ABSTRACT

Breeding double low restorer lines for the Ogu-INRA Cytoplasmic Male Sterility system in rapeseed (Brassica napus L.) has been a major objective during the past few years. After introgressing a restorer gene, Rfo, from radish into rapeseed, backcross and pedigree breeding was achieved and resulted in restorer lines with an improved female fertility and regular meiotic behaviour. However, the use of these restorer lines in double low F1 hybrid breeding has been slowed down because of a tight linkage between the radish introgression and high glucosinolate content. Double low restorer lines have now been selected and are being used to produce double low restored F1 hybrids.

INTRODUCTION 

Breeding restorer lines for the Ogu-INRA Cytoplasmic Male Sterility (CMS) system (Pelletier et al 1983, 1987) in rapeseed (Brassica napus L.) has been a major objective for the last years. A restorer gene Rfo was introgressed from radish (Raphanus sativus L.) into rapeseed (Heyn 1976) through intergeneric hybridisation. Extensive backcross and pedigree breeding (Delourme et al 1991) were necessary to improve the low female fertility of the restorer lines which was attributed to a long radish genetic information remaining around the Rfo gene or elsewhere in the genome (Pellan-Delourme and Renard 1988). This breeding resulted in restorer lines (2n=38) of which female fertility was equal to the one of rapeseed maintainer lines and meiotic behaviour was greatly stabilised, thus leading to a more regular transmission of the restorer gene through backcross or self pollination (Delourme et al 1995).

Some radish DNA still remains around the introgressed Rfo gene. A radish isozyme allele at Pgi-2 locus was found to be tightly linked to the Rfo gene (Delourme and Eber 1992). RAPD and RFLP markers were then identified, the polymorphic DNA fragments being associated either with the restorer allele or with the sterility maintainer allele (Delourme et al 1994; 1998). Some lines were identified to have lost the Pgi-2 allele of radish, which indicated that the introgression can be modified (Delourme and Eber 1992).

The use of the restorer lines in double low F1 hybrid breeding has been slowed down because of a tight linkage between the radish introgression and high glucosinolate content (Delourme et al 1995; 1998). However, different low-glucosinolate restorer lines have been selected since 1992. These lines still carry or have lost the radish Pgi-2 allele.

The origin, the characteristics and the molecular characterisation of the different restorer lines obtained are described in this paper.

MATERIAL AND METHODS

Plant material

The origin of the restored B. napus lines carrying the Rfo radish restorer gene was previously described (Pellan-Delourme and Renard 1988). In 1989, an improved family ('R20') was selected, giving rise to a progeny with a good female fertility (Delourme et al, 1991). Breeding of this material was continued through self pollination and backcrosses with double low lines.

RAPD analyses

The RAPD markers identified in Delourme et al (1994; 1998) were tested on the different restorer lines in order to characterise the rearrangements that have occurred in the introgression. The procedure for RAPD analyses was as described in Foisset et al (1996).

RESULTS AND DISCUSSION

Breeding double low restorer lines

'R40', an homozygous single low winter rapeseed restorer line was derived from the ‘R20’ family improved for female fertility (Delourme et al 1991) through pedigree breeding (F6) (see Fig 1.). This winter restorer line was supplied to private breeding companies in 1992. Two spring restorer lines were also supplied in 1992 and 1994. They were derived from backcrosses first, with ‘Samouraï’, a winter double low line and then, with spring double low lines. One of these spring restorer lines had lost the Pgi-2 allele of radish (Fig 1). Through successive backcrosses with ‘Samouraï’, some low glucosinolate winter restorer lines were obtained in 1995. These lines still carry or not the Pgi-2 allele of radish.

As previously stated in Delourme et al (1995), the female fertility of the restored plants was not affected by the loss of radish Pgi-2 allele when they are at the heterozygous stage but the homozygous restored plants which have lost Pgi-2 allele of radish showed a very poor seed set. This was explained by the fact that these plants lack a rapeseed chromosomal segment (they have not recovered the rapeseed Pgi-2 allele).

