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Crop Science 42:31-36 (2002)
© 2002 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Inheritance and Interaction of Low Palmitic and Low Linolenic Soybean

Valerio S. Primomoa, Duane E. Falka, Gary R. Ablettb, Jack W. Tannera and Istvan Rajcan*,a

a Dep. of Plant Agriculture, Crop Science Division, Univ. of Guelph, Guelph, ON, N1G 2W1 Canada
b Ridgetown College, Univ. of Guelph, Ridgetown, ON, N0P 2C0 Canada

* Corresponding author (irajcan{at}uoguelph.ca)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Decreasing the palmitic and linolenic acid content of soybean [Glycine max (L.) Merrill] oil would help improve its nutritional quality and oxidative stability. The altered fatty acid profile in soybean germplasm lines with decreased levels of palmitic and linolenic acid have been developed at the University of Guelph, Canada, by combining different mutant alleles through hybridization. The objectives of this study were to determine the inheritance and interaction of palmitic and linolenic acid levels in RG3 and RG1, and the effects of these altered fatty acid levels on other fatty acids. RG3 and RG1 (low palmitic {approx}45 g kg-1 and linolenic {approx}40 g kg-1) were crossed reciprocally to several soybean lines with altered or normal fatty acid profiles. Analysis of the reciprocal F2 generations indicated no maternal or cytoplasmic effects for palmitic or linolenic acid content. Chi-square analyses of the F2 generation demonstrated that RG3 and RG1 contained two alleles, fap1 and fapx, that controlled palmitic acid content. In addition, RG1 had a third allele, fan, which reduced its linolenic acid content. Calculation of gene substitution values indicated additive gene action at the fap1 and fan loci, whereas the fapx locus involved partial dominance. Correlation coefficients indicated no association between palmitic and linolenic acid. Decreases in palmitic and linolenic acid content were associated primarily with increases in linoleic acid content. Since fap1, fapx, and fan were inherited independently of each other and appeared to behave in an additive manner, RG3 and RG1 can be used in breeding programs as additional valuable sources of germplasm with altered fatty acid profiles.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
SOYBEAN is the most widely grown source of edible oil for human consumption. It is consumed predominantly in the form of margarine, shortening, and salad and frying oils. Palmitic acid has been shown to increase blood cholesterol levels by increasing the undesirable LDL (low-density lipoprotein) (Khosla and Sundram, 1996), which has been associated with increased risk of heart disease (Hu et al., 1997), and may also contribute significantly to higher rates of breast, colon, and prostate cancers (Harvei et al., 1997). Although soybean oil has a moderate level of palmitic acid (110 g kg-1), further reduction of this saturated fatty acid would improve the nutritional quality by lowering the total palmitic acid intake.

By comparison, the relatively high level of linolenic acid (80–100 g kg-1) in commercial soybean is considered an unstable component responsible for poor flavor and undesirable oil odor, particularly in cooking oils. Partial hydrogenation is used to enhance soybean oil stability by reducing the linolenic acid level to 30 g kg-1. However, this process is expensive and generates transisomers of unsaturated fatty acids, which are associated with an increased risk of coronary disease (Beare-Rogers, 1995). Decreasing the linolenic acid content of soybean oil genetically could provide a mechanism to increase its shelf life and, eliminate or reduce the need for hydrogenation.

Natural genetic variability for palmitic and linolenic acid content in commercial soybean is limited. The most effective method for modifying fatty acid composition in soybean oil has been by mutagenesis (Sigurbjörnsson, 1983). In the past decade, several single locus mutants that affect specific fatty acid levels have been identified. Examples of reduced palmitic acid mutants are germplasm lines C1726 (Erickson et al., 1988), A22 (Schnebly et al., 1994), J3 (Takagi et al., 1995), and ELLP2 (Stojsin et al., 1998a). Examples of low linolenic acid mutants include C1640 (Wilcox and Cavins, 1985), A5 (Hammond and Fehr, 1983), A23 (Fehr et al., 1992), A26 (Ross et al., 2000), M-5 (Rahman et al., 1996), M-24 (Rahman et al., 1998), KL-8 (Rahman et al., 1996), and RG10 (Stojsin et al., 1998b). Allelism tests indicated that the low palmitic or linolenic acid levels were controlled by single major alleles at different loci in which most of them have been designated permanent names (Table 1). In the case of ELLP2 and J3, the alleles have not been assigned permanent names because their relationship to fap1 and fap3 has not been determined.


