¹ Molecular Plant Genetics and Germplasm Development Group, Joint Centre for Crop Improvement, Department of Crop Production, The University of Melbourne, Parkville VIC 3052

² Department of Applied Biology & Biotechnology, RMIT University, Melbourne Vic 3000

ABSTRACT

Blackleg is a major disease of oilseed brassicas and remains a constant threat to the canola industry in Australia. The expression of blackleg symptoms is greatly affected by many factors such as inoculum type, inoculum dose, environmental factors etc. Therefore, there is a need to develop a reliable and efficient method of selecting blackleg resistance genotypes. Analysis of 13 F₃ families from a cross between genotype R13, possessing B. juncea blackleg resistance, and the blackleg susceptible B. napus cultivar Tower, indicated that blackleg resistance was controlled by three genes. Each of these genes have been separated into different lines in order to find the markers for each gene. Bulked segregant analysis was performed using RAPD and EcoR1/Mse1 based AFLP analysis to identify markers linked to the resistance loci. There were no polymorphisms observed between the bulks from 297 RAPD primers screened. However, nine polymorphisms were found between the bulks out of the 16 AFLP primer combinations screened. These markers will be further investigated on the individual plants in the bulks. Once a tightly linked marker is found it will be cloned and sequenced and a more robust screening method will be established.

INTRODUCTION:

Brassica napus (Canola) has become an important crop in Australia as a source of mono-unsaturated edible oil, animal feed and as a break crop in rotation with cereal and legumes. Blackleg disease caused by the virulent form of Leptosphaeria maculans (Desm.) Ces & De Not. is one of the most economically important diseases of oilseed rape (B.napus) crops. The incorporation of blackleg resistance into rapeseed lines with desirable agronomic qualities is a major objective in breeding programs worldwide. Even though cultural practices and chemical means assist in reducing the spread and severity of the disease, genetic resistance remains the most reliable method of control.

Two types of blackleg resistance, early (seedling) and late (adult plant), referring to the plant growth stage when the resistance to the pathogen is expressed have been identified in Brassica. The lack of adequate seedling resistance in B.napus to blackleg has prompted efforts to transfer this resistance from related species such as B.juncea which are shown to be resistant at both seedling and adult plant stage. The successful transfer of resistance from an Indian accession (BJ168) of B.juncea to B.napus was first reported by Roy (1978). Roy (1984) suggested that the B.juncea blackleg resistance gene(s) came from the B genome since all the species carrying this genome showed the same level of resistance. This results was also confirmed by studies done by Chevre et al. (1997). Even though B genome is acknowledged as a good source of blackleg resistance, so far very little is known about the range of resistance genes available in B.juncea species. Pang and Halloran (1996) described three genes (Bl₁, Bl₂, Bl₃)in F₃ families derived from a cross between a B.juncea like resistance bred by Roy (1984) and a blackleg susceptible B.napus Cultivar. Chevre et al. (1997) using a similar source of B.juncea germplasm to that used by Pang and Halloran (1996) described a single gene for resistance to L. maculans of B.napus-B.juncea lines.

The objective of this study was to identify markers linked to the Bl₁ gene which was identified by Pang and Halloran (1996). This would allow the development of reliable and rapid indirect screening of large segregating populations at a very early stage in the selection process.

MATERIALS AND METHODS:

Plant material: Leaves were selected for DNA extraction from recombinant inbred F₄ lines of B.napus, derived from a cross between “R13” possessing B.juncea like resistance and a highly blackleg susceptible B.napus cultivar “Tower” (Pang and Halloran, 1996). Two lines were selected that were segregation for just one gene (Bl₁). DNA was extracted from the freeze dried leaf samples using the adapted CTAB method (Taylor et al. 1995). Marker identification was performed by bulked segregant analysis from the two lines selected. The bulks were constituted from 10 resistant and 8 susceptible plants and 10 resistant and 5 susceptible plants for the first and second lines respectively.

