Crop Science 41:1731-1736 (2001)
© 2001 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Genetic Background and Environment Influence Palmitate Content of Soybean Seed Oil
G. J. Rebetzke*,a,
V. R. Pantaloneb,
J. W. Burtonc,
T. E. Carter, Jr.c and
R. F. Wilsonc
a CSIRO Plant Industry, P.O. Box 1600 Canberra ACT 2601 Australia
b Dep. Plant and Soil Science, University of Tennessee, Knoxville, TN 37901
c USDA-ARS, and Crop Science Dep., North Carolina State Univ., Raleigh, NC 27695
* Corresponding author (G.Rebetzke{at}pi.csiro.au)
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ABSTRACT
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Dietary concerns over high saturates contained in edible vegetable oils has stimulated development of soybean [Glycine max (L.) Merr.] cultivars with reduced palmitate content. Little is known of factors that might influence phenotypic expression of palmitate content among soybean populations varying for presence of a major reduced palmitate allele. The objective of this study was to investigate how environment and genetic background influence palmitate content when introducing the reduced palmitate trait into adapted backgrounds. Crosses were made between reduced palmitate germplasm, N87-2122-4 (53 g kg-1 palmitate) and normal palmitate cultivars, A3733, Burlison, Kenwood, P9273, and P9341 (103–123 g kg-1 palmitate). For each cross, F4:6 lines homozygous for major reduced or normal palmitate alleles were bulked separately into Maturity Groups (MG) II, III, IV, and V, and evaluated in 10 contrasting field environments during 1993. Palmitate content varied between 82 and 90 g kg-1 across southern U.S. and Puerto Rican environments. Much of this environmental variation was associated with changes in minimum temperature during the growing season. Genetic background effects were highly significant (P < 0.01) with cross means for palmitate content ranging between 81 and 93 g kg-1. Across different maturity groups, palmitate content of the progeny was correlated (r = 0.94–0.99, P < 0.05) with mean content of the normal palmitate parent, such that for every 1 g kg-1 palmitate increase in the normal palmitate parent there was a 0.32 to 0.51 g kg-1 palmitate increase in the progeny. Genetic background effects were presumed to be associated with action of minor alleles transmitted from the normal palmitate parent. Presence of the reduced palmitate allele was associated with significantly (P < 0.01) lower stearate (-6 to -13%) and higher oleate (+4 to +10%) contents across all maturity groups. Selection of low palmitate, high-yielding parents should further decrease palmitate content and produce correlated improvements in stearate and oleate contents to improve overall oil quality in progeny containing reduced palmitate alleles.
Abbreviations: MG, Maturity Group
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INTRODUCTION
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EPIDEMIOLOGICAL EVIDENCE and clinical studies alike have demonstrated that higher saturated fatty acid intake contributes to raised blood serum cholesterol levels, thus increasing the risk of coronary heart disease (Willett, 1994; Uusitalo et al., 1996). Public perception of this potential health issue has provoked a strong trend towards greater consumption of foods containing lower levels of saturated fats (Wilson, 1991; Uusitalo et al., 1996). In turn, the U.S. Food and Drug Authority has proposed labeling regulations indicating "low saturate" vegetable oils must contain no greater than 70 g kg-1 total saturates. Although soybean oil is relatively low in total saturates (
120–200 g kg-1), at least a minimum 50% reduction in saturated fat is needed to enhance the utility of soybean oil in this new market.
Palmitate is the predominant saturated fatty acid in soybean and most other vegetable oils (Weiss, 1983). Major alleles conditioning reduced palmitate content are available in soybean germplasm obtained from recurrent selection (Burton et al., 1994) and chemical mutagenesis (Horejsi et al., 1994; Wilcox et al., 1994). In response to consumer demand for healthful oils, soybean breeders are now actively incorporating the reduced palmitate trait into cultivar development programs (Burton et al., 1996). Given the need to transfer reduced palmitate alleles into a range of adapted, high-yielding genetic backgrounds (Wilson, 1991; Burton et al., 1996), it would be helpful to understand whether palmitate expression is contingent upon reduced palmitate genes contributed by the gene donor only, or genes present within both donor and adapted parents. The interaction of target genes with genes present within recipient genetic backgrounds can be an important consideration when introducing new traits into a commercial breeding program (Hallauer and Miranda, 1988). Further, it would be useful to document the influence of genetic background on expression of the reduced palmitate trait in different maturity zones and soybean production regions of the USA.
The influence of parental selection and genetic background on phenotypic expression for reduced palmitate content has not been reported for soybean. Earlier studies (Horejsi et al., 1994; Rebetzke et al., 1998) showed that palmitate content measured in different soybean populations reflected variation at both major and minor loci. The objective of this study was to evaluate the influence of environment and genetic background on palmitate content in soybean populations developed from crosses between reduced palmitate germplasm and normal palmitate cultivars.
