Crop Science 40:1538-1542 (2000)
© 2000 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
A fap7 Allele for Elevated Palmitate in Soybean
David L. Stoltzfus,
Walter R. Fehr,
Grace A. Welke,
Earl G. Hammond 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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Soybean [Glycine max (L.) Merr.] oil with elevated palmitate content may be useful for food and industrial applications requiring oil with high oxidative stability. The mutant line A30 with 
40 g kg-1 greater palmitate content than conventional soybean oil was developed by treating seeds of the line A89-144026 with N-nitroso-N-methyl urea. This study was conducted to determine the genetic control of elevated palmitate content in A30. Crosses were made between A30, its parent, and lines possessing the fap1, fap2-b, fap3, fap4, fap5, or fap6 alleles for altered palmitate. The F1 seeds from reciprocal crosses between A30 and A89-144026 did not exhibit maternal effects or dominance for palmitate content. The phenotypic and genotypic analyses of F2 seeds and F3 progeny indicated that A30 had an elevated palmitate allele, designated fap7, with additive gene action at a single locus. The fap7 locus was independent of fap1, fap2-b, fap3, fap4, and fap5, but closely linked to the fap6 locus. The new allele can be used in combination with other alleles that control fatty ester composition to obtain unique oils for possible food and industrial applications.
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INTRODUCTION
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THE PALMITATE CONTENT of soybean oil has been altered by the formation of mutant alleles through the use of chemical mutagens. Erickson et al. (1988) and Fehr et al. (1991a) described the development of the mutant lines C1726 (fap1) and A22 (fap3), each of which contain an allele for reduced palmitate content of 70 g kg-1. Fehr et al. (1991b), Schnebly et al. (1994), Stoltzfus et al. (2000b), and Narvel et al. (2000) described the mutant lines A21 (fap2-b), A24 (fap4), A27 (fap5), and A25 (fap6), each of which contain an allele for elevated palmitate content of 160 to 200 g kg-1. Soybean oil with 250 g kg-1 palmitate was developed by combining the fap2-b and fap4 alleles (Fehr et al., 1991b). Oil with elevated palmitate has greater oxidative stability for food and industrial applications than conventional soybean oil that has only 110 g kg-1 palmitate.
A mutant line A30 with 160 g kg-1 palmitate was developed at Iowa State University by treatment of seeds of line A89-144026 with N-nitroso-N-methyl urea. The inheritance of elevated palmitate in the mutant line has not been reported. The purpose of this research was to describe the inheritance of elevated palmitate in the mutant line A30.
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Materials and methods
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A30 was crossed with C1726, A21, A22, A24, A27, and A25 at the Agricultural Engineering and Agronomy Research Center near Ames, IA, during 1996. The soil type at the location is a Nicollet loam (Fine-loamy, mixed, superactive, mesic Aquic Hapludoll). C1726 has the fap1 allele and A22 the fap3 allele for reduced palmitate. A21 has fap2-b, A24 fap4, A27 fap5, and A25 fap6 for elevated palmitate. A reciprocal cross was made between A30 and its parent, A89-144026, to evaluate maternal effects and dominance of the alleles that control palmitate content.
A randomized complete-block design was used to analyze the palmitate content of the F1 and parent seeds. Each replication consisted of a hybrid seed and a selfed seed from each parent. The seeds were cut into two portions with a razor blade, and the portion of the seed that lacked the embryonic axis was used for fatty ester analysis. Fatty ester content was measured by gas chromatography as described by Hammond (1991).
The portion of the F1 and parent seeds containing the embryonic axis were planted at the Iowa State University-University of Puerto Rico research nursery at Isabela, PR, in October 1996. The soil type is a Coto clay (Very-fine, koalinitic, isohyperthermic, Typic Haplorthox). Each F1 and parent plant was harvested individually. A random sample of 110 F2 seeds from the reciprocal cross and 188 F2 seeds from each A30 x mutant cross were cut into two portions and analyzed for palmitate content in Ames during January 1997. Five seeds from each of six plants of each parent grown in the same environment also were split and analyzed. The portion of the F2 and parent seed containing the embryonic axis were planted in Puerto Rico in February 1997. Each F2 and parent plant was harvested individually. A progeny test of 11 individual F3 seeds of 50 random F2 plants and five seeds from each of six plants of each parent were analyzed for palmitate content for the genotypic evaluation of the F2 plants.
