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Inheritance of Reduced Stearic and Palmitic Acid Content in Sunflower Seed Oil.

American consumers have become concerned with reducing the saturated fat content of their diets, particularly more so in the past few years. Studies have indicated that high levels of saturated fat consumption may contribute to increased blood serum cholesterol and that high blood serum cholesterol increases the risk of coronary heart diseases (Mensink et al., 1994; Willett, 1994). Current dietary guidelines (USDA, 1992) recommend a reduction in the consumption of saturated fats by the American public as a deterrence to heart and cardiovascular disease. The saturated fatty acid content of sunflower oil is considered moderate ([approximately equals] 130 g [kg.sup.-1]) with the principal saturated fatty acids being palmitic (16:0) and stearic (18:0) acids. However, a further reduction to 60 to 80 g [kg.sup.-1] saturated fatty acids would increase consumer acceptance of sunflower oil and benefit the sunflower industry.

The induction of genetic variability by mutagenesis has altered the fatty acid content in the seed lipids of oilseed crops. Fehr et al. (1991) used chemical mutation with NMU to lower the palmitic acid content in soybean [Glycine max (L.) Merrill] seed. The allele controlling low palmitic acid found in this study, when combined with an additional allele controlling low palmitic acid reported previously (Erickson et al., 1988), produced a soybean genotype with approximately 44 g [kg.sup.-1] palmitic acid. Inheritance studies indicated that the low palmitic content was controlled by different alleles at two independent loci, both exhibiting additive gene action. Rebetzke et al. (1998) found that the role of minor genes was important in control of reduced palmitic and stearic acid content in soybean. An undetermined number of genetic modifiers were associated with a major palmitic acid locus. In addition, the mutant allele was associated with a 15% reduction in stearic acid content. Reduction of the linolenic (18:3) acid level in flax (Linum usitatissimum L.) from 450 to 650 g [kg.sup.-1] to 20 to 30 g [kg.sup.-1] was achieved by mutagenesis of the Australian flax cultivar Glenelg with EMS (Green, 1986). Progeny of [F.sup.2] plants derived by crossing two mutant strains with low linolenic acid were assigned to five linolenic acid content classes. The number of plants in each class was in agreement with the segregation ratio of 1:4:6:4:1 expected for two unlinked loci exhibiting additive gene action. Combining the two alleles from the two mutant strains achieved the 20 to 30 g [kg.sup.-1] linolenic acid level. Mutagenesis was also used to increase the level of fatty acids of oilseed crops. Irradiation of dry seeds of sunflower by gamma-rays was used to increase the palmitic acid content from a normal level of 70 g [kg.sup.-1] to 310 g [kg.sup.-1] (Ivanov et al., 1988). Genetic studies determined that this high palmitic acid was controlled primarily by additive gene action. Osorio et al. (1995) obtained a mutant sunflower line having a five-fold increase in palmitic acid (252 g [kg.sup.-1]). A bimodal distribution in fatty acid content among [M.sub.2] seeds suggested that a major recessive gene was controlling the high palmitic acid expression. Another line, CAS-3, had a stearic acid content of 260 g [kg.sup.-1], significantly higher than the level of 55 g [kg.sup.-1] of the nontreated line. The genetic control of this high level of stearic acid was more complex due to nondiscrete distributions among the [M.sub.2] seeds. Graef et al. (1985) determined the inheritance of high stearic acid content in three mutant soybean lines. The Fas allele for low stearic acid content in the parental line was found to be partially dominant to the allele for high stearic acid content in the respective mutant lines. Crosses among the mutant lines indicated that each possessed a different allele at the same locus.

The objective of this investigation was to determine the inheritance of low stearic and low palmitic acid content in three sunflower mutant lines.