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MATERIALS AND METHODS
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Reduced palmitate germplasm, N87-2122-4 (53 g kg-1 palmitate), was used as a female in crosses to randomly selected midwestern cultivars, A3733, Burlison, Kenwood, P9273, and P9341 to generate five segregating populations. N87-2122-4 is of MG V and traces its pedigree to the reduced palmitate germplasm release, N79-2077-12 (Burton et al., 1994). The parental cultivars ranged in maturity from MG II to III and represented some of the highest-yielding midwestern cultivars at the time of the study. The F1 plants were grown in Puerto Rico and harvested F2 seed sown at Clayton, NC, in 1991. A 5-g seed sample was obtained from individually harvested F2 plants and analyzed for fatty acid composition following Wilson et al. (1981). Seed containing either major alleles for reduced or normal palmitate can be genotyped from the quantity of palmitate found in their oil (Wilcox et al., 1994; Rebetzke et al., 1998). Eighty reduced and 80 normal palmitate progeny from each cross were selected and inbred through single-seed descent to the F4:5 generation. The reduced and normal palmitate classes were presumed to differ for presence or absence of the reduced palmitate allele described by Wilcox et al. (1994) and Rebetzke et al. (1998).
In 1992, a single row of each F4:5 line was grown at Clayton, NC, with MG I to VI check cultivars for the purpose of maturity designation. Seed was harvested and the putative reduced or normal palmitate genotype verified for each line by fatty acid analysis of seed oil following Rebetzke et al. (1998). Equal numbers of seed were then bulked for each cross from F4:6 lines of similar maturity (maximum maturity range in each bulk was 5 d) and either reduced or normal for palmitate content. Individual bulks were composites of between 12 and 15 lines. The inclusion of several random lines produced bulks that were putatively near-isogenic for maturity and major palmitate alleles but were random for minor palmitate alleles and alleles conditioning other traits.
The eight bulks per cross representing four maturity groups (MG I, II, IV, and V) and contrasting palmitate level were evaluated in 1993 along with parental (MG II–III) and nonparental (MG IV–V) check lines. Studies were conducted at three midwestern (Johnson City, IA; Napoleon, OH: St. Joseph, IL) and seven southern (Union City, TN; early and late sowing at Clayton, NC; Fletcher, NC; Plymouth, NC; Greenville, MS; Isabella, Puerto Rico) environments to provide a broad range of conditions during the growing season (Table 1). The MG II and III bulks were sown in the Midwest and at Union City, TN, while the MG IV and V bulks were sown in all seven southern environments. The experimental design at each location was a randomized complete block with three replicates. Three-row plots, 4.8 m in length and with 0.38 m between rows, were used at all locations except Fletcher (2.4-m-long plots); Greenville, Union City, and the Midwest (four-row plots, 6.1 m long). Seed was harvested at maturity from the center row(s) at each location. Maturity was determined for all plots as the date at which 95% of pods in the row obtained mature color. Seed from each plot was analyzed for oil composition following Rebetzke et al. (1998) and for seed oil content using near-infrared analyses by the USDA National Center for Agricultural Utilization Research (NCAUR), in Peoria, IL. Mean daily minimum and maximum temperatures were obtained for the growing season at all sites (Table 1).
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Table 1. Environment means for seed oil quality characteristics measured in 1993 on soybean bulk populations representing soybean Maturity Groups (MG) II to V.
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A combined analysis of variance over environments was conducted on each maturity group for seed oil traits using the SAS procedure GLM (SAS Institute, 1990). Error variances for each environment were deemed homogenous following the Fmax test for homogeneity of error variances (Sokal and Rohlf, 1981). Palmitate content (reduced vs. normal bulks) was deemed a fixed effect, and crosses and environments random effects in deriving appropriate errors for statistical testing. Protected least significant differences (LSD) were calculated for comparisons among entries using the entry x environment interaction mean square as the error from the expected mean squares.
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RESULTS AND DISCUSSION
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Time to maturity averaged over bulks in all populations was 113, 123, 119, and 126 d from sowing for MG II, III, IV, and V, respectively. Resulting differences in phenology combined with the breadth of environments over which studies were conducted produced a broad range of growing conditions from which response could be observed for all oil characteristics (Table 1). In turn, growing conditions appeared to have a large effect on all traits measured. Oil quality varied considerably between midwestern and southern growing regions, and among environments within each region. Seed oil content was generally greater for soybean bulks grown in the South (Table 1).
