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Production of cDNA Clones from the MAV Isolate of Barley Yellow Dwarf Virus

D. J. Barbara

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907, U.S.A.

A library of cDNA clones was produced from the approximately 6 kb RNA of the MAV isolate of barley yellow dwarf virus (BYDV) in bacteriophage gt11, by a method that involved random priming and cloning ds cDNAs of between 1.0 and 2.5 kbp. Screening with antiserum to the dissociated coat protein of the MAV isolate showed that approximately 2.5% of the recombinants were capable of expressing this protein. After subcloning some inserts into the plasmid pUC18, restriction endonuclease mapping showed that they collectively represented at least 85% (a total of 5.1 kbp) of the BYDV genome. We did not attempt to determine which, if any, of the immunologically positive clones expressed the entire coat protein, but of the nine examined, all shared a region of approximately 1000 bp, located between 750 bp and 1750 bp from the 3' terminus of the restriction map. Sequence homology among different isolates of BYDV was examined by using selected MAV cDNA clones as probes in viral nucleic acid hybridization studies. Hybridization specificity varied according to the origin of the clones within the BYDV genome. Those from the putative coat protein-coding region hybridized well only to the homologous MAV isolate; those from elsewhere hybridized also with another isolate from the same subgroup (P-PAV). No clones hybridized significantly to a third isolate (RPV), which is in another subgroup of BYDV. The sensitivity of detection was related to probe size; the larger clones detected as little as 70 pg of purified virus (1.4 ng/ml in a 50 µl sample), and with these the sensitivity of virus detection in plant extracts by dot-blot hybridization was greater than that of ELISA.

Keywords: BYDV, cDNA clones, cross-hybridization

Present address: East Malling Research Station, East Malling, Maidstone, Kent ME19 6BJ, U.K.

Received 8 January 1987; accepted 2 June 1987.