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  		Table 1. Mutants and their alleles for reduced palmitic or linolenic acid content in different soybean lines.

 
Combining mutant alleles should result in soybean lines with novel fatty acid profiles, if these loci segregate independently. Furthermore, the mutant alleles can be readily incorporated into agronomically superior soybean cultivars since simply inherited genes control them. A limited number of soybean lines with 40 g kg-1 palmitic acid have been developed by combining different fap alleles. Examples include A18 (Fehr et al., 1991; Wilson, 1991), C1943 and N94-2575 (Burton et al., 1998). One major drawback with these lines is that they are late maturing and hence not suitable for growing in southern Ontario, Canada.

Soybean oil with combined low palmitic and linolenic acid levels would be beneficial to consumers because of the reduced saturated and trans fatty acids in this oil, and the increased oxidative stability. It has been shown that fap1 segregates independently of fan (Nickell et al., 1991), but it is not known if fapx segregates independently of fan. Combining these alleles should result in a soybean line with reduced levels of palmitic ({approx}40 g kg-1) and linolenic ({approx}40 g kg-1) acids.

RG3 and RG1 are new soybean germplasm lines that provide additional sources of soybean with altered fatty acid profiles. These lines are early maturing (Group II maturity) and, therefore, suitable for growing in southern Ontario. Furthermore, the significantly improved oil quality of RG3 and RG1 are more desirable to producers and consumers, and should increase the use of soybean oil. The inheritance of the altered fatty acid content of RG3 and RG1 has not been studied. The objectives of this study were to determine the inheritance and interaction of the altered fatty acid levels in RG3 and RG1, and the effects of altered levels of palmitic and linolenic acids on other fatty acids.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
In 1987, approximately 2000 seeds of the low linolenic line C1640 were treated with ethyl methanesulfonate (EMS) as described by Stoj in et al. (1998b). The single seed descent method was used to advance this population to the M4 generation, in which individual plants were subsequently analyzed for fatty acid content. In 1992, a line designated as CLP1 was identified with a significantly lower palmitic acid level (86 g kg-1) than C1640 (100 g kg-1). Similarly, the lines ELLP2 and ELLP3 were derived by mutation of the soybean cultivar Elgin87, as described by Stoj in et al. (1998a). In 1992, the crosses CLP1 x ELLP3 and C1726 x ELLP2 were made. From 1992 to 1996, further generations were advanced in the field (Ridgetown, ON, Canada) and the greenhouse. At the F6 generation, a line with {approx}40 g kg-1 palmitic acid was designated RG3 from the C1726 x ELLP2 population, and a line with {approx}40 g kg-1 palmitic and {approx}40 g kg-1 linolenic acids was designated RG1 from the CLP1 x ELLP3 population. C1726 was developed at Purdue University (Erickson et al., 1988).

In the winter of 1998, reciprocal crosses RG3 x ELLP2, RG3 x C1726, RG3 x Elgin87, RG1 x ELLP3, RG1 x CLP1, and RG1 x Elgin87 were made. The F1 generations were planted in a growth room (28°C/22°C day/night temperatures, relative humidity of 70%, and 16-h photoperiod, with a photon flux density of about 350 mol m-2 s-2) at the University of Guelph (Guelph, ON, Canada) in the fall of 1998. Growth rooms were equipped with a mixture of fluorescent (blue and cool-white) and incandescent lights. All parental and F1 plants were grown in 25-cm-diam pots containing Sunshine LA4 Mix Aggregate Plus soil from SunGro Horticulture Inc. (Bellevue, WA). The soil was topped off with Holiday Vermiculite from Vil Vermiculite Inc. (Toronto, ON). Mineral nutrients were provided by watering with a solution containing Plant-Prod 20 (N)-20 (P)-20 (K) from Plant Products Co. Ltd. (Brampton, ON). Seeds were coated with HiStick soybean inoculant Bradyrhizobium japonicum strain 532C from Micro Bio Limited (Hemel Hempstead, UK) to promote nitrogen fixation.

