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Genetic Relationships between Loci Controlling the High Stearic and the High Oleic Acid Traits in Sunflower.

THE QUALITATIVE MANIPULATION of seed oils involves the modification of their fatty acid composition, which determines the chemical properties responsible for the particular end use of the oil. Several alternatives to produce novel seed oil fatty acid profiles are being used. In particular, mutation breeding followed by combination of the mutant lines has been successfully used during the last 30 yr (Robbelen, 1990). In flax (Linum usitatissimum L.) (Green, 1986) and soybean [Glycine max (L.) Merrill] (Fehr et al., 1992; Rahman et al., 1998), a further reduction of the linolenic acid (C18:3) concentration was achieved by crossing single mutants. Following a similar approach, Nickell et al. (1991) in soybean, Ntiahmoah et al. (1995) in flax, and Ladd and Knowles (1971) in safflower (Carthamus tinctorius L.) combined mutations affecting the concentrations of two different fatty acids. In the case of soybean and flax, mutations for high or low palmitic acid (C16:0) and low linolenic acid were combined. In safflower, genotypes combining high stearic (C18:0) and high oleic (C18:1) were developed.

Sunflower mutants with increased levels of oleic acid (C18:1 [is greater than] 750 g [kg.sup.-1] compared with 200-500 g [kg.sup.-1] in commonly grown cultivars; Soldatov, 1976) and stearic acid (C18:0 [is greater than] 220 g [kg.sup.-1] compared with 50 g [kg.sup.-1] in standard sunflower seed oil; Osorio et al., 1995) have been developed. The combination of the seed oil phenotypes of both types of mutants would associate the oxidative stability and heart-healthy properties of the C18:1 with a higher plasticity of the oil because of the C18:0, which would result in a novel oil quality of great value for the food industry (Purdy, 1986; Wardlaw and Snook, 1990; Ascherio and Willet, 1997).

Studies on the genetic control of the high C18:0 and the high C18:1 traits in sunflower have been carried out separately. The inheritance of the high C18:1 trait has been widely studied in crosses between high C18:1 lines derived from the Soldatov mutant and different types of low C18:1 lines. The first studies reported a single partially dominant (Fick, 1984) or dominant (Urie, 1984) gene controlling the high C18:1 character, designated Ol (Fick, 1984). In addition to this major gene, Urie (1985) detected the presence of modifiers as well as an unexplained reversal in the dominance. Miller et al. (1987) found a second gene, Ml, modifying the C18:1 content determined by the Ol gene. High C18:1 levels were only expressed in genotypes Ol_mlml. Fernandez-Martinez et al. (1989) identified two additional dominant genes, complementary to Ol, controlling the high C18:1 trait. The genes were named Old, 012, and 013. Phenotypes having a high C18:1 content carried three dominant alleles (genotype [Ol.sub.1]_[Ol.sub.2]_[Ol.sub.3]_), while the two dominant alleles [Ol.sub.1] and [Ol.sub.2] with the third gene in recessive homozygosis (genotype [Ol.sub.1]_[Ol.sub.2]_[ol.sub.3][ol.sub.3]) expressed intermediate C18:1 levels. The authors concluded that some of the Ol alleles could be present in the low C18:1 lines used in the crossing study, which resulted in segregations for one gene [[Ol.sub.1] or [Ol.sub.2], [F.sub.2] ratio of 1:3 (low: high)] or for two complementary genes [[Ol.sub.1] and [Ol.sub.2], [F.sub.2] ratio of 7:9 (low and intermediate: high)], instead of three complementary genes.

The genetic control of the high C18:0 trait in sunflower has been studied in the mutant line CAS-3. Perez-Vich et al. (1999) reported that this character was determined by partially recessive alleles at two loci. The loci were designated Es1 and Es2, with the Es1 locus having a greater effect on the C18:0 content than the Es2 locus.

The objectives of the present research were (i) to study the genetic relationships between the loci controlling high C18:0 and high C18:1 concentrations in the mutant CAS-3 and in a high C18:1 line and (ii) to combine both traits in a single line.

