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Inheritance of High Oleic/Low Ricinoleic Acid Content in the Seed Oil of Castor Mutant OLE-1
Mar a Ferna ndez-Mart nez* Pilar Rojas-Barros, Antonio de Haro, and Jose ABSTRACT
A mutant line of castor (Ricinus communis L.), OLE-1, was recently identified. It has a 20-fold increase in oleic acid (C18:1, about 780 g kg1) and a six-fold decrease in ricinoleic acid content (C18:1-OH, about 140 g kg1) compared with standard castor oil (C18:1, about 40 g kg1; C18:1-OH, about 870 g kg1). The objective of this research was to determine the inheritance of the high oleic/low ricinoleic trait in this mutant. Reciprocal crosses were made between the mutant OLE-1 and castor line A74/18/10 with standard composition. Although a slight maternal effect for oleic and ricinoleic content was observed in the analysis of F1 seeds, the genetic control was mainly embryonic. The standard oleic/ricinoleic content was dominant over high oleic/low ricinoleic content. Oleic acid content of F2 seeds segregated in bimodal patterns, each consistent with a ratio of 13 to 3 for low-intermediate oleic content (110 g kg1) to high oleic content (650 g kg1), respectively. This segregation was consistent with the action of two independent major genes (ol, Ml) with epistatic interaction. The high oleic/low ricinoleic phenotype was homozygous for the genotypes with the recessive allele ol, and heterozygous or homozygous for the dominant allele Ml. The dominant allele Ml would release the action of the recessive allele ol, controlling the oleic and ricinoleic content. This mo_el was confirmed in the BC1F1 to OLE-1, which segregated following a 1:1 (low-intermediate:high) ratio, and F3 segregations. The information provided by this genetic study will facilitate the transfer of the high oleic/low ricinoleic trait to castor cultivars.

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

he quality of seed oils both for food and nonfood applications is largely determined by their fatty acid composition. The seed oil of castor normally contains about 900 g kg1 of ricinoleic acid (D-12-hydroxyoctadec-cis-9-enoic acid) (Brigham, 1993) and is too high for use as an edible oil but gives the oil its traditional industrial usage in manufacture of polymers, lubricants, polyurethane coatings, cosmetics, plastics, and other things (Bonjean, 1991; Brigham, 1993). However, a natural mutant of castor, OLE-1, with significantly less ricinoleic acid (C18:1-OH, about 140 g kg1 compared with about 900 g kg1 in commonly grown cultivars) and increased oleic acid (C18:1, about 780 g kg1 compared with about 40 g kg1 in standard castor bean oil) has been developed by selection from a germplasm accession with high oleic content (Rojas-Barros et al., 2004). This mutant probably has altered gene(s) encoding for the oleoil-12-hydroxylase enzyme which catalyses the hydroxylation of oleic to ricinoleic acid (Lin et al., 1996, 1998). Since high oleic acid levels are associated with oxidative stability (Friedt, 1988), the oil of the high oleic/
ndez-Mart nez, Instituto P. Rojas-Barros, A. de Haro, and J.M. Ferna rdoba, de Agricultura Sostenible (CSIC), Apartado 4084, E-14080 Co Spain. Received 16 Apr. 2004. *Corresponding author (cs9femaj@ uco.es). Published in Crop Sci. 45: (2005). Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA

