Crop Science 40:635-639 (2000)
© 2000 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Inheritance of Elevated Palmitate in Soybean Seed Oil
James M. Narvel,
Walter R. Fehr,
Jane Ininda,
Grace A. Welke,
Earl G. Hammond,
Daniel N. Duvick and
Silvia R. Cianzio
Dep. of Food Science and Human Nutrition, Iowa State Univ., Ames, IA 50011 USA
wfehr{at}iastate.edu
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ABSTRACT
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Elevated palmitate in soybean [Glycine max (L.) Merr.] seed oil may be useful for some food and industrial products. Four mutant alleles, fap2, fap2-b, fap4, and fap5, each elevate palmitate to 
170 g kg-1 compared with 110 g kg-1 for common soybean cultivars. A new mutant line, A25, with a palmitate content of 170 g kg-1 was developed by treatment of seeds of `Kenwood' with ethyl methanesulfonate. The objective of our study was to determine the genetic control of elevated palmitate in A25. A25 was crossed reciprocally to lines possessing fap1, fap2, fap2-b, fap3, fap4, or fap5. The analysis of reciprocal F1 and parent seeds from the crosses indicated no maternal effect or dominance for palmitate content. The phenotypic analysis of F2 seeds and the genotypic analysis of F2 plants indicated that elevated palmitate in A25 was controlled by an allele, designated fap6, at a single locus that was independent of fap1, fap2, fap3, fap4, and fap5. The combination of fap2-b, fap4, and fap6 resulted in a genotype with seeds that contained up to 398 g kg-1 palmitate.
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INTRODUCTION
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THE RELATIVE AMOUNT of saturated fatty acids in soybean oil influences its oxidative stability. Soybean oil with elevated palmitate content would have greater oxidative stability than normal oil and could reduce the need for hydrogenation in producing plastic fats, such as shortening and margarine (Shen et al., 1997). The palmitate content of common soybean cultivars is 110 g kg-1. The palmitate content in soybean has been modified through chemical mutagenesis and genotypes with elevated or reduced levels have been developed.
Erickson et al. (1988) developed the lines C1726 (fap1fap1) with reduced palmitate content (85 g kg-1) and C1727 (fap2fap2) with elevated palmitate content (175 g kg-1) by treatment of seeds of `Century' with ethyl methanesulfonate (EMS). Fehr et al. (1991b) developed the line A21 with elevated palmitate content (200 g kg-1) by treatment of seeds of `A1937' with N-nitroso N-methyl urea (NMU). They determined that elevated palmitate in this mutant was controlled by a single allele (fap2-b) that occurred at the same locus or a tightly linked locus as the fap2 allele in C1727. The line A22 (fap3fap3) with reduced palmitate content (75 g kg-1) was developed by treatment of seeds of A1937 with NMU (Fehr et al., 1991a; Schnebly et al., 1994). The line A24 (fap4fap4) with elevated palmitate content (170 g kg-1) was developed by treatment of seeds of `Elgin' with EMS (Fehr et al., 1991b; Schnebly et al., 1994). Fehr et al. (1991b) developed the line A19 with >280 g kg-1 palmitate by crossing A21 with A24 to combine the fap2-b and fap4 alleles. Wilcox et al. (1994) identified two lines, N79-2077-12 and N90-2013, that had reduced palmitate (60 g kg-1). They evaluated the genetic control of reduced palmitate in the two lines in crosses with C1726. They determined that reduced palmitate content in both lines was controlled by an allele different from fap1 in C1726. The genetic relationship of reduced palmitate in these lines with alleles at other loci that alter palmitate content has not been determined.