MATERIALS AND METHODS

The lines used in this study were the high C18:0 mutant CAS-3, obtained after treatment of the line RDF-1-532 with ethylmethane sulfonate (Osorio et al., 1995), and the high C18:1 inbred line HAOL-9 (Fernandez-Martinez et al., 1993), developed from the inbred line HA-89 after backcrossing with a high C18:1 line (Soldatov, 1976). HA-89, with standard fatty acid composition in its seed oil and near-isogenic to HAOL-9, was used as a check. Half seeds of CAS-3 and HAOL-9 were analyzed for fatty acid composition to ensure that the plants used in the crosses had either high C18:0 or high C18:1 content. Fatty acid methyl esters were obtained as described by Garces and Mancha (1993) and analyzed on a gas-liquid chromatograph with a 2-m-long column packed with 3% (v/v) SP-2310/2% (v/v) SP-2300 on Chromosorb WAW (Supelco Inc., Bellefonte, PA). The oven, injector, and flame ionization detector were held at 190, 275, and 250 [degrees] C, respectively.

Plants of CAS-3 and HAOL-9 derived from half seeds were reciprocally crossed under greenhouse conditions [35/15 [degrees] C (day/night) with 16-h day length] at the Instituto de Agricultura Sostenible of Cordoba (southern Spain) in November 1994. At flowering, each head was covered with a paper bag to avoid contamination with external pollen. Crossing was achieved through the emasculation of florets of the female parent followed by pollination of their stigmas with pollen from the male parent. The fatty acid composition of [F.sub.1] half seeds from each cross was analyzed by GLC. The t-test for unpaired observations was used to determine significant differences between means of reciprocal [F.sub.1]s. Because the results did not reveal maternal effects, the fatty acid composition of segregating generations was analyzed on single half seeds.

The [F.sub.1] reciprocal half seeds and half seeds from the parents were germinated in May 1995 and, after 15 d in a growth chamber [25/15 [degrees] C (day/night), with a 16-h photoperiod], transplanted in a field nursery at the experimental farm of the Instituto de Agricultura Sostenible at Cordoba (sandy loam, deep alluvial, Typic Xerofluvent). The distance between plants within a row was 40 cm and between rows 85 cm. [F.sub.1] plants were self-pollinated to obtain the [F.sub.2] seed. Backcrosses to both parents were also made to produce the [BC.sub.1][F.sub.1] seed. Reciprocal crosses between the two parents were repeated to obtain reciprocal [F.sub.1] seeds in the same environment as the [F.sub.2] and [BC.sub.1][F.sub.1] seed. A total of 742 [F.sub.2] seeds, 192 [BC.sub.1][F.sub.1] to HAOL-9 seeds, and 144 [BC.sub.1][F.sub.1] to CAS-3 seeds were analyzed by GLC. Eighteen [F.sub.2] half seeds, representing all the different [F.sub.2] classes, were selected on the basis of their fatty acid composition and transplanted in the field in the spring of 1996. The [F.sub.2] plants were self-pollinated to obtain the [F.sub.3] seed. The study of the [F.sub.3] generation was performed through the analysis of 60 half seeds from each segregating [F.sub.2] plant and of about 12 half seeds from each non-segregating [F.sub.2] plant.

The C18:0 and C18:1 contents of [F.sub.2], [BC.sub.1][F.sub.1], and [F.sub.3] seeds were assigned to phenotypic classes based on the values found for the parents grown under the same environment and on previous reports studying the genetics of the high C18:1 and the high C18:0 traits (Urie, 1985; Fernandez-Martinez et al., 1989; Perez-Vich et al., 1999). The observed proportions within each phenotypic class were compared to those expected on the basis of appropriate genetic hypotheses. Chi-square goodness of fit was used to compare observed and expected ratios.

