A SINGLE DOMINANT ALLELE FOR RESISTANCE TO BLACKLEG IN SEEDLINGS OF BRASSICA NAPUS CV. SURPASS4. Caixia Li 1, Wallace Cowling 1,2, Michael Francki 1,2 and Andrew Easton 3 1 Plant Sciences, Faculty of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley WA 69; 2 Canola Breeders Western Australia Pty Ltd., 15/219 Canning Highway, South Perth, WA 6151; and 3 Pacific Seeds Pty. Ltd., 268 Anzac Avenue, Toowoomba QLD 435 ABSTRACT The inheritance of resistance to blackleg (caused by Leptosphaeria maculans) was examined in crosses between highly resistant (R) Brassica napus cv. Surpass4 and highly susceptible (S) cv. Westar, and between Surpass4 and moderately resistant cv. Mystic, with Surpass4 as the male parent. Each genetic population consisted of selfed mother, a bulk of Surpass4, and F 1 and F 2 derived from four individual mother. Seeds were germinated in jiffy pots and seedlings were transplanted 2 weeks later to a screenhouse blackleg disease nursery. Blackleginfected canola stubble was distributed between rows 2 weeks after transplanting. Disease severity was assessed on seedlings 5 weeks after transplanting at the 3-4 leaf stage by counting the number of lesions per leaf on leaves 1 and 2. Lesions were present on all S mother and approximately ¼ of each F 2 population. In contrast, there were no lesions on leaves of the R parent, on F 1, or on approximately ¾ of the F 2 in each population. Some S F 2 had small defined lesions without pycnidia, whereas most Westar and Mystic had large lesions with pycnidia. The S F 2 also had lower average disease severity and greater variance relative to the mean compared with S mother. This suggests that minor resistance genes are also segregating in these crosses. Segregation in the F 2 showed a good fit to the expected 3:1 ratio for a dominant resistance allele at a single locus in Westar/Surpass4 ( 2 = 1.65, df = 1, P =.199) and Mystic/Surpass4 ( 2 =.59, df = 1, P =.44). Surpass4 is the source of a dominant major allele for blackleg resistance at the seedling stage in B. napus. KEYWORDS Leptosphaeria maculans, blackleg disease nursery INTRODUCTION Resistance to blackleg in Brassica napus L., caused by Leptosphaeria maculans (Desm.) Ces. & de Not., has been reported to be under major gene and polygenic control. A single major locus for resistance (LEM1) was reported on linkage group 6 of biennial cv. Major based on wound inoculation of seedlings (Ferreira et al., 1995). Field resistance in adult was identified in spring cv. Crésor and a major gene was designated as LmFr 1 based on QTL mapping (Dion et al., 1995). Quantitative variation in field resistance to blackleg was present in doubled haploid progeny derived from the cross Darmor-bzh x Yudal (Pilet et al., 1998). Major gene resistance was not clearly identified in spring types of B. napus from Australia (Pang & Halloran, 1996a) and appeared to be controlled by quantitative genetic factors in B. napus cv. Maluka (Pang & Halloran, 1996b) in wound-inoculated. This contrasts with a report from Canada that Australian cvv. Maluka and Shiralee have a single major locus controlling resistance, when were wound-inoculated at the seedling stage (Mayerhofer et al., 1997).
Under field conditions in Australia, Canadian or European spring cultivars are normally susceptible to blackleg and Australian B. napus varieties vary from moderately susceptible to moderately resistant. Recently, a cultivar was released in Australia that has achieved the maximum rating of 9 (highly resistant) (Canola Association of Australia, 21). This is Surpass4, which forms the basis of this study of the genetics of blackleg resistance under field conditions in Australia. MATERIALS AND METHODS Glasshouses and disease nurseries were located out at The University of Western Australia Field Station at Shenton Park, Western Australia. Crosses were made between blackleg-resistant (R) Brassica napus cv. Surpass4 and highly susceptible (S) cv. Westar (cross N154), and between Surpass4 and moderately resistant cv. Mystic (cross N153), using Surpass4 as the male parent. Individual mother of Westar and Mystic were identified by the letters A, B, C, etc. Selfed and F 1 seed was harvested from four mother for inclusion in this experiment. One F 1 seedling from each mother plant was grown for production of F 2 seeds. The remaining selfed mother plant and F 1 seed was placed in cold storage until May 21, when seed from a bulk of Surpass4, selfed mother, F 1, and F 2 were germinated in jiffy pots and seedlings were grown in a glasshouse for 2 weeks. Seedlings were then transplanted to a screenhouse in a fully randomized design