Two new mutant soybean lines with elevated palmitate content, A25 (170 g kg-1) and A27 (160 g kg-1) were developed at Iowa State University by treatment of seeds of Kenwood with EMS. The allele in A27 was designated fap5 that has been patented by Iowa State University (Fehr and Hammond, 1998). In an independent study at Iowa State University, the elevated palmitate content in A27 was found to be under separate genetic control from A25 and from fap1, fap2, fap3, and fap4 (Stoltzfus et al., 2000). The objective of this study was to determine if the genetic control for elevated palmitate content in A25 was at a locus different from fap1, fap2, fap3, and fap4.
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Materials and methods
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A25 was an M4 plant selection from the treatment of seeds of Kenwood with EMS. Reciprocal crosses of A25 with Kenwood, C1726 (fap1), C1727 (fap2), A21 (fap2-b), A22 (fap3), A24 (fap4), and A19 (fap2-b, fap4) were made at the Agricultural Engineering and Agronomy Research Center near Ames, IA, in July 1994. Plants of the parents used for crossing were identified and selfed seeds were harvested from a node adjacent to one from which F1 seed was obtained. A randomized complete-block design was used for the analysis of F1 and parent seeds. Each replicate consisted of one selfed seed of each of the parents and one seed from each of the reciprocal F1 hybrids (Table 1)
. Each seed was cut into two portions with a razor blade. The portion of each seed that lacked the embryonic axis, about one-third of a whole seed, was used for fatty acid analysis, as described by Hammond (1991). The portion of each seed that contained the embryonic axis was retained for planting. To determine the presence of maternal effects and dominance relationships, the palmitate contents of reciprocal F1 and parent seeds were compared by using standard analysis of variance for a randomized complete-block design.
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Table 1 Mean palmitate content of F1 seeds from the reciprocal soybean crosses and parent seeds used to study the inheritance of elevated palmitate in oil
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The part of the F1 and parent seeds with the embryonic axis was planted at the Iowa State University–University of Puerto Rico breeding nursery at Isabela, PR, in October, 1994 in the same order as used for fatty acid analysis. Each F1 and parent plant was harvested individually. For all crosses, except A19 x A25, 55 random F2 seeds from each reciprocal mating and 10 seeds of each parent were analyzed for fatty acid composition as split seeds by the procedure used for the F1 seeds. For the A19 x A25 cross, 200 random F2 seeds of each reciprocal mating and 10 seeds of each parent were analyzed for fatty acid composition. The part of each seed with the embryonic axis was planted at Puerto Rico in February 1995 and each plant was harvested individually. For the genotypic evaluation of F2 plants from all crosses except A19 x A25, up to 11 F3 seeds of each of 50 random F2 plants and 10 seeds of each parent were analyzed as split seeds. For A19 x A25, up to 11 F3 seeds from each of 200 random F2 plants and 10 seeds of each parent were analyzed.
To evaluate the segregation in each cross, a standard deviation was calculated for the palmitate content of the 10 seeds of each parent of a cross grown in the same environment as the F1 and F2 plants. The range of each parent was considered equal to ±2 SD from the mean. Segregation ratios were evaluated with the Chi-square test at P = 0.05.
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Results and discussion
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No significant differences in palmitate content were observed between the reciprocal F1 seeds of any of the crosses (Table 1). The absence of maternal effects indicated that the genotype of the seed determined its palmitate content and not the genotype of the maternal plant or cytoplasmic factors. Selection for palmitate content among individual seeds on a heterozygous plant should be possible. The mean palmitate content of the reciprocal F1 seeds was not significantly different from the midparent value for any of the crosses, indicating that the alleles for reduced or elevated palmitate acted in an additive manner (Table 1). The absence of dominance would facilitate selection of homozygous seeds or plants from a segregating population.
The segregation for palmitate content among F2 seeds of the Kenwood x A25 cross satisfactorily fit a 1:2:1 ratio (Table 2) . The progeny test of F2 plants also fit a 1:2:1 ratio, which indicated that A25 differed from Kenwood by an allele at a single locus controlling elevated palmitate (Table 3)
. The other crosses in the study were used to determine if the allele in A25 was at new locus (fap6) for elevated palmitate content.
