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Published online 20 May 2008
Published in Crop Sci 48:1000-1006 (2008)
© 2008 Crop Science Society of America
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Genetic Variation and Genotype x Environment Interactions of Phytosterol Content in Three Doubled Haploid Populations of Winter Rapeseed

Samija Amar, Heiko C. Becker and Christian Möllers*

Dep. of Crop Sciences, Plant Breeding, Georg-August-Universität Göttingen, Von-Siebold-Str. 8, 37075 Göttingen, Germany

* Corresponding author (cmoelle2{at}gwdg.de).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Phytosterols are natural constituents of vegetable oils and are known for their cholesterol-lowering properties. The oil of rapeseed (Brassica napus L.) is one of the richest natural sources of phytosterols. Genetically enhancing the phytosterol content could give an added value to the rapeseed oil and derived products. Our objectives were to develop a gas-liquid chromatographic method for the analysis of phytosterol content in seeds of oilseed rape, to determine the genetic variation and the genotype x environment interactions, and to estimate correlations between phytosterols and other important seed quality traits in three doubled haploid populations of winter rapeseed. The populations were tested during several years in three to four environments. Sitosterol and campesterol were detected as the two major phytosterols followed by brassicasterol, avenasterol, and stigmasterol. Large differences were found in total phytosterol content (2.57 to 4.15 g kg–1 seed), with predominant genetic variance components resulting in high heritabilities ranging from 0.84 to 0.91. Phytosterol content was not negatively correlated with oil content and there were no close correlations to protein and glucosinolate content. The large genetic variation along with high heritabilities indicate that an effective breeding for enhanced phytosterol content and modified composition should be possible without negative impacts on oil, protein, or glucosinolate content.

Abbreviations: DH, doubled haploid • GLC, gas-liquid chromatographic


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
THE ECONOMIC IMPORTANCE of canola (Brassica napus L.) has increased in the past decades because of major improvements in the seed oil content as well as in the oil and meal quality. More recently, attention has focused on minor oil-constituents like tocopherols, phytosterols, and carotenoids, which are recognized for their antioxidative, cholesterol-lowering, and other health-benefiting potentials (Marwede et al., 2004; Gordon and Miller, 1997; Shewmaker et al., 1999). Canola oil has the second highest phytosterol content among vegetable oils, only surpassed by corn oil (Gordon and Miller, 1997). The phytosterol content of rapeseed oil typically ranges between 0.5 and 1% and has been found to be about twice as high as in soybean and sunflower oil (Vlahakis and Hazebroek, 2000).

As essential components of all eukaryotic membranes, phytosterols play a vital role in membrane-associated metabolic processes. As precursors of plant hormones they are involved in plant growth and development (Hartmann, 1998). Whereas animal and fungi contain only one major sterol, cholesterol and ergosterol, respectively, plants have a variety of more than 40 different phytosterols (Law, 2000). Most abundant phytosterols are sitosterol, campesterol, and stigmasterol. Other phytosterols, like avenasterol, are synthesized earlier in the biosynthetic pathway and usually occur only in relatively smaller amounts, or are, like brassicasterol, typical for only one plant family. Phytosterols occur either in the free form, as esters with fatty acids or phenolic acids, and as conjugates with glucose (Moreau et al., 2002). Quantitatively, contents of different phytosterol forms may vary with tissue and plant species (Piironen et al., 2000).