low ricinoleic mutant OLE-1 could have industrial uses requiring high oxidative stability such as for biofuel, or pharmaceutical applications requiring lower ricinoleic levels than the standard castor oil. Moreover, this mutant signifies an important advance toward the development of ricinoleic acid free/high oleic acid castor oil germplasm with potential for the edible oil market. One requisite for the commercial use of the new oil is the incorporation of the modified biosynthetic pathway into commercial cultivars with good agronomic performance, which requires a knowledge of the genetic behavior of the trait. The inheritance of high oleic/low ricinoleic content in castor remains unexplained to date. However, the genetic control of high oleic content has been found to be simply inherited in different mutants of several oilseed crops. In soybean [Glycine max (L.) Merr.] and safflower (Carthamus tinctorius L.), it is controlled by multiple recessive alleles at a single locus (Takagi and Rahman, 1996; Knowles and Hill, 1964). The content of oleic acid in the corn oil (Zea mays L.) was shown to be controlled by single major gene (Widstro m and Jellum, 1984) or by two independent genes (De la Roche et al., 1971). In peanut (Arachis hypogaea L.), Moore and Knauft (1989) found two loci, designed Ol1 and Ol2, controlling the high oleic/low linoleic ratio in seed oil. In sunflower (Helianthus annuus L.), the high oleic acid content was found to be controlled by a single partially dominant gene designated Ol (Fick, 1984) or a dominant gene (Urie, 1984). However, later studies demonstrated that the genetic control of the high oleic acid trait in sunflower was more complex. Urie (1985) reported the existence of reversal in dominance and modifying genes. Additionally, Miller et al. (1987) described a second locus, Ml, whose recessive alleles mlml were necessary for the expression of the high oleic acid ndez et al. (1999) also postulated a twotrait, and Ferna gene (Ol and Ml) model, in which the high oleic acid phenotypes are the result of the expression of the geno ndez-Mart nez et al. (1989) type ololMlMl. Finally, Ferna identified three complementary dominant genes (Ol1, Ol2, and Ol3) controlling the high oleic acid trait in sunflower seed oil. The objective of this study was to determine the inheritance of the high oleic/low ricinoleic acid content of the castor mutant line OLE-1. MATERIALS AND METHODS Plant Materials
The castor germplasm used in this study were (i) OLE-1, a high oleic/low rinoleic mutant line developed by selection from a germplasm accession with high oleic/low ricinoleic conAbbreviations: GLC, Gas-liquid Chromatography.

CROP SCIENCE, VOL. 45, JANUARYFEBRUARY 2005

Table 1. Mean fatty acid content and their standard deviations of the oil for the castor line A74/18/10, mutant OLE-1, and their reciprocal F1 seeds, based on analysis of half-seeds from plants grown in Cordoba (Spain) during 1999.
Fatty acid (g kg1) Germplasm A74/18/10 F1 (A74/18/10 OLE-1) F1 (OLE-1 A74/18/10) OLE-1 n (p ) 109 98 49 206 (5) (2) (1) (2) C16:0 12 12 10 17 2 1 3 3 b b c a C18:0 15 18 14 10 2 4 1 1 b a b c C18:1 33 6 c 36 7 c 68 28 b 742 23 a C18:2 57 49 55 32 6a 3b 7a 5c C18:3 51b 61a 4 1 bc 42c C18:1-OH 870 870 840 183 10 a 9a 25 b 21 c

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

C16:0 palmitic acid, C18:0 stearic acid, C18:1 oleic acid, C18:2 linoleic acid, C18:3 linolenic acid, C18:1-OH ricinoleic acid. Number of half-seed (number of single-plants) analysed. Mean values of parents and reciprocal crosses for each fatty acid that have the same letter are not significantly different (LSD, p 0.05).

tent (Rojas-Barros et al., 2004) and (ii) the line A74/18/10, with a standard seed oil fatty acid profile (low oleic/high ricinoleic) selected from breeding material of PROTOSEMENCES Toulouse (France). The fatty acid composition of these materials is shown in Table 1.