with 15 cm apart in rows 85 cm apart, with crosses in separate blocks. Blackleg-infected canola stubble was distributed between rows 2 weeks after transplanting. Disease severity was recorded 3 weeks later at the 3-4 leaf stage by counting the number of lesions per leaf on leaves 1 and 2 and calculating the average. At this time, lesions on susceptible varieties contained pycnidia of L. maculans. Plants were allocated to a category of resistance (R and S) on the basis of disease severity. Lesions were normally not present on Surpass4 or F 1. Disease severity on progeny of selfed mother followed a normal distribution, and the variation between mother was assessed by single factor analysis of variance. In each cross, there was a discontinuity between disease severity on Surpass4 and the susceptible parent. For the purposes of this experiment, Mystic is considered S although it is moderately resistant under Australian conditions (Canola Association of Australia, 21). Plants were classified R if disease severity was less than half the lowest recorded severity on the S parent, and S if severity was greater than this value. For cross N154, R had 2 and S >2 lesions per leaf, and for cross N153, R had 1.5 and S 1.5 lesions per leaf. The ratio of R:S in the F 2 from each mother plant and pooled R:S data for each cross were tested for goodness-of-fit to a hypothesis for a single dominant allele. Heterogeneity between F 2 progeny from different mother was evaluated using a contingency test. RESULTS There was a clear separation of disease severity between the R and S parents of crosses N153 and N154 (Fig. 1A, 1B). Leaf lesions were present on all S parent and approximately ¼ of each F 2 population (Fig. 1C, 1D). In contrast, there were no leaf lesions on the R parent, on F 1, or on approximately ¾ of the F 2 in each population. Disease severity on S parents was approximately normally distributed (Fig. 1A, 1B). Westar appeared to have more lesions per leaf on average than Mystic (Fig. 1A, 1B; Table 1), and there was no effect of mother plant source on disease levels in selfed progeny (Table 1). Disease severity on the S F 2 progeny of each cross was lower on average than on the S parent, and the error variance of S F 2 progeny was higher relative to the mean than the S parent (Table 1). Some S F 2 had small lesions with definite borders and no pycnidia, especially in cross N153 Table 1.
Comparison of disease severity (average number of lesions on leaves 1 and 2) on susceptible parent and susceptible F 2 progeny from cross N154 (Westar/Surpass4) and cross N153 (Mystic/Surpass4). Mother Plant Cross N154 (Westar/Surpass4) Selfed mother Susceptible F 2 Mother Plant Cross N153 (Mystic/Surpass4) Selfed mother Susceptible F 2 WestarA 12.4 11. MysticA 9. 3.4 WestarC 15.5 1.4 MysticB 8.2 4.3 WestarE 13.9 1.5 MysticC 5.4 4.1 WestarH 11.8 11.5 MysticD 9.1 4.8 Mean s.d. 13.4 4.1 1.9 5.5 Mean s.d. 8.1 4. 4.3 3.8 MS(error) 15.8 31.6 MS(error) 21.3 15.5 F-test (mother ) F {3,36} = 1.758 F {3,49} =.95 F-test (mother ) F {3,34} = 1.371 F {3,48} =.223 Table 2. Resistance of parents and progeny of cross N154 (Westar/Surpass4) Selfed mother Surpass4 bulk F 1 F 2 Mother Plant R 1 S 1 R S R S R S Total 2 (3:1) df P WestarA 1 9 41 21 62 2.62 1.17 ns WestarC 1 13 57 7 64 6.75 1.9 ** WestarE 1 15 46 14 6.89 1.766 ns WestarH 1 9 5 11 61 1.579 1.29 ns Pooled 4 13 46 194 53 247 1.653 1.199 ns Heterogeneity 1.42 3.15 * 1 Resistant (R) have 2 and susceptible (S) >2 lesions per leaf (average of leaves 1 and 2), as described in Materials and Methods. Table 3. Resistance of parents and progeny of cross N153 (Mystic/Surpass4) Selfed mother Surpass4 bulk F 1 F 2 Mother Plant R 1 S 1 R S R S R S Total 2 (3:1) df P MysticA 9 2 36 7 43 1.744 1.187 ns MysticB 1 7 49 13 62.538 1.463 ns MysticC 1 7 48 14 62.194 1.66 ns MysticD 9 3 43 18 61.661 1.416 ns Pooled 38 14 37 176 52 228.585 1.444 ns Heterogeneity 2.72 3.437 ns 1 Resistance (R) is defined as 1.5 and susceptibility (S) is defined as 1.5 lesions per leaf averaged across leaves 1 and 2, as described in Materials and Methods.
A (N154 parents) 16 14 12 1 8 6 4 2 2 4 6 B (N153 parents) 8 1 12 14 16 18 2 22 24 16 14 12 1 8 6 4 2 2 4 C (N154 F 2 ) 6 8 1 12 14 16 18 2 22 24 5 4 3 2 1 2 4 D (N153 F 2 ) 6 8 1 12 14 16 18 2 22 24 5 4 3 2 1 2 4 6 8 1 12 14 16 18 2 22 24 Average No.Lesions/Leaf Figure 1. Disease severity on parents (A, B) and F 2 progeny (C, D) of crosses N154 (Westar/Surpass4) and N153 (Mystic/Surpass4). In Figs. 1A and 1B, Surpass4 bars are solid black, and Westar and Mystic bars are grey. Actual values for Surpass4 in the -1 category are 177 (C) and 176 (D).
