Crop Science 42:379-384 (2002)
© 2002 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Genetic Variation of Palmitate and Oil Content in a Winter Oilseed Rape Doubled Haploid Population Segregating for Oleate Content
Christian Möllers* and
Antje Schierholt
Institut für Pflanzenbau und Pflanzenzüchtung, Georg-August-Universität, Von-Siebold-Str. 8, D-37075 Göttingen, Germany
* Corresponding author (cmoelle2{at}gwdg.de)
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ABSTRACT
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Increasing the oil content and reducing the content of saturated fatty acids in seed oil are important breeding goals for rapeseed (Brassica napus L.). The objective of the study was to investigate the influence of a major change in oleate content on the palmitate and oil content. An oilseed rape doubled haploid (DH) population (n = 60) segregating for one major gene mutation, that increases the oleate content in the seed oil by 11%, was used in a field experiment in five environments. Subdividing the DH lines into normal and high oleate classes showed that the mutation caused a significant reduction in the saturated fatty acids palmitate and stearate and of the ratio of total C16 to total C18 fatty acids (C16/C18). The high oleate class had a significantly increased oil content and an identical protein content, compared with the normal oleate class. Both oleate classes showed a significant negative correlation for oleate and palmitate and a significant positive correlation for oleate and oil content, and oil content was negatively correlated with palmitate. The results show that the high oleate mutation has a pleiotropic effect on palmitate and oil content. The ß-ketoacyl-acyl carrier protein (ACP) synthase II (KAS II) activity reflected by the C16/C18 ratio has been used to explain this effect. Results indicated that palmitate content can be reduced either by recurrent selection for increased oleate content or continued selection for high oil content.
Abbreviations: ACP, acyl carrier protein • DAG, diacylglycerol • DH, doubled haploid • DM, dry matter • KASII, ß-ketoacyl-ACP synthase II • PC, phosphatidyl choline • PA, phosphatidic acid • rS, Spearman rank • TAG, triacylglycerol
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INTRODUCTION
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THE SEED OIL of modern oilseed rape cultivars contains 
60% oleate (C18:1), 20% linoleate (C18:2), 10% linolenate (C18:3), and small amounts of palmitate (C16:0, 4%) and stearate (C18:0, 2%) (Lühs and Friedt, 1993). During the past decade, one major goal in oilseed rape quality breeding has been to increase oleate at the expense of polyunsaturated fatty acids linoleate and linolenate. High oleate rapeseed cultivars would produce a highly nutritional oil with additional markets and potential applications as an industrial raw material. From a nutritional point of view, the content of the saturated fatty acids should also be as low as possible (Willett, 1994; Scarth and McVetty, 1999). Food products with <7% total saturated fatty acids (C16:0 + C18:0 + C20:0 + C22:0) can be promoted as "low in saturated fatty acids" in the USA (Scarth and McVetty, 1999).
Breeding material and cultivars with oleate contents of 75 to almost 90% have been developed not only in rapeseed (Wong et al., 1991, 1998; Auld et al., 1992; De Bonte and Hitz, 1998; Schierholt and Becker, 2001) but also in other important oil crops, like sunflower (Helianthus annuus, Fernández-Martínez et al., 1993), and soybean (Glycine max L. Merr., Kinney et al., 1998). Some authors have reported a negative correlation between palmitate and oleate content in the seed oil, for example in sunflower (Miller et al., 1987; Fernández-Martínez et al., 1993) and in soybean (Carver et al., 1986; Rebetzke et al., 1996, 1998). Kinney et al. (1998) reported that transgenic high oleate soybean had reduced levels of both palmitate and stearate. Following chemical mutagenesis in rapeseed, Auld et al. (1992) found in M3 seeds that higher concentrations of palmitate and stearate were associated with lower levels of oleate (r = -0.67, P < 0.01 and r = -0.53, P < 0.01 respectively). Similar results also were reported by Schierholt and Becker (2001) for a rapeseed DH population segregating for oleate content.