Genetic Study
Seeds of OLE-1 and A74/18/10 were individually analyzed for fatty acid composition by the half seed method, described for castor by Rojas-Barros et al. (2004), to ensure that the plants used in the genetic study bred true for seed oil fatty acid composition. A distal portion of the seed was removed with a scalpel and used to determine the fatty acid composition of seed lipids by gasliquid chromatography (GLC). The remaining portion of the seed containing the embryo, with a known fatty acid profile was used for planting. As the trait high oleic/low ricinoleic is associated with very poor germination (Rojas-Barros et al., 2004), mature embryos of half seeds with this trait, were rescued by in vitro culture with Knudson C Modified Orchid Medium (Knudson, 1946). Plants of OLE-1 were reciprocally crossed with plants of A74/18/10 in the greenhouse in 1999. In all cases, paper bags were placed over racemes to prevent cross-pollination with external pollen. Crossing was done by emasculating immature flower buds in the raceme of the female parent followed by immediate pollination of their stigmas with fresh pollen from open flowers of the male parent. F1 half seeds from reciprocal crosses as well as seeds from the parents were analyzed for fatty acid composition from plants grown in the greenhouse in 2000. F1 plants from reciprocal crosses were self-pollinated to obtain F2 seeds and also backcrossed to both parents. Reciprocal crosses were repeated again to obtain F1 seeds grown under the same environment as the F2 and BC1F1 seeds. An evaluation of the fatty acid composition of F1 plants was made by averaging the GLC analyses of the F2 seeds from each individual F1 plant. Fatty acid composition was determined on a total of 999 individual F2 seeds, 272 BC1F1 to A74/18/10 seeds, and 204 BC1F1 to OLE-1 seeds. A total of 21 F2 half-seeds representing all the classes for oleic acid concentration detected in this generation were selected, germinated, and grown in a field screenhouse in 2001 to obtain the F3 generation. The study of this generation was performed through the analysis of 100 to 200 F3 seeds from each segregating F2 plant and about 20 to 100 seeds for each nonsegregating F2 plant.

was assigned to phenotypic classes on the basis of the appearance of discontinuities in the frequency distribution and the values found in the parentals grown under the same environmental conditions. The proportion of seeds observed in each phenotype class was compared with those expected on the basis of appropriate genetic hypotheses. The goodness of fit to tested ratios was measured by the 2 statistic. Heterogeneity 2 for families within a cross was nonsignificant so that data for families for the same cross were pooled for analysis.

Fatty Acid Analyses

The fatty acid composition of the seed oil was determined by simultaneous oil extraction and methyl esterification following the procedure described by Rojas-Barros et al. (2004) then analyzed by GLC on a PerkinElmer Autosystem gasliquid chromatograph (PerkinElmer Corporation, Norwalk, CT) equipped with a flame ionization detector and with a 2-m-long column packed with 3% SP-2310/2% SP-2300 on Chromosorb WAW (Supelco Inc., Bellefonte, PA). The injector, and flame ionization detector were held at 275 and 250C, respectively. The gas chromatograph was programmed for an initial oven temperature of 190C maintained for 10 min, followed by an increase of 5C m1 up to 225C, holding for 7 min.

RESULTS AND DISCUSSION

The average oleic and ricinoleic acid content of the mutant line OLE-1 were 20-fold higher and five-fold lower, respectively, than those of the standard low oleic/ high ricinoleic line A74/18/10 (Table 1). The oleic and ricinoleic acid content in reciprocal F1 seeds differed significantly indicating the presence of partial maternal effects for these traits (Table 1). However, these differences were much smaller than those between the F1 and each of the parents indicating that the genetic control of the contents of these fatty acids was mainly embryonic with a minor maternal effect. The differences between reciprocal F1 seeds were not observed between F1 plants (F2 seeds averaged) (Table 2) revealing an absence of cytoplasmic effects for both fatty acids. Since no significant cytoplasmic effects could be detected the data from reciprocal F2 seeds in Fig. 1 and F1, F2, and BC1F1 seeds in Fig. 2 were combined. Similar results, embryonic control with a partial maternal effects of low magnitude in some crosses and the absence of cytoplasmic effects, have been reported for oleic acid content in safflower (Knowles and Hill, 1964), sunflower (Urie, 1984, 1985; Fick, 1984; ndez-Mart nez et al., 1989), Miller et al., 1987; Ferna rapeseed (Schierholt et al., 2001), and soybean (Takagi and Rahman, 1996; Rahman et al., 1994). There are no

Statistical Analyses
Mean oleic and ricinoleic acid content was calculated for the parental lines, the F1, and F2 generations and compared by the LSD test. Since the results did not reveal important maternal effects for oleic and ricinoleic acid content, the fatty acid composition of segregating generations was analyzed on single seeds. The oleic acid content of BC1F1, F2, and F3 seeds

