S. Roger Rimmer1, M. Hossein Borhan1,2, Bin Zhu1,2

 and Daryl Somers1

1Agriculture and AgriFood Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK Canada, S7N 0X2

2Department of Plant Science, University of Manitoba, Winnipeg, MB Canada R3T 2N2



Black leg, caused by Leptosphaeria maculans, is one of the more important diseases of oilseed brassica crops worldwide.  Control is generally through the cultivation of resistant cultivars.  Plant breeders have used a number of sources of resistance genes for this purpose but the relationship of these genes is not known.  To clarify this situation we have done comparative mapping of resistance to Leptosphaeria maculans (RLM) in populations derived from crosses of various resistance source parents with Brassica napus cv Westar as the common susceptible parent.  Resistant parents were B.  napus cvs. Cresor, Maluka, and RB87-62.  Doubled haploid (DH) mapping populations were derived from single F1 plants (120-150 DH lines per cross).  An isolate of L. maculans (pl86-12) was used to determine the interaction phenotype for black leg on both cotyledons and adult plants for all populations except Cresor H Westar.  At least two RLM for PG2 isolates have been previously mapped on linkage group 6 (LG6) of Brassica napus.  RFLP markers, wg8g3, wg2a11 and tg2b4 from LG6, were linked to a cotyledon resistance gene in Maluka (cRLMm) when mapped in a population of DH lines from a cross of Maluka H Westar.  Bulked segregant analysis was used to identify AFLP markers linked to cRLMm.  Two AFLP markers were mapped to the interval between the RFLP markers and RLM.   In addition, two RAPD markers BL4 and BL9, linked to a gene conferring resistance in Cresor at the adult plant stage (aRLMc) were also linked to cRLMm. Markers linked to cRLMm were also linked to genes associated with resistance at the cotyledon and adult plant stages in B. napus line RB87-62, when tested in a DH population of Westar X RB87-62.  This information indicates the importance of LG6 as a source of resistance genes for breeding programs and genetic studies.


KEYWORD   Black leg, AFLP, RFLP, RAPD, gene cluster




Leptosphaeria maculans (Desm) Ces. & de Not. is the cause of black leg of brassica crops.  Resistance to the pathogen has been identified and introduced into breeding lines (e.g. Brassica napus winter rape cv. Jet Neuf from Europe). Inheritance of resistance has been reported to be monogenic or polygenic based on the cultivar and the age of the plant (reviewed by Rimmer and van den Berg, 1992).   Genomic location of the some of the resistance genes to L. maculans has been determined using molecular markers.  A locus for resistance to black leg in B. napus cv. Major was mapped to linkage group six (LG6) using a population of 105 doubled haploid (DH) lines and L. maculans isolate PHW 1245 (Ferreira et al. 1995).  Dion et al. (1995) mapped adult plant resistance in cv. Cresor.  Mayerhofer et al. (1997) mapped a cotyledon resistance gene in cv. Shiralee, conferring resistance to L. maculans isolates from western Canada.


Despite genetic studies on interactions between L. maculans and Brassica spp., little is known about the genomic organization of black leg resistance genes.  To determine the possibility of linkage among resistance genes to L. maculans (RLM) in B. napus cultivars we have mapped RLM genes in resistant B. napus cvs. Maluka, Cresor and RB 87-62.  Here we report the map location of these genes in DH populations derived from crosses between the susceptible B. napus cv. Westar with resistant lines of cv. Maluka, Cresor and RB87-62.




Plant Materials:

To produce DH mapping populations, the susceptible B. napus cv. Westar was crossed with resistant cvs. Maluka, Cresor and RB87-62. The method of Coventry (1988) was used to produce DH lines from microspores of F1 plants.  Information on the number of DH lines for each cross and pedigree of the parental lines is presented in Table 1.  DH plants were selfed to produce seeds for pathology tests at the cotyledon and adult plant stage.  Conditions for growing plants were as described by Keri et al. (1997).


Table 1.  Mapping populations developed in crosses between the B. napus cv Westar (susceptible) and resistant accessions.


Resistant lines      Pedigree                                                                      No of DH lines


Maluka                      Haya/Zephyr/Bronowski/3/Chisaya/Zephyr/Bronowski           76

Cresor                       Canbra X Cresus*3                                                                  242

RB87-62                   Chikuzen *2/Zephyr X Bronowski                                       107



Pathogen inoculation and evaluation of disease reaction

The interaction phenotype of the plant response to L. maculans was determined using L. maculans isolate pl86-12 (pathogenicity group 2 (PG2)).  Pycnidiospores were used as inoculum. Pycnidial inoculum was prepared according to the methods described in Mengistu et al 1993.   Cotyledons of one week old seedling were inoculated according to the method described by Williams (1985).  Interaction phenotype (IP) of cotyledons were determined after 10 days using a rating scale of 0 to 9 (Williams 1985).  Parental lines were used as controls in each test.  To determine the adult plant IP, stem inoculation was carried out at the early bolting growth stage (GS 3 to 3.2; Harper and Berkenkamp, 1975). A 10 ml droplet of pycnidiospore inoculum was injected into the stem between the second and the third node using a hypodermic needle.  Adult plant IP was based on the percentage of discoloration on a section of the stem 5 mm above the inoculation point and on a scale of 0 to 5 (0 resistant and 5 fully susceptible).  The methodology for evaluation of the adult plant interaction phenotype for 242 Cresor ´ Westar derived DH lines was described by Dion et al. (1995).


