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Published online 8 September 2006
Published in Crop Sci 46:2195-2199 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP BREEDING & GENETICS

Genotype x Environment Interactions, Heritability, and Trait Correlations of Sinapate Ester Content in Winter Rapeseed (Brassica napus L.)

Thomas zum Felde, Heiko C. Becker and Christian Möllers*

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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Improving the meal and protein quality for feed and food purposes is of increasing importance in canola (Brassica napus L.). The phenolic acid ester content contributes to the bitter taste, astringency, and dark color of rapeseed meal products. The predominant phenolic acid esters are sinapate esters (SE), which make up 1 to 2% of the seed dry matter. The objective of the present study was to analyze the genetic variation and the genotype x environment interactions for SE content and composition in three populations of doubled haploid lines. The populations were grown in three to four environments in Germany. The following SE were analyzed by HPLC: sinapoylcholine (sinapine), sinapoylglucose, and a minor group of other SE which includes sinapate. The three populations showed a highly significant variation for the total SE content, and sinapine was the predominant sinapate ester compound. The analysis of variance showed highly significant effects for the genotype (G), the environment (E) and the G x E interactions for all three populations. In two of the populations the G x E interaction variance components were less than half of the genetic variance, in one population it was slightly higher. The estimates for heritability of the individual and total SE were generally high and ranged from 0.57 to 0.93. A reduction of sinapate ester content was not associated with a change in oil, protein, and glucosinolate content.

Abbreviations: E, environment • G, genotype • SE, sinapate esters


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OIL SEED rape is the most important oil crop in temperate regions and ranks second amongst oilseed crops produced worldwide. Besides being cultivated for its high oil content, the oil extracted meal is a valuable feed stuff. It contains up to 40% protein, which has a well-balanced amino acid composition (Ohlson, 1978) and high biological value (Campbell et al., 1981). Since the worldwide demand for food grade vegetable proteins is steadily increasing, attempts to improve the quality of the rapeseed protein have been started (Leckband et al., 2002). Soybean [Glycine max (L.) Merr.] derived proteins are currently dominating the food market, and any other vegetable protein has to meet existing quality standards to achieve a significant market share. Attempts to use rapeseed proteins in food production has been limited by the presence of undesirable compounds, like glucosinolates, tannins of the black seed coat, and phenolic acid esters. The seed glucosinolate content has been drastically reduced to contents of 10 µmol g–1 seed and below (Raney et al., 1999) by conventional plant breeding, taking advantage of spontaneously arisen mutants. Oilseed rape lines with a yellow seed coat have also been developed and are currently used in breeding programmes to develop high yielding yellow seeded B. napus cultivars (Rahman, 2001). Compared to these achievements little has been done to reduce the phenolic acid ester content in rapeseed, which has been reported to be about 30 times higher than in soybean (Kozlowska et al., 1990; Shahidi and Naczk, 1992).