ROJAS-BARROS ET AL.: INHERITANCE OF HIGH OLEIC CONTENT IN CASTOR

Table 2. Oleic acid content of F1 half-seeds and mean and range of oleic and ricinoleic acid content of the oil of F2 seeds from individual F1 plants of reciprocal crosses between castor lines OLE-1 and A74/18/10 grown in Cordoba (Spain) during 2000.
F1 plant (F2 averaged seeds) or parent seed A74/18/10 OLE-1 F11 (A74/18/10OLE-1) F12 (A74/18/10OLE-1) F13 (A74/18/10OLE-1) F14 (OLE-1A74/18/10) F15 (OLE-1A74/18/10) F16 (OLE-1A74/18/10) F17 (OLE-1A74/18/10) C18:1 content in F1 half-seeds C18:1 content in F2 seeds n 63 100 150 149 100 150 150 150 150 Mean g kg1 31 751 199 210 197 146 174 186 173 1946 685795 21798 19835 24787 19802 17833 20792 31812 872 176 710 697 709 769 741 729 734 843893 151239 117936 86887 13288.8 115902 103899 141892 117879 Range C18:1-OH content in F2 seeds Mean Range

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

45 25 29 109 110 19 25

a a a a a a a

a a a a a a ab

Number of analysed half-seeds; C18:1 oleic acid; C18:1-OH ricinoleic acid. Means values for each fatty acid of the reciprocal in F2 families that have the same letter are not significantly different (LSD test, p 0.05).

Fig. 1. Scatter plot of oleic and ricinoleic acids content in the oil of F2 seeds of reciprocal crosses between castor A74/18/10 and OLE-1.

previous studies on maternal and cytoplasmic effects on the content of ricinoleic concentration. The average oleic-acid content of F1 seeds from reciprocal crosses (52 g kg1) was very close to that of the standard low line A74/18/10 (Table 1) and much lower than the midparent value (387 g kg1) indicating almost complete dominance of the standard low over the increased oleic acid content. This result is in agreement with those obtained in safflower (Knowles and Hill, 1964). However, in sunflower several authors reported dominance of high oleic over low oleic acid content (Miller et ndez-Mart nez et al., 1989). The average al., 1987; Ferna ricinoleic-acid content in F1 generation (855 g kg1) was similar to that of the standard low oleic/high ricinoleic line A74/18/10 (Table 1) and higher than the mid-parent value (526 g kg1) suggesting dominance of standard over reduced ricinoleic acid concentration. The analysis of fatty acid composition of individual F2 seeds from reciprocal crosses between OLE-1 and A74/18/10, showed a considerable variation for oleic and ricinoleic acids (Fig. 1), which were strongly and negatively correlated (r 0.99, P 0.001). There was no substantial variation for the other fatty acids. This indicated that the relative proportions of oleic and ri-

cinoleic acids are under the control of one genetic system. The oleic acid values of the F2 seeds from OLE-1 A74/18/10 and A74/18/10 OLE-1 crosses revealed a clear bimodal distribution (Fig. 1 and 2). The first class with seeds having 110 g kg1of oleic acid was assigned to the combined category low-intermediate with an oleic acid range between 19 and 110 g kg1 and the second class to the high category (oleic acid 650 g kg1). The observed data satisfactorily fit a phenotypic ratio 13 low-intermediate: 3 high for these classes (Table 3). This segregation suggests that the high oleic/low ricinoleic content is determined by an interaction between a recessive allele at one locus and a dominant allele at a second locus (dominant and recessive epistasis). These alleles were designated ol and Ml respectively, following the symbols previously assigned to genes controlling oleic acid content in sunflower (Miller et al., 1987). The proposed genotypes for the high oleic-low ricinoleic acid mutant line OLE-1 was ololMlMl and for the low oleichigh ricinoleic acid line A74/18/10, OlOlmlml. With this genetic model the genotypes with high levels of oleic acid would be homozygous for the recessive allele ol. The olol is recessively epistatic to Ml locus and genotypes with these alleles (ololMl_) would produce high

CROP SCIENCE, VOL. 45, JANUARYFEBRUARY 2005

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

Fig. 2. Frequency of distribution of oleic acid (C18:1) in oil from individual seeds of castor A74/18/10, OLE-1 and their F1, F2 and BC1F1 populations of combined data of reciprocal crosses.

