Published online 2 December 2005
Published in Crop Sci 46:22-29 (2006)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Inheritance of High Stearic Acid Content in the Sunflower Mutant CAS-14
Begoña Pérez-Vich,
Leonardo Velasco,
Juan Muñoz-Ruz and
José M. Fernández-Martínez*
Instituto de Agricultura Sostenible, CSIC, Apartado 4084, E-14080 Córdoba, Spain
* Corresponding author (cs9femaj{at}uco.es)
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ABSTRACT
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A sunflower (Helianthus annuus L.) oil with high concentration of stearic acid has important applications in the food industry. Two sunflower mutants with high stearic acid content, CAS-3 and CAS-14, have been developed. In contrast to CAS-3, high stearic acid expression in CAS-14 seeds is temperature dependent and nonuniformly distributed in the seed. The trait in CAS-3 has been found to be governed by two genes, Es1 and Es2. The objective of the present research was to study the inheritance of high stearic acid content in CAS-14 through crosses with P21, a nuclear male sterile (NMS) line with a wild-type fatty acid profile, and CAS-3. The genetic analysis included the evaluation of the F1, F2, F3, BC1F1, and BC1F2 seed generations. Crosses between P21 and CAS-14 revealed that the high stearic acid trait was recessive and controlled by a single gene, designated Es3. The analysis of the F3 and BC1F2 to P21 generations demonstrated a repulsion-phase linkage between the Es3 and the Ms loci, the latter conferring the NMS trait. The frequency of recombination between Es3 and Ms was estimated to be 0.09. Crosses between CAS-3 and CAS-14 demonstrated that both lines possess alleles for high stearic acid content at different loci, as transgressive segregants with low stearic acid content were observed in all generations. Genetic recombination of es1 and es3 alleles did not result in an increment of the maximum stearic acid content in the seeds compared with the maximum levels produced by the es3 alleles alone.
Abbreviations: GLC, gas-liquid chromatography • NMS, nuclear male sterile/sterility • r, recombination frequency
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INTRODUCTION
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VEGETABLE OILS with high concentrations of saturated fatty acids are characterized by a high viscosity, forming a semisolid fat at room temperature. They are desired by the food industry for producing a wide range of margarine and spread products without need for chemical transformations such as hydrogenation, which produces trans fatty acids that are expected to raise the risk of coronary heart disease (Katan, 1998).
Several sources of high saturated fatty acid content have been developed in sunflower. They include germplasm with high palmitic acid content (Ivanov et al., 1988; Osorio et al., 1995; Fernández-Martínez et al., 1997), as well as germplasm with high stearic acid content (Osorio et al., 1995; Fernández-Moya et al., 2002). Compared with a typical wild-type stearic acid content of 45 g kg–1, the mutant lines CAS-4 and CAS-8 produced medium-high levels of stearic acid (113 g kg–1 and 99 g kg–1, respectively) and CAS-3 produced a high stearic acid content of 260 g kg–1 (Osorio et al., 1995), whereas the mutant line CAS-14 showed a very high stearic acid content of up to 430 g kg–1 (Fernández-Moya et al., 2002). The high stearic acid content in the CAS-14 mutant was found to be strongly influenced by the temperature during seed maturation, with average values ranging from 142 g kg–1 at 30/20°C (day/night) to 369 g kg–1 at 39/24°C (Fernández-Moya et al., 2002). Additionally, the high stearic acid content was not uniformly expressed in CAS-14 seeds, but exhibited a strong longitudinal gradient starting from the embryo (97 g kg–1) up to the end of the cotyledon (346 g kg–1) (Fernández-Moya et al., 2003). Such a seed gradient and strong temperature influence were not observed in the high stearic acid mutant CAS-3 (Pérez-Vich et al., 1998; Fernández-Moya et al., 2003).
