ABSTRACT

Blackleg, caused by Leptosphaeria maculans, is one of the most serious disease of rapeseed in most producing areas in the world. Increasing blackleg resistance is a major objective of rapeseed breeding programs. Resistant rapeseed cultivars such as 'Jet Neuf' were bred in France and used as sources of resistance in many countries. The adult plant resistance of 'Jet Neuf', which is partial, non specific and polygenic, has been durable despite the wide use of this cultivar in Europe. We have developed a project to study the genetics of blackleg resistance present in the highly resistant cv 'Darmor', which is derived from 'Jet Neuf' through backcross breeding. Mapping of the genomic regions associated with blackleg resistance was undertaken in two genetic backgrounds with the study of populations derived from two crosses: 'Darmor.bzh x Yudal' (152 DH lines) and 'Darmor x Samouraï' (134 DH lines and 185 F3 families). Several genomic regions were found to be associated with blackleg resistance. In this paper, we report on the number and effects of QTL in the two crosses, on the stability of these QTL across the years and the genetic backgrounds.

INTRODUCTION

Blackleg, caused by the fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [anamorph Phoma lingam (Tode : Fr.) Desm.], is the most economically important disease of Brassica species in Australia, Europe and North America. Since the disease cannot be efficiently controlled chemically, increasing blackleg resistance has become a major objective of rapeseed breeding programs. For blackleg resistance, an important intraspecific variability has been found in rapeseed. A wide range of inheritance from monogenic to polygenic and dominant to recessive has been reported, depending on the genitor used and the inoculum composition (Cargeeg and Thurling, 1979, Delwiche, 1980, Hill, 1991, Pang and Halloran, 1996a, 1996b, Rimmer and Van Den Berg, 1992, Sawatsky, 1989, Sippell, et al., 1991, Stringam, et al., 1992). In recent years, molecular markers have been used to map blackleg resistance genes in rapeseed. In a DH population derived from the cross 'Major' x 'Stellar', Ferreira et al. (1995a) identified a total of 7 genomic regions associated with blackleg resistance in greenhouse (cotyledon and stem) and field conditions. A major locus (LEM1) was involved in the resistance at the cotyledon stage. In the cross 'Westar' x 'Crésor', Dion et al. (1995) detected a major field resistance QTL (LmFr1) expressed in four different environments. In another study, Mayerhofer et al. (1997) mapped a major locus (LmR1) associated with resistance to L. maculans at the cotyledon stage in the cultivar 'Shiralee'.

In our group, a research project on mapping blackleg resistance has been undertaken at the adult plant stage. We have used the cultivar 'Darmor' as the resistance genitor. 'Darmor' is a double low line, derived from the winter single low cultivar 'Jet Neuf' through backcrosses. The adult plant resistance of 'Jet Neuf' (Renard and Brun, 1979) is partial, non-specific and polygenic. This resistance is interesting because it has been durable despite the wide use of 'Jet Neuf' in western and eastern Europe for about ten years (Hammond and Lewis, 1987a, Humpherson-Jones, 1983). Mapping QTL for field resistance to L. maculans has been performed, first, in the cross 'Darmor-bzh' x 'Yudal' for two years ('bzh', the dwarf gene) (Pilet, et al., 1998), and second, in the cross 'Darmor' x 'Samouraï' for one year. 'Yudal' is a spring korean line very susceptible to blackleg. 'Samouraï' is a double low line close to 'Bienvenu' and is less susceptible to blackleg than 'Yudal'. In the present paper, we synthetize the results obtained in the two crosses. We discuss stability of the resistance QTL across the years and the genetic backgrounds.

MATERIAL AND METHODS

Plant material

A doubled haploid (DH) population was derived from the F1 'Darmor-bzh' x 'Yudal'. A total of 152 DH lines were used for QTL mapping.

From the cross 'Darmor' x 'Samouraï', two progenies were studied. The first one was composed of 134 DH lines and the second one included 185 F_2:3 families.

Genetic maps

The genetic map established from the DH population 'Darmor-bzh' x 'Yudal' was previously described by Foisset et al. (1996). At the time of QTL mapping, this map comprised 288 markers (predominantly RAPD and RFLP markers) distributed on 19 linkage groups and covered 1954 cM (Kosambi).

The genetic map elaborated from the DH population 'Darmor' x 'Samouraï' includes, at present, a total of 250 markers distributed on 1650 cM, among which predominantly RAPD and RFLP markers. From the F_2:3 progeny, the map consists of 83 markers, essentially RFLP markers, and covers 635 cM.

Field experiment

The three progenies were evaluated in field experiments with three replicates, at Le Rheu, France, in intensified conditions of contamination. The 'Darmor-bzh' x 'Yudal' population was tested in 1995 and 1996, and the two 'Darmor' x 'Samouraï' progenies, in 1998. Controls were used in each experiment. Crown canker severity was assessed in each plot using a mean disease index (I) calculated from the scorings of 30 plants per plot. The 1-9 scoring scale applied was based on the extent of external and internal necrosis at the crown.

Statistical analysis and QTL mapping

For each year and progeny, the experimental resistance data were analysed using a generalized linear model of the Statistical Analysis System program (SAS-Institute, 1989). Heritability (h²) of the resistance in our test system was estimated from a one-way ANOVA.