The Midwest and southern environments were characterized by similar palmitate and stearate contents (Table 1). This contrasted with Cherry et al. (1985), who reported large differences for saturated acid content between northern and southern soybean regions. However, such contrasts were confounded with cultivar differences, while bulks in this study represented the same average genetic background, differing only for loci controlling maturity. Variability for palmitate content among locations within the South was greater than for the Midwest (Table 1). Palmitate content ranged between 82 and 90 g kg-1 among southern environments compared with 82 to 86 g kg-1 in the Midwest. Oleate content was about 25% greater in the South than in the Midwest, while reduced oleate in the Midwest was compensated for by increased desaturation to linoleate and linolenate. In turn, linolenate content was greater in the Midwest than in the South.
Variation in oil composition appeared to reflect differences in temperatures prevailing at each environment. The higher proportion of oleate in oil collected from warmer southern environments was consistent with high oleate in soybean seed developing under warmer temperatures in controlled environment (Rennie and Tanner, 1989) and sowing date (Kane et al., 1997) studies. High oleate content follows from inhibition of the oleoyl desaturase system at high air temperatures (Cheesbrough, 1989). Equally, high linolenate content at cooler temperatures follows from other studies (e.g., Wilcox and Cavins, 1992; Kane et al., 1997) and presumably reflects enhanced activity of the lineoyl desaturase system at cooler air temperatures (Cheesbrough, 1989).
Although regional effects exerted minor influence upon palmitate content, certain distinction was noted for location effects particularly in the South (Fig. 1)
. Palmitate content in MG IV and V bulks could be used to separate southern environments into three groups based on mean daily minimum temperature at each location: Fletcher, NC (15.5°C), Puerto Rico (21.9°C), and all other environments (19.1°C). When all southeastern environments were considered, mean daily minimum temperature explained 87 to 94% (P < 0.01) of the MG IV and V among-environment palmitate variance (Fig. 1). This apparent temperature response was similar to findings of Rennie and Tanner (1989), who reported a large increase in palmitate content of soybean grown from 15/12°C (day/night) to 40/30°C. Similarly, Wilcox and Cavins (1992) reported reductions in palmitate content for soybean maturing into increasingly cooler air temperatures.
There was substantial phenotypic variation for palmitate content measured in all maturity groups (Table 2). More than 95% of this variation was attributable to differences in palmitate content among tested entries. Furthermore, among-entry differences were considerably greater than differences associated with the interaction of entries with environments. The relative sizes of entry to interaction mean squares were consistent across maturity groups representing very different production regions within and outside the USA. The lower entry x environment interaction suggests that selection within maturity groups for average response across environments should produce soybean lines with desired palmitate levels. The relatively small entry x environment interaction for palmitate content was consistent with previous studies for lines and cultivars grown in multiple environments (Hawkins et al., 1983; Horejsi et al., 1994; Rebetzke et al., 1996).
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Table 2. Analysis of variance for palmitate content measured on Maturity Groups (MG) II, III, IV, and V reduced and normal palmitate bulk soybean populations.
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Much of the among-entry variation was associated with differences among progeny, particularly between reduced and normal palmitate bulks (Table 2). The latter represents differences among alleles associated with segregation at a single major reduced palmitate locus. Reduced palmitate bulks produced an average 40% less palmitate than normal bulks evaluated in all maturity groups (Tables 3 and 4). Differences in the effect of the reduced palmitate allele were consistent in size with differences observed among F5:7 soybean inbreds varying for reduced and normal palmitate content (Rebetzke et al., 1998). Progeny bulks grown in the MG II and III studies produced significantly (P < 0.01) less palmitate than their midwestern parents (Table 3). For example, P9273 and P9341 produced about 30% greater palmitate content than the mean of their reduced and normal progeny. There were also large differences for palmitate content among progeny bulks homozygous for reduced or normal major palmitate alleles (Table 2). For example, MG V reduced and normal palmitate bulks ranged between 61 and 70 and between 102 and 116 g kg-1 palmitate, respectively (Table 4). The sizes of such differences were repeatable for reduced and normal palmitate bulks grown in all maturity groups (Tables 3 and 4) and appear to reflect variation arising through minor gene differences.
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Table 3. Seed composition for checks and Maturity Group (MG) II and III reduced and normal palmitate soybean bulks grown in the midwestern USA during 1993.
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Table 4. Seed composition for checks and Maturity Group (MG) IV and V reduced and normal palmitate soybean bulks grown in the southern USA and Puerto Rico during 1993.
