Next generation sequencing reveals past and current widespread occurrence of maize yellow mosaic virus in South Africa

DATA AVAILABILITY: All data is available in the manuscript and Electronic Supplementary Material. The MaYMV RSA BR1A and MaYMV RSA SCM genome sequences have been deposited in Genbank (Accessions MG570476; MN943641, respectively). RNA-seq data has been deposited at the NCBI GEO (Gene Expression Omnibus) repository (Accessions GSE94442, GSE99005).Online Resource 1. Bioinformatics pipeline based on de novo assembly of unmapped reads used for discovery of maize yellow mosaic virus in maize RNA‐seq data. (a) Raw reads were assessed with FastQC, trimmed with Trimmomatic based on the FastQC results, and aligned to the reference genomes of maize and C. zeina to collect unmapped reads. (b)Unmapped reads were assembled using Trinity, their protein sequences predicted with TransDecoder and compared against the NCBI nr database using BLASTP.Online Resource 2. Oligonucleotide primers used to amplify and Sanger sequence the complete genomes of Maize yellow mosaic virus isolates RSA BR1A and RSA SCM.Online Resource 3. Percent nucleotide identity for complete genomes between maize yellow mosaic virus from South Africa and other worldwide isolates.Online Resource 4. Comparison of predicted maize yellow mosaic virus proteins of RSA BR1A and RSA SCM from South Africa and MaYMV Yunnan 11 from China.Online Resource 5. RNA-dependent RNA polymerase-based phylogenetic analysis of maize yellow mosaic virus and related Poleroviruses. The evolutionary history was inferred from the RNA-dependent RNA polymerasenucleotide sequences by using the Maximum Likelihood method with the GTRGAMMA model. Bootstrap consensus values are shown at the nodes. The sequences were extracted from complete genome sequences with the following NCBI accession numbers: RSA BR1A (MG570476), RSA SCM (MN943641), Kenya KALRO (MH205607), Kenya MYDV-like (MF974579), Tanzania 76 (MG664790.1), Ethiopia (MF684369), Nigeria (KY684356.1), China Y11 (KU248489.1), China Y1 (KU179221.1), China MYDV-RMV2 (KT992824.1), China SC (MK652149), Brazil (KY940544.1), Ecuador (KY052793), BVG Gimje (KT962089.1) and MYDV-RMV (KC921392.1). The latter two were used as outgroups. The scale bar indicates the number of nucleotide substitutions per site.Online Resource 6. Confirmation by Sanger sequencing that the expected maize yellow mosaic virus RT-PCR product was amplified from maize inbred B73 sample BR1B. Sequences derived from sequencing the 753bp RT-PCR product with the MaYMV-F or MaYMV-R primers were named BR1B.MaYMV.F or BR1B.MaYMV.R, respectively. These sequences were aligned to the corresponding sequence from the assembled MaYMV RSA BR1A genome sequence determined by 5’RACE, 3’RACE, RT-PCR and Sanger sequencing [BR1A (MG570476)], and the reference sequence MaYMV Yunnan 11 from China [KU248489]. The MaYMV-F and MaYMV-R primer sequences are reported in Chen et al. (2016). Non-consensus sites are shown by an asterisk.Online Resource 7. Representative maize leaf symptoms of samples that were positive for maize yellow mosaic virus with the RT‐PCR assay using MaYMV‐F and MaYMV‐R primers. A, B: Mosaic symptoms; C‐D: Yellow streaks; E‐H: Narrow yellow streaks. A‐H samples: 17‐4263, 17‐4267, 17‐4245, 17‐4135, 17‐4275, 17‐4261, 17‐4284, 17‐4172, respectively. Maizegenotypes are not known.Online Resource 8. Actin RT-PCR to confirm RNA and cDNA integrity for samples that were RT-PCR negative for maize yellow mosaic virus. RT-PCR products amplified using the primer pair actinF and actinR were visualized by agarose gel electrophoresis, with an expected 169 bp cDNA actin product. Lane M, O’GeneRuler 100 bp DNA Ladder (Thermo-Fischer, Waltham, USA); lane 1, RT no template control; lane 2, PCR no template control; lane 3, maize healthy control/RNA positive control; lane 4, 16-3308; lane 5, 16-3328; lane 6, 16-3224; lane 7, 16-3252; lane 8, 16-3256. The additional 277 bp product in lanes 3-8 is the actin gDNA product, since the primers flank an intron, indicating presence of some gDNA in the samples.Online Resource 9. Maize B73 reads corresponding to RNA viruses.Maize yellow mosaic virus (MaYMV) is a single-stranded RNA polerovirus first identified in China. MaYMV was recently reported from West and East Africa, however it had not yet been reported from southern Africa. RNA-seq data from South African field-grown fungal-infected maize was mined for viral sequences by de novo assembly of reads that did not map to the maize or fungal genomes. Predicted proteins from the de novo-assembled unmapped reads matched MaYMV proteins with regions of 96–100% identity. MaYMV was detected in maize RNAseq data from 2009, 2012 and 2013. Complete South African MaYMV genome sequences (5642 nt) were determined by RT-PCR and Sanger sequencing of samples from two different maize genotypes, years, and sites. Phylogenetic analysis confirmed the species identity as MaYMV, and showed separate clustering of isolates between Africa, Asia and South America. Some MaYMV positive samples had reads matching Potyviridae (Johnson grass mosaic virus and Sugarcane mosaic virus), and mycoviruses (Setosphaeria turcica hypovirus 1, Bipolaris maydis partitivirus 1, and Pleospora typhicola fusarivirus 1). A 2016/2017 RT-PCR survey of maize plants exhibiting virus-like symptoms, such as yellowing and streaking patterns, revealed MaYMV in 39 samples from six provinces in South Africa. This report documents the earliest known MaYMV infection world-wide, and indicates that the virus is widespread throughout Africa.The National Research Foundation, South Africa, the Department of Agriculture Forestry and Fisheries Research and Technology Fund, South Africa, and the University of Pretoria, South Africa.https://link.springer.com/journal/10658hj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc