Specific Detection Of Barley Yellow Dwarf Viruses And Population Genetic Variation Of BYDV-PAV Species

In China,Barley Yellow Dwarf Virus(BYDVs) caused mainly yellow dwarf diseases of wheat throughout the northern and northwestern provinces,crop losses of 20%-30%had been observed for many years in many provinces.The main objective of this study was specific detection BYDVs of speices type,incidence rates,species distribution and molecular variability,phylogenetic,genetic variation and evolution relationship for BYDV-PAV.281 wheat samples expressing BYDV-like symptoms were collected from four agro-ecological areas in China during 2007,including Northwestern area,Northern area, Central area,and South-western area.Three digoxigenin-labeled cDNA probes complementary to the coat protein and read-through protein(RTP) gene sequences of barley yellow dwarf virus(one each for three species,namely BYDV-GAV,GPV,and PAV) were synthesized for developing a specific and sensitive dot-blot hybridization detection assay for total RNA extracts from field-infected wheat plants.The sensitivity limit for BYDV-GAV, GPV,and PAV probes corresponded to 25 ug,31.25 ug,and 62.5 ug tissue/spot,respectively. Nucleic acid spot hybridization showed that 190 samples had positive reaction,from which GAV(41.99%) was the most prevalent species in all samples by contrast with PAV(19.22%) and GPV(6.41%),GAV occured mostly in Jiaozuo,Datong,Yinchuan and Wunumuqi regions, PAV occured mostly in Luoyang,Zhengzhou,and Tianshui regions,however,GPV,existed as particular strain in China,had less infection and predominated mostly in specific regions, such as Yuncheng and Hancheng city.It seemed that the incidence rates of GAV may reduce and that of PAV would has been ascending tendency according to the quantity of positive GAV and PAV samples compared with reported data during 2004-2006 from our laboratory. The high sensitivity and reliability of the molecular hybridization assay described introduce an important altemative to serological methods for detecting BYDV.Nucleic acid spot hybridization and reverse transcription polymerase chain reaction (RT-PCR) were made to amplify the CP gene of BYDV-GAV and BYDV-PAV using the RNA extract of samples.The product of RT-PCR were cloned and sequenced,the molecular variability of GAV and PAV were analysed through the multiple sequence alignments. Comparison of 11 nucletide sequences of GAV revealed high conservation of the CP gene, phylogenetic tree constructed based on the sequences of GAV CP genes showed that there was no obvious cluster,ttowever,PAV was another case,179 substitutions in 600 nt showed high variality,especially in 3'terminal sequence,the phylogenetic tree constructed based on the sequences of PAV CP genes formed three branches(A,B and C),A and B group were both belong to PAV-CN isolate,C group was comprised of isolates 05GG2 and 06KM25,even from the same branch,the variability of sequences of three groups is obvious,which resulted in the diversity clusters.Isolates 05GG2 and 06KM25 were different from other PAV samples, the CP gene comprised 603 nucleotides,and were very similar to those of BYDV PAV-aus, the nucleotide and deduced amino acid sequences showed that there was a high degree of identity between 05GG2/06KM25 and PAV-aus(93.6%identity),according to the level of sequence similarities,05GG2 and 06KM25 would be closely related to BYDV-PAV-aus.The complete genomic sequences of a selection of 30 BYDV-PAV isolates were cloned and determined,which were comprised of 5652-5709 nucleotides(GenBank accession number: EU332307-EU332336),sharing 75.1%-99.7%identities at the nucleotide level. Phylogeography of PAV was reconstructed from the genomic RNA sequences of these isolates togethered with other PAV isolates reported previously.The variability among BYDV-PAV isolates in China was great,phylogenetic analysis detected four major groups, from which a new cluster of distinct variant was discovered,it is proposed to belong to a new species in China,sharing high sequence identity of individual ORF and UTR with isolates from USA,it suggested the high genetic diversity level and speciality of genetic structure for PAV in China.In contrast,the diversity within PAV isolates from USA was low(97% sequence identity),also when compared to isolates from Sweden,Germany,Australia and Japan.Altogether,it is supported the view that the origin centre of diversification for BYDV-PAV may be Gansu and Shaanxi province and that the virus spread subsequently from Northwest to North,East,Centre and South across China,using bioinformatics methods.