A total of 127 to 200 F2 seeds of each cross were analyzed for fatty acid content using a small sample of cotyledon tissue. For fatty acid extraction, small pieces of cotyledon tissue were placed in separate glass test tubes (13 by 100 cm). The samples were dried in an oven overnight at 90°C. Each seed sample was crushed individually with a clean glass rod. To each test tube 0.6 mL of 0.25 M KOH solution (250 mL methanol, 250 mL ethyl-ether, and 7.01 g KOH) was added. The samples were vortexed for 15 s. The samples were heated in a hot water bath, at 60°C for 1 min and were allowed to cool for 5 min. To each reaction test tube 2.0 mL of saturated NaCl solution and 1.5 mL of iso-octane was added. Each sample was vortexed for 30 s. The phases were allowed to separate for 45 min. Then, 0.85 mL of supernatant was pipetted into a 12 by 32 mm (2 mL) autosampling vial capped with an aluminum seal. A Hewlett-Packard 6890 Series Gas Chromatograph (Mississauga, ON) fitted with a capillary column (15 m long and 0.25 mm diameter) from J&W Scientific (Folsom, CA) was used for fatty acid analyses. The oven, injection, and detector temperatures were adjusted to 180, 290, and 330°C, respectively. The air, hydrogen, and helium (carrier gas) flow rates were set to 400, 30, and 0.7 mL/min, respectively. Standard fatty acid mixtures (D102 and D103) from Serdary Research Laboratories (London, ON) were used as controls. Peaks were integrated and reported as normalized percentages of the fatty acids by the Hewlett-Packard ChemStation Software (Missisauga, ON).

Chi-square analyses were calculated for distribution of frequencies in the F2 generations in order to test the goodness-of-fit of the data to hypothesized genetic ratios. The distribution of frequencies for F2 seeds was assigned to classes on the basis of the palmitic or linolenic acid levels of the parents grown in the same environment. F2 seeds that had values of palmitic or linolenic acid within the range for each parent (mean ± 2 standard deviations) were considered to be of the parental genotype. Seeds with palmitic or linolenic acid levels intermediate to the parents were considered as heterozygote genotypes. Reciprocal populations were pooled for chi-square analysis because their mean values did not differ significantly. The crosses RG3 x ELLP2, RG3 x C1726, RG1 x ELLP3, RG1 x CLP1, and RG1 x Elgin87 were used to calculate relative gene substitution values as described by Stojsin et al. (1998a).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
No maternal or cytoplasmic effect for palmitic or linolenic acid content was observed in analyses of F2 seeds from the reciprocal crosses (Table 2). In all crosses, the mean F2 palmitic and linolenic acid contents were approximately intermediate between the parents. These results indicate that the genotype of the embryo of the seed determined the palmitic or linolenic acid content rather than the genotype of the maternal parent. Therefore, F2 seeds from reciprocal crosses were combined and used to evaluate the inheritance of palmitic and linolenic acid content.


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  		Table 2. Segregation for palmitic and linolenic acid among F2 seeds in six soybean crosses.

 
The evaluation of segregation in each cross was based on the frequency distribution patterns (Fig. 1) and the mean palmitic acid content (±2 SD) of the seeds from the parents grown in the same environment (Table 2). The F2 seeds from the crosses RG3 x ELLP2, RG3 x C1726, RG1 x ELLP3, and RG1 x CLP1 segregated into three palmitic acid classes (Table 2). A chi-square test on each of the four crosses indicated a satisfactory fit to a 1:2:1 ratio for palmitic acid content (Table 2), which suggested segregation at a single locus with additive effects. Because we are assuming that RG3 and RG1 have inherited the alleles fapx fapx from ELLP2 and ELLP3, respectively, the segregation is likely occurring at the Fap1 locus in the crosses RG3 x ELLP2 and RG1 x ELLP3. Similarly, segregation is probably occurring at the Fapx locus in the crosses RG3 x C1726 and RG1 x CLP1 since RG3 and RG1 also inherited fap1 fap1 from C1726 and CLP1, respectively.