Since high serum LDL-cholesterol level has been identified as the main risk factor for cardiovascular diseases in Western countries, efforts were undertaken by the food industry to develop functional food products enriched with natural phytosterols, thereby having a beneficial effect on health. Today, milk and dairy products fortified with natural phytosterols are available in many countries. In most cases, vegetable oils are being used as a source for phytosterol extraction; they are obtained as a by-product during vegetable oil refining. However, enhancing the phytosterol content and modifying its composition in rapeseed oil could give an added value to the oil and oil-derived products. Even so, only a limited number of studies report on genetic variation, or environmental effects on phytosterol content in oilseed rape (Appelqvist et al., 1981; Gordon and Miller, 1997; Hamama et al., 2003); whereas possible correlations between phytosterols and other seed quality traits, or inheritance of phytosterol content, have not been investigated. A possible reason could be that a rather sophisticated extraction and derivatisation method is required for phytosterol identification and that there is, so far, no simple gas-liquid chromatographic analysis protocol available for their accurate quantification, suitable for plant breeding purposes. The major objectives of this study were to develop a gas-liquid chromatographic method for accurate and high throughput analysis of phytosterol content and composition in seeds of oilseed rape, to determine the genetic variation, the genotype x environment interactions and the heritability of phytosterol content in three different doubled haploid populations of winter oilseed rape, grown in different environments. Furthermore, correlations between phytosterol content and other economically important seed quality traits were studied.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Material and Field Experiments
Three doubled haploid (DH) populations were grown in different environments over a period of several years. Population I consisted of 148 DH lines, derived from a cross between two DH lines obtained from two winter rapeseed cultivars, the French cultivar Samourai (low in erucic acid and glucosinolates) and the old Dutch cultivar Mansholt's Hamburger Raps (high in erucic acid and glucosinolates). All DH lines were tested in a field trial without additional N-fertilizer in a randomized block design with two replicates during two years at two locations. In 1999, the two locations were two fields at Reinshof (4 km southwest of Göttingen, Germany) with different soil types. In 2000, one location was Reinshof and the other Weende (5 km northwest of Göttingen). Seeds from three open-pollinated plants were harvested and bulked for the analyses (Gül, 2002). Population II consisted of 49 DH lines obtained from a cross between the high-oleic acid mutant line 19508 and the low-linolenic mutant line 2293E. Population II was grown in 2000 in a randomized block design with three replicates at three different locations, Reinshof, Weende, and Hohenlieth (northwest of Kiel, Germany). One self-pollinated plant per plot was used for the analysis. Population III was composed of 284 DH lines derived from the cross between the old German cultivar Sollux and the old Chinese landrace Gaoyou. Both cultivars have high erucic acid and high glucosinolate content. Population III was grown in 2000 at four locations, two in Germany (Reinshof and Weende) and two in China: Xian (western China) and Hangzhou (eastern China) in a randomized complete block design with two replicates. Population III showed a large segregation for oil content (Zhao et al., 2005). From this population, 20 lines each with lowest and highest oil contents and equally high erucic acid contents were selected and seeds from five self-pollinated plants per plot were bulked and used for analysis.

Analysis of Phytosterol Content and Other Quality Traits
A capillary column gas-liquid chromatographic (GLC) method was developed and used for an accurate assessment of phytosterol content and composition in a large number of seed samples. The method was based on the modified sample preparation method for quantitative analysis of tocopherols (Ulberth et al., 1992). Phytosterol extraction and preparation for the GLC was performed directly on the seeds in three major steps: alkaline hydrolysis, extraction, and derivatisation to trimethyl-silyl ethers. Seed material (200 mg) was measured on an analytical balance (0.1 mg accuracy, M2P Sartorius, Göttingen, Germany) and placed in polypropylene tubes with screw caps (11.5 cm length; 0.9 cm diam., Sarstedt, Nümbrecht, Germany) with one stainless steel rod (1.2 cm length; 0.5 cm diam.) per tube. Two-hundred µL of internal standard solution was added, prepared by dissolving cholesterol (99% purity, Sigma-Aldrich, Germany) in hexane-ethanol (3:2) solution at a concentration of 0.1%. The phytosterol standards sitosterol (40% purity) and stigmasterol (95% purity) were purchased from Sigma-Aldrich, Germany. Brassicasterol was obtained from Dr. Paresh Dutta (Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden) and avenasterol was identified by comparison of the retention time from chromatograms provided by Dr. Paresh Dutta and Dr. Ludger Brühl (Institute for Lipid Research, Münster, Germany). Since stigmasterol was present only in trace amounts (0.01 g kg–1 seed, or 0.4% from the total phytosterol amount), it is not shown separately, but was considered when calculating total phytosterol content. Alkaline hydrolysis was performed with 2 mL of potassium hydroxide (Merck, Darmstadt, Germany) dissolved in ethanol (2%). The samples were homogenized for 60 s in a Mini-Bead-Beater-8 (BioSpec Products, Inc., Bartlesville, OK, USA), with speed chosen to be as high as possible without destroying the tubes, and left for 15 min at 80°C in a water bath. Phytosterols were extracted by briefly vortexing with 1 mL hexane and 1.5 mL water. The upper hexane layer with phytosterols were transferred to a new tube and left on a hot plate at 37.5°C overnight to evaporate. Hexane (100 µL) was added to the dried pellet, transferred to vials together with 50 µL of silylating agent (10% N-methyl-N-trimethyl-silyl-heptafluor(o)butyramid in trimethylchlorosilane) and left in the oven for 15 min at 105°C ± 3°C. Capillary gas-liquid chromatograph (PerkinElmer 8420, San Jose, CA, USA), equipped with an autosampler, flame ionization detector, and split injector, was used with medium polarity, fused silica capillary column (SE-54, 50 m long, 0.1 µm film thickness, 0.25 mm i.d. coated with 5%-phenyl-1%-vinyl-methylpolysiloxane, IVA Analysentechnik, Meerbusch, Germany). The following optimized conditions were used: initial oven temperature of 240°C was increased at 5°C min–1 to final oven temperature of 265°C and held for 20 min. Total analytical time was 25 min. Injection and detection temperature was set at 320°C. Hydrogen (carrier gas) pressure was set at 150 kPa.