In the complex pathway of triacylglycerols biosynthesis, palmitate has different fates. One key enzyme is the ß-ketoacyl-ACP synthase II (KAS II) (Harwood, 1996; Ohlrogge and Browse, 1995). KAS II is exclusively responsible for the condensation of C16:0-ACP with malonyl-ACP to stearoyl-ACP, thus determining the C16/C18 fatty acid ratio of the seed oil (Fig. 1)
. However, palmitate may also be released from palmitoyl-ACP by an acyl-ACP-thioesterase and reesterified on the chloroplast envelope to Coenzyme A (C16:0-CoA, Fig. 1). Alternatively, palmitoyl-ACP may be used within the chloroplast by an acyltransferase to form phosphatidic acid (PA) that can subsequently be desaturated by plastidic enzymes. Hence, the palmitate content found in TAG is determined by the competitive activity of a thioesterase, an acyltransferase, and KAS II.
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Fig. 1. Simplified schematic drawing of the triacylglycerol biosynthetic pathway. ACP = acyl carrier protein; CoA = Coenzyme A; DAG = diacylglycerol; KAS II = ß-ketoacyl-ACP synthase II; PA = phosphatidic acid; PC = phosphatidylcholine; TAG = triacylglycerol.
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The objective of the present study was to investigate the influence of a major change in oleate content on the palmitate and oil content in a B. napus DH population, segregating for one major mutated gene for high oleate content in the seed oil.
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MATERIALS AND METHODS
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Two high oleate mutants, M19508 and M19566 with 74.1 and 74.6% C18:1 in the seed oil, respectively, were selected in an ethyl methanesulphonate mutagenesis program for high contents of C18:1 (Rücker and Röbbelen, 1995) of the winter oilseed rape cv. ‘Wotan’. Both mutants are allelic at the mutated locus (Schierholt et al., 2001) and have been described as one copy of the oleic acid desaturase gene (fad2; Schierholt et al., 2000). Both mutants (M3 generation) were crossed with a low oleate DH line DH11.4 Samourai (58.2% C18:1 in the seed oil) and 60 DH lines were derived from microspore culture (Möllers et al., 2000). The DH population shows a 1:1 segregation pattern with a difference in oleate content of 11% between the mean of high and normal oleate classes, respectively (Schierholt and Becker, 2001). The 60 DH lines and their parental lines were tested in 2 yr (1997–1998 and 1998–1999) across two locations in northern Germany, Göttingen and Hohenlieth, and in Thüle in 1997/1998. In each location, a randomized block design with two replications was used. Three plants were selfed in each DH line per replication and location. Bulked seed samples (200 mg) were analyzed for their fatty acid composition by gas chromatography according to Thies (1971) with minor modifications (Rücker and Röbbelen, 1996). Oil (% dry matter, DM) and protein (% DM) content of selfed seed samples (3 g) were determined by near infrared reflectance spectroscopy (Tillmann, 1997; Tkachuk, 1981) using the calibration Raps97.eqa. Fatty acids are expressed as % of total, and the C16/C18 fatty acid ratio was calculated as follows:
Analysis of variance was based on PLABSTAT (Utz, 1994), considering the available year–location combinations as five environments. The 60 DH lines, five environments, and two replicates were defined as random variables. Broad sense heritabilities (h2) were calculated as
with MSL and MSLE denoting the mean squares of DH lines and the DH lines x environment interaction, respectively. Frequency distributions were tested for normality by calculating the coefficient of skewness (g1) and the kurtosis (g2) (Snedecor and Cochran, 1980) using PLABSTAT. Furthermore, the DH population was subdivided into a high (i = 1, >65%, n = 29) and a normal oleate class (i = 2, <65%, n = 31) solely based on the 1:1 segregation pattern in oleate content of the DH population. Mean values of the genotypes across the five environments were used to calculate the Spearmen rank correlations between the traits. Means of the two classes (i = 1, 2) were tested for significant difference (Student Newman Keuls test). They were also used to calculate the ratio q to describe the size and direction of the effect of the mutation on traits:
xi and yi denoting the mean value of trait x and y for the ith class, that is, the high or normal oleate class.
