DEVELOPMENT OF LOW GLUCOSINOLATE RESTORER AND OGU CMS WINTER RAPE HYBRID

 

JC. Pruvot¹, K. Kräling², D. Charne³, L. Tulsieram³

 

¹ Pioneer Genetique, Frouville, F41290 Oucques

² Pioneer Hi-Bred Northern Europe Gmbh, Wulfshagen, D24214 Gettorf

³ Pioneer Hi-Bred, 12111 Mississauga Road, RR#4, Georgetown, Ontario L7G4S7

 

 

ABSTRACT

For the development of Ogura-CMS based hybrids exhibiting canola quality, an improvement of the INRA restorer (Rf) was required. By recombination and pedigree breeding, Pioneer Hi-Bred (PHI) scientists identified a Rf line with very low glucosinolate content, allowing the production of fully- restored hybrids of canola quality. The stability of expression of the low glucosinolate content, mendelian restorer gene transmission, and meiotic behaviour have been demonstrated over three years in different environments and different genetic backgrounds. The molecular marker profile shows that the length of the original radish introgression is significantly reduced.

 

KEYWORDS:  Winter Brassica napus, Ogura restorer gene, Recombinant, Glucosinolates.

 

INTRODUCTION

INRA, France, introduced the radish “Ogura” cytoplasmic male sterility (CMS) into B. napus Pelletier et al. (1983, 1987). The OGU-INRA-CMS demonstrated stable expression under different environmental conditions and in different genetic backgrounds. By interspecific hybridization, a fertility restorer gene was transferred from Raphanus sativus into Ogura-CMS B. napus (Heyn 1976). Improvement of female fertility of the restorer line, by elimination of radish information, has led to Rf lines with good productivity and stable meiotic behaviour (Delourme et al 1991, 1995).

 

Most European winter canola breeding companies and institutes are using this CMS system in their breeding programs. However, the development of double low F1 hybrids was slowed down due to a tight linkage between the introgressed restorer gene and high glucosinolate content. Molecular marker information indicated that the size of the radish fragment was large. However, the molecular information did not differentiate if the locus controlling seed glucosinolate content was located on the rapeseed genome or in the radish introgression (Delourme et al 1998).

 

Here, we describe the characteristics of a recombinant restorer line exhibiting low glucosinolate content, which was used in the development of PHI’s winter canola hybrids for Europe.

 

MATERIALS AND METHODS

The recombinant line and the segregating restorer populations were evaluated in a nursery in France. Fully restored hybrids were produced between the low glucosinolate restorer and several CMS (male sterile) lines. Yield tests were conducted in Austria, France Germany and the UK in 1998.

 

The seed glucosinolate content was determined using Palladium, NIRS and HPLC on single plants and bulks from the nursery, and on bulks from yield trials.

 

Molecular markers (AFLP, RAPD, RFLP) were developed by PHI in Georgetown, Canada, and mapped to the restorer gene. In 1998, several F2 populations, developed from single crosses involving the low glucosinolate restorer and elite lines or varieties, were observed in the field in France, and single plants were selected for low glucosinolate content. At the same time, F4 lines were produced by SSD. F3 progenies were used to identify homozygous/hemizygous lines.

 

RESULTS

Glucosinolate content

Restorer: In 1996, one restorer line was identified as containing plants with glucosinolate content (7,9µM/g to 12,3µM/g) lower than the variety Bristol, the low glucosinolate check. Progenies fixed for the restorer gene were evaluated in the following two years. A strong influence of the year on the glucosinolate content was found. However, homozygous restorer lines exhibited clearly lower contents than Bristol (Table1).