Genetic studies conducted on the high stearic acid mutant CAS-3 demonstrated the presence of recessive alleles at two independent loci, Es1 and Es2 (Pérez-Vich et al., 1999). The two loci exhibited additive gene action on the stearic acid content of CAS-3 seeds, with es1 alleles contributing about 188 g kg–1 in homozygous condition and es2 alleles contributing about 90 g kg–1 in homozygous condition (Pérez-Vich et al., 2004). The midstearic acid mutant CAS-4 carried the same es2 alleles as CAS-3, but a different allele, es1a, at the Es1 locus (Pérez-Vich et al., 2002a).
The objective of the present research was to study the inheritance of high stearic acid content in the sunflower mutant CAS-14. In addition, the genetic relationships between genes for high stearic acid content in CAS-14 and CAS-3, and between genes for high stearic acid content in CAS-14 and for nuclear male sterility (NMS) in P21 were investigated.
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MATERIALS AND METHODS
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Plant Material
CAS-14 is a high stearic acid mutant developed by chemical mutagenesis. It is characterized by a decreasing longitudinal seed gradient for stearic acid content starting from the embryo (97 g kg–1) and continuing to the end of the cotyledon (346 g kg–1). High stearic acid content in CAS-14 is also highly influenced by temperature (Fernández-Moya et al., 2003). CAS-3 is a high stearic acid mutant with uniform concentration of this fatty acid around 260 g kg–1 throughout the seed (Osorio et al., 1995). High stearic acid content of CAS-3 is not severely affected by temperature (Pérez-Vich et al., 1998; Fernández-Moya et al., 2003). P21 is a line with wild-type seed oil fatty acid profile (stearic acid around 45 g kg–1) possessing a single recessive gene for NMS (Jan, 1992).
Genetic Study
Half seeds of P21, CAS-3, and CAS-14 were analyzed for seed oil fatty acid profile by gas–liquid chromatography (GLC) to ensure that the plants used in the genetic study bred true for this trait. Half seeds were germinated in February 2000 and, after 15 d in a growth chamber (25/15°C with a 16-h photoperiod), the plants were transplanted into pots and grown under greenhouse conditions. At flowering, each head was covered with a paper bag to avoid contamination with external pollen. Reciprocal crosses between plants of P21 and CAS-14, and between plants of CAS-3 and CAS-14 were made in spring 2000. Twenty-four seeds of the F1 and both parents were analyzed for fatty acid composition and the corresponding plants were grown in the greenhouse in spring 2001. F1 plants were self-pollinated to obtain the F2 and also backcrossed to both parents to obtain the BC1F1 seed. Reciprocal crosses between the two parents were repeated in spring 2001 to obtain reciprocal F1 seeds in the same environment as the F2 and BC1F1 seed. Average temperature during the period from flowering to seed maturation was 31.8/19.6°C (day/night).
F2 and BC1F1 half-seeds were analyzed by GLC. The analysis of the P21/CAS-14 cross included two F2 population samples of 240 seeds each, one BC1F1 to P21 population sample of 191 seeds, and one BC1F1 to CAS-14 population sample of 192 seeds. The analysis of the CAS-3/CAS-14 cross included two F2 population samples of 228 and 240 seeds, respectively, one BC1F1 to CAS-3 population sample of 59 seeds, and one BC1F1 to CAS-14 population sample of 192 seeds.
After half-seed analyses, the two F2 population samples from the cross between P21 and CAS-14, and the other two F2 population samples from the cross between CAS-3 and CAS-14 were germinated and the corresponding plants were grown in the field in summer 2002. Similarly, the four BC1F1 population samples (P21/CAS-14//P21, P21/CAS-14//CAS-14, CAS-3/CAS-14//CAS-3, CAS-3/CAS-14//CAS-14) were germinated and grown in the field in summer 2002. Plants of P21, CAS-3, and CAS-14 were grown as checks. Forty-eight F3 or BC1F2 half seeds from each F2 or BC1F1 plant, respectively, were analyzed for fatty acid composition by GLC. In the cases in which the plants produced a lower number of seeds, all the seeds were analyzed, with a minimum of 24 half seeds per plant.
