Inheritance of Increased Oleic Acid Concentration in High-Erucic Acid Ethiopian Mustard

M. Ferna ndez-Mart nez, and Antonio De Haro Leonardo Velasco,* Jose ABSTRACT
In comparison with canola oil, zero-erucic acid Ethiopian mustard (Brassica carinata A. Braun) oil is characterized by a low concentration of the monounsaturated oleic acid and high concentrations of the polyunsaturated linoleic acid and linolenic acid. Because of the low oxidative stability of oils rich in polyunsaturated fatty acids, the increase of oleic acid concentration in zero-erucic acid Ethiopian mustard is needed. Increased oleic acid concentration is currently available only in high erucic acid backgrounds. The objective of the present research was to study the inheritance of increased oleic acid concentration in the high-erucic acid Ethiopian mustard mutant N23591. The mutant was reciprocally crossed with the high-erucic acid line C-101, with the standard composition of C18 fatty acids. Partial maternal and cytoplasmic effects for oleic acid concentration were observed in the analysis of F1 seeds and F1 plants, respectively, from reciprocal crosses. Standard oleic acid concentration in C-101 was partially dominant over increased oleic acid concentration in N23591. Oleic acid concentration of F2 seeds segregated following a 3:1 (standardintermediate: increased) ratio, suggesting monogenic inheritance. This was confirmed in the BC1 to N2-3591, which segregated following a 1:1 (intermediate: increased) ratio. The separation of the standard and intermediate oleic acid classes was not possible, probably because of the partial dominance of standard over increased oleic acid concentration. The monogenic inheritance of increased oleic acid levels in the high-erucic acid N2-3591 line will facilitate the transfer of this trait to zero-erucic acid Ethiopian mustard germplasm.

profile of the zero-erucic acid Ethiopian mustard oils developed so far consists of 330 g kg1 oleic acid, 370 g kg1 linoleic acid, and 210 g kg1 linolenic acid (Alonso ndez-Mart nez et et al., 1991; Getinet et al., 1994; Ferna al., 2001). Polyunsaturated fatty acids are highly susceptible to autoxidation, which involves the production of free radicals, implicated in a number of diseases, tissue injuries, and in the process of aging (Shahidi, 1996). Furthermore, the breakdown products of fatty acid autoxidation are the major source of off flavors in oils, which reduces their shelf life (Tatum and Chow, 1992). As a consequence, the reduction of the levels of polyunsaturated fatty acids and their substitution for the monounsaturated oleic acid is an important goal for the development of higher quality mustard oil (Scarth and McVetty, 1999). A high-erucic acid Ethiopian mustard mutant N23591, exhibiting increased oleic acid concentration, has been developed through mutagenesis (Velasco et al., 1997). The objective of the present research was to study the inheritance of increased oleic acid concentration in this mutant. MATERIALS AND METHODS
The lines used in this study were N2-3591, developed from the treatment of the Ethiopian mustard line C-101 with ethyl methanesulfonate (EMS) (Velasco et al., 1997), and its paren rdoba by selection for agrotal line C-101, developed at Co nomic performance from a population provided by Dr P.F. Knowles, University of California, Davis, CA. Half seeds of N2-3591 (M9 generation) and C-101 were analyzed for fatty acid composition to ensure that the plants used in the genetic study breed true for seed oil fatty acid composition. Plants of both lines were reciprocally crossed in a field screenhouse in spring 1998. Plastic bags were used to prevent cross pollination. Crossing was done by emasculating immature flower buds of the female parent followed by immediate pollination of their stigmas with fresh pollen from open flowers of the male parent. F1 half seeds from reciprocal crosses as well as seeds from both parents were analyzed for fatty acid composition and the corresponding plants grown in a greenhouse in 1999. F1 plants from reciprocal crosses were self-pollinated to obtain F2 seeds and also backcrossed to both parents. Plants of N2-3591 and C-101 were reciprocally crossed again to obtain F1 seeds under the same environment as the F2 and BC1 seeds. Random seed samples of both parents, F1, F2, and BC1 generations were analyzed for fatty acid composition. Since all the generations were grown in the same environment, the fatty acid composition of the parents was used to make the parental classifications. Limits of the parental classes were defined as mean of the parent 2 SD. Evaluation of fatty acid composition at the F1 plant level was performed by averaging the fatty acid composition of F2 seeds from each F1 plant.
Abbreviations: 18:1, oleic acid; 18:2, linoleic acid; 18:3, linolenic acid; 22:1, erucic acid.