Table 3. Number of seeds having different oleic acid (C18:1) content and Chi-square analyses in the F2 and BC1F1 seeds from crosses between the standard castor line A74/18/10 and the mutant line OLE-1.
No. of seeds with C18:1 content Generation F2 (A74/18/10OLE-1) F2 (A74/18/10OLE-1) F2 (A74/18/10OLE-1) F2 (OLE-1A74/18/10) F2 (OLE-1A74/18/10) F2 (OLE-1A74/18/10) F2 (OLE-1A74/18/10) Pooled Heterogeneity BC1F1 ((A74/18/10OLE-1)A74/18/10) BC1F1 ((A74/18/10OLE-1)A74/18/10) BC1F1 ((OLE-1A74/18/10)A74/18/10) BC1F1 ((OLE-1A74/18/10)A74/18/10) BC1F1 ((A74/18/10OLE-1)OLE-1) BC1F1 ((OLE-1A74/18/10)OLE-1) BC1F1 ((OLE-1A74/18/10)OLE-1) Pooled Heterogeneity Low-intermediate (110 g kg1) 118 115 78 128 122 121 125 807 74 82 66 50 25 60 28 113 High (650 g kg1) 32 34 22 22 28 29 25 192 2 (p ) 0.60 1.41 0.61 1.99 0.00 0.03 0.46 0.14 4.96 (0.44) (0.24) (0.43) (0.16) (0.98) (0.86) (0.50) (0.70) (0.55)

expected to be in the low-intermediate class in the backcross to the low oleic parent A74/18/10. The data observed (Fig. 2) fitted satisfactorily the theoretical ratios (Table 3) supporting the proposed model. As a further confirmation of the genetic model proposed a progeny test was conducted on crosses by analyzing the F3 from each of 21 F2 plants. These plants were selected on the basis of the oleic acid content of the corresponding F2 half-seeds, which covered the whole range of oleic levels observed in the F2 population. The F3 seeds derived from F2 half-seed plants with low and intermediate oleic values (23-128 g kg1) showed three different patterns (Table 4). Four of them bred true for values of this fatty acid below 50 g kg1. The genotypes of these plants were identified as homozygous either for the ml or the Ol allele (genotypes _ _mlml or OlOl_ _). Five segregated for high (700 g kg1) oleic acid values with a 3:1 (low-intermediate:high) ratio and nine segregated with a 13:3 (low-intermediate:high) ratio. The segregation 3:1 (one locus) would correspond to a genotype OlolMlMl and the segregation 13:3 (two loci) would be expected for the progeny of the F2 genotype OlolMlml. In contrast, all the F3 progenies derived from F2 halfseeds plants with oleic acid content higher than 700 g kg1 showed no segregation for this fatty acid, the oleic acid content of all the F3 seeds being higher than 700 g kg1. The genotypes of these plants were identified as homozygous for ol and Ml allele (genotype ololMlMl). However, according to the genetic model proposed, F2 halfseeds plants with high oleic acid values (700 g kg1)

23 43 25 91

0.08 2.88 0.17 2.40 0.73

(0.77) (0.09) (0.68) (0.12) (0.69)

Ratios tested: F2 generation 13:3 and BC1F1 to OLE-1 generation 1:1, and p is the probability level for significance.