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  		Fig. 1. Frequency distribution patterns of palmitic or linolenic acid in four soybean F2 populations. Black and open vertical bars represent parental and intermediate palmitic or linolenic acid phenotypic classes as described in Table 2.

 
The mean palmitic acid contents of the three phenotypic classes were compared and used to calculate gene substitution values to determine if there was additive or partial dominance inheritance at the segregating loci (Table 3). For the two crosses RG3 x ELLP2 and RG1 x ELLP3, the mean value of the intermediate category was equivalent to the mid-point value between the P1 and P2 palmitic acid groups. This indicated that there was additive gene action between the Fap1 and fap1 alleles. This was equivalent to a relative gene substitution value of 50% for each allele. In the case of RG3 x C1726 and RG1 x CLP1, the mean value of the intermediate group was not equal to the mid-point between the P1 and P2 palmitic acid families. In addition, it was observed that the intermediate class is closer to the P2 class in the frequency distribution pattern (Fig. 1b). This unequal increment of palmitic acid suggested partial dominance of Fapx over the fapx allele. The relative gene substitution values for this locus were 41 and 59%, respectively.


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  		Table 3. Gene substitution values of fap1, fapx, and fan from five soybean F2 populations.

 
RG3 and RG1 were crossed to Elgin87 (homozygous for Fap1 and Fapx) to verify that two separate loci were responsible for the reduced palmitic acid content in these soybean lines. With completely additive gene action, nine phenotypic groups segregating in a 1:2:1:2:4: 2:1:2:1 ratio would be expected for two segregating loci. The distribution frequency patterns of the F2 seeds consisted of six discrete palmitic acid classes for each of these crosses (Fig. 1c). The chi-square test indicated a satisfactory fit to a 1:2:3:5:4:1 ratio for palmitic acid content in the two crosses (Table 2), a ratio previously reported by Stojs in et al. (1998a) using the cross ELLP2 x C1726. Only six phenotypic groups were observed as opposed to nine because Fapx is partially dominant over fapx. Therefore, a 1:2:3:5:4:1 ratio suggested that the two loci are inherited independently and, hence, not linked.

The phenotypic correlation coefficients indicated that palmitic acid was negatively and significantly correlated with linoleic acid in all segregating populations (Table 4). In addition, the correlation coefficients showed that there was a significant, positive correlation with stearic acid but no significant correlation with linolenic acid. Therefore, it should be possible to decrease palmitic and linolenic acid content in soybean simultaneously, as these two fatty acids were not significantly correlated in the germplasm used.


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  		Table 4. Phenotypic correlation coefficients of palmitic and linolenic acid content with other fatty acids for six soybean F2 populations.

 
In the crosses RG1 x ELLP3 and RG1 x Elgin87, we showed that palmitic acid content was segregating at one or two loci, respectively. In the case of linolenic acid content three classes were observed for these two populations. A chi-square test for each of these populations indicated a satisfactory fit to a 1:2:1 ratio (Table 2) suggesting segregation at a single locus with additive effects. The segregation for linolenic acid content was likely occurring at the Fan locus since RG1 was homozygous for fan, and ELLP3 and Elgin87 were homozygous for Fan. Relative substitution values indicated that alleles at this locus had approximately equal effects (Table 3).