 

Table 1: Glucosinolate content of improved restorer lines                         

 
Field 96

Field 97

Field 98

Genotype

HPLC

µM/g

Rf Status

NIRS

NIRS

96FNW1822-1

12.3
Rfrf

 

 

96FNW1822-2

11.8

RfRf

8.7

14.5

96FNW1822-3

8.5

Rfrf

 

 

96FNW1822-4

8.8

Rfrf

 

 

96FNW1822-5

10.9

RfRf

10.2

20.1

96FNW1822-6

11.5

Rfrf

 

 

96FNW1822-7

8.8

RfRf

8.1

19.5

96FNW1822-8

8.5

RfRf

9.8

15.7

96FNW1822-9

7.9

Rfrf

 

 

96FNW1822-1

9.9

Rfrf

 

 

BRISTOL

12.5

 

11.9

19.9

GOELAND

19.1

 

17.8

23.3

 

Hybrids: The glucosinolate content of five hybrids produced from crosses between Ogu-CMS A-lines and the improved restorer are shown in Table 2. The hybrids and checks were tested across Europe at 18 locations. On average, the unrestored hybrid, Synergy, and the open-pollinated variety, Capitol, exhibited the highest glucosinolate contents. Several PHI hybrids had even lower contents than the hybrid Pronto, which is based on the MSL (male sterile Lembke) system. Therefore, as expected in hybrid combination, the low glucosinolate content is maintained.

 

Table 2: Glucosinolate content (NIRS) of 5 restored hybrids in 1998

 

UK (2)ª

France (7)

Germany (6)

Austria (3)

Average (18)

Capitol

14.2

15.6

19.2

19.4

17.4

Pronto

13.2

15.6

16.8

16.8

15.9

Synergy

17.7

20.3

18.3

17.4

18.9
PHI A1/PHI Rf

11.4

14.6

13.0

14.5

13.6

PHI A2/PHI Rf

12.9

15.7

13.8

14.9

14.6

PHI A3/PHI Rf

13.0

15.9

13.9

14.6

14.7

PHI A4/PHI Rf

15.4

16.7

15.5

16.3

16.1

PHI A5/PHI Rf

15.2

16.2

14.7

16.6

15.6

ª Number of location

Meiotic behaviour and gene transmission

In 5 F2 populations consisting of 500 plants, we scored the male fertility/sterility. In F3 progenies, homozygous Rf/Rf lines were identified. In both generations, segregation data was not significantly different from expected Mendelian ratios with one dominant gene: 74.8% of the F2 plants were fertile (expected = 75%), and 35.2% of F3 lines were homozygous (expected = 33%). This material was screened in the field in 1998/1999 and exhibited excellent vigour, equivalent to conventional varieties. Self-pollinations and testcrosses produced with homozygous plants were 100% fertile. Therefore, meiotic behaviour and gene transmission in the improved Rf source are not disturbed.

 

Linkage with glucosinolate content

A comparison of the glucosinolate content of homozygous and heterozygous F2 plants (1159 plants in total) indicates that there is no linkage between fertility restoration and glucosinolate content.

 

Length of the radish fragment

In addition to the PGI II isozyme marker, 5 RAPD and 8 AFLP markers were linked to the Rf gene in the original restorer line obtained from INRA. Characterisation of the radish introgression of our improved restorer shows that the PGI, as well as 9 other markers are absent, indicating that significant radish DNA has been lost.

 

CONCLUSION

PHI has developed a stable, double-low, source of restoration for the OGU-INRA-CMS, exhibiting good agronomic value. Quality traits are no longer a limitation to the production of fully-restored hybrids. Breeders can now focus on selection for specific and general combining ability as well as agronomic characteristics in hybrid products.

 

REFERENCES

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

Delourme R., Eber F. and 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.

Delourme R., Foisset N., Horvais R., Barret P., Champagne G., Cheung W. Y., Landry B. S. and 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.

Heyn F.W., 1976. Transfer of restorer genes from Raphanus to cytoplasmic male sterile Brassica napus. Cruciferae Newslett. 1: 15-16.

Pelletier G., Primard C., Vedel F., Chetrit P., Remy R., Rouselle P. and Renard M., 1983. Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet. 191: 244-250.

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