The phenolic compounds in canola seeds are predominantly sinapate esters. The most prominent one is sinapoylcholine (sinapine) followed by sinapoylglucose. Minor contents are reported for sinapate and other sinapate esters (Kozlowska et al., 1990; Shahidi and Naczk, 1992). Sinapate and the derived esters make up 1 to 2% of the seed dry matter (Bell, 1993) and contribute to the bitter taste, astringency, and dark color of rapeseed products (Sosulski, 1979; Ismail et al., 1981). Being oxidized during seed oil processing, sinapate esters may form complexes with proteins, thus lowering the digestibility of rapeseed meal (Kozlowska et al., 1990; Shahidi and Naczk, 1992; Naczk et al., 1998). This indicates that the reduction of sinapate ester content could be a substantial requirement for establishing oilseed rape as a source for food grade protein. Specific breeding programmes aimed at developing canola cultivars with low sinapate ester content have so far not yet been started. However, several studies report on the existence of a large genetic variability of sinapate ester content and composition in seeds of B. napus (Kerber and Buchloh, 1980; Kräling et al., 1991; Bouchereau et al., 1991; Wang et al., 1998; Velasco and Möllers, 1998). More recently, transgenic approaches have been successful to reduce the sinapate ester content much below the level naturally found (Nair et al., 2000; Hüsken et al., 2005a, 2005b). With the development of a Near Infrared Reflectance Spectroscopical (NIRS) calibration an efficient and nondestructive tool for the identification of genotypes with a low sinapate ester content in breeding programs is available (Velasco et al., 1998). However, only very little is known about the G x E interactions (Kräling et al., 1991;Wang et al., 1998) and possible correlations to other agronomically important seed quality traits. The objective of the present study was to analyze the relative importance of genotypes and environments, and their interactions on sinapate ester content and composition in three populations of doubled haploid lines of oilseed rape and to analyze correlations to agronomically relevant seed quality traits.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Field Experiments
Three doubled haploid populations of winter rapeseed were tested in different years and at different locations. Population I consisted of 142 doubled haploid lines derived from the cross between doubled haploid lines of the two rapeseed cultivars, Mansholt's Hamburger Raps and Samourai (Uzunova et al., 1995; Gül, 2002). Population I was grown at two locations in 1999 and 2000 in a randomized block design with two replications. In 1999 the two locations were two fields at Reinshof (4 km south of Göttingen, Germany); in 2000 one location was Reinshof, the other was Weende (5 km northwest of Göttingen). Population II consisted of 49 doubled haploid lines from the cross between the high oleic acid mutant line 19508 and the low linolenic acid line 2293E. Population II was tested in 2000 at Hohenlieth (located in Northern Germany), Reinshof, and Weende in a randomized block design with three replications. Population III consisted of 46 doubled haploid lines derived from the cross between the line Sva 0565 and a doubled haploid line derived from the cv. Samourai. Population III was tested in 2001 and 2002 at Reinshof and at Hohenlieth in a randomized block design with two replications. In Population I, seeds from three open pollinated plants were harvested, in Populations II and III, three plants per plot were selfed. Seeds of the plants were bulked for analysis.

Analysis of Sinapate and Sinapate Esters
The method applied for the analysis of sinapate and sinapate esters was based on zum Felde (2005). The two major sinapate esters, sinapoylglucose and sinapoylcholine (sinapine), and sinapate were identified by HPLC chromatographic comparison with standard compounds. Seed material (20 mg) was extracted with 400 µL of a methanol–water mixture (4:1) in 2-mL safe-lock tubes by vigorous shaking in the presence of zirconia beads (1 mm in diameter) using a bead beater (Bio Spec Products, Bartlesville, OK). The resulting homogenates were cleared by centrifugation and aliquots of the supernatants transferred into HPLC autosampler vials. Reversed phase HPLC (Gynkotec HPLC with UV-detector) was performed using a 5-µm Nucleosil C18 column (250 by 3 mm i.d., Macherey-Nagel, Düren, Germany). A 20-min linear gradient was applied at a flow rate of 1.2 mL min–1 from 10 to 90% solvent B (acetonitrile) in solvent A (1.5% o-phosphoric acid in water). Sinapate esters were photometrically detected at 330 nm and quantified by external standardization with authentic compounds. Sinapate and the other minor SE were identified by their for sinapate specific absorption maxima at 240 and 330 nm. Their total content was calculated as milligrams per gram of sinapate and their sum is given as other SE. Total sinapate and SE content was calculated as sinapate equivalents (mg g–1 sinapate). Oil, protein, and glucosinolates were determined in whole seed by near-infrared reflectance spectroscopy (NIRS) using the calibration raps2001.eqa developed by Tillmann (2006).

Statistical Analysis
Analysis of variance was performed by the Plant Breeding Statistical Program (PLABSTAT, Version 2N [Utz, 2006]) using the following model: Yijk = µ + gi + ej + rjk + geij + {varepsilon}ijk with Yijk = observation of genotype i in environment j in replication k, µ = general mean, gi = effect of genotype i, ej = effect of environment j, rjk = effect of replication k in the environment j, geij = G x E interaction of genotype i with environment j, {varepsilon}ijk = residual error of genotype i in environment j in replication k. All factors were considered as random. Broad sense heritability (h2) for mean values over environments was calculated following Hill et al. (1998) from components of variance: h2 = {sigma}2g/({sigma}2g + {sigma}2ge/E + {sigma}2{varepsilon}/ER) with {sigma}2g, {sigma}2ge, and {sigma}2{varepsilon} as variance components for g, ge, and {varepsilon}, and E and R number of environments and replicates, respectively, using PLABSTAT (Utz, 2006).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All three doubled haploid populations showed a significant variation for the total sinapate ester (total SE) content (Table 1). The largest variation for total SE content was found in Population I, which ranged from 5.30 to 9.93 mg sinapate g–1 seed. This population also showed the largest variation for sinapoylglucose, sinapine and for sum of the other SE. In all three populations sinapine was the predominant sinapate ester compound (Table 2). Calculated on the basis of sinapate, the mean relative sinapine contents ranged from 58% in Population III to 73% in Population II of the total SE content. The mean sinapoylglucose content contributed between 17% in Population II and 20% in Population III to the total SE content. The other SE content accounted for 10% in Population II over 18% in Population I to 21% in Population III of the total SE content. Within the doubled haploid populations a significant variation for the contribution of the individual SE constituents to the total SE content was found (Table 2). For instance, in the doubled haploid lines of Population I the relative sinapine content ranged from 49% to 81%.