DNA extraction and Southern hybridization

Leaves from 6-8 weeks old plants were harvested and freeze dried.  Freeze dried tissue (0.5 g ) was used for DNA extraction according to the method described by Sharp et al. (1995).  DNA was blotted onto N+ nylon membranes according to the instructions of the manufacturer (Amersham).  Hybridization was carried out as described by Sharp et al. (1995).



Restriction fragment length polymorphism (RFLP) markers were provided by TC Osborn, University of Wisconsin, Madison, USA.  Polymorphism was detected by digesting DNA with EcoRI or HindIII. DNA inserts of RFLP probes were amplified by polymerase chain reaction (PCR) as described by Ferreira et al. (1994).  Amplified DNA was radio-labeled with [32P] by random priming (Feinberg and Vogelstein 1983).  Amplified fragment length polymorphism (AFLP) was carried out on EcoRI and MseI digested DNA essentially as described by Vos et al. (1995).  Linked AFLP markers were first identified by bulked segregant analysis (BSA) (Michelmore et al. 1991) then mapped to a population of resistance and susceptible DH lines.  Randomly amplified DNA polymorphism (RAPD) markers were generated with primers obtained from the University of British Columbia, Canada.  The PCR reaction contained 10 ng DNA, 0.5 U Taq DNA polymerase (BRL, Mississauga, Canada), 50 mM KCl, 2.5 mM MgCl2 , 200 μM of each dNTP and 0.2 μM primer.  Amplification of DNA was carried out on a PCT-200 DNA Engine thermocycler (MJ Research, INC. U.S.A).  The cycle parameters were 95 1C -1:30 min (1 cycle); 95 1C- 20s, 36 1C -1 min, 72 1C -1 min (35 cycles); 72 1C B 7 min (1 cycle). PCR products were separated on a 2 % (w/v) agarose gel in 1 X TAE by electrophoresis at 100 V for 3 h. Gels were stained in ethidium bromide and photographed on a digital gel-documentation system.


Linkage analysis

Linkage analysis and mapping of molecular markers were carried out using MAPMAKER/EXP 3.0 (Lander et al. 1987) with the minimum LOD score of 3.0 and a maximum recombination fraction of 0.3.  Interaction phenotype was mapped as a Mendelian trait in each DH population.




Mapping of RLM loci was initiated by identifying molecular markers linked to the cRLM in Maluka (cRLMm).  BSA was used with 30 DH lines to obtain AFLP markers linked to cRLMm.  Of 84 primer combinations tested, 11 primer combinations gave markers linked to the cRLMm.  These were mapped on the 30 individual DH lines and 5 markers were linked to the resistance locus.  Two AFLP markers, which co-segregated with cRLMm, were mapped using 48 additional DH lines and recombinant families were identified.  In order to determine the genomic location of cLMRm, RFLP markers were used.  RFLP markers on LG6 of B. napus map (Ferreira et al 1994) were used since LG6 contained L.  maculans resistance loci (Ferreira et al.1995; Mayerhofer et al. 1997).  Of all the RFLP markers from LG6 mapped on 76 DH lines, three (wg8g3, tg2b4 and wg2a11) were linked to cLMRm (Figure 1).

Figure 1.  Position of resistance genes to Leptosphaeria maculans in cultivars Maluka, RB87-62 and Cresor in linkage group 6 (LG6) of Brassica napus map.  wg8g3, tg2b4 and wg2a11 are RFLP markers developed by Ferreira et al. (1994).  BL4 and BL9 are RAPD markers.  22 and 25 are AFLP markers. Loci named as cRLMm and cRLMrb confer resistance to L. maculans at the cotyledon stage in cvs. Maluka and RB87-62 respectively.  Loci for adult plant resistance in cultivar Cresor and RB87-62 are indicated as aRLMc and aRLMrb respectively.  The figure is not to scale.


