3.7.26
IDENTIFICATION AND LOCALIZATION OF A PATHOGENICITY GENE IN OPHIOSTOMA NOVO-ULMI

A ET-TOUIL1, L BERNIER1 and CM BRASIER2

1Centre de recherche en biologie forestière, Université Laval, Québec, Qc, Canada, G1K 7P4; 2Forest Research Station, Alice Holt Lodge, Farnham, Surrey, GU10 4LH, UK

Background and objectives
Successive pandemics of Dutch elm disease have caused much destruction in European and North American elm populations. The highly aggressive species Ophiostoma novo-ulmi, which is further subdivided into the Eurasian (EAN) and North American (NAN) races, is associated with the current pandemic [1]. Earlier work [2] suggested that the pathogenicity of O. novo-ulmi is under additive nuclear gene control. The objectives of the study described hereafter were to identify and localize one of these genes.

Material and methods
A highly aggressive (H327) and a moderately aggressive (AST27) EAN strains were crossed in the laboratory. The resulting progeny (n=40) and their parents were inoculated into Commelin elm (U. x Commelin) and English elm (Ulmus procera) in the summers of 1995 and 1996, respectively

.

Total genomic DNA was extracted from parent and progeny strains, and amplified by the polymerase chaine reaction (PCR) using RAPD (Random amplified polymorphic DNA) primers. RAPD markers linked to a putative pathogenicity gene were identified by bulked segregant analysis (BSA) and mapped by analyzing segregation data for all 40 progeny with MapMaker V2.0 Software, using the LOD score procedure

.

Chromosomal DNA was isolated from spheroplasts of 5-7 day old budding cell suspensions and separated by pulse-field chef electrophoresis (PFGE). Chromosomal DNA was transferred from PFGE gels to charged nylon membranes by capillarity and hybridized overnight with a 32P-labelled probe prepared from a RAPD fragment linked to the putative pathogenicity gene

.

Results and conclusions
Field inoculation of elms indicated that the highly and moderately aggressive phenotypes segregated 1:1 in the 40 F1 progeny recovered from the cross H327 x AST27, suggesting that the difference in aggressiveness was controlled by a single nuclear gene, named Pat1. To the best of our knowledge, this is the first pathogenicity gene to be identified in O. novo-ulmi. Ten RAPD markers linked to Pat1 were identified by BSA and mapped on both sides of the pathogenicity gene. Southern hybridization of PFGE-separated chromosomes with a probe made from a RAPD fragment cosegregating with aggressiveness showed that Pat1 was located on a large (> 5.7 Mb) chromosome. RAPD data further suggested that the moderately aggressive strain AST-27 is an introgressant or the backcross product of a natural hybridization between O. novo-ulmi and the less aggressive species O.ulmi

.

References
1. Brasier CM, 1991. Mycopathologia 115, 151-161.
2. Brasier CM, 1987. Genetics and Plant Pathogenesis, pp. 297-310.