RESULTS AND DISCUSSION

The C18:0 content of CAS-3 and the C18:1 levels of HAOL-9 were five-fold and four-fold higher, respectively, than those of HA-89 (Table 1). Maternal effects for C18:0 or C18:1 contents were not observed, as indicated by the absence of significant differences between reciprocal [F.sub.1] half seeds (Table 1). The C18:0 content in reciprocal [F.sub.1] half seeds (79 g [kg.sup.-1] and 85 g [kg.sup.-1], respectively) was significantly different from that of both parents (230 g [kg.sup.-1] and 45 g [kg.sup.-1], respectively) (Table 1) and lower than the midparent value (137 g [kg.sup.-1]), indicating a partial dominance of the low over the high C18:0 levels, as reported previously (Perez-Vich et al., 1999). In contrast, there was only a minor difference between the C18:1 content in reciprocal [F.sub.1] half seeds (834 g [kg.sup.-1] and 824 g [kg.sup.-1], respectively), and in HAOL-9 (858 g [kg.sup.-1]) (Table 1), suggesting a complete dominance of the high over the low C18:1 content. Urie (1985) and Fernandez-Martinez et al. (1989) reported similar results in their genetic analysis of the high C18:1 trait.

Table 1. Fatty acid composition of the seed oil of sunflower lines CAS-3, HAOL-9, their reciprocal [F.sub.s], and a control line (HA-89) with standard fatty acid composition. Fatty acids are expressed as mean value [+ or -] standard deviation.
Material                         C16:0                 C18:0

                                       g [kg.sup.-1]

Control line (HA-89)         72 [+ or -]          57 [+ or -] 5b
                              6a([dagger])
CAS-3                        75 [+ or -] 7a      230 [+ or -] 16d
[F.sub.1] (CAS-3 x HAOL-9)   54 [+ or -] 3b       79 [+ or -] 6c
[F.sub.1] (HAOL-9 x CAS-3)   53 [+ or -] 4b       85 [+ or -] 7c
HAOL-9                       46 [+ or -] 4c       45 [+ or -] 8a

Material                         C18:1                C18:2

                                       g [kg.sup.-1]

Control line (HA-89)         219 [+ or -] 31b     651 [+ or -] 29a
CAS-3                        132 [+ or -] 19a     562 [+ or -] 25b
[F.sub.1] (CAS-3 x HAOL-9)   834 [+ or -] 6c       33 [+ or -] 3d
[F.sub.1] (HAOL-9 x CAS-3)   824 [+ or -] 16c      37 [+ or -] 19cd
HAOL-9                       858 [+ or -] 21d      50 [+ or -] 14c


([dagger]) Means for each fatty acid followed by the same letter are not significantly different (t-tests, P = 0.05).

Data from the [F.sub.2] seeds of the cross HAOL-9 x CAS-3 were analyzed for the es1, es2, and the Ol alleles separately to determine if the data were consistent with the previously reported genetic ratios for these loci. The C18:1 [F.sub.2] content was assigned to two [F.sub.2] phenotypic classes: low-intermediate (C18:1 [is less than or equal to] 700 g [kg.sup.-1]) and high (C18:1 [is greater than] 700 g [kg.sup.-1]) (Fig. 1). Four of the six [F.sub.2] populations analyzed satisfactorily fit a 1:3 (low-intermediate: high) ratio, while the other two were adjusted to a 7:9 ratio (Table 2). Both genetic ratios have been previously described for [F.sub.2] segregations from crosses between low and high C18:1 lines (Fick, 1984; Urie, 1985; Fernandez-Martinez et al., 1989). The [F.sub.2] C18:0 content was assigned to two phenotypic classes: low-intermediate (C18:0[is less than or equal to] 200 g [kg.sup.-1]) and high (C18:0[is greater than]200 g [kg.sup.-1]) (Fig. 1). All the [F.sub.2] populations analyzed were adjusted to a 15:1 (low-intermediate: high) genetic ratio (Table 2), a modified form of 1:14:1 (low: intermediate: high), which was consistent with previous results (Perez-Vich et al., 1999).