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RESULTS
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Seed oleate (C18:1) content of DH lines varied from 55.7 to 74.9%. The lines also showed a wide range for palmitate (C16:0), palmitoleate (C16:1), palmitolenate (C16:2), stearate (C18:0), linoleate (C18:2), linolenate (C18:3), the ratio of the C16/C18 fatty acids and for the oil and protein contents (Table 1). While the ranges for C16:1 and C16:2 contents were low in absolute terms, they represent a 1-fold and 22-fold increase from the lowest to the highest value, respectively. The ANOVA showed highly significant variation in the DH lines for all traits (Table 2). The variance components showed a main contribution of DH lines for the traits C16:0, C16:1, C18:1, and C18:2, and the C16/C18-fatty acid ratio. The ANOVA revealed furthermore a small but significant DH lines x environment interaction for all traits with exception of C16:0 and C16:1, and a significant environmental effect except for C16:1. Estimated heritabilities were high to very high, and ranged from 0.70 to 0.99 for C16:2 and C18:1, respectively (Table 2). The frequency distribution of oleate content showed the expected 1:1 segregation for normal and high oleate genotypes (Fig. 2a)
. The frequency distribution for C16:0 content also indicates a bimodal segregation with a bias towards the low C16:0 contents (Fig. 2b), but the Chi square test did not accept a 1:1 segregation (
= 21.6). The oil content did not show a normal frequency distribution (Fig. 2c). The distribution was skewed to the low oil contents (g1 = -0.83, P > 0.01) and there was a significant excess (g2 = 1.31, P > 0.05).
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Table 1. Means and range for fatty acids, rapeseed oil and protein content, and the C16/C18 fatty acid ratio of the seeds from 60 doubled haploid lines evaluated in five environments.
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Table 2. Variance components and heritability for fatty acids, the C16/C18 fatty acid ratio, and the oil and protein content of the seeds from 60 doubled haploid rapeseed lines evaluated in five environments.
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Fig. 2. Frequency distribution of (a) oleate, (b) palmitate, and (c) oil content of the doubled haploid rapeseed population (n = 60). The dotted line marks the point of subdivision of the DH population into the two classes with low (<65%) and high ( 65%) oleate content.
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When the DH population was divided at a threshold of 65.0% oleate into two classes, one with a normal (<65.0%) and one with a high (65.0%) oleate content (Fig. 2a, Table 3), the two classes showed highly significant (P 
0.01) differences for C16:0, C18:0, C18:1, C18:2, C18:3, and the C16/C18 fatty acid ratio. The results for C18:1 clearly show that the mutation leads to an increase of 10.6% in C18:1 (Table 3). The two classes were significantly different for oil content (P 0.05) but not for protein content.
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Table 3. Means for fatty acids, the C16/C18 fatty acid ratio, and oil and protein content of the doubled haploid rapeseed lines after division into a normal (<65 g kg-1) and a high (65 g kg-1) oleate class in five environments.
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The Spearman rank (rS) correlations uncovered significantly negative correlations between C16:0 and C18:1 for the normal (rS = -0.72, P < 0.01) and the high C18:1 class (rS = -0.47, P < 0.05; Table 4). In both classes, C16:0 was negatively correlated with oil content and positively correlated with protein content, although not significantly. The negative correlation between the oil content and the C16 fatty acids increased in magnitude from C16:0 over C16:1 to C16:2. C18:1 showed a strong positive correlation with oil content in both classes. The C16/C18 fatty acid ratio was negatively correlated with the oil content in the normal and the high oleate class (rS = -0.43, P < 0.05, and rS = -0.37, respectively). Out of 45 calculated correlations for the two classes, only four did not show an identical direction, most of them were even similar in strength.