oleic/low ricinoleic whereas the ololmlml genotypes would produce low oleic/high ricinoleic. The F2 phenotypic expression of these genotypes would be in the ratio 13 low-intermediate (including genotypes Ol_Ml_, Ol_mlml and ololmlml): 3 high (genotype ololMl_). According to the proposed genetic model, a ratio of 1 low-intermediate (including genotypes OlolMlMl and OlolMlml): 1 high (including genotypes ololMlMl and ololMlml) was to be expected in the backcross to the high oleic plant OLE-1, whereas all the individuals were

heterozygous for the Ml gene (genotype ololMlml) would be expected to segregate 1 low-intermediate (genotype ololmlml): 3 high (genotypes ololMlMl and ololMlml). The absence of this segregation in the progeny of high oleic/low ricinoleic F2 half-seeds plants may be attributed to the fact that only three F3 families from high oleic F2 half seeds were evaluated due to the poor germination of F2 half seeds with this phenotype (Rojas-Barros et al., 2004) and apparently none of them had the ololMlml genotype.

ROJAS-BARROS ET AL.: INHERITANCE OF HIGH OLEIC CONTENT IN CASTOR

Table 4. Number of F3 castor seeds having a different oleic acid (C18:1) content in the analysis of 21 F3 families from the cross A74/ 18/10 OLE-1 and Chi-square (2) analyses.
No. F3 seeds in C18:1 classes F3 family F336 F373 F348 F3678 F3669 F3675 F383 F363 F372 F3664 F3707 F3690 F3698 F3674 F375 F3706 F3686 F3114 F3684 F399 F3702 C18:1 in F2 half-seed g kg1 23 33 37 39 39 39 44 48 51 57 78 87 89 104 104 105 109 128 788 791 815 196 39 178 43 210 269 43 147 40 233 223 171 200 192 217 181 191 190 789 832 751 122 20 121 20 112 78 20 119 20 176 125 128 117 118 113 168 120 118 33 29 36 35 23 61 42 28 32 32 37 43 32 30 148 116 100 0.07 (0.79) 0.00 (0.96) 0.07 (0.80) 0.66 (0.41) 0.60 (0.44) 0.01 (0.92) 0.35 (0.55) 0.48 (0.49) 0.21 (0.65) 0.04 (0.84) 1.89 (0.17) 0.67 (0.41) 0.60 (0.44) 0.03 (0.86) C18:1 of F3 family Low-intermediate High 2 (p ) 1-gene 3:1 2 (p ) 2-genes 13:3

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

Mean value of analysed F3 seeds.

No previous studies have been reported on inheritance of altered oleic acid content in castor. However, similar genetic systems with two genes and epistatic interaction have been proposed for the control of oleic ndez acid content in sunflower (Miller et al., 1987; Ferna et al., 1999). These studies concluded that the desaturation of oleic to linoleic acid was controlled by a major gene which action was modified by a second gene. Marker analyses supported that the oleoyl-PC-desaturase locus (OLD7) was altered in the high oleic sunflower mutant rez-Vich et al., 2002) and other molecular studies (Pe (Lacombe et al., 2001) identified another locus, present only in some genotypes, that suppress the effect of the OLD7 locus on the high oleic trait. In the present study, the recessive gene ol present in the high oleic/low ricinoleic castor mutant OLE-1 could affect the action of the oleoyl-12-hydroxylase enzyme preventing the hydroxylation of oleic acid to synthesize ricinoleic acid. Recessive alleles at the Ml locus would suppress the effect of the ol allele on the oleic/ricinoleic trait. Because of the low number of genes involved in the genetic control of the high oleic/low ricinoleic trait in the mutant OLE-1 a successful transfer of this trait into breeding lines could be performed in a few generations. Furthermore, despite a minor partial maternal effect for oleic acid concentration, the trait appears to be primarily under embryogenic control, which suggests that selection for the high oleic/low ricinoleic trait increased oleic acid (decreased ricinoleic) can be efficiently conducted at the single-seed level by means of the halfseeds technique. The use of this technique will accelerate breeding efforts for this trait. In conclusion, the information provided by this study clarify the genetic control of the high oleic/low ricinoleic content in the castor mutant OLE-1 and establish the basis for effective breeding strategies for the development of hybrid cultivars with these characteristics.

ACKNOWLEDGMENTS This work was supported by the European Commission under the project No AIR3-CT93-2324.

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