[Figure 1 ILLUSTRATION OMITTED]

Table 2. Frequency distributions and Chi-square analyses for C18:0 and C18:1 in [F.sub.2] seeds of sunflower crosses between HAOL-9 and CAS-3.
                    No. of [F.sub.2] in C18:0 and
                        C18:1 classes([dagger])

                L-I 18:0   L-I 18:0    H 18:0     H 18:0
[F.sub.2]       L-I 18:1   H 18:1      L-I 18:1   H 18:1

F2-32            35          118         12         0
F2-34            51           99         15         0
F2-35            16           64          6         0
F2-37            51           79         13         0
F2-43            21           62          7         0
F2-45            25           62          6         0
Pooled          199          484         59         0
Heterogeneity

                         Chi-square (P)

                   C18:0                C18:1

[F.sub.2]       15:1 (L-I:H)         1:3 (L-I:H)

F2-32           0.30 (0.58)          1.07 (0.30)
F2-34           2.29 (0.13)
F2-35           0.07 (0.79)          0.01 (0.90)
F2-37           2.01 (0.15)
F2-43           0.36 (0.55)          1.79 (0.18)
F2-45           0.01 (0.93)          3.44 (0.06)
Pooled          3.66 (0.05)          4.67 (0.03)
Heterogeneity   1.38 (0.95-0.90)     1.64 (0.70--0.50)

                 Chi-square (P)

                    C18:1

[F.sub.2]        7:9 (L-I:H)

F2-32
F2-34            0.94 (0.33)
F2-35
F2-37            0.06 (0.80)
F2-43
F2-45
Pooled           0.30 (0.58)
Heterogeneity    0.70 (0.50--0.30)

                      C:18:0 and C18:1
                     (combined classes)

[F.sub.2]          15:45:1:3          105:135:7:9

F2-32            42.07 (<0.001)
F2-34                               35.93 (<0.001)
F2-35            22.07 (<0.001)
F2-37                               27.39 (<0.001)
F2-43            24.60 (<0.001)
F2-45            18.44 (<0.(8)1)
Pooled          105.76 (<0.(8)1)    61.71 (<0.001)
Heterogeneity     1.42 (0.99)        1.61 (0.70--0.50)


([dagger]) The limits between the different classes for each fatty acid are explained in the text. L = Low, I = Intermediate, H = high.

From the above data, three or four independent genes are expected to segregate in the [F.sub.2] generation of crosses involving HAOL-9 and CAS-3 when both the high stearic and the high oleic acid traits are studied together. Because these characters segregate to 15:1 (high C18:0) and 1:3 or 7:9 (high C18:1) phenotypic classes (Table 2), a 15:45:1:3 (for a 1:3 C18:1 [F.sub.2] ratio) or a 105:135:7:9 (for a 7:9 C18:1 [F.sub.2] ratio) [F.sub.2] ratio was expected in a combined segregation. However, only three phenotypic classes were observed (Fig. 1). The missing group was that containing high C18:0 ([is greater than] 200 g [kg.sup.-1]) levels in a high C18:1 background. As a consequence, the three or four loci hypotheses were rejected in all the [F.sub.2] families studied after the chi-square analyses (Table 2).

All the [BC.sub.1][F.sub.1] to HAOL-9 seeds had a high C18:1 content (Fig. 1). The C18:0 content of these seeds fell into two classes (Fig. 1). The first one, containing one fourth of the seeds, showed C18:0 values below 50 g [kg.sup.-1]. The second one, which included three fourth of the seeds, was characterized by C18:0 levels between 50 and 100 g [kg.sup.-1] (Table 3). In the [BC.sub.1][F.sub.1] to CAS-3 generation, the segregation ratio was 3:1 for the high C18:0 and 1:1 or 3:1 for the high C18:1 when the traits were studied separately (Table 3). However, when both characters were studied for their combined segregation there was not a good fit of the observed to the expected genetic ratios (Table 3).

