THE DEVELOPMENT AND GENETICS OF WHITE RUST

(ALBUGO CANDIDA) RACE 2V-RESISTANT CANOLA QUALITY BRASSICA JUNCEA

 

Coreen Franke, Derek A. Potts and Daryl R. Males

 

Saskatchewan Wheat Pool, Research and Development, 201-407 Downey Road, Saskatoon, Saskatchewan, S7N 4L8, CANADA.  E-mail:  coreen.franke@swp.com, derek.potts@swp.com, daryl.males@swp.com

 

 

ABSTRACT

 

Canadian Brassica napus canola varieties are immune to Albugo candida race 2, an obligate parasite which causes white rust and staghead in B. juncea.  Cultivars of B. juncea which were previously resistant to white rust race 2 are now susceptible to a more virulent race of the fungus (race 2V), a race which has become prevalent across the Canadian prairies. Qualitative, hypersensitive response type resistance to A. candida race 2V has been developed in canola quality B. juncea by interspecific introgression of a resistance gene from B. napus. Resistant selections from an interspecific (canola quality B. juncea X B. napus) F1-derived doubled haploid (DH) population were backcrossed to canola quality B. juncea, and BC1F1 DH populations were created. Resistant selections from BC1F1-derived DH populations were subsequently backcrossed to canola quality B. juncea, and BC2F1-derived DH populations were created.  These populations were screened with A. candida race 2V to determine introgression of the white rust resistance gene.  Although difficulty in obtaining cytogenetically stable populations was experienced (likely due to aneuploidy and irregular meiosis), one population was found to have undergone an introgression event with respect to the white rust resistance trait.  Screening of F2 lines confirmed that resistance was due to a single dominant gene that segregated in Mendelian fashion.  PCR-based molecular markers linked to the white rust resistance trait are being identified via bulked segregant analysis.

 

 

KEYWORDS - white rust resistance, Brassica napus, B. juncea, interspecific introgression

 

 

INTRODUCTION

 

Brassica juncea (L.) Czern. and Coss., a major oilseed Brassica grown worldwide, is well adapted to drier climates and has considerable potential in western Canada (Woods et al. 1991).  Low erucic acid, low glucosinolate (canola-quality) B. juncea was first developed in the early 1990’s (Love et al. 1991) and has since been developed into a commercial variety by Saskatchewan Wheat Pool in collaboration with Agriculture and Agri-Food Canada.  This new canola oil-producing crop can be grown in areas where current canola varieties traditionally do not thrive because it is more heat and drought tolerant than other canola Brassicas.  White rust and staghead, caused by the fungal pathogen Albugo candida (Pers.) Kunze, is one of the most important diseases of B. juncea in Canada and in other parts of the world, and can have a significant impact on seed production (Subudhi and Raut, 1994; Verma and Bhowmik, 1989).  In western Canada, there has been a shift from the predominant biotype of A. candida that affects B. juncea, race 2, to a more virulent biotype of the fungus, race 2V (Rimmer, unpublished data).  B. juncea varieties previously resistant to race 2 of A. candida are susceptible to 2V, including newly developed canola-quality B. juncea.  In contrast, Canadian B. napus varieties have been shown to be immune to white rust 2V infection (Liu et al. 1992).

 

The objective of this research was to introgress white rust resistance from B. napus (genomic formula AACC, 2n=38) into canola-quality B. juncea (AABB, 2n=36) by an interspecific cross and subsequent backcrosses to B. juncea. Verma and Bhowmik (1989) reported that white rust race 2 resistance in B. napus was controlled by dominant genes at two loci, and that the presence of only one allele for resistance was sufficient to condition a hypersensitive response to infection with the pathogen.  White rust race 2-resistance genes are believed to be on the C genome (Subudhi and Raut, 1994). Because the Brassica genome is very plastic, able to exchange chromosomal segments by non-homeologous recombination (Quiros et al. 1994), it is reasonable to expect that transfer of resistance through an interspecific cross is possible.  Evidence of introgression events from B into C genomes (i.e. B. juncea gene(s) transferred to B. napus) has been reported.  Prakash and Chopra (1988) achieved successful interspecific introgression of shattering tolerance from B. juncea into B. napus, and Chevre et al. (1997) selected stable B. napus-B. juncea recombinant lines with complete resistance to blackleg.