Analysis of the F2 data indicated that the loci in the A30 x A25 cross may be linked. An additional 1007 F2 seeds from the cross were cut into two portions and analyzed. The portion of the seeds containing the embryonic axis and seeds of the parents were grown at the Agricultural Engineering and Agronomy Research Center in 1998. An F2 seed was considered a transgressive segregate if its palmitate content was either less or greater than both parents. Each F2 and parent plant was harvested individually. Eleven individual F3 seeds from each F2 plant identified as a transgressive segregate and five seeds from each of six plants of the two parents were analyzed for palmitate content.
The evaluation of segregation in each cross was based on the mean palmitate content ± two standard deviations of the seed from the parents grown in the same environment. The F2 seeds for the A30 x A89-144026 cross were categorized for palmitate content as follows: =A89-144026; >A89-144026 to <A30; and =A30. If there was one allele for elevated palmitate and additive gene action was expressed, a 1:2:1 ratio would be expected for the three classes. The F2 seeds for the A30 x reduced palmitate crosses were categorized as follows: =Parent (P) 1; >P1 to <A30; and =A30. If the alleles for altered palmitate exhibited additive gene action and were at two independent loci, a 1:14:1 ratio would be expected. The F2 seeds for the A30 x elevated palmitate crosses were categorized as follows: < either P; =P; and > either P. If the alleles for elevated palmitate were at two independent loci and had no dominance, a 5:6:5 ratio would be expected.
The F2 plants were genotyped by analyzing the phenotype of 11 random F3 seeds from each F2 plant. For the A30 x A89-144026 cross, the expected F2 genotypic ratio would be 1:2:1 if there was an allele for elevated palmitate at one locus and no dominance: all seeds =A89-144026; segregating; and all seeds =A30. For the A30 x reduced palmitate crosses, the expected F2 genotypic ratio would be 1:4:2:4:4:1 if there were alleles at two independent loci: all seeds =P1; seeds =P1 and >P1 to <A30; all seeds >P1 to <A30; seeds =P1, >P1 to <A30, and =A30; seeds >P1 to <A30 and =A30; and all seeds =A30. For the A30 x elevated palmitate crosses, the expected F2 genotypic ratio would be 1:4:2:4:4:1 if the alleles were at two independent loci: all seeds <P; seeds <P and =P; all seeds =P; seeds <P, =P and >P; seeds =P and >P; and all seeds >P. Segregation ratios were evaluated with a Chi-square test.
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Results and discussion
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No maternal effect for palmitate content was observed in the F1 seeds of the reciprocal cross between A30 and A89-144026 (Table 1)
. The mean palmitate contents of F1 seeds were not significantly different when A89-144026 or A30 was the female parent. The lack of a maternal effect made it possible to evaluate segregation for palmitate among individual selfed seeds on a plant.
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Table 1 Mean palmitate content of F1 and parent soybean seeds from seven crosses of A30 with its parent and lines containing different fap alleles
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The major alleles for palmitate exhibited primarily additive gene action in five crosses because there were no significant differences between the average palmitate content of the F1 seeds and the midparent value (Table 1). The significant differences between the F1 and the midparent values that occurred in the crosses A22 x A30 and A27 x A30 represented minor deviations from an additive model. The lack of dominance would permit the differentiation of homozygous and heterozygous seeds, if the alleles at a locus controlled different palmitate contents.