Seed oil (%), protein (%), and glucosinolates (µmoles g–1), were expressed on seed dry matter basis, and fatty acids (%) were determined using the Near-Infrared Reflectance Spectroscopy (NIRS) with the calibration equation raps2001.eqa developed by Tillmann (2007).

Statistical Analysis
Analysis of variance was performed with PLABSTAT software version 2N (Utz, 1997) using the following model:

Formula
Where: Yijk is observation of genotype i in environment j in replicate k; µ is general mean; gi, ej, and rjk are effects of genotype i, environment j, and replicate k in the environment j, respectively; geij is genotype x environment interaction of genotype i with environment j, and {varepsilon}ijk is residual error of genotype i in environment j in replicate k. Genotypes, environments, and replicates were considered as random variables.

Broad-sense heritability (H2) of mean values over environments was calculated using PLABSTAT software version 2N (Utz, 1997), following Hill et al. (1998) from components of variance:

Formula
Where: {sigma}2g, {sigma}2ge, and {sigma}2{varepsilon} are variance components for g, ge, and {varepsilon}, and E and R are number of environments and replicates, respectively. Genetic correlation coefficients were calculated using PLABCOV software version 1B (L) (Utz, 1994). Genetic coefficient of variation was calculated as genetic variance ({sigma}2g) divided by the mean.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Phytosterol Analysis
The developed GLC analysis protocol was suitable for the identification and quantification of sitosterol, campesterol, brassicasterol, avenasterol, and stigmasterol in seeds of oilseed rape (Fig. 1 ). Phytosterol quantification was based on the internal standard method (peak area and retention time). In the present study, as in a number of other studies (Piironen et al., 2002; Hamama et al., 2003), cholesterol was used as an internal standard, despite the small amounts present in rapeseed (Appelqvist et al., 1981). The reason for doing so was that cholesterol is structurally very similar to phytosterols and hence shows the same extraction characteristics; it completely dissolved in the hexane-ethanol mixture. One of the advantages of the present method compared with others (Dutta and Normen, 1998; Fiebig et al., 1998) was the direct alkaline hydrolysis performed on the seed-meal avoiding a separate step of quantitative oil extraction. Analysis of a reference seed sample in two other laboratories (Dr. Paresh Dutta, Uppsala, Sweden, and Dr. Ludger Brühl, Münster, Germany) proved accuracy of the developed method (data not shown). The simplified method is therefore suitable for the analysis of a large number of samples as it is the case in breeding programs. The alkaline hydrolysis as performed in the present study allowed the quantification of free phytosterols and phytosterol fatty acid esters. However, total phytosterol content may still be underestimated, because phytosterol glycosides are not detected with the present method. Their analysis would have required an additional acid-hydrolysis step (Yang et al., 2003), which has the disadvantage that it may lead to destruction of phytosterols, due to the strong acidic conditions (Piironen et al., 2000).