(Mystic/Surpass4), whereas most Westar and Mystic had large lesions with pycnidia. This indicated possible segregation for minor resistance genes in these crosses. Segregation in the F 2 showed a good fit to the expected 3:1 (R:S) for a major dominant allele for resistance at a single locus (Tables 2 and 3). Most families conformed to this hypothesis, except F 2 progeny of mother plant WestarC which failed to segregate 3:1 due to an excess of resistant types. This caused significant heterogeneity within cross N154 (Table 2), but did not affect the 3:1 hypothesis based on pooled data. DISCUSSION The results indicate that Surpass4 is the source of a major dominant allele for blackleg resistance at the seedling stage in B. napus, and this allele masks the effect of other minor genes for resistance segregating in these crosses. While Mystic is normally considered moderately resistant to blackleg, it clearly lacks the allele present in Surpass4 that prevents lesion formation on seedlings. Mystic appears to be more resistant than Westar based on seedling disease severity, which correlates with mature plant resistance. Later in 21, we will measure lesion size and other traits that may further distinguish Mystic and Westar. Surpass4 has the highest resistance rating of all cultivars in Australia (Canola Association of Australia, 21), hence we are confident that the resistance we have measured at the seedling stage will be strongly correlated with resistance later in the growing season. This will be confirmed in this disease nursery in 21. These data confirm previous unpublished results of one of us (AE), from field experiments in Australia, that resistance to blackleg in Surpass4 is controlled by a single dominant allele. The F 2 of both crosses showed wide variation in disease severity and lesion phenotype. Some S F 2, especially in cross N153 (Mystic/Surpass4), had small lesions with defined borders and no signs of pycnidia. This differed from Westar, which had large lesions and abundant pycnidia. Susceptible F 2 also had fewer lesions on average than the S parent. Both Surpass4 and the S parents may have contributed to polygenic variation for resistance in the F 2, which was visible in S progeny but masked in R progeny by the major allele for resistance. The ancestry of Surpass4 includes synthetic B. napus derived from crosses between wild B. rapa ssp. sylvestris and B. oleracea ssp. alboglabra. Some accessions of B. rapa ssp. sylvestris were found to be resistant to blackleg, and this resistance was transferred by interspecific and backcrossing to B. napus where it held up under field conditions in the UK and Australia (Crouch, 1994; Crouch et al., 1994). This major allele for blackleg resistance in Surpass4 is inherited through the nuclear genome. We base this conclusion on the fact that it was derived most likely from wild B. rapa by a complex process of interspecific crossing and backcrossing to B. napus. Also, mother in our crosses were susceptible, and F 1 were always highly resistant. Resistance was therefore transferred through the pollen and was not cytoplasmically inherited. ACKNOWLEDGEMENTS One of us (CL) was awarded an International Postgraduate Research Scholarship at the University of Western Australia to undertake this research. The Export Grains Centre Ltd. contributed part salary (WAC and MF) and operating support. We thank Milton Sanders for high quality technical input to the project.
REFERENCES Canola Association of Australia, 21. CAA 21 blackleg resistance ratings. Crouch, J.H., 1994. Resistance to Leptosphaeria maculans (Desm.) Ces. & de Not. in Brassica L. PhD Thesis. University of East Anglia, Norwich, UK. Crouch, J.H., B.G. Lewis, & R.F. Mithen, 1994. The effect of A genome substitution on the resistance of Brassica napus to infection by Leptosphaeria maculans. Plant Breeding 112:265-278. Dion, Y., Gugel, R.K., Rakow, G.F.W., Séguin-Swartz, G., & Landry, B.S., 1995. RFLP mapping of resistance to the blackleg disease [causal agent, Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.). Theoretical and Applied Genetics 91:119-1194. Ferreira, M.E., S.R. Rimmer, P.H. Williams, & T.C. Osborn, 1995. Mapping loci controlling Brassica napus resistance to Leptosphaeria maculans under different screening conditions. Phytopathology 85:213-217. Mayerhofer, R., V.K. Bansal, M.R. Thiagarajah, G.R. Stringam, & A.G. Good, 1997. Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus. Genome 4:294-31. Pang, E.C.K. & Halloran, G.M., 1996a. The genetics of blackleg [Leptosphaeria maculans (Desm.) Ces. et de Not.] resistance in rapeseed (Brassica napus L.). 1. Adult-plant resistance in F 2 and first-backcross populations. Theoretical and Applied Genetics 93:932-94. Pang, E.C.K. & Halloran, G.M., 1996b. The genetics of blackleg [Leptosphaeria maculans (Desm.) Ces. et de Not.] resistance in rapeseed (Brassica napus L.). 2. Seedling and adult-plant resistance as quantitative traits. Theoretical and Applied Genetics 93:941-949. Pilet, M.L., R. Delourme, N. Foisset, & M. Renard, 1998. Identification of loci contributing to quantitative field resistance to blackleg disease, causal agent Leptosphaeria maculans (Desm.) Ces. et de Not., in winter rapeseed (Brassica napus L.). Theoretical and Applied Genetics 96:23-3.