José M. Fernández-Martínez

Stearoyl-ACP and oleoyl-PC desaturase genes cosegregate with quantitative trait loci underlying high stearic and high oleic acid mutant phenotypes in sunflower

Abstract The genetic control of the synthesis of stearic acid (C18:0) and oleic acid (C18:1) in the seed oil of sunflower was studied through candidate-gene and QTL analysis. Two F2 mapping populations were developed using the high C18:0 mutant CAS-3 crossed to either HA-89 (standard, high linoleic fatty acid profile), or HAOL-9 (high C18:1 version of HA-89). A stearoyl-ACP desaturase locus (SAD17A), and an oleoyl-PC de-saturase locus (OLD7) were found to cosegregate with the previously described Es1 and Ol genes controlling the high C18:0 and the high C18:1 traits, respectively. Using linkage maps constructed from AFLP and RFLP markers, these loci mapped to LG1 (SAD17A) and to LG14 (OLD7) and were found to underlie the major QTLs affecting the concentrations of C18:0 and C18:1, explaining around 80% and 56% of the phenotypic variance of these fatty acids, respectively. These QTLs pleiotropically affected the levels of other primary fatty acids in the seed storage lipids. A minor QTL affecting both C18:0 and C18:1 levels was identified on LG8 in the HAOL-9×CAS-3 F2. This QTL showed a significant epistatic interaction for C18:1 with the QTL at the OLD7 locus, and was hypothesized to be a modifier of Ol. Two additional minor C18:0 QTLs were also detected on LG7 and LG3 in the HA-89×CAS-3 and the HAOL-9×CAS-3 F2 populations, respectively. No association between a mapped FatB thioesterase locus and fatty acid concentration was found. These results provide strong support about the role of fatty acid desaturase genes in determining fatty acid composition in the seed oil of sunflower.

Survey of safflower (Carthamus tinctorius L .)germplasm for variants in fatty acid composition and other seed characters

SummaryTwo hundred safflower accessions, originated in 37 countries, and multiplied in two environments, were evaluated for fatty acid composition of the seed oil and other seed characters. Overall mean values of stearic and palmitic acids were similar in both environments but differed for seed weight and oil content. Oleic and linoleic acids showed also similar overall mean content in both environments but some entries with intermediate contents of these acids displayed significant variation among environments. Oleic and linoleic acids showed a tremendous range of variation, from 3.1 to 90.60% and from 3.9 to 88.8%, respectively. The ranges of variation observed for stearic, oleic and linoleic acids indicate that all the reccessive genes, already discovered, controlling high content of these acids, st, ol and li, are present in the collection. Moreover, the upper values of oleic, ten points higher than the published values for the high oleic genotype olol, suggest than other genes controlling such levels may be present.

Sunflower mutant containing high levels of palmitic acid in high oleic background

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil.

Genetic and environmental variation for tocopherol content and composition in sunflower commercial hybrids

Tocopherols are the most important compounds having antioxidant activity in sunflower seeds. The objective of the present research was to study the genetic and environmental variation for tocopherol content and composition in sunflower. Thirty-six sunflower hybrids were grown at 13 locations across all major areas of sunflower cultivation in southern Spain. Seed yield, 100 seed weight, oil content and tocopherol content and composition were determined. Tocopherol content ranged from 314-5 to 1024.5 mg/kg seed and from 562.8 to 1872.8 mg/kg oil. The tocopherol fraction was largely composed of alpha-tocopherol, which accounted for 88.4 % to 96.3 % of the total tocopherols. Both genotypic and environmental effects were significant for tocopherol content and composition. For alpha-, beta- and total tocopherol content, the effect of the genotype was larger than that of the environment, whereas the latter had a greater effect on gamma-tocopherol content. Genotype x location interaction was significant for alpha-, gamma- and total tocopherol content. Tocopherol content was not correlated with seed oil or seed yield, indicating the possibility of selecting for this trait without affecting the performance of the genotypes.