thiopian mustard is a promising oilseed crop for semiarid areas where it has a better agronomic performance than its close relative rapeseed, B. napus L. (De Haro et al., 1998). Similar to other Brassica species, naturally occurring Ethiopian mustard forms are characterized by the presence of a high concentration of erucic acid in their seed oil (Velasco et al., 1998), which is considered harmful for human consumption (Ackman and Loew, 1977). Breeding efforts on this crop have resulted in the elimination of erucic acid from the seed oil (Alonso et al., 1991; Getinet et al., 1994; ndez-Mart nez et al., 2001). This process, however, Ferna was in all cases paralleled by a considerable increase of the concentration of the polyunsaturated linoleic acid and linolenic acid, which had not occurred in the development of zero-erucic acid forms of rapeseed (Stefansson et al., 1961), turnip rape (B. rapa L.) (Downey, 1964), and Indian mustard (B. juncea [L.] Czern.) (Kirk and Oram, 1981). Thus, the typical canola (zero-erucic, low-glucosinolate cultivars of Brassica spp.) oil profile for C18 unsaturated fatty acids is 610 g kg1 oleic acid, 210 g kg1 linoleic acid, and 110 g kg1 linolenic acid (Scarth and McVetty, 1999), whereas the average oil
rdoba, Instituto de Agricultura Sostenible, Apartado 4084. E-14080 Co Spain. Received 26 Nov. 2001. *Corresponding author (ia2veval@ uco.es). Published in Crop Sci. 43:106109 (2003).

106

VELASCO ET AL.: INHERITANCE OF INCREASED OLEIC ACID IN ETHIOPIAN MUSTARD

107

The chi-square test was used to evaluate proposed segregation ratios. Reciprocal F1 means were compared by independent t tests. The fatty acid composition of the seed oil was determined s by simultaneous oil extraction and methyl esterification (Garce and Mancha, 1993) followed by gas-liquid chromatography of fatty acid methyl esters on a Perkin-Elmer Autosystem gasliquid chromatograph (Perkin-Elmer Corporation, Norwalk, CT) equipped with a 2-m long column packed with 3% SP2310/2% SP-2300 on Chromosorb WAW (Supelco Inc., Bellefonte, PA). A temperature program of 190C for 10 min, increasing 2C min1 up to 220C was used. The injector and flame ionization detector were held at 275 and 250C, respectively.

