Differentiation of potyviruses and their strains by hybridization with the 3' non-coding region of the viral genome.
Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.
Frenkel
M J
MJ
CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Jilka
J M
JM
Shukla
D D
DD
Ward
C W
CW
D015139:Blotting, Southern; D016679:Genome, Viral; D007438:Introns; D008722:Methods; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D012367:RNA, Viral