MATERIALS AND METHODS

Two USDA maintainer inbred lines, HA 821 and HA 382, and two USDA pollen fertility restorer inbred lines, RHA 274 and RHA 801, were selected for chemical mutagenesis treatment. Two chemical mutagens (NMU and EMS) were evaluated at rates of 1 and 2 g [kg.sup.-1] and EMS at rates of 1 and 2 g [kg.sup.-1]. Rates of 4, 6, and 8 g [kg.sup.-1] of both mutagens were found to be lethal for sunflower seed. A total of 56 000 seeds were treated, or 14000 seeds per inbred line (four lines) and 3500 per mutagen treatment (two levels of two mutagens). Treatment procedures were similar to those used by Williams et al. (1985) for wheat (Triticum aestivum L.). Approximately 875 seeds at a time were placed in a 1-L Erlenmeyer flask with 500 mL of 0.1 M K-phosphate pH 8.0. The seeds were swirled on a rotary shaker for 8 h, and the buffer was decanted. A new buffer containing the mutagen was then added. After swirling the seeds with the mutagen solution on a rotary shaker for 16 h, the solution was decanted and the seeds rinsed with distilled water. The water was decanted and the seeds were blotted on paper toweling to remove excess water. They were further air dried for 30 to 40 min until they no longer adhered to one another.

The seeds were planted the same day, with a mechanical planter, in a field nursery at Fargo, ND (clay loam, Vertic Haplaquol). The seeding rate was 60 seeds per plot, with plots 6 m long and 91 cm between rows. In October 1993, [M.sub.2] seeds from approximately 8000 [M.sub.1] heads were harvested from the Fargo nursery and, utilizing a single-seed descent breeding method, a single seed from each head was planted and self-pollinated in a winter nursery in Hawaii (fine, kaolinitic, isohyperthermic, Oxic Ustropept). The [M.sub.3] seeds from each [M.sub.2] plant grown in the Hawaii nursery were planted at Moorhead, MN (coarse-silty, frigid, Aeric Calciaquoll), in June 1994, self-pollinated, and harvested in October 1994. [M.sub.4] seeds from each selected [M.sub.3] plant grown in the Moorhead nursery were planted in a winter nursery near Santiago, Chile, and a single [M.sub.4] plant was selected and self-pollinated. Approximately 6800 [M.sub.4] plants from Chile were harvested to obtain [M.sub.5] seed. The [M.sub.5] seeds were then analyzed for fatty acid content.

The fatty acid content of approximately 20 [M.sub.5] seeds derived from each selected M4 plant was determined by gas chromatography. Seeds to be analyzed were placed in envelopes and pulverized with a hammer. A small portion of the pulverized seeds (10-20 mg) was transferred to a disposable filter column and eluted with 3.5 mL of diethyl ether. The oil in the diethyl ether solution was converted to methyl esters (Metcalfe and Wang, 1981) by the addition of 200 [micro]L of tetramethylammonium hydroxide (100 g [kg.sup.-1] in methanol), followed by vortexing. After 30 min, water was gently added to the reaction mixture, and the upper diethyl ether layer was transferred to a glass vial and capped. The sample was injected into a gas chromatograph to determine fatty acid content.

Remnant [M.sub.5] seed from three [M.sub.4] plants which produced seed low in stearic or palmitic acid content were selected for the inheritance study (Table 1). HA 821 LS-1, a maintainer line low in stearic acid content, RHA 274 LS-1, a restorer line low in stearic acid content, and RHA 274 LP-1, a restorer line low in palmitic acid content, were grown in the field nursery at Fargo, ND (clay loam, Vertic Haplaquol), in 1996. HA 821 LS-1 resulted from the NMU 2 g [kg.sup.-1] treatment, RHA 274 LS-2 from the NMU 1 g [kg.sup.-1] treatment, and RHA 274 LP-1 from the EMS 2 g [kg.sup.-1] treatment. Reciprocal crosses were made with their respective original lines, HA 821 and RHA 274. Fatty acid content was determined for the [F.sub.1] seeds from the reciprocal crosses after harvesting the seed in October, 1996. Remnant [F.sub.1] hybrid seeds were planted in the field at Fargo, ND (clay loam, Vertic Haplaquol), in 1997. The right one-half of a selected [F.sub.1] plant was emasculated at anthesis and crossed with pollen from the respective mutant line. The left one-half of the same [F.sub.1] plant was emasculated at anthesis and crossed with pollen from the respective original line. These two crosses created the [BC.sub.1][F.sub.1] or testcross populations. One [F.sub.1] plant was allowed to self-pollinate to produce [F.sub.2] seeds. After each individual head was harvested, seeds produced on the outer one-third of the head were used for fatty acid analysis. Fifty individual seeds were selected for fatty acid analysis for the [F.sub.2] population and 20 individual seeds were selected for fatty acid analysis for each testcross ([BC.sub.1][F.sub.1]) population. The ratios of fatty acid classes were used to determine the genetic relationships, and classes were determined by comparing individual seed fatty acid contents with their respective parents. Theoretical segregation ratios for each [F.sub.2] and testcross family were tested using Chi-square tests for goodness of fit. The Yates correction factor was used for ratios with 1 degree of freedom.