RESULTS AND DISCUSSION

N2-3591 exhibited a higher oleic acid concentration (196 g kg1) and a lower linoleic acid concentration (62 g kg1) than C-101 (64 g kg1 and 171 g kg1, respectively) (Table 1). Linolenic acid concentration was similar in both lines, averaging 123 g kg1 in N2-3591 and 130 g kg1 in C-101. N2-3591 was originally selected for several generations under field conditions and it consistently exhibited a considerably reduced linolenic acid concentration in comparison with C-101, averaging 66 g kg1 and 116 g kg1 linolenic acid, respectively in the M5 generation (Velasco et al., 1997). This difference was not observed in the present study conducted under greenhouse conditions. It has been reported that environmental conditions have a more marked influence on linolenic acid content than on oleic and linoleic acid contents in rapeseed (Rakow and McGregor, 1973; Kondra and Thomas, 1975). The average oleic acid concentration in the F1 seeds (98.5 g kg1) was significantly (P 0.05) different from both parents (Table 1) and was lower than the midparent value (130 g kg1), suggesting a partial dominance of standard oleic acid concentration in C-101 over increased oleic acid concentration in N2-3591. This result is not in agreement with those obtained in zero-erucic acid rapeseed by Kondra and Thomas (1975) and Rakow and McGregor (1973), who reported partial dominance for high oleic/low linoleic acid. F1 seeds from reciprocal crosses differed significantly (P 0.05) for oleic acid concentration, indicating the presence of a partial maternal effect for this trait (Table
Table 1. Fatty acid composition of seed oil (major fatty acids) of Ethiopian mustard lines N2-3591, C-101, and their reciprocal F1 seeds and F2.
Material N2-3591 C-101 F1 seed: F1 (N2-3591 C-101) F1 (C-101 N2-3591) F2 seed: F1 (N2-3591 C-101) F1 (C-101 N2-3591) N 4 4 1 1 n 18:1 18:2 18:3 22:1 48 196 22 62 5 123 15 476 17 48 64 7 171 12 130 10 502 21 72 107 11 134 12 150 8 72 90 8 154 9 136 7 478 16 484 18 F2 or backcross F2(N2-3591 C-101) F2(C-101 N2-3591) Pooled Heterogeneity BC to N2-3591

Fig. 1. Histograms of oleic acid concentration (g kg1 oil) in F2 populations from the cross between the Ethiopian mustard lines N23591 C-101 (A) and the reciprocal cross C-101 N2-3591 (B).

1). At the F2 level, oleic acid concentration also differed significantly (P 0.05) in reciprocal crosses, indicating a partial cytoplasmic effect (Table 1). Thomas and Kondra (1973) identified maternal effects on oleic and linoleic acid concentrations in two out of three crosses involving zero-erucic strains of rapeseed with contrasting oleic and linoleic acid contents. The same authors found no cytoplasmic effects in any of the reciprocal crosses. Similarly, Rakow and McGregor (1973) found maternal effects and no cytoplasmic effects on oleic and linoleic acid concentrations in zero-erucic acid rapeseed. The analysis of oleic acid concentration in reciprocal F2 populations revealed a bimodal distribution (Fig. 1), with about one fourth of the F2 seeds having the increased oleic acid phenotype of the N2-3591 parent. Chi-square tests (Table 2) confirmed that the observed segregations were not significantly different from a 3:1 ratio (N2-3591: N2-3591), suggesting that increased oleic acid concentration in N2-3591 was controlled by alleles at one locus. Monogenic inheritance was confirmed in the backcross to N2-3591, which segregated following a 1:1 (N2-3591: N2-3591) ratio (Table 2; Fig. 2). The classes corresponding to the phenotype of C-101 (standard oleic acid) and to the heterozygote (intermediate oleic acid) could not be separated in the F2 populations (Fig. 1) probably because of the partial dominance of standard over increased oleic acid concentration. The wild-type allele present in C-101, conferring standard oleic acid levels has been designated Ol, whereas the
Table 2. Frequency distribution for oleic acid concentration in F2 and BC1 populations from crosses between Ethiopian mustard lines N2-3591 and C-101.
Number of seeds with oleic acid N2-3591 97 78 175 70 N2-3591 263 282 545 74 1:3 0.73 2.13 0.19 2.67 P 0.39 0.14 0.66 0.10 0.11 0.74 2 1:1 P

2 360 134 36 147 45 120 17 470 26 2 360 125 34 141 40 128 15 475 26

N number of analyzed plants; n number of analyzed seeds; 18:1 oleic acid; 18:2 linoleic acid; 18:3 linolenic acid; 22:1 erucic acid. Fatty acids, given as mean standard deviation, are expressed in g kg1 seed oil.