 

 

MATERIALS AND METHODS

 

Breeding Procedure.  An interspecific cross was made between a homozygous canola quality (CQ) B. juncea line (J90-4316, A. candida race 2-resistant, race 2V-susceptible) used as the female parent, and a homozygous low linolenic acid B. napus cultivar (Apollo, A. candida race 2 and 2V-resistant) used as the male parent (Figure 1).  An F1-derived doubled haploid (DH) population was created and screened with A. candida race 2V.  Homozygous resistant selections, as characterized by hypersensitive response, were backcrossed to canola quality DH B. juncea lines chosen for good agronomic performance. A BC1F1-derived DH population was screened with white rust 2V, and resistant selections were subsequently backcrossed to canola quality B. juncea to create BC2F1.  Two segregating populations from each of six BC2F1s were created to test introgression of white rust resistance (R) and stability of inheritance: BC2F1-derived DH populations were expected to

 

 

CQ B. juncea (2V susceptible)             X           B. napus (2V resistant)

                                                         AABB                                                                AACC

               

 

                F1                                                                                         AABC

 


                                                                                F1-derived DH 2V-resistant selections

 


                BC1                                                                                                         X             CQ B. juncea (2V susceptible)

 

                                                                                                                BC1F1

 


                                                                                BC1F1-derived DH 2V-resistant selections

 


                BC2                                                                                                         X             CQ B. juncea (2V susceptible)

                                                                                               

                                                                                                                BC2F1

 


                                BC2F1-derived DH segregating populations                   BC2F2 segregating populations

 


Figure 1.  Development of white rust race 2V-resistant CQ (canola quality) B. juncea.

segregate 1R:1S, and BC2F2 populations were expected to segregate 3R:1S in accordance with a one-gene hypothesis.  It was assumed that white rust resistance introgressed into B. juncea would behave as it does in B. napus, with the presence of only one of the two R alleles necessary to condition resistance.  It was also assumed that the likelihood of both genes being transferred together was very small, and therefore a one-gene hypothesis was tested.  A total of eighteen BC2F1-derived DH populations and six BC2F2 populations (those with sufficient seed) were screened with A. candida race 2V to determine introgression of the resistance trait.

 

Disease evaluation.  Seeds of  DH lines were sown in soiless mix in 4-inch plastic pots and placed in an environmentally controlled growth chamber with day/night temperatures of 21/18°C, and a 16 hour photoperiod.  Two replicates consisting of five plants/rep (for a total of ten plants per DH line) were screened in a completely randomized design.  Parents of each cross along with appropriate checks (B. juncea cv Cutlass, race 2 resistant, 2V susceptible; B. napus cv Apollo, race 2 and 2V resistant) were included in each disease evaluation. Nine days after sowing, plants were tested for white rust resistance by inoculating seedlings (misting until runoff) with a suspension of 105 germinating zoospores per ml, and incubating in a mist chamber for 24 to 36 h.  Plants were scored as resistant (R) or susceptible (S) seven days after inoculation.  Resistance was defined as complete absence of pustules, and susceptibility as the presence of even a single small pustule on the leaves.  Samples of the BC2F2 populations (53 to 179 plants depending on seed availability and germination) were evaluated in the same manner.

 

Statistical analysis.  Chi-square analyses to test goodness-of-fit were applied to analyze data from segregating BC2F1-derived DH and BC2F2 populations at a=0.05 level of significance.

 

 

RESULTS AND DISCUSSION

 

BC2F1-derived DHs had very small population numbers.  Nine of eighteen of the DH populations created had <20 lines per population, and of the remaining, only four had populations of >50 DH lines.  Segregation ratios for all but two of the DH populations did not fit (P<0.05) the ratio expected for segregation of a single dominant (introgressed) gene.  Of the two that did have a good fit (P>0.05) to the 1R:1S ratio, one (XJ96-099C) had a population size of only 14 lines, and white rust screening of the BC2F2 revealed a poor fit (P<0.05) to the expected 3R:1S ratio.  However, the observed segregation ratios of resistant to susceptible phenotypes for the DH and F2 populations for XJ96-098A were not significantly different than 1:1 and 3:1 respectively at P=0.05 (Table 1).  This suggests that an introgression event had occurred and that the introgressed gene was segregating in Mendelian fashion.  DH lines derived from XJ96-098A resembled the B. juncea parent phenotypically in all aspects except white rust susceptibility, with some small differences in leaf morphology (Figure 2).   For other crosses, low DH population numbers, segregation within some

 

 


Table 1.  Chi-square test of a one-gene hypothesis for segregation of introgressed white rust resistance based on absence (R) or presence (S) of pustules in BC2F1-derived DH and BC2F2 populations.

 

BC2F1

 

Segregating

Population

 

Observed

R         S

 

Ratio

Tested

 

c2

 

Segregating

Population

 

Observed

R         S

 

Ratio

Tested

 

c2

XJ96-097A

BC2F1-DH

  13      41

1:1

13.50**

 

 

 

 

XJ96-097C

BC2F1-DH

   0       57

1:1

  N/A

 

 

 

 

XJ96-098A

BC2F1-DH

  37      54

1:1

 2.16

BC2F2

   126    43

3:1

  0.02

XJ96-099C

BC2F1-DH

   5         9

1:1

 0.64

BC2F2

     26    27

3:1

18.58**

XJ96-100B

BC2F1-DH

   4       44

1:1

28.60**

 

 

 

 

 ** Sig at a=0.01

DH lines (data not presented), and failure to fit expected ratios for one-gene introgression in BC2F1-

derived DH and BC2F2 populations indicated that there was cytogenetic instability.