Table 3. Frequency distributions and Chi-square analyses for C18:0 and C18:1 in [BC.sub.1][F.sub.1] seeds of sunflower crosses between HAOL-9 and CAS-3.
                                 No. of [BC.sub.1][F.sub.1] seeds
                                      in C18:0 and C18:1
                                        classes([dagger])

                                  L 18:0    L 18:0    I 18:0
[BC.sub.1][F.sub.1]               L-18:1    H 18:1   L-I 18:1

- [BC.sub.1][F.sub.1] to HAOL-9
BCOL-1                                        31
BCOL-2                                        32
Pooled                                        63
Heterogeneity

- [BC.sub.1][F.sub.1] to CAS-3
BCC3-1                                                  14
BCC3-2                                                  17
Pooled                                                  31
Heterogeneity

                                  No. of [BC.sub.1][F.sub.1] seeds
                                      in C18:0 and C18:1
                                        classes([dagger])

                                  I 18:0    H 18:0    H 18:0
[BC.sub.1][F.sub.1]               H 18:1   L-I 18:1   H 18:1

- [BC.sub.1][F.sub.1] to HAOL-9
BCOL-1                              65
BCOL-2                              64
Pooled                             129
Heterogeneity

- [BC.sub.1][F.sub.1] to CAS-3
BCC3-1                              18        16        0
BCC3-2                              58        21        0
Pooled                              76        37
Heterogeneity

                                     Chi-square (P)

[BC.sub.1][F.sub.1]                     C18:0

                                    Chi-square (P)

- [BC.sub.1][F.sub.1] to HAOL-9      1:3 (L:I)
BCOL-1                               2.72 (0.10)
BCOL-2                               3.55 (0.06)
Pooled                               6.25 (0.01)
Heterogeneity                        0.02 (0.9-0.7)

- [BC.sub.1][F.sub.1] to CAS-3       3:1 (I:H)
BCC3-1                               1.78 (0.18)
BCC3-2                               0.50 (0.48)
Pooled                               0.04 (0.85)
Heterogeneity                        0.62 (0.5-0.3)

                                          Chi-square (P)

[BC.sub.1][F.sub.1]                           C18:1

- [BC.sub.1][F.sub.1] to HAOL-9
BCOL-1
BCOL-2
Pooled
Heterogeneity

- [BC.sub.1][F.sub.1] to CAS-3     3:1 (L-I:H)      1:1 (L:H)
BCC3-1                             4.00 (0.05)
BCC3-2                                             4.16 (0.04)
Pooled
Heterogeneity

                                            Chi-square (P)

                                            C18:0 and C18:1
[BC.sub.1][F.sub.1]                       (combined classes)

- [BC.sub.1][F.sub.1] to HAOL-9
BCOL-1
BCOL-2
Pooled
Heterogeneity

- [BC.sub.1][F.sub.1] to CAS-3      9:3:3:1            3:3:1:1
BCC3-1                            23.70 (<0.001)
BCC3-2                                             42.22 (<0.001)
Pooled
Heterogeneity


([dagger]) The limits between the different classes for each fatty acid are explained in the text. L = Low, I = Intermediate, H = high.

An [F.sub.2] family (F2-32) showing a two-gene segregation for C18:0 and a one-gene segregation for C18:1 (Table 2) was selected because of its simple C18:1 segregation. The progenies of 18 [F.sub.2] half seeds from this [F.sub.2] family representing all the [F.sub.2] classes were evaluated (Fig. 2). The [F.sub.3] families derived from [F.sub.2] half seeds with low C18:1 levels ([is less than] 500 g [kg.sup.-1]) and a low C18:0 content ([is less than] 50 g [kg.sup.-1]) (families F3-1 and F3-2; Fig. 2g) bred true for low values of both fatty acids (Table 4). Similarly, the [F.sub.3] progenies from [F.sub.2] seeds with low C18:1 values and a C18:0 content above 200 g [kg.sup.-1] (families [F.sub.3]-3 and F3-4; Fig. 2i) or with high C18:1 ([is greater than] 700 g [kg.sup.-1]) and low C18:0 contents (family F3-5; Fig. 2d) bred true for high C18:0 or high C18:1 values, respectively (Table 4). In those [F.sub.3] populations where only one of the studied fatty acids was segregating while the other one was at low concentrations (families F3-6; Fig. 2f, and F3-7, F3-8 and F3-9; Fig 2h), a one-gene segregation for the high C18:0 or the high C18:1 traits was found (Table 4). This kind of segregation was also found in those [F.sub.3] populations segregating for C18:0 in a high C18:1 background (family F3-10; Fig. 2a; Table 4).