The adult plant resistance gene in Cresor (aRLMc) was tagged using RAPD markers. A population of 242 DH lines from a cross of Westar ´ Cresor was used for mapping.  Two RAPD markers (BL4, BL9) flanked the aRLMc locus.  BL9 was closer to aRLMc with 3 recombinant families and  6 recombinant families for BL4.  These RAPD markers when applied to DH lines of Westar ´ Maluka population provided evidence of linkage between cRMLm and aRLMc.  BL4 and BL9 co-segregated with resistance and showed 1 recombinant DH line out of 76 DH lines tested.  The markers linked to aRLMc and cRLMm were then used to map cotyledon and adult plant resistance genes in RB87-62 (cRLMrb, aRLMrb).  RFLP and AFLP markers linked to cRLMm locus were also linked to cRLMrb when mapped on 30 DH lines of Westar ´ RB87-62 cross.  The RAPD markers BL4 and BL9 were mapped on 107 DH lines of Westar ´ RB87-62 cross.  BL4 and BL9 co-segregated but two recombinant families were detected between these markers and cRLMrb. The IP of RB87-62 for adult plant resistance to L. maculans were tested in the green house.  Comparison of these data for 107 DH lines with cotyledon resistance of the same DH lines showed that aRLMrb is linked to cRLMrb and only 7 DH lines showed recombination between these two loci.  The position of resistance genes in cultivars Maluka, Cresor and RB87-62 are shown in Figure 1.




In cvs. Cresor and Maluka, resistance is controlled by a single locus as indicated by segregation ratio of 1 resistance : 1 susceptible in DH lines (for aRLMc: X2: 5.19, P:0.01-0.025; for cRLMm X2: 1.37, P: 0.1-0.25).  For RB87-62 segregation for resistance in F2 individuals indicated that resistance, both at the cotyledon stage and for adult plants, is monogenic.  However segregation distortion occurred in the DH lines of this cross as more than 80 % of the lines were susceptible when tested at the cotyledon or adult plant stage.  Segregation distortion has been reported for Brassica populations developed by microspore culture (Ferreira et al. 1994; Mayerhofer et al. 1997).


The resistance genes in the three accessions of B. napus that we studied are all located in LG6 of the B. napus map developed by Ferreira et al. (1994).  At least two other B. napus resistance genes to L. maculans have been located to LG6.  Ferreira et al (1995) identified a major locus (LEM1) controlling cotyledon resistance to isolate PHW 1245 of L. maculans in B. napus cv. Major.  This gene was located between RFLP marker wg2a3b and tg5d9b on LG6 south of the cRLMm loci mapped in the present study.  A cotyledon resistance gene in B. napus cv. Shiralee (LmR1) to a PG2 isolate of L. maculans also mapped on LG6.  Since the markers used by Mayerhofer et al. (1997) for mapping LmR1 are different from ours, it is not possible to determine the position of resistance loci identified in our study with respect to the LmR1 locus.  However, considering the pedigrees of Maluka (Haya // Zephyr/Bronowski/3/Chisaya // Zephyr/Bronowski) and Shiralee (Haya // Zephyr/Bronowski/3/RC33 // BJ168/Cresus-o-Precose) it is likely that LmR1 and the cotyledon resistance gene in Maluka (cRLMm) are allelic as proposed by Mayerhofer et al. (1997).


It is also probable that cRLMm and cRLMrb loci are allelic as Maluka and RB87-62 share common parents in their breeding pedigrees (Table 1).  Molecular markers used for mapping cRLMm and cRLMrb showed the same polymorphic patterns in both populations indicating a high level of similarity for this part of the genome between Maluka and RB87-62.  However, we consider the two cotyledon resistance alleles in Maluka and RB87-62 to be linked but not allelic (Figure 1).  This is due to the position of AFLP markers which flank cRLMm but are located on one side of the cRLMrb locus.  To resolve the map positions of cRLMm and cRLMrb we are continuing mapping of markers linked to these loci with a larger population.  The adult plant resistance gene in cultivar RB87-62 (aRLMrb) is linked to the cotyledon resistance locus in this cultivar (cRLMrb) but not allelic as 7 recombinant DH families were detected between these two loci.


The adult plant resistance gene in cultivar Cresor (aRLMc) is linked to cRLMm and cRLMrb but not allelic to these loci.  This is evident from the position of two RAPD markers BL4 and BL9, which flank aRLMc but map to one side of cRLMm and cRLMrb (Fig 1).  Except for RFLP marker wg2a11, most of the markers linked to RLM genes in LG6 are located south of RLM genes (Figure 1).  RFLP probe wg2a11 is distantly (~ 24 cM) linked to the RLM genes.  Difficulty with identifying markers from this interval of LG6 has also been encountered by Mayerhofer et al. (1997) for mapping LmR1.  They proposed that this might be because LmR1 is located toward the end of LG6.




This work was supported in part by grants to SRR from the Western Grains Research Foundation, Agriculture and Agri-Food Canada and the Natural Sciences and Engineering Research Council of Canada.  The technical support of Ms P. Parks is gratefully acknowledged.




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