108

CROP SCIENCE, VOL. 43, JANUARYFEBRUARY 2003

vey, 1963). The use of this technique will accelerate breeding efforts for this trait. Moreover the simple, monogenic inheritance of increased oleic acid in high-erucic acid Ethiopian mustard will facilitate the transfer of the trait to zero-erucic acid germplasm and the development of a Ethiopian mustard oil similar to the canola oil quality profile.
REFERENCES
Ackman, R.G., and F.M. Loew. 1977. The effects of high levels of fats rich in erucic acid (from rapeseed oil) or cetoleoic and cetelaidic acids (from partially hydrogenated fish oil) in a short-term study in a non-human primate species. Fetten Seifen Anstrichmitt. 79:1524. ndez-Serrano, and J. Ferna ndez-Escobar. 1991. Alonso, L.C., O. Ferna The outset of a new oilseed crop: Brassica carinata with low erucic acid. p. 170176. In D.I. McGregor (ed.) Proceedings of the 8th International Rapeseed Congress, Saskatoon, Sask., Canada. 911 July 1991. Groupe Consultatif International de Recherche sur le Colza (G.C.I.R.C.) (GCIRC). Auld, D.L., M.K. Heikkinen, D.A. Erickson, J.L. Sernyk, and J.E. Romero. 1992. Rapeseed mutants with reduced levels of polyunsaturated fatty acids and increased levels of oleic acid. Crop Sci. 32: 657662. nguez, R. Garc a-Ru z, L. Velasco, M. Del R o, De Haro, A., J. Dom ndez-Mart nez. 1998. Registration of six J. Mun oz, and J. Ferna Ethiopian mustard germplasm lines. Crop Sci. 38:558. Downey, R.K. 1964. A selection of Brassica campestris L. containing no erucic acid in its seed oil. Can. J. Plant Sci. 44:295. Downey, R.K., and B.L. Harvey. 1963. Methods of breeding for oil quality in rape. Can. J. Plant Sci. 43:271275. ndez-Mart nez, J.M., M. Del R o, L. Velasco, J. Dom nguez, Ferna and A. De Haro. 2001. Registration of zero erucic acid Ethiopian mustard genetic stock 25X1. Crop Sci. 41:282. s, R., and M. Mancha. 1993. One-step lipid extraction and fatty Garce acid methyl esters preparation from fresh plant tissues. Anal. Biochem. 211:139143. Getinet, A., G. Rakow, J.P. Raney, and R.K. Downey. 1994. Development of zero erucic acid Ethiopian mustard through an interspecific cross with zero erucic acid Oriental mustard. Can. J. Plant Sci. 74: 793795. Kirk, J.T.O., and R.N. Oram. 1981. Isolation of erucic acid-free lines of Brassica juncea: Indian mustard now a potential oilseed crop in Australia. J. Aust. Inst. Agric. Sci. 47:5152. Kondra, Z.P., and P.M. Thomas. 1975. Inheritance of oleic, linoleic and linolenic acids in seed oil of rapeseed (Brassica napus). Can. J. Plant Sci. 55:205210. Rakow, G., and D.I. McGregor. 1973. Opportunities and problems in modification of levels of rapeseed C18 unsaturated fatty acids. J. Am. Oil Chem. Soc. 50:400403. Ru cker, B., and G. Ro bbelen. 1997. Mutants of Brassica napus with altered seed lipid fatty acid composition. p. 316318. In Proc. 12th Int. Symp. Plant Lipids, Toronto, Canada, 812 July 1996. Kluwer Academic Publishers, Dordrecht, The Netherlands. Scarth, R., and P.B.E. McVetty. 1999. Designer oil canola. A review of food-grade Brassica oils with focus on high oleic, low linolenic types. In N. Wratten and P.A. Salisbury (ed.) Proc. 10th Int. rapeseed Congr., Canberra, Australia. 2629 Sept. 1999. Groupe Consultatif International de Recherche sur le Colza (G.C.I.R.C.) (GCIRC). CD ROM. Schierholt, A., and H.C. Becker. 2001. Environmental variability and heritability of high oleic acid content in winter oilseed rape. Plant Breed. 120:6366. Schierholt, A., H.C. Becker, and W. Ecke. 2000. Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.). Theor. Appl. Genet. 101:897901. Schierholt, A., B. Ru cker, and H.C. Becker. 2001. Inheritance of high oleic acid mutations in winter oilseed rape (Brassica napus L.). Crop Sci. 41:14441449. Shahidi, F. 1996. Natural antioxidants: an overview. p. 111. In F. Shahidi (ed.) Natural Antioxidants. Chemistry, health effects, and applications. AOCS Press, Champaign, IL.