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Table 4. Spearman rank correlations and sign of the ratio q, that is, the straight line between the means of the two classes for fatty acids, and oil and protein content of the seeds from 60 doubled haploid rapeseed lines divided in two classes, one with a normal oleate content and the other with a high oleate content, evaluated in five environments.
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The results from the regression analysis of C16:0 and oil content on oleate content (Fig. 3a, b)
and of oil content on C16:0 content (Fig. 3c) gave similar slopes of the same direction for the two classes. The direction of q, that is, the direction of the straight line between the means of the two classes, was identical for these three comparisons. However, the slope of the straight line was different (Fig. 3a, b, c). Out of the 41 comparisons in which the correlations of two classes had the same direction, only four did not show an identical direction as q (Table 4).
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Fig. 3. Regression of the two classes for (a) palmitate and oleate content, (b) oil and oleate content, and (c) oil and palmitate content and the slope (q) of the straight line between the means of the two classes.
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DISCUSSION
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There are several possible explanations for the observed decrease in C16:0 content and the concomitant increase in oil content in high C18:1 rapeseed. A significantly reduced C16/C18 ratio in the high C18:1 material indicates an increased KAS II activity (Harwood, 1996). Since the condensation reaction from C16 to C18 is exclusively catalyzed by this enzyme, its activity reflects the total flux through this pathway. However, the change in the activity is low and it is very unlikely that a second mutation is closely linked to the mutated high C18:1 gene (Schierholt et al., 2000). Rather, it can be assumed that the observed changes are pleiotropic, caused by the high C18:1 mutation. If it is postulated that, for any reason, high C18:1 content leads to a higher oil content, the observed changes could be explained as follows: an increased oil content requires a higher KAS II activity; a higher KAS II activity reduces the pool of palmitoyl-ACP (Fig. 1, Table 3) leading to reduced releases by the activities of the acyl-ACP-thioesterase and the acyl-ACP-transferase, consequently leading to less palmitoyl-CoA exported from the chloroplasts. The increased flux through the pathway also explains the reduced C18:0 content in the seed oil of the high C18:1 class (Table 3). Attempts to overexpress the KAS II enzyme in soybean and rapeseed have been successful but resulted in little or no change in the relative fatty acid composition (Kinney, 1997). Hence, it appears that the KAS II enzyme itself has a low control coefficient on fatty acid flux during oil biosynthesis (Kinney, 1997).
The question is to explain how the presence of one major mutated gene leading to high C18:1 content accounts for the observed changes in the high C18:1 rapeseed. The desaturation of C18:1 to C18:2 is accomplished by a desaturase enzyme (FAD2) acting most effectively on a phosphatidylcholine derivative of C18:1 (Stymne and Stobart, 1987). The transfer of oleoyl CoA from diacylglycerol (DAG) to the phosphatidylcholine (PC) molecule is made by a specific acyl exchange enzyme. There is also an additional pathway that converts DAG to PC in a freely reversible PC-DAG interconversion process. This reaction has been proposed to control TAG synthesis (Töpfer et al., 1995). Furthermore, an exchange between the sn2-position of PC and acyl-CoA has been observed in seeds of some species that enriches the acyl-CoA pool with 18:2-CoA and 18:3-CoA, which are then available for synthesis of PA and acylation of DAG to TAG (Somerville and Browse, 1991). Since the C18:1 desaturase (FAD2) acts only on the oleoyl-PC, it can be assumed that in the high C18:1 mutant the PC-DAG interconversion process is affected which may contribute to a higher TAG synthesis. Mancha and García-Diaz (1998) provided evidence that in sunflower C18:1 can be transferred from TAG to PC and that the C18:1 desaturase activity regulates this acyl exchange. C18:1 desaturase activity may also regulate the PC-DAG interconversion process in the present DH lines. However, since fatty acids desaturated at PC may enter again the acyl pool, it is also possible that the DAG acyltransferase substrate specificity is higher with monounsaturated fatty acids than with polyunsaturated fatty acids, explaining the higher oil content. Rücker and Röbbelen (1996) did not find an increased oil content in rapeseed bulks segregating for C18:3 content, indicating that the effect of increased oil content may be confined to the first step of C18:1 desaturation.