The F2 seeds from the reciprocal crosses between A30 and A89-144026 fit a 1:2:1 ratio that would be expected for two alleles with additive gene action at a single locus (Table 2)
. Of the 50 progeny-tested F2 plants, eight were homozygous for the palmitate content of A89-144026, 34 were segregating, and eight were homozygous for the palmitate of A30. There was not a satisfactory fit to a 1:2:1 ratio
because of a lower than expected number of homozygous F2 plants. The number of homozygous F2 plants with homogeneous F3 progeny was lower than expected because only one of 11 F3 seeds outside the range of ± 2 SD of a parent mean caused the F2 plant to be classified as heterozygous. An F3 seed from a homozygous F2 plant could have a palmitate content greater or less than ± 2 SD of a parent mean due to environmental effects, analytical variation, or modifying genes (Hartmann et al., 1996; Bravo et al., 1999). The observed genotypic frequency did not satisfactorily fit the 1:14:1 ratio
for a two-loci model because the frequency of the parental types was almost threefold greater than expected. The results indicated that A30 contained a single allele for elevated palmitate. The allele in A30 was designated fap7 on the basis of the evaluation of segregation in crosses with other genotypes.
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Table 2 Segregation for palmitate content among F2 seeds from seven crosses of A30 with its parent and lines containing other fap alleles
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The evaluation of segregation from the crosses of A30 with the other mutant lines was based on a model of two independent loci each with additive gene action because each of the parents has a major allele for altered palmitate content. For most of the crosses, the segregation observed for the F2 seeds or for the progeny test of the F2 plants did not satisfactorily fit the expected ratios. When this occurred, the frequency of the parental types was used to determine if the loci were independent or linked. For independent loci, the deviations could have been caused by non-additive gene action, epistatic interactions among the major alleles, modifying genes, environmental effects, or analytical variation (Hartmann et al., 1996; Bravo et al., 1999).
The F2 seeds of the cross between A30 and C1726 (fap1) satisfactorily fit the 1:14:1 ratio that would be expected with additive alleles at two independent loci, but the progeny test of the F2 plants did not fit a 1:4:2:4:4:1 ratio (Tables 2 and 3)
. The deviations from the expected genotypic frequency were not attributed to linkage because the frequencies of the parental types should have exceeded the expected frequencies if the loci were linked, and this did not occur. The phenotypic and genotypic data indicated that the fap7 was at a locus independent of fap1.
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Table 3 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap1 Fap1 fap7 fap7) x C1726 (fap1 fap1 Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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The segregation of F2 seeds of the cross of A30 and A22 (fap3) failed to fit the 1:14:1 ratio for a two-loci model (Table 2). The progeny-tested F2 plants failed to fit the 1:4:2:4:4:1 ratio that would be expected for additive alleles at two independent loci (Table 4)
. Of the F2 plants progeny tested, only two were homozygous for the palmitate content of A22, and none were homozygous for the palmitate of A30. If the alleles in the two parents were linked, the frequency of parental types would have been greater than expected. The results indicated that the fap7 allele was independent of the fap3 locus.
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Table 4 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap3 Fap3 fap7 fap7) x A22 (fap3 fap3 Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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For the A30 cross with A21 (fap2-b), the F2 seeds failed to satisfactorily fit the 5:6:5 ratio, and the genotyped F2 plants did not satisfactorily fit the 1:4:2:4:4:1 ratio expected for two independent loci (Tables 2 and 5)
. There were F2 seeds with greater or less palmitate than either parent, and progeny-tested F2 plants that were homozygous for palmitate less than either parent. The frequency of F2 plants homozygous for the palmitate content of the parents was less than expected and indicated that the fap7 and fap3 loci were not linked. The transgressive segregates indicated that the fap7 allele was not at the fap3 locus.
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Table 5 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap2-b Fap2-b fap7 fap7) x A21 (fap2-b fap2-b Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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The segregation of F2 seeds from the cross between A30 and A24 (fap4) failed to fit the 5:6:5 ratio, and the progeny-tested F2 plants did not satisfactorily fit the 1:4:2:4:4:1 ratio (Tables 2 and 6) . Six transgressive segregates were recovered with palmitate values less than either parent, and one transgressive segregate was recovered with palmitate values greater than either parent, indicating that fap7 and fap4 were at different loci. If the two loci were linked, the frequency of F2 plants homozygous for the palmitate content of the parents would have been greater than the observed frequency. The results indicated that the fap7 and fap4 alleles were at independent loci.
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Table 6 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap4 Fap4 fap7 fap7) x A24 (fap4 fap4 Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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The F2 seeds of the cross between A30 and A27 (fap5) satisfactorily fit a 5:6:5 ratio, and the progeny-tested F2 plants fit a 1:4:2:4:4:1 ratio (Tables 2 and 7)
. The fap7 allele was at a locus independent of fap5.
