جداسازی و واکاوی فرم‌های ناقصِ ژنوم ویروس کوتولگی گندم

Wheat dwarf virus (WDV) is one of the causal agents of yellowing and stunting among wheat and barley cultivations which annually cause economic loss to these crops. The virus is classified into the genus Mastrevirus (family Geminiviridae) and it conventionally has two strains including wheat and barley strains (WDV-Wheat and WDV-Barley, respectively) which share 83-84% nucleotide identity. WDV is naturally transmitted by leafhopper in a persistent circulative manner. The genome of WDV is circular single-stranded DNA which encodes four proteins including two replication-associated proteins (RepA and Rep), coat protein (CP) and movement protein (MP). Additionally, two regulatory regions including large and small intergenic regions (LIR and SIR, respectively) are located on the genome which play role in transcription and replication. The viral genome is replicated by rolling circle mechanism. In order to isolate defective forms of WDV genome, samples were collected from symptomatic winter wheat and barley plants in Masjed Soleinman region, Khuzestan province, and subjected to Indirect-ELISA using polyclonal antibody of WDV. ELISA-positive samples were then subjected to total DNA extraction and viral genome was amplified using rolling circle amplification (RCA) technology. Restriction enzymes including HindIII and EcoRI were used to release full-length (~2.7 kb) and incomplete (0.5-0.7 kb) fragments of the viral genome and they were extracted from the agarose jell and cloned. Two complete fragments and four incomplete fragments were selected for sequencing and the resulting sequences were assembled using SeqMan Pro software (DNASTAR Lasergene v8). Nucleotide comparison of the full-length sequences with other WDV sequences available at GenBank showed that they share high level of identity (96-97%) to Iranian isolates of WDV which had been previously deposited in GenBank. Analysis of defective forms of WDV genome showed that they contain complete, incomplete and/or duplicated coding regions of the parental sequences. Also, non-viral regions with host origins (wheat and barley) were observed in these defective forms. It was found that all defective forms contain LIR/SIR as these regions are required for their replication. It seems that the exchange of genomic fragments by defective forms of WDV genome play a role in genome evolution of their hosts. However, further experiments are necessary to determine the potential role of these defective forms in WDV infection and symptom expression.