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Genotype x environment interactions and heritability of tocopherol contents in canola.

TOCOPHEROLS ARE IMPORTANT natural antioxidants that inhibit fatty acid peroxidation in vegetable oils and act as free radical quenchers. Tocopherols contain a tocochromanol ring and exist in four forms ([alpha]-, [beta]-, [gamma]-, and [delta]-tocopherol) differing in both molecular structure and biological effectiveness. Tocopherols are synthesized only by plants and are important dietary nutrients. All four tocopherols are collectively known as vitamin E; [alpha]-tocopherol has the highest vitamin efficacy, while [gamma]-tocopherol is a strong antioxidant for oxidation-sensitive fatty acids in vegetable oil products (Pongracz et al., 1995; Kamal-Eldin and Appelqvist, 1996). Because of the nutritional value and the importance for oil stability, tocopherol content in seed oil is considered as a value-added compound. In canola, tocopherol proportions of 65% [gamma]-tocopherol and 35% [alpha]-tocopherol are commonly found in the seed oil. Amounts of [delta]-tocopherol are <1% and [beta]-tocopherol is absent (Dolde et al., 1999; Goffman and Becker, 2001). The variation in total tocopherol content ranges from about 80 to 1000 mg [kg.sup.-1] in seed oil (Marquard, 1976; Coors and Montag, 1988; Abidi et al., 1999; Dolde et al., 1999). However, this large variation in tocopherol content is partly due to environmental factors (Dolde et al., 1999; Marquard, 1976, 1990). Previous research revealed a variation of 230 to 1000 mg [kg.sup.-1] for total tocopherol content in seed oil of single plants grown in the same environment and [alpha]/[gamma]-tocopherol ratio was ranged from 0.54 to 1.70 (Marwede et al., 2003). To evaluate genotype X environment interactions and heritability of [alpha]-, [gamma]-, and total tocopherol content and [alpha]/[gamma]-tocopherol ratio, three doubled haploid populations of different genetic origin were grown in three to four field environments. Also, correlations between tocopherol content and other seed quality traits were investigated.

MATERIALS AND METHODS

Plant Material

Three doubled haploid populations of canola were grown over several years and locations. Population 1 consisted of 144 doubled haploid lines derived from the cross between doubled haploid lines of the two canola cultivars, Mansholt's Hamburger Raps and Samourai (Uzunova et al., 1995; Gul, 2002), and the parental lines were grown at two locations in 1999 and 2000 in a randomized block design with two replicates. In 1999, the two locations were two fields at Reinshof (4 km south of Gottingen, Germany), in 2000, one location was Reinshof, the other was Weende (5 km northwest of Gottingen).

Population 2 included 49 doubled haploid lines from the cross between a high oleic acid mutant line 19508 and the low linolenic acid line 2293E, grown in 2000 at Hohenlieth, Reinshof, and Weende in a randomized block design with three replicates.

Population 3 consisted of 46 doubled haploid lines of the cross between the winter canola line 'Sva 0565' and cv. Samourai tested together with the parental lines, grown in 2001 and 2002 at Reinshof and Hohenlieth in a randomized block design with two replicates.

In Population 1, seeds from three open pollinated plants were harvested; in Populations 2 and 3, three plants/plot were selfed. Seeds of the plants were bulked for analysis.

Chemical Analysis

About 1 g of air-dried seed was milled and tocopherols were extracted with isooctane. Tocopherol analysis was performed with isocratic HPLC using a C-18 diol column as described by Thies (1997). The mobile phase was a mixture of isooctane and tert-butyl-methylether (94/6, v/v) at a flow rate of 0.7 mL/ min. The eluate was monitored with a fluorescence detector ([[lambda].sub.ex] = 295 nm and [[lambda].sub.em] = 330 nm). Tocopherols were identified by comparison of retention times and area values with inner standard [beta]-tocopherol. Total tocopherol content was calculated as the sum of [alpha]-, [gamma]-, and [delta]-tocopherol and expressed in tocopherol contents in air-dried seed. Oil, protein, and glucosinolate contents were determined in whole seed by near-infrared reflectance spectroscopy (NIRS).