RAPD analysis: 297 decamer oligonucleotide primers (Operon Technologies Inc., USA) were tested. Reaction volumes of 25μl were optimized to contain 40ng of DNA template, 0.75units of Taq DNA polymerase (Boehringer Mannheim Biochemica, Germany), 0.24mmol each of dATP, dCTP, dGTP and dTTP, 0.4μM of primer and PCR buffer with a final concentration of 0.01M Tris-HCl/3mM MgCl₂/0.05M KCl/0.1mg/ml gelatin, pH 8.3. The PCR amplification and electrophoresis was performed as described by Ford et al. (1997).

AFLP analysis: Genomic DNA was digested with restriction endonucleases EcoR1 and Mse1. After ligation of double stranded adaptors to the ends of the restriction fragments pre-amplification was performed with primers specific for the EcoR1, Mse1 adaptors including one selective nucleotide. This was followed by selective amplification with similar primers with three selective nucleotides. The EcoR1 primer was end labelled with γ³³P ATP. The amplification fragments were separated on 5% denaturing polyacrylamide gels. The gels were dried and autoradiography was carried out with Kodak BioMAX MR X-Ray film for 2-3 days.

RESULTS AND DISCUSSION:

Plants of the two lines selected were used to perform bulked Segregant analysis as described by Michelmore et al. (1991). Among the 297 primers tested none of the primers revealed markers present either in the resistant bulk or in the susceptible bulks. Chevre, et al. (1997) who also used the same breeding material as used in this experiment, noted that 400 primers were necessary to identify 3 markers that is totally linked to the resistance gene. Chevre et al. (1997) also indicated that even if the markers were totally linked to the resistance gene, a marked discrepancy may exist between the genetic and physical distance due to the lack of information available on the size of the introgression.

The lack of polymorphism observed using the RAPD technique lead to the search for a more sensitive technique. AFLP technique is robust and reliable because stringent reaction conditions are used for primer annealing and also it combines the reliability of RFLP technique with the power of PCR technique. Sixteen AFLP primer combinations were screened among the two sets of bulks with 9 producing markers present in the resistant or the susceptible bulks. Two of the primer combinations which produced the polymorphism were tested on the individual plants in the bulks and found to be not tightly linked to the resistance gene. The other possible markers are being tested on the individuals. Once a tightly linked marker is found it will be cloned and sequenced and a more robust screening method established.

ACKNOWLEDGMENTS:

I would like to thank the Grains Research Development Corporation (GRDC) for funding this project.

REFERENCES:

1. Chevre, A.M., Barret, P., Eber, F., Dupuy, P., Brun, H., Tanguy, X., and Renard, M. (1997) Selection of stable Brassica napus-B.juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1. Identification of molecular markers, chromosomal and genomic origin of introgression. Theoretical and Applied Genetics 97:95 (7) 1104-1111.

2. Ford, R., Pang, E.C.K. and Taylor, P.W.J. (1997) Diversity analysis and species identification in Lens using PCR generated markers. Euphytica 96: 247-255.

3. Michelmore, R.W., Paran, I., Kesseli, R.V. (1991) Identification of marker linked to disease-resistance genes by bulk Segregant analysis: a rapid method to detect markers in specific genomic regions by using segregation populations Proceedings of the National Academy of Science USA 88:9828-9832.

4. Pang, E.C.K and Halloran, G.M. (1996) The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B.napus. Theoretical and Applied genetics 92: 382-387.

5. Roy, N.N. (1978) A study on disease variation in the populations of an interspecific cross of Brassica juncea L. X B.napus L. Euphytica 27:145-149.

6. Roy, N.N. (1984) A study on disease variation in the population of an interspecific cross of Brassica juncea L. x B.napus L. Euphytica 27: 145-149.

7. Taylor, P.W.J., Fraser, T.A., Ko, H-L and Henry, R.J. (1995) RAPD analysis of sugarcane during tissue culture. In: M. Terzi, Cella, R. and Falavigan, A. (Eds), Current issues in Plant Molecular and Cellular Biology, pp 241-246. Kluwer Academic Int., Dordrecht, The Netherlands.