ABSTRACT

Introgression of resistance genes to blackleg, due to Leptosphaeria maculans, from black mustard (Brassica nigra, BB, 2n=16) and brown mustard (B. juncea (AABB, 2n=36) into oilseed rape genome (B. napus, AACC, 2n=38) was carried out by sexual crosses. The B. nigra recombinant line was obtained from the addition line containing the chromosome B4, by selfing and backcrossing to oilseed rape. The recombinant line containing the B. juncea resistance was provided by Dr. N.N. Roy (department of Agriculture, South Perth, Australia). Along the generations obtained for each origin of material, plants were analyzed on the basis of their chromosome number and of their meiotic behavior ; the most resistant stable recombinant lines were selected. Each blackleg resistance was under monogenic control and efficient at the cotyledon stage and at adult stage under field conditions. However, molecular markers of each introgression were different as well as their location on the oilseed rape genetic map.

In order to know if it is possible to produce stable material with the two sources of resistance, the meiotic behavior of plants containing the resistance genes alone or cumulated was established from one progeny and compared. The effects of the oilseed rape genetic background on the resistance level and of the selection pressure on L. maculans populations due to the resistance genes will be discussed.

INTRODUCTION

Blackleg, caused by Leptosphaeria maculans (Desm) Ces. & De Not., is one of the most damaging diseases of oilseed rape (Brassica napus L., AACC, 2n=38) crops. Either oligo-genic specific resistance or polygenic partial resistance are available within oilseed rape germplasm at the cotyledon and adult stages, respectively. On the contrary, all the Brassica species containing the B genome i.e. the diploid species B. nigra L. Koch (BB, 2n=16) and the two derived amphidiploids, B. juncea L. Czern (AABB, 2n=36) and B. carinata A. Br. (BBCC, 2n=34) carry a complete resistance efficient throughout the life of the plant (Rimmer and van den Berg 1992). The introduction of resistance genes carried by the B genome into oilseed rape was attempted by several authors (Roy 1984 ; Sacristan and Gerdmann 1986 ; Sjödin and Glimelius 1989 ; Chèvre et al. 1996,1997 ; Struss et al. 1996).

From the material provided by Dr Roy, we were able to select a stable recombinant line carrying a B. juncea resistance gene, Jlm1, efficient at the cotyledon and adult stage (Chèvre et al. 1997). The potential durability of such resistance was assessed under field conditions during three years under high selection pressure (Somda 1996). From this experiment, single ascospore isolates, obtained from pseudothecia on stem base residues of few damaged tissues of recombinant line, can overcome the JLm1 resistance gene at the cotyledon stage (Somda et al. 1999). This data suggests that the resistance is unlikely to be durable and so, new sources of resistance have to be investigated.

The resistance of B . nigra carried by the chromosome B4 was described by Chèvre et al. (1996) from B. napus-B. nigra addition lines. It was shown that this plant material was resistant at the cotyledon and adult stage. The introduction of this resistance into oilseed rape genome was performed by sexual crosses and presented in this paper.

Characteristics of each of these two sources of resistance, from B. juncea and from B.nigra and the possibility to cumulate both resistances in a single plant are reported.

B. napus-B. juncea RECOMBINANT LINE

A stable and blackleg resistant recombinant line (MXS) (2n=38) in a winter oilseed rape genetic background, ‘Samouraï, was selected by Chèvre et al. (1997). The results reported in this study showed that the resistance at the seedling stage is dominant and corresponds to the Mendelian segregation of one gene. The efficiency of this resistance at the cotyledon stage was confirmed using the highly virulent 314 isolate belonging to the A-group. This isolate was obtained from oilseed rape leaf lesion (figure 1). Under field conditions, the resistance was efficient at the adult stage (Chèvre et al. 1997). However, the resistance was overcame at the cotyledon stage with a single ascospore isolate, MX4.3, obtained from the residues of few damaged tissues of the MXS recombinant line by Somda et al. (1999) (figure 1). Chèvre et al. (1997) demonstrated that the Jlm1 gene of B. juncea is on the B genome and carried by the chromosome B8. Using a ‘to and fro’ strategy, Barret et al. (1998) localized the B. juncea introgression carrying the Jlm1 gene on the DY 17 linkage group of the oilseed rape genetic map.