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  		Table 1. Variation (mg g–1) for the content of sinapate esters (SE) in three doubled haploid populations of rapeseed (Brassica napus L.)

 

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  		Table 2. Relative contents of sinapate esters (SE; %, sum = 100%) in three doubled haploid populations of rapeseed (Brassica napus L.), calculated on the basis of sinapate.

 
For all three populations the analysis of variance showed highly significant effects for the genotype, the environment and the G x E interactions (Table 3). For Populations I and III the variance components showed for all traits a predominant effect of the genotype in comparison to the G x E interaction effect. The opposite was observed for Population II. Here, the G x E variance components were much bigger in comparison to the genotypic effects. In general, the experimental error varied with the constituent, but was high for total SE in all three populations. Accordingly, high heritabilities for all traits were obtained in Population I and III, whereas only medium to low heritabilities were found in Population II (Table 4). High heritabilities for all three populations were obtained for the glucosinolate content. The heritability for oil content was only high in Population I and II. Comparatively lower heritabilities were found for the protein content in all three populations.


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  		Table 3. Components of variance of the sinapate ester (SE) content (mg g–1) in three doubled haploid populations of rapeseed (Brassica napus L.)

 

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  		Table 4. Heritability for the content of sinapate esters and other traits in three doubled haploid populations of rapeseed (Brassica napus L.).

 
The coefficients of correlations among the different sinapate esters and the oil, protein and glucosinolate content for the three populations are shown in Table 5. The total SE content was highly significantly positively correlated with the contents of the individual SE constituents. Between sinapine and sinapoylglucose significant negative correlations were found in Populations II and III. In all three populations the other SE were not significantly correlated to sinapine but were highly significantly positively correlated to sinapoylglucose. Except for the other SE in Population II, there was no significant correlation between the oil content and any of the individual constituents or the total SE content, although highly significant genotypic differences in oil content were found in each of the three populations (data not shown). Similar results were found for the correlations to protein content. No correlation between the glucosinolate content and the total SE were found in all three populations, although highly significant genotypic variation was found (data not shown). In Population II and III a significant positive correlation between sinapine and glucosinolates was found. In Population III a highly significant negative correlation between sinapoylglucose, the other SE and the glucosinolate content was found. Population II segregated for oleic acid (18:1), linoleic (18:2), and linolenic acid (18:3; data not shown). Here, 18:1 was highly significantly positively correlated with sinapine (r = 0.56, P ≤ 0.01) and highly significantly negatively correlated with sinapoylglucose (r = –0.55, P ≤ 0.05). Opposite correlations were found for 18:2 (r = –0.55, P ≤ 0.01 and r = 0.45, P ≤ 0.01, respectively). No correlation was found to the other SE and total SE content. In contrast to this, 18:3 was highly significantly positive correlated to the other SE (r = 0.60, P ≤ 0.01) and to the total SE (r = 0.49, P ≤ 0.01), but no significant correlations to 18:1 and 18:2 were found.