Quantitative trait loci for broomrape (Orobanche cumana Wallr.) resistance in sunflower

Broomrape (Orobanche cumana Wallr.) is a root parasite of sunflower that is regarded as one of the most important constraints of sunflower production in the Mediterranean region. Breeding for resistance is the most effective method of control. P-96 is a sunflower line which shows dominant resistance to broomrape race E and recessive resistance to the very new race F. The objective of this study was to map and characterize quantitative trait loci (QTL) for resistance to race E and to race F of broomrape in P-96. A population from a cross between P-96 and the susceptible line P-21 was phenotyped for broomrape resistance in four experiments, two for race E and two for race F, by measuring different resistance parameters (resistance or susceptibility, number of broomrape per plant, and proportion of resistant plants per F3 family). This population was also genotyped with microsatellite and RFLP markers. A linkage map comprising 103 marker loci distributed on 17 linkage groups was developed, and composite interval mapping analyses were performed. In total, five QTL (or1.1, or3.1, or7.1 or13.1 and or13.2) for resistance to race E and six QTL (or1.1, or4.1, or5.1, or13.1, or13.2 and or16.1) for resistance to race F of broomrape were detected on 7 of the 17 linkage groups. Phenotypic variance for race E resistance was mainly explained by the major QTL or3.1 associated to the resistance or susceptibility character (R2=59%), while race F resistance was explained by QTL with a small to moderate effect (R2 from 15.0% to 38.7%), mainly associated with the number of broomrape per plant. Or3.1 was race E-specific, while or1.1, or13.1 and or13.2 of were non-race specific. Or13.1, and or13.2 were stable across the four experiments. Or3.1, and or7.1 were stable over the two race E experiments and or1.1 and or5.1 over the two race F experiments. The results from this study suggest that resistance to broomrape in sunflower is controlled by a combination of qualitative, race-specific resistance affecting the presence or absence of broomrape and a quantitative non-race specific resistance affecting their number.

Genetic analysis of yield and related traits in sunflower (Helianthus annuus L.) in dryland and irrigated environments

The definition of a suitable breeding strategy in drought-prone environments is an important task for sunflower breeders. To achieve this task, reliable information on heritability and gene effects on yield and related traits under these conditions is necessary. Thirty six sunflower hybrids were produced by factorial cross of six male-sterile and six restorer lines. Parents and their hybrids were evaluated in eight environments. Six environments consisted of two adjacent trials in the experimental area, the first under irrigation and the second under dryland conditions, during 1987, 1988 and 1992. The other environments were: one early planting trial in dryland conditions, conducted during 1987, and a winter trial planted in January during 1988. Estimates of female variance (σf) were significant for seeds per head, seed weight, head sterile center, days to blooming and oil content. Female × male interactions (σ2fm) were significant for all characters except harvest index and index of susceptibility to drought. Estimates of narrow sense heritabilities, calculated with information from analyses combined across environments, were 0.65 for yield, 0.80 for seeds per head, 0.84 for seed weight, 0.81 for head diameter, 0.60 for sterile head center, 0.72 for oil content, 0.61 for harvest index, 0.72 for biomass, 0.94 for days to bloom, and 0.42 for drought susceptibility index. Heritability estimates for individual environments showed more variation for yield than for other traits. Estimates for heritability of canopy temperature were high (0.68–0.79). Rainfed yield was positively correlated with yield components and negatively correlated with canopy temperature and susceptibility index. It is concluded that an efficient breeding strategy for sunflower under moderate drought-stressed conditions is the simultaneous selection for seed yield in both rainfed and irrigated environments together with selection for canopy temperature and stem diameter.

Screening of wild Helianthus species and derived lines for resistance to several populations of Orobanche cernua

Twenty-six different perennial species of Helianthus, 18 wild annual species of the same genus, and 29 lines tracing to wild species were evaluated for resistance to three highly virulent populations of broomrape (Orobanche cernua). Evaluations were carried out in pots containing soil mixture infested with broomrape seeds. Most of the perennial Helianthus species were immune to the populations of broomrape used in the tests. Some wild annual species and wild derived lines were resistant. The resistance found in the wild species, introgressed to cultivated sunflower, could provide unique resistance to the parasite.

