Crop Science 43:106-109 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Inheritance of Increased Oleic Acid Concentration in High-Erucic Acid Ethiopian Mustard
Leonardo Velasco*,
José M. Fernández-Martínez and
Antonio De Haro
Instituto de Agricultura Sostenible, Apartado 4084. E-14080 Córdoba, Spain
* Corresponding author (ia2veval{at}uco.es)
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ABSTRACT
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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 N2-3591. 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 N2-3591. Oleic acid concentration of F2 seeds segregated following a 3:1 (standard–intermediate: 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.
Abbreviations: 18:1, oleic acid • 18:2, linoleic acid • 18:3, linolenic acid • 22:1, erucic acid
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INTRODUCTION
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ETHIOPIAN 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; Fernández-Martínez et al., 2001). This process, however, 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 kg-1 oleic acid, 210 g kg-1 linoleic acid, and 110 g kg-1 linolenic acid (Scarth and McVetty, 1999), whereas the average oil profile of the zero-erucic acid Ethiopian mustard oils developed so far consists of 330 g kg-1 oleic acid, 370 g kg-1 linoleic acid, and 210 g kg-1 linolenic acid (Alonso et al., 1991; Getinet et al., 1994; Fernández-Martínez et 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 N2-3591, 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.
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MATERIALS AND METHODS
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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 parental line C-101, developed at Córdoba by selection for agronomic 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. 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 by simultaneous oil extraction and methyl esterification (Garcés and Mancha, 1993) followed by gas-liquid chromatography of fatty acid methyl esters on a Perkin-Elmer Autosystem gas-liquid chromatograph (Perkin-Elmer Corporation, Norwalk, CT) equipped with a 2-m long column packed with 3% SP-2310/2% SP-2300 on Chromosorb WAW (Supelco Inc., Bellefonte, PA). A temperature program of 190°C for 10 min, increasing 2°C min-1 up to 220°C was used. The injector and flame ionization detector were held at 275 and 250°C, respectively.
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RESULTS AND DISCUSSION
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N2-3591 exhibited a higher oleic acid concentration (196 g kg-1) and a lower linoleic acid concentration (62 g kg-1) than C-101 (64 g kg-1 and 171 g kg-1, respectively) (Table 1). Linolenic acid concentration was similar in both lines, averaging 123 g kg-1 in N2-3591 and 130 g kg-1 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 kg-1 and 116 g kg-1 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).
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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.
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The average oleic acid concentration in the F1 seeds (98.5 g kg-1) was significantly (P < 0.05) different from both parents (Table 1) and was lower than the midparent value (130 g kg-1), 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 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.
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Fig. 1. Histograms of oleic acid concentration (g kg-1 oil) in F2 populations from the cross between the Ethiopian mustard lines N2-3591 x C-101 (A) and the reciprocal cross C-101 x N2-3591 (B).
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Table 2. Frequency distribution for oleic acid concentration in F2 and BC1 populations from crosses between Ethiopian mustard lines N2-3591 and C-101.
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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 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; Rücker and Röbbelen, 1997). Rücker and Röbbelen (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 kg-1 (Alonso et al., 1991; Getinet et al., 1994; Fernández-Martínez et al., 2001), which is about half the oleic acid concentration of standard canola oil (average oleic acid = 610 g kg-1; 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 Harvey, 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.
Received for publication November 26, 2001.
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ABSTRACT
INTRODUCTION