Figure 1 : Results from cotyledon tests performed either with 314 or MX4.3 isolates on B. nigra, ‘Junius’, B. juncea, ‘Picra’ and B. napus, ‘Darmor’, ‘Samouraï’ varieties and on recombinant lines containing either one resistance gene from B. juncea, MXS, or from B.nigra, 74, or both resistance genes, MXS74. The mean score disease was established from the score at each inoculation points ranging from 1 (hypersensitive reaction) to 9 (collapse of tissues) (Williams and Delwiche 1979)

B. napus -B. nigra RECOMBINANT LINE

From the B. napus - B. nigra addition line (2n=39) with the chromosome B4, a recombinant line (2n=38), named B2-74-4, was obtained by selfing. Its meiotic behavior was disturbed with a high frequency of univalents and multivalents (table 1). The selection in the following generations obtained by selfing and backcrosses to recurrent oilseed rape parent, ‘Darmor, was based on three criteria : the stability of meiotic behavior, the presence of a chromosome B4 marker, Pgi-2B (Chèvre et al. 1996) and/or the resistance at the cotyledon stage using the 314 isolate.

All along the different generations, we observed that the percentage of multivalents decreased from 98 to 0% and that the percentage of cells with only 19 bivalents increase from 0 to 95% (table 1).

Table 1 : Cytogenetic analyses of the plants selected at each generation

Cotyledon tests, using the 314 isolate, revealed that the resistance is under monogenic control and that the Pgi-2B marker is linked to the resistance. This resistance gene is also efficient after MX4.3 isolate inoculation (figure 1). Resistance tests under field conditions is in progress to confirm that the recombinant line is as resistant as the original addition line.

The markers localized on the chromosome B4 (Chèvre et al. 1996) are present on the resistant recombinant line, 74. The location of this introgression into the oilseed rape genome is in progress but we already showed that none of the DY17 markers are linked to the introgression (unpublished data).

COMBINATION OF THE B. juncea AND OF THE B.nigra RESISTANCE GENES

We have crossed the 74 line to the MXS one. The selfing progeny of the F1 hybrid containing the two resistances was analyzed. The segregation ratio among the 106 plants studied confirmed that the two introgressions were independent. Cytologic analyses are reported in table 2.

Table 2 : Cytogenetic analyses of the plants obtained in a selfing progeny of an F1 hybrid (74 x MXS) * : 8 plants were observed, among them one was with 2n=39. Status of the insertion : ++, homozygous, + -, heterozygous, - -, no insertion

Whatever the status of the introgressions (homozygous, heterozygous, alone or combined), the meiotic behavior is regular and similar to the one of plants without insertion. We noted that, among the 8 plants presenting the two introgressions at the homozygous stage, one showed 39 chromosomes.

In figure 1, we showed that the recombinant lines, that combine both resistant genes (MXS74), were resistant to the two isolates 314 and MX 4.3, at the cotyledon stage.

DISCUSSION

Our study demonstrates that it is possible to create stable oilseed rape resistant lines containing B. juncea and/or B. nigra resistance genes. Cytogenetic analyses combined with blackleg resistance tests allowed us to select only the resistant plants with 38 chromosomes with regular meiotic behavior. This chromosomal control was performed to eliminate aneuploid plants which were detected at each generation (data not shown). For both recombinant lines containing either B. juncea or B. nigra gene, it was shown that only one gene confers resistance at cotyledon and adult stages.

The two introgressions carrying the resistance gene can be distinguished by their location on the B. nigra genome : B4 and B8 chromosomes for B. nigra and B. juncea resistance, respectively. Moreover, the interspecific introgression of B. juncea was localized on the DY 17 linkage group of the oilseed rape genetic map (Barret et al. 1998) and the B. nigra introgression was not linked to this latter. An other difference is their differential behavior by testing with the MX4.3 isolate, obtained from pseudothecia produced on few damage tissues of MXS recombinant line (Somda et al. 1999), whereas both lines were resistant to the 314 strain. The segregation analyses of the selfing progeny obtained from a F1 hybrid combining the two resistances confirmed that the two insertions are independent.

The two recombinant lines were on different oilseed rape genetic backgrounds, ‘Samouraï’ and ‘Darmor’ varieties for the B. juncea and B. nigra insertions, respectively. These two varieties are both highly susceptible to L. maculans at the cotyledon stage, however ‘Darmor’ shows a partial resistance at the adult stage (Pilet et al. 1998). In our study, we have shown that it is possible to cumulate the two resistance genes in a single plant. In presence of the MX4.3 isolate overcoming the B. juncea resistance gene, the B. nigra resistance gene remains efficient. In order to know the effect of different oilseed rape genetic backgrounds on the level of resistance, crosses are performed to produce five different winter type varieties containing the two genes alone or combined. From this material, further works to assess the potential durability of the resistance sources and the impact of the resistance gene(s) on the structure of the pathogen populations are in progress.

REFERENCES

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