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Table 3 Classification of 50 F2 soybean plants from the cross of Kenwood (Fap6Fap6) x A25 (fab6fap6), and expected F2 genotypes based on the model of two alleles at one locus with additive gene action
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The cross of C1726 x A25 was used to determine if the allele in A25 was at the fap1 locus. The observed phenotypic segregation among F2 seeds satisfactorily fit a 1:14:1 ratio that would be expected for the segregation of two independent loci with additive gene action (Table 2). The F3 progeny test confirmed the presence of transgressive segregates, although the genotypic frequency of F2 plants did not satisfactorily fit a 1:4:2:4:4:1 ratio (Table 4)
.
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Table 4 Classification of 50 F2 soybean plants from the cross of C1726 (fap1 fap1 Fap6 Fap6) x A25 (Fap1 Fap1 fap6 fap6), and expected F2 genotypes based on the model of two alleles each at two loci with additive gene action
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To test the relationship of the allele in A25 to the fap2 locus, the crosses of C1727 x A25 and A21 x A25 were evaluated. If the allele in A25 was at the fap2 locus, none of the F2 seeds would be expected to have palmitate contents outside the range of the parents. There were 52 out of 77 F2 seeds from the C1727 x A25 cross and 62 out of 110 F2 seeds from the A21 x A25 cross with lower or greater palmitate contents than the parents (Table 2). These results indicated that the allele in A25 was not at the fap2 locus. The observed segregation among F2 seeds from the C1727 x A25 cross satisfactorily fit a 5:6:5 ratio based on a model for alleles at two independent loci that act in an additive manner. The observed segregation pattern among F2 seeds from the A21 x A25 cross did not fit a 5:6:5 ratio because of the lower than expected number of seeds with palmitate contents greater than A21. The F3 progeny test confirmed the presence of transgressive segregates for both crosses, although the frequency of F2 plants homozygous for the mutant alleles was less than expected (Tables 5 and 6)
. It is possible that in the Puerto Rico environment in which the F2 plants were grown, the degree of expressivity for one or more of the mutant alleles was not consistent among seeds. Variation in palmitate content also may have been due to the segregation of modifier genes that influence palmitate content.
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Table 5 Classification of 50 F2 soybean plants from the cross of C1727 (fap2 fap2 Fap6 Fap6) x A25 (Fap2 Fap2 fap6 fap6), and expected F2 genotypes based on the model of two alleles each at two loci with additive gene action
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Table 6 Classification of 50 F2 soybean plants from the cross of A21 (fap2-b fap2-b Fap6 Fap6) x A25 (Fap2-b Fap2-b fap6 fap6), and expected F2 genotypes based on the model of two alleles each at two loci with additive gene action
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The relationship of the allele in A25 to fap3 was determined by the A22 x A25 cross. The segregation among F2 seeds satisfactorily fit a 1:14:1 ratio, which indicated that the allele was not at the fap3 locus (Table 2). The genotypic frequency of F2 plants satisfactorily fit the expected ratio for two independent loci that act in an additive manner (Table 7)
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Table 7 Classification of 50 F2 soybean plants from the cross of A22 (fap3 fap3 Fap6 Fap6) x A25 (Fap3 Fap3 fap6 fap6), and expected F2 genotypes based on the model of two alleles each at two loci with additive gene action
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The A25 x A24 cross was used to determine if the allele in A25 was at the fap4 locus. The segregation among F2 seeds satisfactorily fit a 5:6:5 ratio, which indicated that the allele was not at the fap4 locus (Table 2). The genotypic frequency of F2 plants satisfactorily fit the expected ratio for two independent loci that act in an additive manner (Table 8)
.