Figure 1
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  		Figure 1. Gas chromatogram of major phytosterols in a seed sample of the cv. Linetta with cholesterol as internal standard. Peaks: a = cholesterol; b = brassicasterol; c = campesterol; d = stigmasterol; e = sitosterol; f = avenasterol.

 
Genetic Variation
Highly significant genetic variation was found for sitosterol, campesterol, brassicasterol, avenasterol, and for total phytosterol content in all three DH populations (Table 1 ). Mean total phytosterol content among three populations ranged from 3.1 to 3.7 g kg–1 seed. The largest range of the total phytosterol content within populations was observed for Population I with 2.6 to 4.1 g kg–1 seed. Considering the oil content of the seed samples the range of phytosterol content in the oil was calculated. For all three populations it ranged from 4.5 to 9.4 g kg–1. This range compares well with the variation of phytosterol content detected in nine commercial canola lines ranging from 4.6 to 8.1 g kg–1 oil with an average phytosterol content of 5.8 g kg–1 oil (Vlahakis and Hazebroek, 2000). Gordon and Miller (1997) have analyzed two commercial rapeseed cultivars and found an average phytosterol content of 6.9 g kg–1 oil. Population I showed the largest ranges for the two most prominent phytosterols sitosterol (1.3–2.1 g kg–1 seed) and campesterol (0.6–1.5 g kg–1 seed), which is in accordance with the largest variation for total phytosterol content of this population. A tenfold variation in avenasterol content was observed in Population III. Appelqvist et al. (1981) reported more than threefold difference of avenasterol content in two summer canola cultivars.


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  		Table 1. Variation in phytosterol content (g kg–1 seed) in three DH populations of Brassica napus L.

 
Of the three DH populations, sitosterol was the most prominent phytosterol, accounting for more than 50% of the total phytosterol content (Table 2 ), followed by campesterol (28–31%), brassicasterol (11–15%), and avenasterol (3–4%). Similar relative mean contents of individual phytosterols in rapeseed were also observed in other studies (Appelqvist et al., 1981; Warner and Mounts, 1990; Hamama et al., 2003). Considerable variation in relative phytosterol composition was found within the populations, indicating the possibility to develop material with a modified phytosterol composition in breeding programs. From a nutritional point of view the contents of sitosterol should be high and the contents of campesterol should be low. Although they have equal cholesterol lowering effects, sitosterol is taken up in the small intestine to a somewhat lower extent than campesterol (Miettinen, 2001).


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  		Table 2. Relative content of phytosterols (total phytosterols = 100%) in three DH populations of Brassica napus L.

 
In all three populations the analysis of variance revealed predominant and highly significant effects of the genotypes on total and individual phytosterol content (Table 3 ). Only in Population III did the variance components show a larger effect of the environment on avenasterol and on total phytosterol content, which probably results from the considerably different test environments in China and Germany. The average amount of avenasterol content measured at the locations in Germany (0.21 g kg–1 seed) was more than three times higher than the content measured for the locations in China (0.06 g kg–1 seed), whereas the total phytosterol content in Germany (3.40 g kg–1 seed) was only slightly higher than in China (2.99 g kg–1 seed). In a previous study (Zhao et al., 2005) the same DH population showed higher average oil content for the locations in Germany (51.5%) compared with those in China (44.5%). However, at present, very little is known about the influence of specific environmental factors on phytosterol content. In one study a 2.5-fold variation in phytosterol content was detected in 12 commercial soybean lines grown in three different temperature regimes (Vlahakis and Hazebroek, 2000). It was shown that total phytosterol content increased at elevated temperatures, while its composition significantly changed with proportionally more campesterol at the expense of sitosterol. In another study with eleven canola genotypes grown during one year at two locations in the mid-Atlantic region of the United States, no effect of the environment on total phytosterol content was observed (Hamama et al., 2003).


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  		Table 3. Components of variance for phytosterol content (g kg–1 seed) in three DH populations of Brassica napus L.