To test whether fan segregates independently of the fap alleles, palmitic and linolenic acid contents were considered together for the crosses RG1 x ELLP3 and RG1 x Elgin87 by dividing each palmitic acid class further into three linolenic acid classes (Table 5). For the cross RG1 x ELLP3, nine phenotypic groups were observed (Table 5). The interaction chi-square test indicated a satisfactory fit to the expected ratio ({chi}2 = 8.68, 0.30 < R < 0.50) indicative of independent assortment. This indicated that fap1 and fan segregate at independent loci. Because the soybean lines RG1 and ELLP3 were homozygous for fapx, segregation was not observed at this locus. The results from this study agreed with the findings in the cross C1726 x CX1022-90 made by Nickell et al. (1991) which showed that fap1 and fan behaved in an additive manner and were at separate loci. In the cross RG1 x Elgin87, 18 phenotypic groups were observed (Table 5). The interaction chi-square test indicated a satisfactory fit to the expected ratio ({chi}2 = 13.99, 0.50 < R < 0.70). The results demonstrated that the alleles at the Fan locus assort independently of the alleles at the Fap1 and Fapx loci and that the effects are additive. In addition, the segregation of linolenic acid did not interfere with the palmitic acid 1:2:3:5:4:1 segregation ratio observed in the cross RG1 x Elgin87 providing further evidence that fan segregates independently of fapx and fapx.


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  		Table 5. Frequency distribution for combined palmitic and linolenic acid in F2 seeds from the soybean crosses RG1 x ELLP3 and RG1 x Elgin87.

 
Correlation coefficients between palmitic and linolenic acids from the RG1 x ELLP3 and RG1 x Elgin87 F2 populations (Table 4) were not significant, supporting earlier reports that these two fatty acids were not significantly correlated (Brossman and Wilcox, 1984; Hawkins et al., 1983). Instead, palmitic and linolenic acids were correlated negatively and significantly with linoleic acid. As a result, RG1 has a very high linoleic acid content (69 g kg-1) in comparison to normal levels (54 g kg-1).

The reduced palmitic acid contents of RG1 and RG3 were attributed to two major alleles, fap1 and fapx, that are inherited independently and behave in an additive manner. The additive nature of the fan allele with the fap alleles and the lack of interaction between palmitic and linolenic acids made it possible to combine these traits in the line RG1. To our knowledge, this is the first published report on the inheritance of a soybean line with a combined fatty acid profile {approx}45 g kg-1 palmitic and {approx}40 g kg-1 linolenic acids; however, Burton and Wilson (personal communication) have released a new low palmitic low linolenic variety in 2000. Preliminary results from a continuing study have suggested that fap3 is at the same locus as fapx. Therefore, additional mutant alleles that segregate independently of fapx and fapx are required if breeders are to further decrease palmitic acid by traditional breeding methods. Incorporating other fan alleles such as fan2 or fan3 should reduce the low linolenic acid level of RG1 to 1%. Alternatively, fap alleles combined with fan-b should also improve the fatty acid profile of soybean oil.

Agronomic traits and genotype x environment interactions of RG1 and RG3 have been studied (Primomo et al., 2002). Although the results indicated that the fatty acid profiles are stable over several locations and years, RG1 and RG3 did not yield as high as commercially available varieties. However, it has also been reported that decreases in palmitic and linolenic acids do not significantly reduce seed yield (Horejsi et al., 1994; McClure, 1999). Further studies should concentrate on increasing seed yield in these novel soybean lines. Perhaps, future research may involve crossing of RG3 and RG1 to high yielding cultivars to improve seed yield and other important agronomic traits, which would make these unique soybean lines more appealing and profitable for producers and the industry.


   ACKNOWLEDGMENTS
 
The technical assistance of Yesenia Salazar and Julia Zilka is greatly appreciated. We thank Dr. J.R. Wilcox for providing C1726 and ‘Century’, and Dr. W.R. Fehr for providing ‘Elgin87’. Dr. Bruce Luzzi is acknowledged for his initial encouragement and suggestions provided to the senior author. Appreciation is extended to the Ontario Ministry of Agriculture, Food, and Rural Affairs, the Ontario Soybean Growers, and the Natural Science and Engineering Research Council of Canada (NSERC) for their generous financial support.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This work was submitted by V.S. Primomo in partial fulfilment for the M.Sc. degree from the Dep. of Plant Agriculture, Crop Science Division at the Univ. of Guelph.

Received for publication October 11, 2000.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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