Recombination frequencies between the Es and Ms loci were calculated using the maximum likehood procedure (Sánchez-Monge and Jouve, 1981) based on the fatty acid profile of F3 and BC1F2 to P21 seeds from male-fertile F2 and BC1F1 plants. Fatty acid data of F2 and BC1F1 seeds resulting in male-sterile plants were not used for calculation of recombination frequencies because phenotypic classes could not be clearly separated without the information obtained from the analysis of F3 and BC1F2 seeds. In the F2 generation, MsMs and Msms genotypes, both of them producing male-fertile plants, were combined in a single class because they could not be separated. The expected genotype frequencies of classes used to derive maximum likehood formulas are given in Table 1.
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Table 1. Expected frequencies and observed number of genotypes at Es3 (increased stearic acid) and Ms (nuclear male sterile) loci in F2 and BC1F1 population samples from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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Seed Oil Fatty Acid Analyses by Gas–Liquid Chromatography
The fatty acid composition of the seed oil was analyzed in all cases on a seed portion distal to the embryo of about one fourth of the seed length. The excised seed portion was of the same relative size in all the analyzed seeds to minimize the effect of the seed gradient observed in the CAS-14 mutant.
Fatty acid analyses were done following the method described by Garcés and Mancha (1993), using a gas-liquid chromatograph PE Autosystem XL (PerkinElmer Corporation, Norwalk, CT) equipped with a 2-m-long column packed with 3% SP-2310/2% SP-2300 on Chromosorb WAW (Supelco, Inc., Bellefonte, PA). The oven, injector, and flame ionization detector were maintained at 195, 275, and 250°C, respectively.
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RESULTS
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Crosses between P21 and CAS-14
Seeds of the wild-type line P21 averaged 46.0 ± 8.4 g kg–1 stearic acid, whereas those of the high stearic acid mutant CAS-14 had 262.0 ± 62.5 g kg–1 stearic acid. As expected, the CAS-14 mutant was characterized by a broad range of variation for stearic acid content, from 153.2 to 347.7 g kg–1. Such a wide range of variation is typical of this mutant, as the fatty acid composition of its seed oil is strongly affected by small changes of temperature (Fernández-Moya et al., 2002). F1 seeds from the cross P21/CAS-14 averaged 50.1 ± 9.4 g kg–1 stearic acid, whereas those of the reciprocal cross averaged 63.7 ± 8.7 g kg–1, indicating dominance of wild-type over high stearic acid content.
Two F2 seed population samples from the cross P21/CAS-14 were analyzed for stearic acid content (Fig. 1). In both cases, two different classes could be distinguished: a narrow class including stearic acid values <70 g kg–1 in the first population (Fig. 1A) and <85 g kg–1 in the second population (Fig. 1B), and a broad class, encompassing stearic acid values from 82 to 454 g kg–1 in the first population and from 95 to 414 g kg–1 in the second population. The narrow class corresponded approximately to the observed ranges of variation of the P21 parent and the F1, whereas the broad class was similar to the observed range of variation for the CAS-14 parent. Both populations followed 3:1 (narrow to broad class) segregation patterns (
2 = 0.02, P = 0.76 in the first population; 2 = 2.22, P = 0.10 in the second population), which was interpreted as the segregation of recessive alleles at a single locus. The locus was tentatively named Esx. In the F2 distribution, the narrow class corresponded to both EsxEsx and Esxesx genotypes, whereas the broad class corresponded to esxesx genotype. The parents P21 and CAS-14 were expected to have the allelic configurations EsxEsx and esxesx, respectively.
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Fig. 1. Histograms of stearic acid concentration in two F2 population samples (A and B) from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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BC1F1 to P21 seeds had uniformly low stearic acid phenotypes, with a range of variation from 20.8 to 67.2 g kg–1 (Fig. 2). The lack of phenotypic segregation in the BC1F1 to the low stearic acid parent would be expected for a single recessive gene. BC1F1 to CAS-14 seeds ranged from 26.3 to 196.1 g kg–1 (Fig. 2), with no clear class separation.