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Table 8 Classification of 50 F2 soybean plants from the cross of A24 (fap4 fap4 Fap6 Fap6) x A25 (Fap4 Fap4 fap6 fap6), and expected F2 genotypes based on the model of two alleles each at two loci with additive gene action
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The A19 x A25 cross resulted in F2 seeds with higher palmitate contents than have been reported previously (Table 2). The transgressive segregation confirmed that the allele in A25 was not at the same locus as fap2-b and fap4. On the basis of a genetic model for three independent loci with additive gene action, a 7:50:7 model would have been expected for the F2 seeds. The observed segregation did not satisfactorily fit the proposed model because of a lower than expected number of seeds with palmitate contents greater than A19 and a greater number of seeds with palmitate contents lower than A25. In the genotypic evaluation of F2 plants, the three-locus model was supported by the presence of F2 plants that produced F3 seed with palmitate contents less than A25 or greater than A19. There were 27 F2 families that produced at least one F3 seed with a palmitate content greater than A19 and 102 F2 plants that produced at least one F3 seed with a palmitate content less than A25. There were two F2 plants in which all F3 seed was less than A25, which indicated they were homozygous for the normal alleles. There were no F2 plants that had all of their F3 seed greater than A19, which may have been due to variation in expressivity of mutant alleles for elevated palmitate or the influence of modifier genes.
Our results indicated that the allele in A25 was at a different locus than the alleles previously reported and was designated fap6. The combination of the fap6 allele with the fap2-b and fap4 alleles resulted in the highest palmitate content that has been reported in soybean. The highest palmitate content observed among F2 seeds from the cross of A25 x A19 was 360 g kg-1 and among F3 seeds was 398 g kg-1. The impact of elevated palmitate on agronomic and seed traits needs to be evaluated to determine the commercial potential of this type of soybean.
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NOTES
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Journal Paper no. J-17926 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA 50011. Projects no. 2799 and 3107.
Received for publication April 5, 1998.
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REFERENCES
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- Erickson E.A., Wilcox J.R., Cavins J.F. Inheritance of altered palmitic acid percentage in two soybean mutants. J. Hered. 1988;79:465-468.[Abstract/Free Full Text]
- Fehr W.R., Welke G.A., Hammond E.G., Duvick D.N., Cianzio S.R. Inheritance of reduced palmitic acid content in seed oil of soybean. Crop Sci. 1991;31:88-89 a.
- Fehr W.R., Welke G.A., Hammond E.G., Duvick D.N., Cianzio S.R. Inheritance of elevated palmitic acid content in seed oil of soybean. Crop Sci. 1991;31:1522-1524 b.[Abstract/Free Full Text]
- Fehr, W.R., and E.G. Hammond. 1998. Elevated palmitic acid production in soybeans. U.S Patent Number 5 750 846. Date issued: 12 May.
- Hammond E.G. Organization of rapid analysis of lipids in many individual plants. In: Linskins H.F., Jackson J.F., eds. Essential oils and waxes. Modern methods of plant analysis; new series. Vol. 12. Berlin: Springer-Verlag, 1991:321-330.
- Schnebly S.R., Fehr W.R., Welke G.A., Hammond E.G., Duvick D.N. Inheritance of reduced and elevated palmitate in mutant lines of soybean. Crop Sci. 1994;34:829-833.[Abstract/Free Full Text]
- Shen N., Fehr W.R., Johnson L., White P. Oxidative stabilities of soybean oil with elevated palmitate and reduced linolenate contents. J. Am. Oil Chem. Soc. 1997;74:299-302.[Web of Science]
- Stoltzfus D.L., Fehr W.R., Welke G.A., Hammond E.G., Cianzio S.R. A fap5 allele for elevated palmitate in soybean. Crop Sci. 2000;40:647-650 (this issue).[Abstract/Free Full Text]
- Wilcox J.R., Burton J.W., Rebetzke G.J., Wilson R.F. Transgressive segregation for palmitic acid in seed oil of soybean. Crop Sci. 1994;34:1248-1250.[Abstract/Free Full Text]
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TOP
ABSTRACT
INTRODUCTION