Epistatic interaction among loci controlling the palmitic and the stearic acid levels in the seed oil of sunflower

Abstract Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in their seed oil have been identified and studied extensively. The mutant line CAS-5 has high concentrations of palmitic acid (C16:0) (>25% compared with 7% in standard sunflower seed oil) and low-C18:0 values (3%). CAS-3 is characterized by its high levels of stearic acid (C18:0) (>22% compared with 4% in standard sunflower seed oil) and a low-C16:0 content (5%). CAS-5 also possesses elevated levels of palmitoleic acid (C16:1) (>5%), which is absent in standard sunflower seed oil. The objective of this study was to determine the relationships between the loci controlling the high-C16:0 and the high-C18:0 traits in these mutants. Plants of both mutants were reciprocally crossed. Gas chromatographic analyses of fatty acids from the seed oil of F1, F2, F3 and the BC1F1 to CAS-5 generations indicated that the loci controlling the high-C16:0 trait exerted an epistatic effect over the loci responsible for the high-C18:0 character. As a result, the phenotypic combination containing both the high-C16:0 levels of CAS-5 and the high-C18:0 levels of CAS-3 was not possible. However, phenotypes with a saturated fatty acid content of 44% (34.5% C16:0+9.5% C18:0) were identified in the F3 generation. These are the highest saturated (C16:0 and C18:0) levels reported so far in sunflower seed oil. When F3 C16:0 segregating generations in both a high- and a low-C18:0 background were compared, the high-C16:1 levels were not expressed as expected in the high-C18:0 background (CAS-3 background). In this case, the C16:1 content decreased to values below 1.5%, compared with >5% in a low-C18:0 background. As the stearoyl-ACP desaturase has been reported to catalyze the desaturation from C16:0-ACP to C16:1-ACP, these results suggested that a decrease in its activity was involved in the accumulation of C18:0 in the high-C18:0 mutant CAS-3.

Mapping minor QTL for increased stearic acid content in sunflower seed oil

Increased stearic acid (C18:0) content in the seed oil of sunflower would improve the oil quality for some edible uses. The sunflower line CAS-20 (C18:0 genotype Es1Es1es2es2), developed from the high C18:0 mutant line CAS-3 (C18:0 genotype es1es1es2es2; 25% C18:0), shows increased C18:0 levels in its seed oil (8.6%). The objective of this research was to map quantitative trait loci (QTL) conferring increased C18:0 content in CAS-20 in an F2 mapping population developed from crosses between HA-89 (wild type Es1Es1Es2Es2; low C18:0) and CAS-20, which segregates independently of the macromutation Es1 controlling high C18:0 content in CAS-3. Seed oil fatty acid composition was measured in the F2 population by gas-liquid chromatography. A genetic linkage map of 17 linkage groups (LGs) comprising 80 RFLP and 19 SSR marker loci from this population was used to identify QTL controlling fatty acid composition. Three QTL affecting C18:0 content were identified on LG3, LG11, and LG13, with all alleles for increased C18:0 content inherited from CAS-20. In total, these QTL explained 43.6% of the C18:0 phenotypic variation. Additionally, four candidate genes (two stearate desaturase genes, SAD6 and SAD17, and a FatA and a FatB thioesterase gene) were placed on the QTL map. On the basis of positional information, QTL on LG11 was suggested to be a SAD6 locus. The results presented show that increased C18:0 content in sunflower seed oil is not a simple trait, and the markers flanking these QTL constitute a powerful tool for plant breeding programs.

Genetic control of high stearic acid content in the seed oil of the sunflower mutant CAS-3.

Abstract A sunflower mutant, CAS-3, with about 25% stearic acid (C18:0) in the seed oil was recently isolated after a chemical-mutagen treatment of RDF-1-532 seeds (8% C18:0). To study the inheritance of the high C18:0 content, CAS-3 was reciprocally crossed to RDF-1–532 and HA-89 (5% C18:0). Significant reciprocal-cross differences were found in one of the two crosses, indicating possible maternal effects. In the CAS-3 and RDF-1–532 crosses, the segregation patterns of the F1, BC1, and F2 populations fitted a one-locus (designated Es1) model with two alleles (Es1, es1) and with partial dominance of low over high C18:0 content. Segregation patterns in the CAS-3 and HA-89 crosses indicated the presence of a second independent locus (designated Es2) with two alleles (Es2, es2), also with partial dominance of low over high C18:0 content. From these results, the proposed genotypes (C18:0 content) of each parent were as follows: CAS-3 (25.0% C18:0) =es1es1es2es2; RDF-1–532 (8.0% C18:0) =Es1Es1es2es2; and HA-89 (4.6% C18:0) =Es1Es1Es2Es2. The relationship between the proposed genotypes and their C18:0 content indicates that the Es1 locus has a greater effect on the C18:0 content than the Es2 locus. Apparently, the mutagenic treatment caused a mutation of Es1 to es1 in RDF-1–532.