Table 1. Fatty acid content of seed oil and their standard errors for the sunflower inbred lines, HA 821 and RHA 274, and three lines derived from mutagen treatments.
                           Fatty acid content (g [kg.sup.-1])
Genotype              Palmitic         Stearic        Oleic
Conventional lines
HA 821                50 [+ or -]3    68 [+ or -] 2  194 [+ or -] 10
RHA 274               63 [+ or -]4    48 [+ or -] 4  219 [+ or -] 10

Mutagen lines
HA 821 LS-1           56 [+ or -] 2   41 [+ or -] 5  202 [+ or -] 15
RHA 274 LS-2          86 [+ or -] 2   20 [+ or -] 3  108 [+ or -] 10
RHA 274 LP-1          47 [+ or -] 4   54 [+ or -] 8  238 [+ or -] 41

Genotype              Linoleic
Conventional lines
HA 821                644 [+ or -] 8
RHA 274               646 [+ or -] 42
Mutagen lines
HA 821 LS-1           674 [+ or -] 11
RHA 274 LS-2          750 [+ or -] 12
RHA 274 LP-1          637 [+ or -] 37


RESULTS AND DISCUSSION

Sixteen of the 6800 [M.sub.5] lines analyzed significantly deviated for altered fatty acid content from their respective parental genotypes. Five lines were lower in palmitic acid content, seven lines were lower in stearic acid content, two lines had over 800 g [kg.sup.-1] oleic acid, and one line was classified as a mid-oleic genotype (525 g [kg.sup.-1]). Only one line had an elevated level of a saturated fatty acid, with the palmitic acid content reaching 132 g [kg.sup.-1], compared with 59 g [kg.sup.-1] in the original HA 382 genotype. Also found was a mutant line of RHA 274 having a high linoleic acid content of 786 g [kg.sup.-1], with the linoleic acid content of the parental genotype 646 g [kg.sup.-1]. No significant reductions were found in the arachidic (20:0), behenic (22:0), or lignoceric (24:0) saturated fatty acids.

Treatment with either NMU or EMS was effective in producing lines with altered fatty acids. We did not observe any lines with both low palmitic and low stearic acid content in lines with a normal oleic and linoleic acid profile. Lines having low palmitic and low stearic acid content appeared to be produced by independent mutation events, with different alleles controlling the two saturated fatty acids. Since HA 821 was naturally lower in palmitic acid content and RHA 274 was naturally lower in stearic acid content, we wondered whether the fatty acids in these lines could be further decreased by mutagenesis. The mutagen line RHA 274 LS-2 was significantly lower in stearic acid than RHA 274, indicating that an additional allele controlling low stearic acid was identified (Table 1).

No maternal effect for stearic or palmitic acid content was observed in analysis of [F.sub.1] seeds from the reciprocal crosses. In all crosses, the mean [F.sub.1] stearic and palmitic acid contents were intermediate between the parents. Ivanov et al. (1988) and Osorio et al. (1995) also found that [F.sub.1] seeds of reciprocal crosses or nonfixed [M.sub.1] plants did not differ for palmitic acid content in sunflower. These results indicate that the genotype of the embryo of the seed determined the stearic or palmitic acid content and not the genotype of the maternal plant. Therefore, seeds obtained by self-pollinating the [F.sub.1] hybrid plant could be used in single-seed analysis to evaluate the inheritance of fatty acid content.