Fig. 2. Histogram of oleic acid concentration in the BC1 to the Ethiopian mustard mutant N2-3591.

mutated allele in N2-3591, conferring increased oleic acid levels has been designated ol. No previous studies have been reported on inheritance of altered oleic acid concentration in Ethiopian mustard. In zero-erucic acid rapeseed, several sources of high oleic acid content have been developed (Wong and Swanson, 1991; Auld et al., 1992; Ru cker and Ro bbelen, 1997). Ru cker and Ro bbelen (1997) reported that a single gene controlled high oleic acid content in the seed oil of rapeseed mutants, which was confirmed by Schierholt and Becker (2001). Marker analysis supported that fad2 (microsomal oleic acid desaturase) was the gene altered in the mutants (Schierholt et al., 2000). In a comparative analysis of eight high oleic acid mutant lines of rapeseed, Schierholt et al. (2001) identified a mutation at a second locus, present only in one of the lines, which affected the oleic acid concentration in the seed oil. Zero-erucic acid Ethiopian mustard germplasm developed so far possess an average oleic acid concentration of 330 g kg1 (Alonso et al., 1991; Getinet et al., ndez-Mart nez et al., 2001), which is about 1994; Ferna half the oleic acid concentration of standard canola oil (average oleic acid 610 g kg1; Scarth and McVetty, 1999). Such a low oleic acid concentration is associated with a high degree of polyunsaturation in the oil, which has a marked detrimental effect on its oxidative stability (Scarth and McVetty, 1999). As a consequence, breeding for increased oleic acid levels is an important goal for developing higher quality Ethiopian mustard oil. Despite a partial maternal effect for oleic acid concentration, this trait appears to be under embryogenic control, which suggests that selection for increased oleic acid can be efficiently conducted at the single-seed level by means of the half-seed technique (Downey and Har-

VELASCO ET AL.: INHERITANCE OF INCREASED OLEIC ACID IN ETHIOPIAN MUSTARD

109

Stefansson, B.R., F.W. Hougen, and R.K. Downey. 1961. Note on the isolation of rape plants with seed oil free from erucic acid. Can. J. Plant Sci. 41:218219. Tatum, V., and C.K. Chow. 1992. Effects of processing and storage on fatty acids in edible oils. p. 337351. In C.K. Chow (ed.) Fatty acids in foods and their health implications. Marcel Dekker, Inc., New York. Thomas, P.M., and Z.P. Kondra. 1973. Maternal effects on the oleic, linoleic, and linolenic acid content of rapeseed oil. Can. J. Plant Sci. 53:221225.

ndez-Mart nez, and A. De Haro. 1997. Induced Velasco, L., J.M. Ferna variability for C18 unsaturated fatty acids in Ethiopian mustard. Can. J. Plant Sci. 77:9195. Velasco, L., F.D. Goffman, and H.C. Becker. 1998. Variability for the fatty acid composition of the seed oil in a germplasm collection of the genus Brassica. Genet. Res. Crop Evol. 45:371382. Wong, R.S.C., and E. Swanson. 1991. Genetic modification of canola oil: high oleic acid canola. p. 153164. In C. Haberstrohn and C.F. Morris (ed.) Fat and cholesterol reduced foods: technologies and strategies. Portfolio Publ. Co., The Woodlands, TX.