[Figure 2 ILLUSTRATION OMITTED]

Table 4. Number of seeds having different C18:0 and C18:1 content in the analysis of [F.sub.3] sunflower populations from the cross HAOL-9 x CAS-3 and Chi-square ([chi square]) analyses.
                            C18:0 and C18:1    Range C18.0 and
                            in [F.sub.2]           C18:1 in
                            half seed          [F.sub.3] seeds

                 Id. in
[F.sub.3]        Fig. 2    C18:0   C18:1       C18:0      C18:1

                                 g [kg.sup.-1l]

F3-1                g       62      282        51-77     203-371
F3-2                g       69      476        49-80     313-504
F3-3                i       251     216       216-256    131-164
F3-4                i       253     244       240-276    127-171
F3-5                d       37      894        30--46    897-924
F3-6                f       84      660        52-98     285-890
F3-7                h       101     480        63-261     96-435
F3-8                h       110     410        55-255    102-438
F3-9                h       116     303        57-281     95-500
F3-10               a       78      834        37-150    765-911
F3-13               c       122     801       118-153    762-818
F3-14               e       149     727       130-294     98-788
F3-15               e       146     731       108-270    107-809
F3-16               e       180     699       157-286    104-759
F3-17               e       184     513        90-235    131-820
F3-18               e       196     704       142-241    100-792

                       No. [F.sub.3] seeds in C18:0
                             and C18:1 classes

                 L 18:0           L 18:0             I 18:0
[F.sub.3]       L-I 18:1          H 18:1            L-I 18:1

F3-1               12
F3-2               47
F3-3
F3-4
F3-5                                12
F3-6               16               28
F3-7               12                                  25
F3-8               23                                  50
F3-9               15                                  21
F3-10                               12
F3-13
F3-14
F3-15
F3-16
F3-17
F3-18

                       No. [F.sub.3] seeds in C18:0
                             and C18:1 classes

                 1 18:0           H 18:0             H 18:0
[F.sub.3]        H 18:1          L-I 18:1            H 18:1

F3-1
F3-2
F3-3                                11
F3-4                                12
F3-5
F3-6
F3-7                                11
F3-8                                22
F3-9                                10
F3-10              24                             11([dagger])
F3-13              47
F3-14              73               23
F3-15              71               25
F3-16              35               13
F3-17              71               25
F3-18              30               15

              [chi square]     [chi square]      [chi square]
                   (P)              (P)               (P)
[F.sub.3]          1:3             1:2:1              3:1

F3-1
F3-2
F3-3
F3-4
F3-5
F3-6           3.03 (0.08)
F3-7                            0.12 (0.94)
F3-8                            0.28 (0.87)
F3-9                            1.43 (0.45)
F3-10                           0.06 (0.97)
F3-13                                             0.05 (0.81)
F3-14                                             0.05 (0.81)
F3-15                                             0.05 (0.81)
F3-16                                             0.11 (0.74)
F3-17                                             0.05 (0.81)
F3-18                                             1.67 (0.20)


([dagger]) The C18:0 limits between classes in F3-10 were: L: <60 g [kg.sub.-1], which are the limits of a [F.sub.2] segregating genotype Esles1Es2Es2[Ol.sub.1][Ol.sub.1][Ol.sub.2][Ol.sub.2],