The [F.sub.2] and testcross populations derived from the crosses between HA 821 and HA 821 LS-1 produced seeds with low, intermediate, and high stearic acid contents. The range of seeds with low stearic was 38 g [kg.sup.-1] to 46 g [kg.sup.-1], the range of seeds with intermediate stearic was 48 to 56 g kg-1, and the range of seeds with high stearic was 60 g [kg.sup.-1] to 71 g [kg.sup.-1]. The observed ratio of low, intermediate, and high stearic acid seeds in the [F.sub.2] population indicated a 1:2:1 segregation ratio (Table 2). Segregation in the testcross between the [F.sub.1] plant and HA 821 produced seeds in the intermediate and high stearic classes, with numbers in each class fitting a 1:1 ratio (Table 3). The range of seed with intermediate stearic was 45 to 52 g kg-1 and the range of seeds with high stearic was 57 to 59 g [kg.sup.-1]. Segregation in the testcross between the [F.sub.1] plant and HA 821 LS-1 produced seeds in the low and intermediate stearic classes, with the numbers in classes also fitting a 1:1 ratio (Table 3). The range of seeds with low stearic was 28 to 40 g [kg.sup.-1] and the range of seeds with intermediate stearic was 44 to 52 g [kg.sup.-1] These results indicate that low stearic acid content in HA 821 LS-1 was controlled by one gene with additive gene action. This allele will be designated fas1, with the heterozygous genotype Fas1 fas1 producing an intermediate stearic acid content.

The [F.sub.2] and testcross populations from crosses between RHA 274 and RHA 274 LS-2 produced very few seeds that were low (17-21 g kg-1) or high (48-56 g [kg.sub.-1]) stearic content. The intermediate stearic class was the predominant class. The range of stearic acid content in seeds in the intermediate class of the [F.sub.2] population was 33 to 44 g [kg.sup-1]. with the stearic acid content in a nearly continuous distribution. An attempt was made to divide the intermediate class into three additional groups to test an [F.sub.2] ratio of 1:4:6:4:1. However, it was impossible to distinguish between the three intermediate classes. Therefore, a ratio of 1:14:1 was tested and a good fit to this ratio was found (Table 2). Segregation in the testcross between the [F.sub.1] plant and RHA 274 produced seeds in the intermediate (31-44 g [kg.sup.-1]) and high (47-55 g [kg.sup.-1]) stearic classes, with numbers indicating a good fit to the ratio of 3 intermediate to 1 high (Table 3). Segregation in the testcross between the [F.sub.1] plant and RHA 274 LS-2 produced seeds in the low (17-25 g [kg.sup.-1]) and intermediate (37-44 g [kg.sup.-1]) stearic classes, with the numbers fitting a ratio of 1:3, respectively (Table 3).

Table 2. Chi-square ([chi square]) goodness-of-fit values for [F.sub.2] populations of crosses HA 821/HA 821 LS-1, RHA 274/RHA 274 LS-2, and RHA 274/RHA 274 LP-1 evaluated for stearic or palmitic acid content.
                                   [F.sub.2] data
                       Ratio         No. of seeds
Cross                  tested      Fatty acid class

                                   Low       Intermediate
                                 stearic      stearic
HA 821/HA 821 LS-1     1:2:1        12           32
RHA 274/RHA 274 LS-2   1:14:1        6           43
                                   Low       Intermediate
                                 palmitic     palmitic
RHA 274/RHA 274 LP-1   1:2:1         8           31

Cross                           [chi square]    P([dagger])
                        High
                       stearic
HA 821/HA 821 LS-1        6           5.36      0.05-0.10
RHA 274/RHA 274 LS-2      1           4.13      0.10-0.20
                        High
                       palmitic
RHA 274/RHA 274 LP-1     11           3.24      0.10-0.20


([dagger]) Probability of a larger [chi square] value due to chance.