Our observations are supported by findings of other groups working on oil composition of sunflower and soybean. Fernandez-Martinez et al. (1993) found that, in high C18:1 sunflower hybrids, the contents of both C16:0 and C18:0 were significantly lower. The mean seed yield and oil content of high C18:1 sunflower hybrids were higher or similar when compared with those of their low C18:1 isogenic counterparts.
In soybean, Rebetzke et al. (1996) observed a strong negative correlation between the C18:1 and C16:0 content and concluded that a direct selection for increases in monounsaturated C18:1 produces a concomitant reduction in C16:0 content. However, Rebetzke et al. (1998) found that selection for reduced C16:0 content produced significant increases in seed oil content only in one of two crosses within a normal C16:0 content population. The report of Kinney et al. (1998) shows that a reduced content of the saturated fatty acids C16:0 and C18:0 are also found in transgenic high C18:1 soybean plants expressing an antisense gene against the C18:1 desaturase, confirming that the reduction in saturated fatty acids is indeed associated with the high C18:1 phenotype.
The reduction of C16:0 content with increasing C18:1 contents can be drawn from regression formulas (Fig. 3a). A 10% increase in C18:1 content leads to a 1.5% and 1% decrease in C16:0 content for the normal and the high C18:1 classes, respectively. For the mutation, a 10% increase in C18:1 leads to a 0.6% decrease in C16:0 (q = -0.057; Fig. 3a), which is a considerably lower value compared with the correlated responses to selection in any of the two classes. An increase in C18:1 content by 10% leads to an increase in oil content by 3.3% and 4.1% for the normal and the high C18:1 class, respectively (Fig. 3b). In contrast to this, a 10% increase in C18:1 content caused by the mutation leads only to an increase in oil content of 1% (q = 0.094; Fig. 3b). An increase in C16:0 content by 1% leads to a decrease in oil content of 1.4% and 1% for the normal and the high C18:1 class, respectively (Fig. 3c). The mutation leads to a 1.7% decrease in oil content per 1.0% increase in C16:0 content (q = -1.667; Fig. 3c). The comparison of the size of q with the size of the regressions of the two classes indicates that additional regulatory factors affect the C16:0 content (Fig. 3a) and the oil content (Fig. 3b) in the lines carrying the mutation. However, the high correspondence between the sign of q and the sign of the correlation coefficients in the two classes (Table 4) indicates that the alleles at the mutant locus do not differ markedly in their indirect effect on fatty acid and oil content, from the indirect effect of the other, yet unknown minor genes segregating in this cross.
The present results show that a given locus responsible for a major variation in fatty acid content of the seed oil also represents a QTL for oil content. In the present DH population, the fatty acid composition can be used to predict the oil content. The situation may be more complex in crosses involving genotypes with additional QTLs for oil content that may interact in different ways.
It can thus be concluded that high C18:1 rapeseed oil has a significantly lower content of the saturated fatty acids C16:0 and C18:0, making the oil more attractive from a nutritional point of view. The content of C16:0 is negatively correlated with high C18:1 content in both the normal and the high C18:1 class, showing that the C16:0 content in rapeseed can be further reduced by recurrent selection for increased C18:1 contents. Equally, a continued selection for high oil content should contribute to a reduction of the C16:0 content. On the other hand, a selection for reduced C16:0 contents could result in higher C18:1 and higher oil contents.
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ACKNOWLEDGMENTS
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The authors are indebted to Wolfgang Link and Heiko C. Becker for their critical reading of the manuscript and many helpful suggestions, to Norddeutsche Pflanzenzucht and Deutsche Saatveredlung for carrying out field experiments, and to Fachagentur Nachwachsende Rohstoffe and Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung for financial support.
Received for publication January 30, 2001.
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ABSTRACT
INTRODUCTION