Characterisation of the radish introgression in different restorer lines

RAPD markers carried by the radish introgression were tested on several restorer lines. Table 1 summarises the different types observed. It shows that some markers were lost in PGI - or/and in low glucosinolate restorer lines. RAPD6 and RAPD14 were lost in low glucosinolate restorer lines, independently of the presence or absence of Pgi-2 allele of radish, indicating that the elimination of radish Pgi-2 allele is not necessary to get low glucosinolate restorer lines.

Table 1: Presence/absence of RAPD markers of the radish introgression in different types of restorer lines.

(a) GLS content was assessed through HPLC; High, intermediate and low GLS content correspond to more than 35, between 25 and 35 and less than 20 µmoles/g seed, respectively.

Breeding double low restored F1 hybrids

A double low winter restorer line was used to produce restored F1 hybrids. These F1 hybrids were tested in field trials and assessed for glucosinolate content. It showed that we are able to obtain winter restored F1 hybrids with a glucosinolate content ranging from 9 to 18 µmoles/g seed depending on the female parent and on the year. This line is being used in backcross breeding in order to diversify the winter and spring restorer lines.

To develop early sowing, to improve resistance to lodging and resistance to frost as well as to simplify harvesting, INRA, in collaboration with SERASEM, is breeding semidwarf F1 hybrids. Thus a semidwarf restored F1 hybrid (B017) was produced with the double low winter restorer line and is now in second year of official trials for registration in France and Great Britain.

CONCLUSION

Our results indicate that it is possible to get double low restored lines through conventional breeding and to use these lines in double low F1 hybrid breeding. However, the radish introgression is still large in these lines. It still needs to be reduced in order to decrease the probability of rearrangements which could lead to material with a poor agronomic value. Thus, programs aiming at cloning the Rfo gene or at reducing the size of the introgression are in progress.

REFERENCES

1. Delourme R, Eber F, Renard M (1991) Radish cytoplasmic male sterility in rapeseed: Breeding restorer lines with a good female fertility. Proc of the 8th Int Rapeseed Cong, Saskatoon, Canada : 1506-1510.

2. Delourme R, Eber F (1992) Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.). Theor Appl Genet 85: 222-228.

3. Delourme R, Bouchereau A, Hubert N, Renard M, Landry BS (1994) Identification of RAPD markers linked to fertility restorer gene for the Ogura radish cytoplasmic male sterility of rapeseed (Brassica napus L.) Theor Appl Genet 88: 741-748.

4. Delourme R, Eber F, Renard M (1995) Breeding double low restorer lines in radish cytoplasmic male sterility of rapeseed (Brassica napus L.). Proc 9th Int Rapeseed Cong, Cambridge, UK 1: 6-8.

5. Delourme R., Foisset N., Horvais R., Barret P., Champagne G., Cheung W.Y., Landry B.S., Renard M. (1998) Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.). Theor Appl Genet 97: 129-134.

6. Foisset N, Delourme R, Barret P, Hubert N, Landry BS, Renard M (1996) Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled haploid progeny. Theor Appl Genet 93: 1017-1025.

7. Heyn FW (1976) Transfer of restorer genes from Raphanus to cytoplasmic male sterile Brassica napus. Cruciferae Newslett 1: 15-16.

8. Pellan-Delourme R and Renard M (1988) Cytoplasmic male sterility in rapeseed (Brassica napus L.): female fertility of restored rapeseed with "Ogura" and cybrids cytoplasms. Genome 30:234-238.

9. Pelletier G, Primard C, Vedel F, Chétrit P, Rémy R, Rousselle P, Renard M (1983) Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol Gen Genet 191: 244-250.

10. Pelletier G, Primard C, Vedel F, Chétrit P, Renard M, Pellan-Delourme R (1987) Molecular, phenotypic and genetic characterization of mitochondrial recombinants in rapeseed. Proc of the 7th Int Rapeseed Cong, Poznan, Pologne: 113-118.