Table 3. Chi-square ([chi square]) goodness-of-fit values for testcross populations between [F.sub.1] crosses and their respective parental lines and evaluated for stearic or palmitic acid content.
                                                Testcross data
                                   Ratio          No. of seeds
Cross                              tested      Fatty acid class
                                              Low      Intermediate
                                             stearic      stearic

HA 821/HA 821 LS-1/HA 821           1:1                       13
HA 821/HA 821 LS-1/HA 821 LS-1      1:1          8            12
RHA 274/RHA 274 LS-2/RHA 274        3:1                       15
RHA 274/RHA 274 LS-2/RHA 274 LS-2   1:3          5            15

                                               Low      Intermediate
                                             palmitic      palmitic
RHA 274/RHA 274 LP-1/RHA 274         1:1                       6
RHA 274/RHA 274 LP-1/RHA 274 LP-1    1:1         8            12

Cross                                            [chi square]
                                      High
                                    stearic
HA 821/HA 821 LS-1/HA 821               7            1.25
HA 821/HA 821 LS-1/HA 821 LS-1                       0.45
RHA 274/RHA 274 LS-2/RHA 274            4            0.05
RHA 274/RHA 274 LS-2/RHA 274 LS-2                    0.00
                                       High
                                     palmitic

RHA 274/RHA 274 LP-1/RHA 274            14           0.10
RHA 274/RHA 274 LP-1/RHA 274 LP-1                    0.45

Cross                                 P([dagger])

HA 821/HA 821 LS-1/HA 821               0.20-0.30
HA 821/HA 821 LS-1/HA 821 LS-1          0.50-0.70
RHA 274/RHA 274 LS-2/RHA 274            0.70-0.90
RHA 274/RHA 274 LS-2/RHA 274 LS-2         >0.95
RHA 274/RHA 274 LP-1/RHA 274            0.70-0.90
RHA 274/RHA 274 LP-1/RHA 274 LP-1       0.50-0.70


([dagger]) Probability of a large [chi square] value due to chance.

These results indicate that low stearic acid content in RHA 274 LS-2 was controlled by two genes with additive gene action. The first allele controlling low stearic acid content will be designated fas2. The second allele in RHA 274 LS-2 will be designated fasx until appropriate allelism tests have been conducted between fas1 derived from HA 821 LS-1 and fasx derived from RHA 274 LS-2.

The [F.sub.2] and testcross populations derived from crosses between RHA 274 and RHA 274 LP-1 produced seeds which were low palmitic, intermediate palmitic, and high palmitic acid content. The range of seeds with low palmitic was 47 g [kg.sup.-1] to 54 g [kg.sup.-1], the range of seeds with intermediate palmitic was 55 to 58 g [kg.sup.-1], and the range of seeds with high palmitic acid was 61 g [kg.sup.-1] to 65 g [kg.sup.-1] in the [F.sub.2] population. The observed ratio of low, intermediate, and high palmitic acid content in the [F.sub.2] population was consistent with a ratio of 1:2:1 (Table 2). Segregation in the testcross between the [F.sub.1] plant and RHA 274 produced seeds only in the intermediate (54-59 g [kg.sup.-1]) and high (68-76 g [kg.sup.-1]) palmitic classes, with the numbers in each class indicating a ratio of 1:1 (Table 3). The range of the high palmitic acid content in this testcross was higher than in the [F.sub.2] population. Segregation in the testcross between the [F.sub.1] plant and RHA 274 LP-1 produced seeds in the low (47-49 g [kg.sup.-1]) and intermediate (53-58 g [kg.sup.-1]) palmitic acid classes, with the numbers in each class also corresponding to a ratio of 1:1 (Table 3). The range of the low palmitic acid content in this testcross also was slightly higher than expected, but still was lower than the parental palmitic acid content of 63 [+ or -] 4 g [kg.sup.-1]. These results indicate that low palmitic acid content in RHA 274 LP-1 was controlled by one gene with additive gene action. This allele will be designated fap1.