[F.sub.3] seeds of [F.sub.2] plants derived from [F.sub.2] half seeds with intermediate C18:0 levels and a high C18:1 concentration showed three different behaviors (Fig. 2b, 2c, 2e). A first group segregated for both fatty acids (Fig. 2b), following a similar pattern to that described for the [F.sub.2] generation. A second group did not segregate (family F3-13; Fig. 2c), with all the [F.sub.3] seeds having intermediate C18:0 and high C18:1 values (Table 4). Finally, a third group of [F.sub.3] seeds, which was derived from the [F.sub.2] half seeds with the highest [F.sub.2] C18:0 content (150 g [kg.sup.-1] to 196 g [kg.sup.-1]) in a high C18:1 background (families F3-14, F3-15, F3-16, F3-17, F3-18; Fig. 2e) was not stable and segregated for both fatty acids. The segregation for C18:1 in this latter group fit a 3:1 (high: low) ratio (Table 4), with the high C18:1 seeds having C18:0 levels from 130 g [kg.sup.-1] to 190 g [kg.sup.-1] and the low C18:1 seeds having C18:0 values higher than 200 g [kg.sup.-1].

According to the genetic systems proposed to explain the inheritance of the high C18:0 trait in CAS-3 (Perez-Vich et al., 1999) and of the high C18:1 trait in different high C18:1 lines (Fick, 1984; Urie, 1985; Miller et al., 1987; Fernandez-Martinez et al., 1989), the genotype of CAS-3 would be es1es1es2es2[ol.sub.1][ol.sub.1][ol.sub.2][ol.sub.2] or es1es1es2es2[ol.sub.1][ol.sub.1][Ol.sub.2][Ol.sub.2] and that of HAOL-9 Es1Es1Es2Es2[Ol.sub.1] [Ol.sub.1][Ol.sub.2][Ol.sub.2]. Such genetic configurations were confirmed in the present study by the observed segregation for each trait independently. The existence of two different C18:1 segregations for one or two complementary genes in crosses with CAS-3 (Table 2) suggested the presence of C18:1 dominant alleles in some genotypes of CAS-3, as reported previously for other low C18:1 lines (Fernandez-Martinez et al., 1989).

Whereas the study of the segregations of the high C18:0 and the high C18:1 traits independently was consistent with previous reports (Perez-Vich et al., 1999; Fernandez-Martinez et al., 1989), their combined segregation was significantly different from that expected for an independent inheritance. The combination of the high C18:0 mutant CAS-3 and the high C18:1 line HAOL-9 did not produce [F.sub.2] or [BC.sub.1][F.sub.1] to CAS-3 recombinant phenotypes with high C18:0 levels (similar to those found in CAS-3, from 200 g [kg.sup.-1] to 280 g [kg.sup.-1]) in a high C18:1/low C18:2 background.

Previous studies on the genetic relationships between loci controlling high or low concentrations of different seed oil fatty acids in other oil crops also reported the lack of a recombinant class combining the fatty acid levels found in the parental lines (Ladd and Knowles, 1971; Ntiamoah et al., 1995). This fact was attributed to the existence of modifying genes or maternal effects (Ladd and Knowles, 1971). However, in those studies, [F.sub.3] seeds with a stable recombinant phenotype belonging to the [F.sub.2] missing group were recovered from [F.sub.2] seeds. This was not the case of the present study, where the [F.sub.2] seeds with the highest C18:0 values found (from 150 g [kg.sup.-1] to 196 g [kg.sup.-1]) in high C18:1 background did not lead to stable [F.sub.3] high C18:0/high C18:1 values, segregating to low C18:1 values in the [F.sub.3] generation, as described above (Fig. 2e). These results suggested that the [F.sub.2] seeds of this group had a genotype that was always heterozygous, and could not be fixed.