Statistical Analysis

Analysis of variance was performed by the Plant Breeding Statistical Program (PLABSTAT), Version 2N (Utz 1997) using the following model:

[Y.sub.ijk] = [mu] + [g.sub.i] + [e.sub.j] + [r.sub.jk] + [ge.sub.ij] + [[epsilon].sub.ijk]

with [Y.sub.ijk] = observation of genotype i in environment j in replication k, [mu] = general mean, [g.sub.i] = effect of genotype i, [e.sub.j] = effect of environment j, [r.sub.jk] = effect of replication k in the environment j, [ge.sub.ij] = genotype x environment interaction of genotype i with environment j, [[epsilon].sub.ijk] = residual error of genotype i in environment j in replication k.

All factors were considered as random. Broad sense heritability ([h.sup.2]) for mean values over environments was calculated following Hill et al. (1998) from components of variance:

[h.sup.2] = [[sigma].sup.2.sub.g]/([[sigma].sup.2.sub.g] + [[sigma].sup.2.sub.ge]/ E + [[sigma].sup.2.sub.[epsilon]]/ER)

with [[sigma].sup.2.sub.g], [[sigma].sup.2.sub.ge], and [[sigma].sup.2.sub.[epsilon]] as variance components for g, e, and [epsilon], and E and R number of environments and replicates, respectively.

RESULTS AND DISCUSSION

Table 1 shows the variation of [alpha]-, [gamma]-, and total tocopherol contents and [alpha]/[gamma]-tocopherol ratio in three doubled haploid canola populations. Means of populations varied between 78 and 97 mg [kg.sup.-1] for [alpha]-tocopherol, while [gamma]-tocopherol ranged between 148 and 187 mg [kg.sup.-1]. The range observed for total tocopherol content was between 234 and 274 mg [kg.sup.-1] and [alpha]/[gamma]-tocopherol ratios varied between 0.43 and 0.58. Population means for oil content ranged between 438 and 447 g [kg.sup.-1], for protein content between 195 and 205 g [kg.sup.-1], and for seed glucosinolate content between 17.6 and 43.8 [micro]mol [g.sup.-1].

Table 2 gives the variance components from the analysis of variance. Significant or highly significant genotypic differences occurred for all traits, but genotype x environment interaction effects were always larger than the genotypic variance except for total tocopherol content in Population 3. A relatively high experimental error was found for all traits in the three populations.

Table 3 indicates the low heritabilities for tocopherols between 0.23 for [alpha]-tocopherol and 0.50 for [gamma]-tocopherol in population 1. The heritabilities were higher for all other seed traits except for protein content in population 3. Seed yield, which was only measured in Population 2, had a higher heritability than tocopherol content.

The analysis of the three doubled haploid canola populations revealed highly significant genetic variation, but identified genotype x environment interactions as a major source of variation for tocopherol traits. This resulted in a low heritability for all tocopherol traits. Consequently, more locations or years are required to evaluate different genotypes for tocopherol content as compared to oil, protein and glucosinolate contents. The heritability for oil- and protein content in Population 3 was notably lower compared to Populations 1 and 2. The reason for this lies in a relatively large experimental error for these traits (data not shown). In 2002, at Reinshof and Hohenlieth treatment plants experienced adverse weather conditions during flowering and ripening, which probably affected number of seeds per plant and seed quality. Under such stress conditions, there might have been greater nongenetic variation among plants of the same genotype and the sampling procedure used could be improved: only three plants per plot were harvested and sampling a larger number of plants per plot should be recommended to reduce the experimental error.

In agreement with the results of this study, earlier studies also identified genotype x environment interactions as being a major factor in determining tocopherol variation (Marquard, 1976). However, these results were derived from the comparison of a small number of cultivars. Environmental conditions, temperature in particular, strongly affected tocopherol contents and tocopherol composition. Marquard (1990) pointed out that tocopherol contents have strong interactions with temperature and light exposure. Dolde et al. (1999) reported the great effect of environmental conditions, temperature in particular, when discussing breeding efforts to modify tocopherol contents.

The [alpha]-tocopherol content was not significantly correlated with any other quality trait except for a negative correlation with oil content in Population 2 (Table 4). The same was true for [gamma]-tocopherol except for a negative correlation with oil content in Population 1 and glucosinolates in Population 2. Total tocopherol content showed a highly significant positive correlation with [alpha]- and [gamma]-tocopherol in all populations.