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Fig. 2. Histograms of stearic acid concentration in BC1F1 to P21 and BC1F1 to CAS-14 population samples from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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Because of the atypically broad segregation range of the CAS-14 class, the inheritance of the increased stearic acid content in the P21/CAS-14 cross was further investigated through the analysis of the F3 and BC1F2 seed generations. Class separation was accomplished by considering both the average and the maximum stearic acid content of F3 or BC1F2 seeds for every F2– or BC1F1–derived family, respectively. Following this criterion, three classes were clearly distinguished in the two F3 populations evaluated (Fig. 3). According to the initial hypothesis proposed after the analysis of the F2 seed generation, the classes should correspond to the F2 genotypes EsxEsx (both average and maximum stearic acid content below 100 g kg–1), Esxesx (average below 250 g kg–1, maximum above 250 g kg–1), and esxesx (both average and maximum stearic acid content above 250 g kg–1). The maximum stearic acid content in some families of the EsxEsx class was greater than the maximum stearic acid content of the low stearic acid parent P21 grown in the same environment (67.3 g kg–1), which suggested the possible presence of additional genes with minor effect in the CAS-14 mutant. The distribution of the F2–derived F3 (F2:3) families in the three classes was 8:68:35 (EsxEsx–Esxesx–esxesx) in the first F3 population sample (Fig. 3A), and 5:69:21 (EsxEsx–Esxesx–esxesx) in the second F3 population sample (Fig. 3B). However, such frequency distributions were far from the expected 1:2:1 segregation ratio.
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Fig. 3. Scatter plot of average stearic acid vs. maximum stearic acid concentration of two samples of F2–derived F3 families (48 F3 seeds analyzed per F2:3 family) from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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Even though the initial F2 seed populations were in both cases composed of 240 seeds and all the seeds were sown to produce the F3 generation, F3 seeds were only obtained from 111 F2 plants in one population, and from 95 F2 plants in the other population. The reasons for such a reduction were threefold: lack of germination, plant death, and segregation for male sterility. The latter occurred because the P21 parent carries recessive alleles ms at a single locus, Ms, that produce NMS (Jan, 1992), resulting in about one-fourth of the F2 plants being unable to produce seeds following exclusive self-pollination. Accordingly, the numbers of the F2 seeds that did not germinate, produced plants that did not reach maturity, or produced NMS plants, were taken into consideration to identify the causes of the unexpected segregation.
The first population (Fig. 1A) contained 240 F2 seeds with an average stearic acid content of 107.4 g kg–1. A subpopulation of 149 F2 seeds that germinated and produced viable plants that reached maturity had an average stearic acid content of 106.3 g kg–1, which did not differ significantly from the original F2 population (t = 0.09, P = 0.93). Mature F2 plants included both male-fertile (n = 111, average stearic acid content = 123.8 g kg–1) and male-sterile plants (n = 38, average stearic acid content = 51.5 g kg–1), which differed significantly for stearic acid content (t = 3.23, P < 0.01). Only one of the NMS plants derived from an F2 seed with increased stearic acid phenotype (>82 g kg–1), whereas the other 37 plants derived from F2 seeds with low stearic acid phenotype (<70 g kg–1). The second population (Fig. 1B) contained 240 F2 seeds with an average stearic acid content of 97.4 g kg–1. The subpopulation of seeds that germinated and produced viable plants did not differ significantly for stearic acid content from the original population (mean = 94.8 g kg–1; t = 0.24, P = 0.81). The subpopulation included 95 male fertile plants (average stearic acid content = 106.3 g kg–1) and 28 male-sterile plants (average stearic acid content = 53.3 g kg–1), which differed significantly for stearic acid content (t = 2.69, P < 0.01). Only one of the NMS plants derived from an F2 seed with increased stearic acid phenotype (>82 g kg–1). The extremely low proportion of plants that combined a high stearic acid with a male-sterile phenotype (genotype esxesx msms) suggested the existence of linkage in repulsion between both Ms and Esx genes. Frequencies of recombination (r) were estimated according to the expected and observed frequencies of the Esx and esx alleles in male fertile plants of both F2 population samples using the maximum likelihood method (Sanchez-Monge and Jouve, 1981). The estimated r values were 0.11 ± 0.02 and 0.07 ± 0.02, respectively (Table 1).
