCV isolates. (a) Maximum likelihood phylogenetic tree of genome sequences for 33 SPFMV isolates including six isolates from this study indicated by orange color. SPVC was used as an outgroup. (b) Maximum likelihood phylogenetic tree of genome sequences for 28 SPVC isolates including seven isolates in this study indicated by orange color. SPFMV was used as an outgroup. (c) Maximum likelihood phylogenetic tree of genome sequences for 12 SPVG isolates including two isolates in this study indicated by orange color. SPV2 was used as an outgroup. (d) Maximum likelihood phylogenetic tree of genome sequences for eight SPLV isolates including three isolates in this study indicated by orange color. Plum pox virus (PPV) was used as an outgroup. (e) Maximum likelihood phylogenetic tree of genome sequences for 14 SPV2 isolates including four isolates in this study indicated by orange color. SPVG was used as an outgroup. (f) Maximum likelihood phylogenetic tree of genome sequences for 14 SPLCV isolates including four isolates in this study indicated by orange color. SPGVaV was used as an outgroup. For phylogenetic analyses, we retrieved only complete genome sequences for each virus from GenBank based on a BLASTN search. We used not only complete viral genome sequences with respective accession numbers but also nearly complete viral genome sequences (Table S2) without accession numbers in this study. Accession number, isolate name, and virus name were described. Orange color indicates virus genomes obtained from this study. We used bootstrap replication values of 1,000, and bootstrap values over 70% are shown.

Figure 5

Viral genome assembly and SNP analyses for six assembled viruses including SPVF, SPVE, SPVG, SPLCV, and SPLV. Genome organization of assembled virus genome, mapping results of sequence reads on the assembled virus genome, and positions of identified SNPs for SPVF isolate GS (a), SPVE isolate GS (b) SPVG isolate IS (c), SPLCV DNA A isolate GS (d), SPVG isolate YJ (e), and SPLV isolate YJ-B (f) were visualized. Based on assembled virus-associated contigs and mapping of sequence reads on the reference virus genome, viral genomes were obtained. Positions of individual ORFs were indicated. In addition, mapping results on the assembled individual virus genome were used for SNP identification. The positions of identified SNPs in each virus genome were visualized by the Tablet program.

Figure 6

Viral genome assembly and SNP analyses for three SPFMV isolates and three SPVC isolates and mutation analyses of 12 virus genomes. Genome organization of assembled virus genome, mapping results of sequence reads on the assembled virus genome, and positions of identified SNPs for SPFMV isolate IC (a), SPVC isolate GS (b) SPFMV isolate YJ (c), SPVC isolate IC (d), SPFMV isolate YJ-B (e), and SPVC isolate IS (f) were visualized. Number of identified SNPs (g) and frequency of SNPs for the 12 virus genomes. Based on assembled virus-associated contigs and mapping of sequence reads on the reference virus genome, viral genomes were obtained. Positions of individual ORFs were indicated. In addition, mapping results on the assembled individual virus genome were used for SNP identification. The positions of identified SNPs in each virus genome were visualized by the Tablet program.

Figure 7

Confirmation of 11 identified viruses infecting sweet potato by RT-PCR. (a) Position of amplicon on individual virus genome was indicated by gray bar with respective size of amplicon. The detailed information of primer pairs can be found in Table S3. (b) Agarose gel electrophoresis results by RT-PCR with newly designed primer pairs. Full-length gels of RT-PCR results can be found in Fig. S1 in the Supplementary Information. Actin gene of sweet potato was used as positive control. We used the same total RNA for both NGS and RT-PCR. Green color indicates RT-PCR primer pairs for two novel viruses, SPVE and SPVF, as well as a variant of SPVG.