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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » National Germplasm Resources Laboratory » Research » Publications at this Location » Publication #315608

Research Project: CHARACTERIZING, DETECTING, AND ELIMINATING PATHOGENS TO ENABLE THE SAFE INTRODUCTION OF PLANT GENETIC RESOURCES

Location: National Germplasm Resources Laboratory

Title: Simultaneous identification and molecular characterization of viruses associated with an apple tree with mosaic symptom

Author
item Li, Ruhui
item Cao, Mengji - Southwest University
item Pu, Lingling - South China Agricultural University
item Kinard, Gary
item Zhou, Changyong - Southwest University
item Bateman, Margarita - Animal And Plant Health Inspection Service (APHIS)

Submitted to: International Conference on Graft Transmissible Diseases of Fruit Crops
Publication Type: Abstract Only
Publication Acceptance Date: 4/6/2015
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: We conducted genomic sequencing to identify viruses associated with mosaic disease of an apple tree using the high-throughput sequencing (HTS) Illumina RNA-seq platform. The objective was to examine if rapid identification and characterization of viruses could be effectively achieved by RNA-seq analysis. A seedling of apple cv. Spy was grafted with dormant buds from an apple tree infected by Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV). Total RNA was isolated from the grafted Spy tree, which also developed symptoms, three months after grafting and used for HTS. Raw sequence reads (33 million) of 101 nucleotides (nt) were assembled and mapped with DNA databases of plant (mainly Malus spp.) and virus genomes using the CLC Genomics Workbench bioinformatics tool. Three viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and an ilarvirus similar to Tulare apple mosaic virus (TApMV), were identified. A large contig of 7535 nt (99.7% coverage) had the highest nt identity of 87.2% with the QD-13 isolate of ACLSV (KJ522695). Five large contigs of 2868 nt (30.7% coverage) – 9298 nt (99.6%) shared 71.7-77.0% with each other and 78.0% to the known ASPV isolates, indicating they are distinct APSV strains. A contig of 953 nt shared the highest identity of 67% with the coat protein gene of TApMV and Spinach latent virus at the nt sequence level, suggesting it might be a novel species. The sequence coverage of these contigs was estimated to be 20- to 40-fold. Infections were confirmed by RT-PCR cloning and sequencing. This platform can simultaneously detect and differentiate multiple viruses and strains in a single sample. The RNA-seq analysis offers an efficient diagnostic method for indexing plant germplasm in quarantine and clean stock programs.