The above data were interpreted as the existence of a genetic linkage between the allele Es2 and one of the Ol alleles ([Ol.sub.1] or [Ol.sub.2]). The Es2 locus was hypothesized to be involved in the genetic linkage instead of Es1 because the contribution of es2 allele to the C18:0 content is lower than that of the es1 allele (Perez-Vich et al., 1999). As the alleles [Ol.sub.1] or [Ol.sub.2] are complementary and they have the same effect in the control of the high C18:1 trait (Fernandez-Martinez et al., 1989), either of them could be involved in the genetic linkage. For the discussion, it has been considered that the allele [Ol.sub.1] was the one linked to Es2. Such a linkage would modify the combined segregation of the two traits studied, but not their independent segregation, because of a change in the proportion of the parental classes (high C18:0/low C18:1 and low C18:0/high C18:1) in relation to the recombinant classes (low C18:0/low C18:1 and high C18:0/ high C18:1). The lethality of the embryos expressing a phenotype double high (high C18:0/high C18:1) was ruled out because it would have modified the independent segregation of the high C18:0 and the high C18:1 traits when they were studied separately. According to the proposed Es2-[Ol.sub.1] linkage model, the highest [F.sub.2] C18:0 values in a high oleic acid background would be the result of the expression of a genotype eslesles2Es2 [Ol.sub.1][Ol.sub.1][Ol.sub.2][Ol.sub.2]. This [F.sub.2] genotype is always heterozygous, and will segregate to low C18:1 values in the [F.sub.3] generation (Fig. 2e). Thus, the genetic linkage determines the absence of a recombinant [F.sub.2] genotype es1es1es2es2[Ol.sub.1][Ol.sub.1][Ol.sub.2][Ol.sub.2] and the lack of [F.sub.2] or [F.sub.3] recombinant phenotypes completely fixed for high values of both fatty acids.

In addition, the linkage Es2-[Ol.sub.1] would only lead to [F.sub.3] one-gene segregations for the C18:0 content either in low or high C18:1 backgrounds (Fig 2h, 2a). These segregations would correspond to the segregation of the [F.sub.2] genotypes Es1es1es2es2[ol.sub.1][ol.sub.1][Ol.sub.2][Ol.sub.2] (Fig. 2h) and Es1es1Es2Es2[Ol.sub.1][Ol.sub.1][Ol.sub.2][Ol.sub.2] (Fig. 2a). [F.sub.2] genotypes such as EsleslEs2es2[Ol.sub.1][Ol.sub.1][Ol.sub.2][Ol.sub.2] or EsleslEs2es2[ol.sub.1][ol.sub.1][Ol.sub.2] [Ol.sub.2] (segregating for two C18:0 genes in high or low C18:0 backgrounds) would not be found (Fig. 2).

An objective of this research was to create a novel sunflower seed oil profile with a high C18:0 in high C18:1 background. This phenotype was not recovered, as has been described. However, [F.sub.2] seeds with C18:0 values from 80 g [kg.sup.-1] to 150 g[ kg.sup.-1] and with high C18:1 levels were obtained (Table 4), and some of them led to stable intermediate C18:0/high C18:1 values (130 g [kg.sup.-1] / 800 g [kg.sup.-1]) in the [F.sub.3] generation (Fig. 2c). Taking into account the proposed model, these [F.sub.3] seeds have the genotype es1es1Es2Es2Ol1Ol1Ol2Ol2.

According to the results of the present study, further strategies are needed to obtain higher C18:0 levels in a high C18:1 background. First, the use of biochemical or molecular approaches will give us further information about the nature of the relationships between the high C18:0 and the high C18:1 traits. Second, the analysis of even larger segregating populations will increase the probability of recovering high recombinant values of both C18:0 and C18:1. Finally, the use of other high C18:1 backgrounds, different from that of HAOL-9 might also contribute to overcome the limitations imposed by the Es2-[Ol.sub.1] linkage detected in this research.

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Begona Perez-Vich, Rafael Garces, and Jose Maria Fernandez-Martinez(*)

B. Perez-Vich and J.M. Fernandez-Martinez, Instituto de Agricultura Sostenible (CSIC), Apartado 4084, E-14080 Cordoba, Spain; R. Garces, Instituto de la Grasa (CSIC), Apartado 1078, E-41080 Sevilla, Spain. Received 25 June 1999. (*) Corresponding author (cs9femaj @uco.es).
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Author:Perez-Vich, Begona; Garces, Rafael; Fernandez-Martinez, Jose Maria
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Date:Jul 1, 2000
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