The analysis of BC1F2 seeds from the backcross to P21 allowed the separation of the BC1F1 genotypes EsxEsx (both average and maximum stearic acid content below 100 g kg–1) and Esxesx (maximum stearic acid content above 300 g kg–1), which were found in a proportion 9:73 (EsxEsx–Esxesx; Fig. 4), also far from the expected 1:1 ratio. Such a deviation was attributed to the genetic linkage between the Ms and Esx genes. On the basis of the expected frequencies of the Esx and esx alleles in male fertile plants (genotype Msms), the recombination frequency was estimated as 0.09 ± 0.02 (Table 1), very close to the estimates obtained using the F2 populations.
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Fig. 4. Scatter plot of average stearic acid vs. maximum stearic acid concentration of BC1F1 to P21 families (48 BC1F2 seeds analyzed per BC1F1 family) from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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The analysis of BC1F2 seeds from the backcross to CAS-14 allowed the separation of the BC1F1 genotypes esxesx (minimum stearic acid content above 140 g kg–1) and Esxesx (minimum stearic acid content below 60 g kg–1) (Fig. 5). Both genotypes were found in a proportion 59:54 (Esxesx–esxesx), which was not significantly different (2 = 0.22; P = 0.64) from the expected 1:1 ratio for the segregation of a single recessive gene.
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Fig. 5. Scatter plot of minimum stearic acid vs. maximum stearic acid concentration of BC1F1 to CAS-14 families (48 BC1F2 seeds analyzed per BC1F1 family) from the cross between the sunflower lines P21, with wild-type seed oil fatty acid profile, and CAS-14, with increased stearic acid concentration.
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Crosses between CAS-3 and CAS-14
Seeds of the high stearic acid mutant CAS-3 averaged 290.0 ± 20.4 g kg–1 stearic acid, whereas those of the high stearic acid mutant CAS-14 had 262.0 ± 62.5 g kg–1. Both high stearic acid mutants clearly differed for their ranges of variation, from 255.8 to 324.4 g kg–1 in CAS-3 and from 153.2 to 347.7 g kg–1 in CAS-14. F1 seeds from the cross CAS-3/CAS-14 averaged 128.5 ± 18.7 g kg–1 stearic acid, whereas those of the reciprocal cross averaged 127.2 ± 15.8 g kg–1. The mean and lower range of the F1 generation was lower than both high stearic acid parents (Fig. 6), suggesting differences in the genetic control of the high stearic acid content in both mutants.
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Fig. 6. Histograms of stearic acid concentration in the high stearic acid sunflower lines CAS-3, CAS-14, and their F1 seeds.
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The analysis of F2 seeds from two different F1 plants, both of them from the cross CAS-3/CAS-14, revealed broad ranges of variation for stearic acid, from 20.9 to 429.3 g kg–1 in one F2 population, and from 18.9 to 454.6 g kg–1 in the other one (Fig. 7). Such broad ranges of variation had transgressive boundaries compared with both parents, indicating the presence of alleles for high stearic acid at different loci in both mutants. No clear phenotypic classes could be separated in both F2 populations.
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Fig. 7. Histograms of stearic acid concentration in two F2 population samples from the cross between the high stearic acid sunflower lines CAS-3 and CAS-14.
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Seeds of the BC1F1 to CAS-3 segregated from 45.5 to 304.9 g kg–1, whereas those of the BC1F1 to CAS-14 showed a range of variation between 42.4 and 449.6 g kg–1 (Fig. 8). The occurrence of a phenotypic class clearly below the lower boundary of both parents (<150 g kg–1) was confirmed in both backcrosses, which supported the presence of different loci for high stearic acid in CAS-3 and CAS-14 mutants. In both cases, segregation followed 1:1 (<150:>150 g kg–1) ratios (2 = 2.86, P = 0.09 for BC1F1 to CAS-3; 2 = 2.52, P = 0.11 for BC1F1 to CAS-14), which suggested the presence of a single major gene in each mutant.
