3.4.4S
APPLICATION OF DOUBLED HAPLOID TECHNOLOGY AND DNA MARKERS IN BREEDING FOR CLUBROOT RESISTANCE IN BRASSlCA OLERACEA

DAC PINK and GJ KING

Horticulture Research International, Wellesboume, Warwick, CV35 9EF, UK

Background and objectives
Clubroot (Plasmodiophora brassicae) is an important disease of brassicas. It is characterised by an undifferentiated proliferation of root tissue to form galls (clubs). These reduce the efficiency of the root system so that infected plants will more lodge more readily. Severely affected plants may be stunted resulting in loss of yield and marketability. Soft-rot bacteria and other organisms may infect galls, resulting in a further reduction of root efficiency . The disease is difficult to control because of the longevity of the resting spores in the soil. Soil sterilisation can eradicate these but is prohibitively expensive on a field scale. Traditionally the disease has been controlled on a field scale by the application of lime to raise soil pH but high application rates are often necessary. Clubroot resistant cultivars are therefore an attractive means of control.

Host resistance to P. brassicaeis available in B. oleracea. With the exception of resistance in the cabbage cv. Badger Shipper controlled by a single dominant gene, resistance appears to be a recessive quantitative character [1]. Sources of resistance to clubroot are found amongst agronomically poor cabbages and kales. Attempts to utilise these in backcross breeding programmes have been largely unsuccessful and relatively few "clubroot resistant' cultivars exist. The use of novel breeding techniques enables more effective and efficient breeding for clubroot resistance. Some of the difficulties of assessing a quantitative recessive character can be overcome by the production of homozygous doubled haploid (DH) lines. The use of molecular markers linked to resistance can also improve the effectiveness of a selection programme since they are not subject to environmental influences.

Materials and methods
The landrace cabbage Bohmerwaldkohl was used as a source of non-specific, durable clubroot resistance[2]. Anther culture was used to produce DH lines from the F1between the clubroot susceptible cv. Hawke and Bohmerwaldkohl. All DH lines were assessed for resistance to three isolates of P. brassicae in a glasshouse and a subset were assessed in the field. Bulked segregant analysis was used to identify four RAPD markers linked to clubroot resistance, which were then cloned and converted to CAPs markers. The loci were positioned on a B.oleraceamap [3]. Additional PCR markers are being used to shuttle between reference maps and the original population in order to establish tighter linkage. DH lines have been made from the F1 between a clubroot resistant cabbage DH line and a susceptible broccoli DH line. These will be used to assess the effectiveness of a marker assisted selection programme.

Results
Responsiveness to anther culture was additive; the cv. Hawke showed a consistently high response, Bohmerwaldkohl was poor and the F1 intermediate. 151 Hawke x Bohmerwaldkohl DH lines were produced. There was significant variation for resistance to three isolates of P. brassicae among the DH lines. 11% of DH lines had resistance comparable to Bohmerwaldkohl in the glasshouse Comparing results from the glasshouse and field identified an isolate x line interaction. Three of the resistant DH lines had significantly improved morphology compared to Bohmerwaldkohl. Five DNA-RAPD loci were linked to clubroot resistance, three in repulsion and two in coupling. The markers mapped to more than one linkage group.

References
1. Crute IR et. al. Plant Breeding Abstracts 50,91-104. 2. Crute IR, Pink DAC 1989. Aspects of Applied Biology 23, 57-60 3. Bohoun EJR et. al. Theoretical and Applied Genetics 93, 833-9.