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Phenotypic variation was also attributed to significant (P < 0.01) differences in mean palmitate content among crosses (Table 2). Cross means ranged between 81 and 93 g kg-1 palmitate (Fig. 2) . Because a single reduced palmitate donor was used to generate all populations tested, all of the differences between cross means must have arisen through genetic differences among normal palmitate parents. This is supported by evidence gained from observations on both parents and progeny. First, large significant (P < 0.01) differences were observed among normal palmitate parents in the MG II and III studies (Table 2). Mean palmitate contents averaged over four midwestern locations were 109, 103, 123, 108, and 116 g kg-1 for parental cultivars A3733, Burlison, Kenwood, P9273, and P9341, respectively. Kenwood produced about 20% greater palmitate content than Burlison, but only 6% higher palmitate than P9341. Second, palmitate content varied among reduced and normal palmitate bulks homozygous at the major reduced palmitate locus. Relationships between palmitate content of each normal palmitate parent and mean palmitate content of its progeny were strong (r = 0.94–0.99, P < 0.05) and repeatable across bulks representing the broad range of maturity groups and environments sampled (Fig. 2). Furthermore, these relationships were linear such that there was a 0.32 to 0.51 g kg-1 palmitate increase in the progeny for every 1 g kg-1 palmitate increase in the normal palmitate parent. This suggests that development of soybean populations from normal palmitate parents producing 30 g kg-1 lower palmitate content can reduce mean palmitate content in the progeny by up to 16 g kg-1, or 1.6% of total oil.
Rebetzke et al. (1998) demonstrated that reduced palmitate content in soybean oil was conditioned by both major and minor alleles. Effects associated with modifying gene action were small but heritable in homozygous reduced and normal inbred lines for a range of crosses representing different genetic backgrounds. The linear relationship observed between parent and progeny in this study corroborated earlier evidence that phenotypic differences among parents reflect variation for the presence of minor genes. Furthermore, the strong nature of this linear relationship suggests that genetic effects are accumulated in an additive manner.
An understanding of the influence of palmitate content on seed oil quality is necessary for breeders wishing to introduce the reduced palmitate trait into soybean cultivars. Observations on associated traits were made simple in this study through use of near-isogenic bulk populations contrasting for a single major reduced palmitate allele. Significant (P < 0.01) reductions in palmitate content produced concomitant changes in oil quality (Tables 3 and 4). Oil content remained unchanged for reduced and normal palmitate bulks grown in the Midwest, while oil content was greater for reduced palmitate bulks grown in the South. Selection for reduced palmitate content produced no change in seed protein content (Tables 3 and 4) or seed quality appearance (data not shown). Stearate, the other major saturated fatty acid in soybean oil, was significantly (P < 0.05) lower, and the monounsaturated oleate significantly (P < 0.05) higher, for reduced palmitate bulks grown in both the Midwest and South (Tables 3 and 4). Furthermore, oleate was significantly (P < 0.01) greater for all bulks than commercial checks evaluated in midwestern and southern environments. Resulting changes in stearate and oleate contents through selection for reduced palmitate content should enhance the overall nutritional quality of soybean oil (Grundy, 1986). Differences in linolenate content between reduced and normal palmitate bulks were contingent on the maturity group in which bulks were evaluated. Quantity of palmitate had little or no effect on linolenate content of bulks grown in the South, while reduced palmitate bulks grown in the Midwest were generally higher in linolenate content. However, the small nature of this increase (75 vs. 80 g kg-1 oil averaged over bulks) is unlikely to have any greater affect on oil stability than for oil collected from normal bulks (Wilson, 1991).
In conclusion, any factor that increases palmitate content will increase total saturates. This study has shown that environment and genetic background have the potential to increase palmitate content above the 70 g kg-1 saturate threshold necessary for reduced saturate vegetable oils. Warmer temperatures will increase the level of palmitate in the processed oil, the extent being somewhat predictable based on regional temperatures up to physiological maturity. Breeders must also consider the effect of the high-yielding parent when making crosses aimed at the development of high-yielding, reduced palmitate cultivars. Higher palmitate cultivars will produce higher palmitate progeny owing to minor genes contributing to variation for palmitate content. Selection of lower palmitate, high-yielding parents when incorporating reduced palmitate alleles will produce populations with decreased palmitate content. Correlated improvements in stearate and oleate contents together with reduced palmitate should improve overall oil quality.
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ACKNOWLEDGMENTS
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The authors would like to acknowledge Mr. E. Huie, F. Farmer, R. McMillan, and W. Novitzky for their advice and help with aspects of this study. Thanks are also extended to Dr. C. Brownie for assistance with statistical analyses, and to Mr. W.E. Rayford and staff at NCAUR, Peoria for seed oil and protein determinations. We would also like to thank Pioneer HiBred Seeds for funding this research, and Pioneer collaborators at Greenville, MS, Johnston City, IA, Napoleon, OH, St. Joseph, IL, and Union City, TN for assistance in conducting field experiments. We would also like to thank USDA collaborators at Isabella, Puerto Rico for their assistance with aspects of this study.
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NOTES
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Cooperative investigations of the USDA-ARS, and North Carolina Agric. Res. Serv., Raleigh, NC.
Received for publication May 22, 2000.
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REFERENCES
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TOP
ABSTRACT
INTRODUCTION
) and V (
) soybean bulks grown in the southeastern USA and Puerto Rico in 1993.
) and V (