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Fig. 8. Histograms of stearic acid concentration in BC1F1 to CAS-3 and BC1F1 to CAS-14 population samples from the cross between the high stearic acid sunflower lines CAS-3 and CAS-14.
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The F3 and BC1F2 generations were analyzed to confirm the number of major genes involved and their relationships. The first F2 population sample (Fig. 7A) included 228 seeds with an average stearic acid content of 128.9 g kg–1, which did not differ from the subpopulation of 83 seeds that germinated and produced mature plants (mean = 128.9 g kg–1). F3 seed analysis in the latter showed that five F2:3 families did not segregate for high stearic acid content (>120 g kg–1), 48 F2:3 families showed segregation for stearic acid in F3 seeds, and the other 30 F2:3 families showed no segregation for low stearic acid content (<120 g kg–1). Such an F2 distribution followed the expected 1:8:7 (low–segregating–high stearic acid) ratio for the segregation of two independent genes for high stearic acid (2 = 2.12, P = 0.35). The second F2 population sample (Fig. 7B) contained 240 seeds with an average stearic acid content of 120.2 g kg–1, which was not significantly different from the subpopulation of 164 seeds that germinated and produced mature plants (mean = 108.1 g kg–1; t = 1.81, P = 0.07). F3 seed analysis revealed 14 low stearic, 81 segregating, and 69 high stearic F2:3 families, which also fit the expected 1:8:7 ratio (2 = 1.49, P = 0.47).
A set of twenty-two BC1F1 to CAS-3 plants was obtained. The average stearic acid content of their corresponding BC1F1 seeds (163.1 g kg–1) did not differ significantly from the original population (Fig. 8), with an average stearic acid content of 158.5 g kg–1 (t = 0.26; P = 0.80). Eight BC1F1 families produced BC1F2 seeds showing uniformly high stearic acid content (>150 g kg–1), whereas the other 14 families showed wide segregation at the BC1F2 seed level. These results confirmed the 1:1 segregation ratio observed after the analysis of BC1F1 seeds (2 = 1.64, P = 0.20). Similarly, a set of 94 BC1F1 to CAS-14 plants were obtained from the initial population of 192 BC1F1 seeds (Fig. 8). However, this subpopulation had a stearic acid content of 139.8 g kg–1, significantly lower than the original BC1F1 population (198.2 g kg–1; t = 3.44, P < 0.01). The difference was caused by a lower germination rate in the high stearic acid seeds, which was not observed in the other populations used in this study. The analysis of the BC1F2 seeds showed that all the families derived from BC1F1 seeds with stearic acid content above 150 g kg–1 did not segregate for low stearic acid content (<120 g kg–1) at the BC1F2 seed level, whereas families derived from seeds with stearic acid content below 150 g kg–1 showed a segregation for low stearic acid levels. These results confirmed the 1:1 segregation ratio observed in the BC1F1 seed generation (Fig. 8).
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DISCUSSION
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Crosses between CAS-14 and P21 revealed that the high stearic acid content in the sunflower mutant CAS-14 is controlled by recessive alleles. Moreover, transgressive segregation in F1, F2, and BC1F1 seeds from crosses between CAS-14 and CAS-3 proved that this locus is different from Es1 and Es2 (Pérez-Vich et al., 1999). Accordingly, the locus in CAS-14 was named Es3. Additionally, CAS-14 might carry additional genes with minor effect for increased stearic acid content, as in some F2 and BC1F1 families of the P21/CAS-14 cross, Es3Es3 genotypes showed a greater stearic acid content (around 100 g kg–1) than that typical of the low stearic parent P21 (<70 g kg–1). Both CAS-3 and CAS-14 were obtained after chemical mutagenesis of the line RDF-1–532, using ethyl methanesulfonate in the former case (Osorio et al., 1995) and sodium azide in the latter (Fernández-Moya et al., 2002). In crosses between CAS-3 and RDF-1–532, Pérez-Vich et al. (1999) found that es2 alleles were already present in RDF-1–532, while mutagenesis only produced the es1 mutation in CAS-3. From this mutant, Pérez-Vich et al. (2004) developed the line CAS-20, which contained the es2 alleles as the only source of increased stearic acid content. CAS-20 showed a range of variation for stearic acid from 61 to 99 g kg–1, which is the upper boundary found in the Es3Es3 genotypic class. This evidence, together with the lack of segregation for genes other than Es1 and Es3 in the CAS-3/CAS-14 cross, suggests that the Es2 gene, with a minor effect on increasing stearic acid content, might be present in CAS-14.
