(19) & (11) EP 2 220 930 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 25.08.2010 Bulletin 2010/34 A01H 5/10 (2006.01) C12N 15/82 (2006.01) (21) Application number: 10159412.5 (22) Date of filing: 13.06.2008 (84) Designated Contracting States: • Pleines, Stephen AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 32130 Enger (DE) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Coque, Marie RO SE SI SK TR 31140 Aucamville (FR) • Gielen, Jan (30) Priority: 13.06.2007 EP 07290741 31620 Bouloc (FR) (83) Declaration under Rule 32(1) EPC (expert (74) Representative: Schaberg, Ulf Günther solution) Syngenta Participations AG Seeds IP Basel (62) Document number(s) of the earlier application(s) in Schwarzwaldallee 215 accordance with Art. 76 EPC: 4058 Basel (CH) 08010783.2 / 2 002 711 Remarks: (71) Applicant: Syngenta Participations AG This application was filed on 08-04-2010 as a 4058 Basel (CH) divisional application to the application mentioned under INID code 62. (72) Inventors: • Stiewe, Gunther 32657 Lemgo (DE) (54) New hybrid system for brassica napus (57) SUMMARY OF THE INVENTION lines. Further provided are methods for the production of This invention relates to a nuclear conditional male those lines. Further embodiments of the invention relate sterility system in Brassica napus. Embodiments of the to markers associated to the sterility, fertility and main- invention provide for the (male sterile) prebasic female tainer alleles and the use of those markers in providing (MsMsrfrf), the (male fertile) maintainer line (msmsrfrf), a hybrid system. the (male sterile) basic female line (Msmsrfrf), and hybrid EP 2 220 930 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 220 930 A2 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to a nuclear conditional male sterility system in Brassica napus. Embodiments of the present invention provide for the prebasic (male sterile) female (MsMsrfrf), the (male fertile) maintainer line (msmsrfrf), the basic (male sterile) female line (Msmsrfrf), and hybrid lines. Further provided are methods for the pro- duction of those lines. Further embodiments of the present invention relate to markers associated to the sterility, fertility and maintainer alleles, respectively, and the use of those markers in providing a hybrid system. 10 BACKGROUND OF THE INVENTION [0002] Oilseed from Brassica plants is an increasingly important crop. As a source of vegetable oil, it presently ranks only behind soybeans and palm in commercial importance and it is comparable with sunflowers. The oil is used both as 15 a salad and cooking oil, and play an increasingly important role in biofuels (biodiesel). [0003] In its original form, Brassica oil, known as rapeseed oil, was harmful to humans due to its relatively high level of erucic acid. Erucic acid is commonly present in native cultivars in concentrations of 30-50% by weight based upon the total fatty acid content. This problem was overcome when plant scientists identified a germplasm of low erucic acid (Stefansson, 1983). Although these varieties with less than 2% of erucic acid in their total fatty acid profile (single zero 20 quality) yielded edible oil, the continuing presence of sulfur compounds called glucosinolates (GSLs) in the high protein meal remained a major constraint to further market expansion. Wide acceptance of rapeseed meal for animal nutrition is hampered by the presence of GSLs in the seed. Furthermore, glucosinolates are also undesirable since they can lead to the production of antinutritional breakdown products (e.g., thiocyanates, isothiocyanate and nitrite) upon enzymatic cleavage during oil extraction and digestion when acted upon by the endogenous enzyme myrosinase during crushing. 25 In consequence, so-called "double-low" varieties (low in erucic acid in the oil as well as low in glucosinolates in the solid meal after oil extraction) were developed, which have an erucic acid content of less than 2% by weight based upon the total fatty acid content, and a glucosinolate content of less than 30 Pmol/gram of the oil-free meal. These high quality forms of rape, first developed in Canada, are known as canola. At present the maximum threshold set by European law is 25 Pmol total glucosinolate (GSL) per gram (g) of seed at 8.5% moisture, as measured by HPLC (EU decree 2294/92). 30 Double low spring canola varieties cultivated in Canada need to have GSL levels of less than 30 Pmoles GSLs per gram of air-dried oil-free meal at 0% moisture, as measured by TMS. The GSL levels of commonly cultivated double zero oilseed rape varieties in Europe and Canada varies significantly below the threshold levels at 60% of the official threshold level or even lower. However, many countries are requiring even lower levels of glucosinolates in order to register canola varieties. 