Genetics, Vol. 166, 99-111, January 2004, Copyright © 2004

Marker-Based Cloning of the Region Containing the UhAvr1 Avirulence Gene From the Basidiomycete Barley Pathogen Ustilago hordei

R. Linninga, D. Lind, N. Leed, M. Abdennadhere, D. Gaudeta,b, P. Thomasa,c, D. Millse, J. W. Kronstadd, and G. Bakkerena
a Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia V0H 1Z0, Canada,
b Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada,
c Cereal Research Centre, Winnipeg, Manitoba R3T 2M9, Canada,
d Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
e Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331

Corresponding author: G. Bakkeren, Pacific Agri-Food Research Centre, Highway 97, Summerland BC V0H 1Z0, Canada., bakkereng{at}agr.gc.ca (E-mail)

Communicating editor: M. SACHS

Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940s. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain 54 progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a nonrepetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an ~80-kb region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.