Expression of high stearic acid in seeds of CAS-14 is very different from that in CAS-3 seeds. Stearic acid content in CAS-14 is nonuniformly distributed in the seeds and strongly influenced by temperature (Fernández-Moya et al., 2002). Both characteristics are not observed in CAS-3 (Pérez-Vich et al., 1998). Through a candidate-gene strategy, Pérez-Vich et al. (2002b) demonstrated that Es1 cosegregated with a stearoyl-acyl carrier protein (ACP) desaturase gene. However, the role of Es3 in the fatty acid biosynthetic pathway is unknown.
The present research was conducted using the conventional half-seed analysis, which consists in the analysis of a small portion of the seed distal to the embryo, where the maximum stearic acid content in the seed is located. After the analysis of F3 seeds from F2 generations of the crosses P21/CAS-3 and CAS-3/CAS-14, it was found that the maximum average stearic acid content in F2 generations from the cross P21/CAS-14 was similar to the maximum values found in F2 generations from the cross CAS-3/CAS-14, in both cases around 450 g kg–1. These data suggest that the maximum potential to accumulate stearic acid conferred by the es3 alleles is not further increased by an additive action of es1 alleles. Previous studies in sunflower indicated that the increased stearic acid levels of the CAS-3 and CAS-4 mutants were controlled by two independent genes, Es1 and Es2, acting in an additive manner (Pérez-Vich et al.,1999, 2002a). In soybean [Glycine max (L.) Merr.], Bubeck et al. (1989) found that the recombination of recessive alleles for high stearic acid at two different loci produced transgressive segregation for higher stearic acid levels than the parents. Similar results were obtained by Rahman et al. (1997). Even though a similar additive action between es1 and es3 alleles was not observed in the present research, a major goal from a breeding point of view is to determine whether es1 alleles may play a role in attenuating the seed gradient and environmental instability of CAS-14 mutant.
Little is known about the effect that modified fatty acid profiles may exert on the germination capacity of sunflower seeds. In soybean, seeds with a high stearic acid content above 276 g kg–1 showed good germination, but plantlets died after few days. Additionally, the seeds were abnormally irregular in shape and size (Rahman et al., 1997). In the present research, we compared four F2 and two BC1F1 seed populations segregating for stearic acid content with their corresponding subpopulations that germinated and produced mature plants. In most cases, the stearic acid content of the final subpopulation was not different from the original population, indicating no detrimental effect of high stearic acid levels on seed germination or plant viability.
The Es3 locus was found to be closely linked in repulsion to the Ms locus that produces NMS in the P21 line (Jan, 1992), that is, high stearic acid is linked with male fertility. Three different estimates of the frequency of recombination between both loci resulted in r values of 0.07, 0.09, and 0.11, which resulted in an average estimate of 0.09. Breeding programs including the high stearic acid trait from the Es3 gene could benefit from such a close linkage, since elimination of NMS plants would result in an indirect selection against low stearic acid genotypes.
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ACKNOWLEDGMENTS
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The authors thank Antonia Escobar, Cristóbal Prieto, and Angel L. Benito for excellent technical assistance. The work was funded by Advanta Seeds B.V.
Received for publication December 13, 2004.
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REFERENCES
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