35 [0004] In addition, plant scientists have attempted to improve the fatty acid profile for rapeseed oil (Röbbelen, 1984; Ratledge et al., 1984; Röbbelen, 1975; Rakow & McGregor, 1973). [0005] Especially winter oilseed rape (Brassica napus L. ssp. oleifera (Metzg.), Brassicaceae) is an important crop for the production of oilseed in temperate agricultural regions. In Germany in 2006 approximately 1.5 million hectares (12% of the total agricultural area) were sown with oilseed rape. 40 [0006] Oilseed rape is a predominantly self-pollinated crop with about one-third outcrossing (Becker et al., 1992). Breeding of rapeseed plants have been centered on open-pollinated seeds by taking advantage of high self-compatibility affinity of said plants. The significant heterosis for seed yield in oilseed rape has created interest in the development of hybrid cultivars (Riaz et al., 2001). Heterosis means the growth and yield advantage of hybrids in comparison to their parents gained by the crossing of two genetically different, homozygous genotypes (Shull, 1922). Rapeseed hybrids 45 always show a significant heterosis in yield. Considerable heterosis for seed yield in F1 hybrids of oilseed rape ( Brassica napus L.) has been reported by various authors at the beginning of hybrid breeding in rapeseed (Schuster, 1969; Röbbelen, 1985; Grant & Beversdorf, 1985; Lefort-Buson et al., 1987; Brandle & McVetty, 1989; Paulmann & Frauen, 1991; Stefansson, 1983; Brandle & McVetty, 1990; Shen, 2005). Principally the heterosis level in spring type rapeseed can be as high as 20% to 30%, and in winter type rapeseed about 30% to 40%. 50 [0007] An effective production of hybrid seeds requires both the identification of heterotic groups (genetic distinct genepools; Melchinger & Gumber, 1998, Becker & Link, 2000) and a method for targeted crossing of those heterotic groups. [0008] In winter oilseed rape the first hybrid varieties that were registered in Europe are hybrid line- associations using the INRA ogura system and fully restored hybrids using the MSL system (see below for details). However, in comparison 55 to elite inbred lines the heterosis effect is still moderate. The relatively low gain in yield is, however, not caused by the inefficiency of the hybrid system, but by the lack of genetic diverse gene pools in rapeseed in consequence of decades of inbreeding and governmental regulations (e.g., glucosinolate or erucic acid content), which still cause limited germ- plasm variability. 2 EP 2 220 930 A2 [0009] More diverse gene pools will lead to higher heterosis effects but their development was initiated only recently. Nevertheless, a commercially functional hybrid system is the predominant prerequisite for achieving significant yield increases in rapeseed in the future. Those increases are not only required by the increased demand for food purpose but the rapidly increasing demand in biofuels (biodiesel). 5 [0010] Beside chemical-induced male sterility (CHA), three genetics based hybrid system principles have been ex- plored in Brassica varieties: Breeders use self-incompatible (SI), cytoplasmic male sterile (CMS), and nuclear male sterile (NMS; formerly also genetic male sterility, GMS) Brassica plants as the female parent (for review of hybrid systems in vegetables see Kumar et al., 2004). SI plants are not able to self pollinate due to their genetic constitution and CMS as well as NMS female plants are incapable of producing pollen. Thus, all these plants must be cross-pollinated by a 10 male fertile parent. In using these plants, breeders are attempting to improve the efficiency of seed production and the quality of the F1 hybrids and to reduce the breeding costs. When hybridisation is conducted without using Si, CMS or NMS plants, it is more difficult to obtain and isolate the desired traits in the progeny (F1 generation), because the parents are capable of undergoing both cross-pollination and self-pollination. [0011] A simple and efficient pollination control system is the key step for utilizing heterosis in commercial hybrid seed 15 production. If one of the parents is a SI, CMS or NMS plant that is not able to self- pollinate or is incapable of producing pollen, only cross pollination will occur. By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality, provided that the parents are of uniform quality and the breeder conducts a single cross. [0012] Self-incompatibility systems: So far no commercially useable SI system for rapeseed has been developed. 20 Canadian patent CA 2,143,781 describes a hybrid breeding method for crop plants in the family Brassicaceae in which an F1 seed is produced by crossing the female parent of a self- incompatible male sterile line with a male parent. However, the main question for SI is the reproduction of SI parent lines in large scale, which makes this system difficult for commercial applications. [0013] Cytoplasmic male sterility (CMS): CMS is a maternally inherited phenomenon, the genetic determinants of 25 which are located in the genome of the cytoplasmic organelles, the mitochondria.