QTL were mapped from each progeny by 'Interval Mapping', using the computer program Mapmaker/QTL 1.1 and 1.9 (Lincoln, et al., 1990, 1992). Markers associated with blackleg resistance were checked by multiple regression of the resistance data on the markers at a significance level threshold of P<5.10^-2.

RESULTS AND DISCUSSION

Blackleg resistance data

The frequency distributions observed in the three years showed the polygenic nature of the field resistance to L. maculans. In the 'Darmor-bzh' x 'Yudal' DH population, within years heritabilities for blackleg resistance were high and similar over the two years (h² = 0.89 and 0.88 in 1995 and 1996, respectively). In the 'Darmor' x 'Samouraï' cross, heritabilities for the mean disease index (I) assessed in 1998 were 0.53 and 0.00 for DH and F_2:3 progeny, respectively. It could particularly be attributed to the low level of contamination observed in 1998, as demonstrated by controls scores.

Mapping QTL for field resistance to L. maculans

The results of QTL mapping are summarized in Table 1.

Table 1 : Characteristics of blackleg resistance QTL detected by 'Interval Mapping' fore the mean disease index (I) for different years, crosses and progenies : position, linkage group, LOD score, individual additive (a) and dominant (d) effects, total contribution to the resistance variation (R²).

Position (cM)	Linkage group	LOD	Weight (a)	Dominance (d)	R² (%)
'Darmor-bzh' x 'Yudal' DH progeny - 1995
169.1	DY2	3.1	-0.38	-
22.1	DY9	+ 3.8	0.36	-
117.3	DY3	+ 3.2	0.35	-
71.9	DY5	+ 2.9	0.27	-
100.8	DY11	+ 2.9	0.26	-
28.8	DY10	+ 2.8	0.27	-
92.1	DY6	+ 3.2	0.27	-	57.1
'Darmor-bzh' x 'Yudal' DH progeny - 1996
117.9	DY6	10.3	0.50	-
115.3	DY3	+ 2.8	0.25	-
173.1	DY2	+ 2.6	-0.27	-
40.7	DY8	+ 2.7	0.21	-
117.0	DY1b	+ 2.3	-0.21	-	50.7
'Darmor' x 'Samouraï' DH progeny - 1998
0.0	DS3	3.2	0.19	-
19.9	DS11	3.1	0.21	-
0.0	DS8	+ 2.1	-0.16	-
9.0	DS2?	+ 1.9	0.17	-
16.0	DS6	+ 1.9	-0.16	-	40.8
'Darmor' x 'Samouraï' F2:3 progeny - 1998
11.7	DS3	2.4	0.13	-0.16
24.0	DS11	2.4	0.18	-0.30
22.5	DS6	2.3	-0.10	0.42
6.0	DS1a	2.3	-0.10	-0.02	41.2

The QTL position from the first marker of the group is expressed in centiMorgans (Haldane function). When a QTL was detected with a multiple-QTL model, the increase in the total LOD with the new QTL is indicated with the sign '+'. The weight of each QTL corresponds to the substitution effect of one 'Darmor-bzh' or 'Darmor' allele by one 'Yudal' or 'Samouraï' allele. Note : bold-type font indicates the common QTL between the two crosses.

In the cross 'Darmor-bzh' x 'Yudal', 7 genomic regions were associated with the index resistance in 1995, and 5 in 1996, collectively accounting for 57 % and 51 % of the variation, respectively. Two QTL, revealed on the DY2 and DY3 groups have important additive effects, were common to the two years. The one located on the DY2 group is from the susceptible parent. A major QTL was detected in 1996 close to the dwarf gene (bzh) on the DY6 group. It seems that the dwarf trait acted upon the expression or the evaluation of the resistance in 1996 field conditions, masking other QTL detection.

In the cross 'Darmor' x 'Samouraï', 5 and 4 resistance QTL were detected from the DH population and the F_2:3 progeny, respectively. They explained 41 % of the variation in each progeny. Three of them were common to the two populations, among which one, on the DS6 group, came from the parent 'Samouraï'. The other two common QTL were from 'Darmor' and had dominant and superdominant effects for the resistance.

Over all the experiments and the two crosses, the number of QTL revealed and the R² explained were different and several regions were specific to one environment or one cross. Three reasons could explain these differences. First, the intensity of the contamination was not the same in 1995, 1996 and 1998 and was particularly low in 1998. Second, the genetic distance between 'Darmor-bzh' and 'Yudal' was higher than between 'Darmor' and 'Samouraï'. Third, the 'Darmor' x 'Samouraï' genetic maps do not cover the genome as well as the 'Darmor-bzh' x 'Yudal' map. Yet, two major genomic regions were stable over the two crosses 'Darmor-bzh' x 'Yudal' and 'Darmor' x 'Samouraï'. They were revealed on the linkage groups 3 and 11. Such regions are potentially interesting for developing breeding strategies.

CONCLUSION

This study provides a better understanding of the genetic complexity of the polygenic blackleg resistance in the cultivar 'Darmor'. It also brings useful markers which could be exploited in rapeseed marker-assisted selection for blackleg resistance. Another field resistance experiment with the 'Darmor' x 'Samouraï' DH and F_2:3 progenies is performed in 1998/99 in order to validate the identified QTL.

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