The correlations reported here were based on the comparison of homozygous doubled haploid lines of three populations grown under diverse environmental conditions. An advantage of this experimental approach compared with the comparison of different cultivars is the possibility of analyzing genetic correlations between traits within segregating populations. The correlations obtained when comparing a set of cultivars are more coincidental and strongly affected by genotype-specific responses to environments, while correlations among doubled haploid lines within populations are mainly of genetic origin.

This study revealed no significant correlation between [alpha]- and [gamma]-tocopherol in any of the populations. This suggested that [alpha]- and [gamma]-tocopherol synthesis is independently regulated. Newton and Pennock (1971) found that [alpha]-tocopherol was predominantly found in chloroplasts, while other tocopherol derivatives were located elsewhere in the cell, suggesting different sites for tocopherol synthesis and no interactions between the formation of [alpha]- and [gamma]-tocopherol, although [gamma]-tocopherol is known to be the direct precursor of [alpha]-tocopherol in tocopherol synthesis (Schultz, 1990). Goffman et al. (1999) observed no decrease of [gamma]-tocopherol in favor of [alpha]-tocopherol in their study of tocopherol accumulation in developing seeds of canola.

Individual tocopherols and total tocopherol content was not significantly correlated with oil content in most cases, and if correlations were observed, they were below 0.4 and of little practical importance (Table 4). No correlation of tocopherols with oil content or oil composition were also observed by Dolde et al. (1999) and Abidi et al. (1999). Goffman and Becker (2001) reported no correlation of [alpha]-tocopherol content and oil content in nine out of 10 [F.sub.2] winter canola populations, while [gamma]-tocopherol content and oil content were positively correlated in five of the [F.sub.2] populations, which, however, were grown in only one environment.

In conclusion, the results of this study suggest the possibility of developing canola cultivars with increased tocopherol content in the seed oil. Breeding efforts for increased or altered tocopherol levels will not affect oil content and other major quality traits, but will be hampered by low heritability because of large genotype x environment interactions.
Table 1. Variation of [alpha]-, [gamma]- and total tocopherol
contents (mg [kg.sup.-1] in air dried seeds) and [alpha]/
[gamma]-tocopherol ratio in three doubled haploid canola
populations.

            [alpha]-Tocopherol     [gamma]-Tocopherol

                        Population number

             1      2       3       1       2       3

Mean        84.9   78.2    96.7   148.1   186.8   175.1
Min         76.2   71.1    87.0   128.8   159.8   148.9
Max        104.4   86.6   108.4   179.5   217.1   198.3
LSD 0.05     9.6    8.9    11.8    17.0    30.2    20.8

                                    [alpha]/[gamma]-
              Total tocopherol      Tocopherol ratio

                         Population number

             1       2       3      1      2      3

Mean       234.1   267.4   274.0   0.58   0.43   0.57
Min        206.9   233.8   250.0   0.46   0.36   0.48
Max        268.2   306.5   299.2   0.78   0.53   0.72
LSD 0.05    20.6    33.9    22.8   0.09   0.08   0.11

Table 2. Components of variance of [alpha]-, [gamma]-, total
tocopherol contents (mg [kg.sup.-1] in air dried seeds) and
[alpha]/[gamma]-tocopherol ratio in three doubled haploid
canola populations.

                                                           [gamma]-
                              [alpha]-Tocopherol          Tocopherol

                                     Population number

                            1         2          3            1

[[sigma].sup.2.sub.G]
  ([dagger])              3.6 *     8.8 **    11.2 *       37.9 **
[[sigma].sup.2.sub.GE]
  ([double dagger])      23.8 **   14.7 **     3.6 (NS)    66.2 **
[[sigma].sup.2.
  sub.[epsilon]]
  ([section])            47.4      48.3      135.1        166.3

                          [gamma]-Tocopherol     Total tocopherol

                                     Population number

                            2          3          1          2

[[sigma].sup.2.sub.G]
  ([dagger])              65.2 *     49.7 **    38.9 **    75.3 *
[[sigma].sup.2.sub.GE]
  ([double dagger])      215.7 **    58.5 **   102.9 **   288.9 **
[[sigma].sup.2.
  sub.[epsilon]]
  ([section])            397.8      324.1      234.1      443.3

                           Total            [alpha]/[gamma]-
                         tocopherol         Tocopherol ratio

                                     Population number

                             3           1         2         3

[[sigma].sup.2.sub.G]
  ([dagger])              62.1 **      7.1 **    6.8 **   10.9 **
[[sigma].sup.2.sub.GE]
  ([double dagger])      -20.6 (NS)   21.2 **   10.4 **   25.9 **
[[sigma].sup.2.
  sub.[epsilon]]
  ([section])            572.7        35.4      41.4      68.8

* Significant at P [less than or equal to] 0.05.