 
High heritabilities in the range from 0.82 to 0.97 were found in all three DH populations for individual and total phytosterols (Table 4 ). Heritabilities in Population II were as high as in other populations, although only one self-pollinated plant per plot was used for the analysis. This confirmed the strong genetic component for phytosterol content indicating that an effective selection for high phytosterol genotypes in a cultivar development program would be possible with a comparatively low effort with respect to the number of required test environments. In all three DH populations the heritabilities were similar to those for oil (0.88–0.91) and glucosinolate content (0.89–0.95) and somewhat higher than those for protein content (0.70–0.83). Marwede et al. (2004) analyzed tocopherol content in the seed samples of the first two populations and found, as a result of large genotype x environment interactions and a large experimental error, much lower heritabilities (0.41 in Population I and 0.34 in Population II).


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  		Table 4. Heritability of phytosterol content and other quality traits in three DH populations of Brassica napus L.

 
Correlations between Traits
Genetic and phenotypic correlations between different phytosterols and other quality traits were mostly of similar size and sign (Table 5 ). In Population I and II positive correlations were detected among all individual phytosterols, while in Population III most of the individual phytosterols were not correlated to each other, with the exception of a negative correlation between campesterol and brassicasterol (rg = –0.782+). However, in all three DH populations both major phytosterols sitosterol and campesterol were positively correlated with total phytosterol content. Oil content was negatively correlated with total phytosterol content (rg = –0.672+) only in Population I. This population segregated for erucic acid content and the two erucic acid genes have been shown earlier to account for major differences in oil content (Ecke et al., 1995). In the present study a close negative correlation between total phytosterol content and erucic acid content has been found (Spearman rank rS = –0.80**). The correlation between erucic acid and oil content was positive (rS = 0.69**). Applying path coefficient analysis, Amar (2007) found no direct effect of oil content on phytosterol content in this population (for further discussion see Amar, 2007). A positive correlation (rg = 0.592+) between oil and phytosterol content was detected in Population II, segregating for oleic and linolenic acid content (data not shown). In this population, total phytosterol content was to a low extent negatively correlated with oleic acid content (rg = –0.342+) and positively correlated with linolenic acid content (rg = 0.21+). Since oil content was not significantly correlated with oleic (rg = 0.14) and linolenic (rg = –0.14) acid content, this does not provide an explanation for the positive correlation between phytosterols and oil content in this population. The correlation between phytosterol and oil content should be studied in more detail using other plant material. Comparing different canola varieties with their transgenic counterparts having a modified fatty acid composition, Abidi et al. (1999) found that total phytosterol content was decreased twofold in high oleic/low linolenic acid lines, whereas high stearic acid lines had higher levels of phytosterols than the control. In sunflower, increased phytosterol contents were found under conditions of high temperature and water stress (Roche et al., 2006). There is no evidence that increased phytosterol content is associated with a change in other relevant seed quality traits like protein and glucosinolate content since no, or only weak, correlations to protein and glucosinolate content were found. The large genotypic differences and the high heritabilities for the total and individual phytosterol content indicate that an effective selection for high phytosterol genotypes in a cultivar development program would be possible with a comparatively low effort with respect to the number of required test environments.


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  		Table 5. Coefficients of genetic rg (upper part) and phenotypic rp (lower part) correlations for phytosterol content and other quality traits in three DH populations of Brassica napus L.

 

   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical assistance of Uwe Ammermann. The authors are thankful to Dr. Kemal Gül, Dr. Antje Schierholt, and Dr. Jianyi Zhao for providing the seed samples for Populations I, II, and III, as well as to Dr. Paresh Dutta (Swedish University of Agricultural Sciences, Uppsala, Sweden) and Dr. Ludger Brühl (Institute for Lipid Research, Münster, Germany) for helpful advice regarding the method and for performing analysis of a reference seed sample. Samija Amar received a scholarship from the Hans-Böckler-Stiftung.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication October 18, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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This Article
Right arrow Abstract Freely available
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Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amar, S.
Right arrow Articles by Möllers, C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Amar, S.
Right arrow Articles by Möllers, C.
Agricola
Right arrow Articles by Amar, S.
Right arrow Articles by Möllers, C.
Related Collections
Right arrow Canola
Right arrow Crop Genetics

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