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Table 7 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap5 Fap5 fap7 fap7) x A27 (fap5 fap5 Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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The cross between A30 and A25 (fap6) indicated that the fap7 allele was closely linked to the fap6 locus. The F2 seeds did not fit the 1:14:1 ratio that would be expected with additive alleles at two independent loci because only 19 F2 seeds had palmitate contents less than or greater than either parent (Table 2). Of the 50 random F2 plants that were progeny tested, none were homozygous for palmitate content less than or greater than either parent, and more than expected were homozygous for the palmitate content of the parents (Table 8)
. An additional 1007 F2 seeds were cut into two portions and analyzed for palmitate content. Only 38 of 1007 F2 seeds had a palmitate content less than or greater than either parent. In the progeny test of the 38 transgressive segregates, three had the Fap6Fap6Fap7Fap7 genotype for normal palmitate and none had the fap6fap6fap7fap7 genotype for elevated palmitate. However, there were F2:3 lines with F3 individuals that had palmitate contents greater than either parent. The results indicated that the fap7 locus was closely linked to the fap6 locus.
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Table 8 Genotypic evaluation of 50 F2 soybean plants from the cross of A30 (Fap6 Fap6 fap7 fap7) x A25 (fap6 fap6 Fap7 Fap7) based on a model of two independent loci for altered palmitate each with additive gene action
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The fap7 allele of A30 is a new source of elevated palmitate that can be used in conjunction with other alleles that alter the fatty ester composition of soybean oil. A line with the fap2-b, fap4, fap5, and fap7 alleles has been reported with 402 g kg-1 palmitate (Stoltzfus et al., 1999a). Soybean oils with elevated palmitate content may be useful for food and industrial applications that require high oxidative stability and for production of plastic fats by interesterification (Shen et al., 1997; Kok et al., 1999).Stoltzfus Fehr Welke 2000
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NOTES
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Journal Paper No. J-18380 of the Iowa Agric. and Home Econ. Exp. Stn., Ames; Projects No. 2799 and 3107, and supported by the Hatch Act, State of Iowa, and Pioneer Hi-Bred International, Inc.
Received for publication May 17, 1999.
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REFERENCES
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- Bravo J.J., Fehr W.R., Welke G.A., Hammond E.G., Cianzio S.R. Family and line selection for elevated palmitate of soybean. Crop Sci. 1999;39:679-682.[Abstract/Free Full Text]
- Erickson E.A., Wilcox J.R., Cavins J.F. Inheritance of altered palmitic acid percentages 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 soybean seed oil. Crop Sci. 1991;31:1522-1524 b.[Abstract/Free Full Text]
- Hammond E.G. Organization of rapid analysis of lipids in many individual plants. In: Linskens H.F., Jackson J.F., eds. Modern methods of plant analysis. Vol. 12. Berlin: Springer-Verlag, 1991:321-330.
- Hartmann R.B., Fehr W.R., Welke G.A., Hammond E.G., Duvick D.N., Cianzio S.R. Association of elevated palmitate content with agronomic and seed traits of soybean. Crop Sci. 1996;36:1466-1470.[Abstract/Free Full Text]
- Kok L.L., Fehr W.R., Hammond E.G., White P.J. Trans-free margarine from highly saturated soybean oil. J. Am. Oil Chem. Soc. 1999;76:1175-1181.[ISI]
- Narvel J.M., Fehr W.R., Ininda J., Welke G.A., Hammond E.G., Duvick D.N., Cianzio S.R. Inheritance of elevated palmitate in soybean seed oil. Crop Sci. 2000;40:635-639.[Abstract/Free Full Text]
- 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., Johnson L., White P. Oxidative stabilities of soybean oils with elevated palmitate and reduced linolenate contents. J. Am. Oil Chem. Soc. 1997;74:299-302.[ISI]
- Stoltzfus D.L., Fehr W.R., Welke G.A. Relationship of elevated palmitate with soybean seed traits. Crop Sci. 2000;40:52-54 a.[Abstract/Free Full Text]
- 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 b.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