** Significant at P [less than or equal to] 0.01.

(NS) nonsignificant at P [less than or equal to] 0.05, F-test
from analysis of variance

([dagger]) [[sigma].sup.2.sub.G]  = genetic variance.

([double dagger]) [[sigma].sup.2.sub.GE] = variance of genotype
x environment interaction.

([section]) [[sigma].sup.2.sub.[epsilon]] = residual error.

Table 3. Heritability for tocopherol and other traits in three
doubled haploid canola populations.

                                     Trait

                     Tocopherol                           Oil

                                         [alpha]/
Population   [alpha]   [gamma]   Total   [gamma]          %DM

1             0.23      0.50     0.41      0.42     0.90 ([dagger])
2             0.46      0.36     0.34      0.46     0.88
3             0.39      0.47     0.48      0.42     0.56

                                 Trait

                 Protein

Population         %DM         Glucosinolates    Yield

1            0.76 ([dagger])   0.95 ([dagger])
2            0.70              0.91              0.81
3            0.43              0.93

([dagger]) Data provided by Gul (2002).

Table 4. Coefficients of correlation for [alpha]-, [gamma]- and
total tocopherol content, [alpha]/[gamma]-tocopherol ratio and
other quality traits in three doubled haploid canola populations.

                                                [gamma]-
                        [alpha]-Tocopherol     Tocopherol

                             Population number

                       1       2         3         1

[alpha]-Tocopherol                             -0.002
[gamma]-Tocopherol
Total tocopherol
Oil content          -0.13   -0.30 *   -0.18   -0.35 **
Protein content       0.05    0.09     -0.12    0.17
Glucosinolates       -0.08   -0.11     -0.21    0.05

                         [gamma]-
                        Tocopherol       Total tocopherol

                               Population number

                        2        3        1          2

[alpha]-Tocopherol   -0.11     -0.06    0.39 **    0.39 **
[gamma]-Tocopherol                      0.91 **    0.96 **
Total tocopherol
Oil content           0.23     -0.06   -0.35 **    0.13
Protein content      -0.18      0.05    0.17      -0.14
Glucosinolates       -0.30 *   -0.10    0.02      -0.30 *

                       Total             [alpha]/[gamma]-
                     tocopherol          Tocopherol ratio

                                  Population number

                         3           1          2          3

[alpha]-Tocopherol    0.42 **      0.62 **    0.54 **    0.71 **
[gamma]-Tocopherol    0.88 **     -0.77 **   -0.76 **   -0.72 **
Total tocopherol                  -0.46 **   -0.54 **   -0.32 *
Oil content          -0.14         0.25 **   -0.41 **   -0.07
Protein content      -0.002       -0.14       0.24      -0.10
Glucosinolates       -0.18        -0.09       0.19      -0.03

* Significant at P [less than or equal to] 0.05.

** Significant at P [less than or equal to] 0.01.


ACKNOWLEDGMENTS

We thank Priv. Doz. Dr. W. Ecke and Dr. M. K. Gul for providing data on population 1. This work was funded by Bundesministerium fur Bildung und Forschung (BMBF) through the research project "NAPUS 2000-Gesunde Nahrungsmittel aus transgener Rapssaat". The support of Deutsche Saatveredelung, Thule and Norddeutsche Pflanzenzucht, Hohenlieth in conducting of field experiments is gratefully acknowledged.

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Volker Marwede, Antje Schierholt, Christian Mollers, and Heiko C. Becker *

V. Marwede, C. Mollers and H.C. Becker, Institute of Agronomy and Plant Breeding, Georg-August-University Gottingen, Von-Siebold-Str. 8, 37075 Gottingen, Germany; Antje Schierholt, Ernst Benary Samenzucht GmbH, Postfach 1127, 34331 Hann. Munden, Germany. Received 13 May 2003. * Corresponding author (hbecker1@gwdg.de).
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Title Annotation:Crop Breeding, Genetics & Cytology
Author:Marwede, Volker; Schierholt, Antje; Mollers, Christian; Becker, Heiko C.
Publication:Crop Science
Date:May 1, 2004
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