ABSTRACT

Offspring from symmetric and asymmetric hybrids between Brassica napus and Arabidopsis thaliana have been assayed for blackleg resistance. It was found that adult leaf resistance genes were localised on chromosome 3 of A. thaliana and cotyledon resistance was most likely on chromosome 1. Twelve EMS mutants and three T-DNA mutants susceptible to blackleg, have been identified in A. thaliana and are now used for further studies with the aim to clone genes involved in the plant defence to this fungal pathogen .

INTRODUCTION

Leptosphaeria maculans (Desm.) Ces & de Not (anamorph: Phoma lingam (Tode ex Fr.)Desm is the causal agent of the blackleg disease in several Brassica species, resulting in substantial yield loss worldwide (Salisbury et al. 1995). The Brassica B genome species B. carinata, B. nigra and B. juncea exhibit high level of resistance to blackleg (Roy 1978; Sacristán and Gerdemann 1986; Sjödin and Glimelius 1988). By sexual crossings (Roy 1978; 1984; Jahier et al. 1989; Struss et al. 1996) in combination with embryo rescue (Sacristán and Gerdemann 1986) and by somatic hybridisation (Sjödin and Glimelius 1989a; 1989 b), blackleg resistance has been subsequently transferred to the B. napus genome from these related Brassica species. However, little is known about the mechanisms that make plants resistant to L. maculans. In order to characterise key disease resistance components, this plant-pathogen system has been established in Arabidopsis thaliana. This approach became possible when it was shown that A. thaliana possessed resistance to blackleg (Bohman and Dixelius 1997). Various A. thaliana mutants and B. napus lines with introgressed A. thaliana DNA are now under investigation.

RESULTS

B. napus and A. thaliana hybrids

Since A. thaliana and B. napus are more or less sexually incompatible, somatic hybridisation has been used to transfer DNA from A. thaliana to B. napus (Bohman et al. 1999). Both symmetric hybrids, where the complete genomic set of both parents have been fused (Forsberg et al. 1994) and asymmetric hybrids where the A. thaliana genome were fragmentised before fusion to the B. napus genome (Forsberg et al. 1998) were produced. In the symmetric hybrid material, addition lines were found after backcrossing and selfing which consisted of the B. napus genome and extra A. thaliana chromosome pairs. Determination of A. thaliana was performed by utilisation of RFLP markers mapped on its genome (Liu et al. 1996). When this plant material was assayed for blackleg resistance it became clear that chromosome 3 of A. thaliana carried adult leaf resistance genes and chromosome 1 most likely possessed cotyledon resistance. Since no recombination between the chromosomes of the two species were obtained the asymmetric hybrid material carrying pieces of A. thaliana in a oilseed rape background were further studied. In backcrossed lines of the asymmetric hybrids the adult leaf resistance character has been localised to two regions on chromosome 3 of A. thaliana, on each side of the centromere. This material is still rather unstable and larger populations are being screened by PCR and or CAPS markers to obtain further information about the position of the genes.

A. thaliana mutants

We have now identified tvelve EMS mutants in a Landsberg erecta background and three T-DNA mutants with single inserts in the Wassilewskija ecotype that are used in ongoing research. One of the EMS mutants was chosen for a map based cloning approach and crossed to the resistant ecotype Colombia. From the F2 and F3 mapping populations results indicate a strong linkage of the mutation to an area of chromosome 2. A more detailed mapping is now being carried out on this material. One of the three T-DNA mutants with single inserts was also chosen for sequence analysis. Both an IPCR and a plasmid rescue approach have been adopted to pick up flanking plant DNA and this work is now progressing at full pace.

CONCLUSION

In this study, functional genes towards L. maculans have been found within the A. thaliana genome.

The completion of the Arabidopsis genome sequencing project in the near future will mean that oilseed rape breeding will benefit tremendously from this research, due to the genome similarities. Molecular tools created from work such as that presented here will provide us with a deeper understanding of the various mechanisms and processes in the two plants.

ACKNOWLEDGEMENT

This work was supported by grants from the Swedish University of Agricultural Sciences, the Swedish Foundation of Strategic Research and the Swedish Council for Forestry and Agricultural Reseach.

REFERENCES

1. Bohman S and Dixelius C.1997. Positioning and identification of resistance locus towards Phoma lingam (Tode ex Fr) Desm in Arabidopsis thaliana. 5^th Int Congr. of Plant Molecular Biology, 21-27 Sept. Singapore, p 523

2. Bohman S, Forsberg J, Glimelius K and Dixelius C. 1999. Inheritance of Arabidopsis DNA in offspring from Brassica napus and Arabidopsis thaliana hybrids. Theor. Appl. Genet. 98:99-106

3. Forsberg J, Landgren M and Glimelius K. 1994. Fertile somatic hybrids between Brassica napus and Arabidopsis thaliana. Plant Science 95:213-223

4. Forsberg J, Dixelius C, Lagercrantz U, Glimelius K. 1998 UV dose-dependent DNA elimination in asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana. Plant Science 131:65-76

5. Jahier J, Chèvre AM, Tanguy AM and Eber F. 1989. Extraction of disomic addition lines of Brassica napus-Brassica nigra. Genome 32:408-413

6. Liu Y-G, Mitsukawa N, Lister C, Dean C and Whitter RF. 1996. Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines. Plant J 10:733-736

7. Roy NN. 1978. A study of disease variation in the population of an interspecific crosss of Brassica juncea L x B. napus. Euphytica 27: 145-149

8. Roy NN. 1984. Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus. Euphytica 33: 295-303

9. Sacristán MD and Germdemann M. 1986. Different behaviour of Brassica juncea and B. carinata as sources of Phoma lingam resistance in experiments of interspecific transfer to B. napus. Plant Breeding 97:304-314

10. Salisbury PA, Ballinger DJ, Wratten N, Plummer K and Howlett BJ. 1995. Blackleg disease on oilseed Brassica in Australia: a review. Aust. J. Exper. Agric. 35:665-672

11. Sjödin C, Glimelius K (1988) Screening for resistance to blackleg Phoma lingam (Tode ex Fr.) Desm. within Brassicaceae. J Phytopath 123: 322-332

12. Sjödin C, Glimelius K (1989a) Transfer of resistance against P. lingam to Brassica napus L. by asymmetric somatic hybridisation and toxin selection. Theor Appl Genet 78: 513-520

13. Sjödin C, Glimelius K (1989b) Brassica naponigra, a somatic hybrid resistant to Phoma lingam. Theor. Appl. Genet. 77: 651-656

14. Struss D, Quiros CF, Plieske J, Röbbelen G (1996) Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers. Theor. Appl. Genet. 93: 1026-1032