1Cereals Research Department, and 2Sainsbury Laboratory, John lnnes Centre, Colney Lane, Norwich NR4 7UH, UK

Background and objectives
The ascomycete Erysiphe graminis (= Blumeria graminis) f.sp. hordei, the causal agent of powdery mildew of barley, is one of the best understood fungal pathogens of plants in terms of its genetics and pathology. Some 30 avirulence genes have been identified, matching specific host resistance genes, as well as several genes controlling responses to fungicides. A programme aimed at cloning agriculturally important genes from E. graminis f.sp. hordei is under way, with the aim of understanding the following features of the biology of this fungus: (i) the nature, frequency and spread of mutations to virulence and fungicide resistance in the pathogen's population; (ii) the molecular interactions between pathogen avirulence factors and plant resistance factors that induce defence responses; and (iii) the physiology of fungicide resistance. This programme involves detailed molecular mapping of the genome of E. graminis f.sp. hordei, using AFLPTM (amplified fragment length polymorphism), followed by map-based cloning of genes of interest.

Materials and methods
A cross of the E. graminis f.sp. hordei isolates CC52 and DH14 was made, and progeny extracted by established procedures [1]. This cross was chosen for detailed analysis because both parents can be readily crossed and because many avirulence genes segregate in the progeny [2]. Avirulence determinants and fungicide responses were assessed in detached leaf tests by standard methods. DNA for AFLP analysis was extracted from parent and progeny isolates using a CTAB-based protocol. AFLP was performed by a slightly modified version of the standard method. High molecular-weight DNA for library construction was extracted and fractionated on a sucrose gradient. DNA of the appropriate size range was selected for cloning.

Results and conclusions
Approximately 300 AFLP markers have been placed on a genetic map of the E. graminis f.sp. hordei cross CC52xDH14 and assigned to eight large linkage groups. About 15% of AFLP markers form small groups of up to four loci, while several markers cannot yet be assigned to linkage groups. The up-to-date version of the map will be presented.

Eight avirulence (Avr) genes are included on the map. One Avr gene corresponds to each of the four resistance genes M1a9, M1a12, M1a22 and Mih, while two Avr genes correspond to the M1a7 and to the M1a13 resistances. Three pairs of Avr genes are linked to one another (less than 10 cM apart): Avra7-1 with Avrh, Avra7-2 with Avra13-2 and Avra9 with Avra22. The ethirimol resistance gene, Eth 1, is not linked to any Avr gene but is closely flanked by AFLP markers, less than 5 cM away. Two Avr genes are considered to be possible candidates for map-based cloning, on the basis of their being flanked by AFLPs and the ease of distinguishing virulent and avirulent progeny.

Genomic libraries of E. graminis f.sp. hordei isolate DH14 are being constructed by cloning high molecular-weight DNA into cosmid and bacterial artificial chromosome (BAC) vectors. These libraries will be screened using PCR primers derived from the sequences of AFLP markers flanking an Avr gene, in order to identify clones that may contain the Avr gene.

The next stage of genetic mapping will be to link the map of CC52xDH14 with those of other crosses in which economically significant genes segregate. This high-density AFLP map and the high molecular-weight genomic libraries will be the basis of strategies for map-based cloning of other Avr and fungicide resistance genes from E. graminis f.sp. hordei.

This work was supported by MAFF and BBSRC.

1. Brown JKM, Jessop AC, Thomas S, Rezanoor HN, 1992. Plant Pathology 41, 126-135.
2. Brown JKM, Simpson CG, 1994. Current Genetics 26, 172-178.