Genomic Variability among Barley Yellow Dwarf Virus-Pav Infecting Wheat Crop In Pakistan

Abstract

Wheat (Triticum aestivum L.) is the major staple grain food of Pakistan and is prone to many fungal, bacterial and viral diseases. Diseases caused by vriuses are among the biotic factors inflicting huge economic losses. Every year Barley yellow dwarf virus (BYDV) causes substantial losses to wheat crop. A total of 210 samples showing typical barley yellow dwarf virus symptoms were collected from different wheat growing area of Pakistan. The DAS-ELISA technique was used to identify the existing serotype of BYDV. The BYDV-PAV was serologically identified as the most prevalent strain in Pakistan. The overall infection rate in symptomatic plants of BYDV-PAV was 43% whereas highest infection was recorded in Muzaffar Garh area of Punjab province. Different plants species viz. oat (Avena sativa), maize (Zea mays), johnson grass (Sorghum halepense) and Italian rye (Lolium multiform) in the cropping system was tested for BYDV-PAV infection. The maize and grasses showed the highest infection rate of 70% while oats showed comparatively less infection percentage (63%). Total RNA was extracted by Tri- reagent from BYDV-PAV ELISA positive samples. Coat protein gene of BYDV-PAV was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers set and a product of approximately 600 bp was obtained. The overlapping strategy was employed for which 12 primers set were designed, for complete genome sequencing. A maximum of 5654bp complete genome size of Pakistani isolate of BYDV-PAV was obtained after joining contigs. The complete genome sequences were compared with other world isolates of BYDV-PAV such as Asia, Europe and USA by using basic local alignment search tool (BLAST) available on National Center for Biotechnology Information (NCBI) data base. The phylogenetic analysis was done by using Molecular Evolutionary Genetics Analysis (MEGA) Program. The gene wise inter isolate comparison among four Pakistani isolates of wheat was made. Between the RdRp/POL genes (i.e. P1 and P2), P2 gene showed higher identity (94.7-99.8%) as compared with P1 gene. The P3 coat protein gene was found the most conserved region with maximum of 99.2-100% among the isolates. The nucleotide sequence of P4 (putative movement protein gene) has showed identity of 98.6-100% among inter isolate of BYDV-PAV. The nucleotide sequence of aphid transmission gene/RTD (P5) has shown identity of 91.1-99.9% among inter isolate. Similarly the P6 gene, whose function is still unknown, has shown sequence identity of 89.4-99.9% among inter isolate. Similarity index of un-translated region (UTR) i.e. 5', 3' and intergenic regions were 91.4-100%, 93.9-100%, 97.6-100% (P2-P3) respectively while for P5-P6 intergenic region was 93.8-100%. The overall complete genomic comparison among four Pakistani Isolates (KT 252975, KT 252976, KT 252977and KT 252978) has shown divergence ranges from 0.14% to 6.9%. However these isolates have shown identity of 74.4 to 99.9% with other BYDV-PAV isolates of the world. It is concluded from this study that Pakistani isolates of BYDV-PAV are more closely related to USA, Europe and Japan but are distinct from Chinese isolates. Moreover the German isolate seems establishing an interlink bridge between PAV-I and PAV- II clusters based on phylogenetic tree analysis. The sequences of Pakistani isolates clearly indicated their identity as BYDV-PAV.