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  		Table 5. Spearmans coefficients of correlation for sinapate esters (SE) and the oil, protein and glucosinolate (GSL) contents in three doubled haploid populations of rapeseed (Brassica napus L.).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study revealed a large variation and highly significant genotypic differences for the total SE content in three different winter rapeseed populations. The largest variation for total SE was found in Population I, which varied from 5.3 to 9.9 mg sinapate g–1 seed. Calculated on the basis of sinapine, this variation (0.9–1.7%) is about the same as the one reported by Bouchereau et al. (1991) for seed samples from 202 different B. napus types (0.8– 1.5%, winter and spring types, oil and forage types, B. napus var. rapifera Metzg.). Analyzing 1361 samples of rapeseed breeding lines by NIRS using a calibration which was based on reference values for total SE content obtained from a photometric test (Velasco et al., 1998), Velasco and Möllers (1998) reported on a range of total SE content from 0.5 to 1.8%. Using HPLC, zum Felde (2005) determined a variation of total SE content ranging from 0.3% to 1.3% in a set of 549 genotypic diverse winter rapeseed samples. Although these results fit quite well to each other, in all three reports nonreplicated seed samples were analyzed and the material has not been analyzed again after repeated testing in the field. Those data are of limited value because the total SE content has been reported to be considerably influenced by environmental conditions (Bouchereau et al., 1991; Wang et al., 1998). This was also confirmed in the present study, in which highly significant effects of the environment and G x E interactions were found. Therefore, the mean values obtained from several environments in the present study represent a more reliable estimation of the genotypic contents and variation. In Population II the mean sinapine content accounted for 73% of the total sinapate content. This is quite similar to the 70% reported by Bouchereau et al. (1991) and the 65% reported by zum Felde (2005). However, the results of this study show that there is a large genetic variation for the relative contents of the SE constituents among the DH lines of the three populations (Table 2).

High heritabilities were found for individual sinapate esters and for total SE content in Population I and III (Table 4). Population II showed medium heritabilities for sinapate esters, which may be explained by comparatively smaller genotypic variance components in relation to the G x E interactions (Table 3). Although the variation for SE contents in Population II was similar to Population III, the G x E interactions were much larger in Population II. This could be due to the different environments or may be specific for the tested material. It is noteworthy, that Population III showed low heritabilities for oil and protein content. This has been explained by adverse weather conditions and a relatively large experimental error (see Marwede et al., 2004). These conditions, however, obviously did not affect the heritabilities for secondary compounds like sinapate esters, glucosinolates (Table 4), and tocopherols (Marwede et al., 2004).

The correlations between total SE content and sinapine, sinapoylglucose, and the other SE were all positive and of a medium to high size. This indicates that reductions in total SE can be achieved by crossing genotypes with a low total SE content and with low contents of individual SE constituents. Similar and in some cases even closer correlations among the individual sinapate esters and the total SE content were found by Hüsken et al. (2005a, 2005b), when analyzing segregating transgenic T2-plants with a drastically reduced total SE content (up to 83% reduction). The correlations among the individual SE can be explained by the biochemical pathway of these compounds. Sinapoylglucose is not only the direct precursor of sinapine, but it is likely also the precursor of the other SE (Milkowski et al. [2004], and references therein). Analyzing the T2-plants cultivated in the greenhouse Hüsken et al. (2005a, 2005b) did not find any indication that other important agronomic traits, like oil, protein, fatty acid, and glucosinolate content of the seeds are affected by decreasing sinapate ester accumulation. These results have been confirmed in the present study with nontransgenic material, in which no significant correlation between total SE and oil, protein, and glucosinolate content was found.

In conclusion, genotypic differences and medium to high heritabilities for the total SE indicate that an effective selection for low sinapate ester genotypes in a cultivar development program would be possible with a comparatively low effort with respect to the number of required test environments. There is no evidence that a reduction of sinapate ester content coincides with a change in other relevant seed quality traits. The NIRS technology together with the calibration equations developed by zum Felde (2005) provide an effective analytical tool, because it allows the nondestructive, fast and sufficiently accurate determination of the sinapate ester content, simultaneously to the prediction of other relevant seed quality traits like oil and glucosinolate content and fatty acid composition.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Alfred Baumert (IPB Halle) for providing his HPLC protocol for the analysis of sinapate and sinapate esters, for providing sinapoylglucose as standard, and for providing a HPLC chromatogram that allowed peak identification. We thank Dr. Wolfgang Ecke and Dr. Kemal Gül for providing seeds of Population I, Dr. Antje Schierholt for providing seeds and data on fatty acid content determined by gas chromatography of Population II, and Nicole Ritgen-Homayounfar and Uwe Ammermann for technical assistance. Many thanks to Norddeutsche Pflanzenzucht Hans-Georg Lembke KG (NPZ), Hohenlieth, for carrying out field experiments. This work was part of the research project "NAPUS 2000—Healthy Food from Transgenic Rape Seeds"; the financial support provided by the Bundesministerium für Bildung und Forschung (BMBF) is gratefully acknowledged.

Received for publication March 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





This Article
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Right arrow Canola

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