 

In general, the interspecific hybrid of a B. juncea (AABB)  X  B. napus (AACC) cross is amphidiploid with one genome derived from the seed parent and one from the pollen parent, resulting in an F1 with the genomic formula AABC.  Usually segregation in the progeny of interspecific crosses, when either selfed or backcrossed, does not fit a Mendelian pattern because meiosis rarely functions normally.   The pairing of B genome chromosomes with those of C is relatively infrequent due to only partial homology (Attia and Robbelen, 1986), and therefore true exchange of genetic material between B and C chromosomes is rare.  Roy (1984) reported the transfer of complete blackleg resistance from B. juncea to B. napus, and although F1s were all resistant, most advanced F7 lines were found to still be segregating for the juncea-type resistance, indicating aneuploidy.  In other attempts to transfer blackleg resistance from B. juncea to B. napus, a breakdown of resistance was also observed, either due to loss of introgression (Salisbury et al, 1995) or to the production of aneuploid lines (Rimmer and van den Berg, 1992). 

 

It is clear from the successful B. juncea-B. napus recombinants selected by Prakash and Chopra (1988) and by Chevre et al (1997), that cytogenetic analysis combined with doubled-haploid technology is very useful in ensuring the recovery of euploid genotypes showing regular meiotic behavior.  In our study, meiotic analysis in the BC1F1-DH white rust 2V-resistant parent, and BC2F1(XJ96-098A)-derived DH lines is underway to determine cytogenetic stability.  We expect that a configuration of 18 bivalents (chromosome pairs) in the pollen mother cells of these lines will verify that white rust 2V resistance is due to introgression of a gene from B. napus.  Also, F3 families from XJ96-098A will be screened with A. candida race 2V to further confirm stability of inheritance of the trait.

 

Figure 2.  Reactions to white rust race 2V infection: A) pustules on susceptible B. juncea leaves; B) hypersensitive reaction in canola quality B. juncea BC2F1-derived DH lines; C) hypersensitive reaction in B. napus.

 

 

 

In Canada, resistance of B. napus cultivars to A. candida appears to be durable in that it has remained effective for over 50 years of exposure to isolates of the pathogen which attack B. juncea and B. rapa (Liu et al. 1992).  Through interspecific hybridization, we have introgressed this valuable resistance from B. napus into canola quality B. juncea.   Material developed from this study will be incorporated into an ongoing breeding program for the improvement of canola quality B. juncea.  PCR-based molecular markers linked to the white rust resistance trait are being sought via bulked-segregant analysis of the BC2F1(XJ96-098A)-derived DH segregating population, and should prove to be a useful tool for marker-assisted selection. 


REFERENCES

 

Attia, T. and G. Robbelen.  1986.  Cytogenetic relationship within cultivated Brassica analyzed in amphihaploids from the three diploid ancestors.  Can. J. Genet. Cytol. 28:323-329.

 

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 the introgression.  Theor. Appl. Genet.  95:1104-1111.

 

Liu, J. Q., Parks, P. and Rimmer, S. R.  1992.  Development of monogenic lines for resistance to Albugo candida from a Canadian Brassica napus cultivar.  Phytopathology 86:1000-1004.

 

Love, H. K., Rakow, G., Raney J. P. and Downey, R. K.  1991.  Breeding improvements towards canola quality Brassica juncea.  Proceedings 8th International Rapeseed Congress, Saskatoon, Canada, pp. 164-169.

 

Prakash, S. and Chopra, V. L.  1988.  Introgression of resistance to shattering in Brassica napus from Brassica juncea through non-homologous recombination.  Plant Breeding 101:167-168.

 

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Rimmer, S. R. and van den Berg, C. G. J.  1992.  Resistance of oilseed Brassica spp. to blackleg caused by Leptosphaeria maculans.  Can. J. Plant Pathol. 14:56-66.

 

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

 

Salisbury, P. A., Ballinger, D. J., Wratten, N., Plummer, K. M. and Howlett, B. J.  1995.  Blackleg disease on oilseed Brassica in Australia: a review.  Aust. J. Exp. Agric. 35:665-672.

 

Subudhi, P. K. and Raut, R. N.  1994.  White rust resistance and its association with parental species type and leaf waxiness in Brassica juncea L. Czern. & Coss. X Brassica napus L. crosses under the action of EDTA and gamma-ray.  Euphytica 74:1-7.

 

Verma, U. and Bhowmik, T. P.  1989.  Inheritance of resistance to a Brassica juncea pathotype of Albugo candida in B. napus.  Can. J. Plant Pathol. 11:443-444.

 

Woods, D. L., Capcara, J. J. and Downey, R. K.  1991.  The potential of mustard (Brassica juncea (L.) Coss.) as an edible oil crop on the Canadian prairies.  Can. J. Plant Sci. 71:195-198.