Incorporation of the fasl, fas2, fasx, and fap1 genes into commercial hybrids will require extensive testing in early generation breeding material for development of parental inbred lines. Utilizing half-seed analysis of segregating seeds derived from one sunflower head will be feasible. For example, a genotype homozygous for the fas1 fas1 fas2 fas2 fap1 fap1 alleles should be easily discerned from a genotype with heterozygous alleles at any locus. The key to selection is testing sufficient numbers of seeds to find the desired genotype.

The combination of the alleles providing low stearic acid content with alleles providing low palmitic acid content could substantially reduce the saturated fatty acid content of seed oil in sunflower. On the basis of results of this study, a sunflower hybrid could be produced with a total saturated fatty acid content of less than 80 g [kg.sup.-1], including the 20:0, 22:0, and 24:0 saturated fatty acids. Future studies will be conducted to assess whether or not physiologically sound lines can be developed with a saturated fatty acid content less than 80 g [kg.sup.-1], without adverse effects derived from combining the alleles identified in this study.

REFERENCES

Erickson, E.A., J.R. Wilcox, and J.F. Cavins. 1988. Inheritance of altered palmitic acid percentage in two soybean mutants. J. Heredity 79:465-468.

Fehr, W.R., G.A. Welke, E.G. Hammond, D.N. Duvik, and S.R. Cianzio. 1991. Inheritance of reduced palmitic acid content in seed oil of soybean. Crop Sci. 31:88-89.

Graef, G.L., W.R. Fehr, and E.G. Hammond. 1985. Inheritance of three stearic acid mutants of soybean. Crop Sci. 25:1076-1079.

Green, A.G. 1986. Development of an edible-oil flax genotype. In Proc. 51st Flax Institute, Fargo, ND. 30-31 Jan. 1986. Flax Institute of the U.S., Fargo, ND.

Ivanov, P., D. Petakov, V. Nikolova, and E. Pentchev. 1988. Sunflower breeding for high palmitic acid content in the oil. p. 463-465. In Proc. 12th Int. Sunflower Conf., Novi Sad, Yugoslavia. 25-29 July. International Sunflower Association, Paris.

Mensink, R.P., E.H.M. Temme, and G. Hornstra. 1994. Dietary saturated and trans fatty acids and lipoprotein metabolism. Ann. Med. 26:461-464.

Metcalfe, L.D., and C.N. Wang. 1981. Rapid preparation of fatty acid methyl esters using organic base-catalyzed transesterification. J. Chromatogr. Sci. 19:530-535.

Osorio, J., J. Fernandez-Martinez, M. Mancha, and R. Garces. 1995. Mutant sunflowers with high concentration of saturated fatty acids in the oil. Crop Sci. 35:739-742.

Rebetzke, G.J., J.W. Burton, T.E. Carter, Jr., and R.F. Wilson. 1998. Genetic variation for modifiers controlling reduced saturated fatty acid content in soybean. Crop Sci. 38:303-308.

Willett, W.C. 1994. Diet and health: What should we eat? Science 264:532-537.

Williams, N.D., L.R. Joppa, M.E. Duysen, and T.P. Freeman. 1985. Inheritance of three chlorophyll-deficient mutants of common wheat. Crop Sci. 25:1023-1025.

U.S. Department of Agriculture, Human Nutrition Information Service. 1992. The food guide pyramid. Home and Garden Bull. 252.

J.F. Miller and B.A. Vick, USDA-ARS, Northern Crop Science Laboratory, PO Box 5677, Fargo, ND 58105. Cooperative investigation between the USDA-ARS and the North Dakota Agric. Exp. Stn., Fargo, ND 58105. Research was supported in part by a grant from the North Dakota Agricultural Products Utilization Commission. Mention of a proprietary product does not constitute a recommendation or warranty of the product by the USDA or North Dakota State University and does not imply approval to the exclusion of other suitable products. Received 6 April 1998.

J.F. Miller, Corresponding author (millerjf@fargo.ars.usda.gov).
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