Inheritance of reduced height in sunflower

Summary Breeding to improve stem strength is a major objective of researchers of sunflower (Helianthus annuus L.). This study was undertaken to investigate genetic factors controlling reduced plant height and increased stem diameter in three sources of sunflower, DDR, Donsky, and Donskoi 47, crossed with a conventional height line, HA 89. As these two characters may lead to improved standability, knowledge of their inheritance will assist researchers in utilizing proper breeding methods. Estimates of additive, dominance, and epistatic genetic effects controlling reduced height indicated that the additive component was most important in two of the three crosses with the additive and epistatic component nearly equal in the third cross. Breeding efforts to reduce height of sunflower hybrids utilizing these lines in crosses could be effective due to the magnitude of additive effects. The dominance component of genetic effects controlling stem diameter was the most important for two crosses, with both dominance and additive components important for the third cross. Epistasis was present, but minor, for controlling stem diameter. The high relative importance of the dominance component indicates that testcross evaluation of lines in early generations could identify lines for producing increased stem diameter in hybrids. Even though the three sources of sunflower with reduced height were different in morphologic and agronomic characteristics, they had similar genetic control of plant height and stem diameter. Each could be utilized in a breeding program to develop lines with reduced height and larger stem diameter.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

A new source of cytoplasmic male sterility in sunflower

Summary Interspecific substitutions of the nucleus of Helianthus annuus (2n=34) cv. Saturn into the cytoplasm of H. petiolaris (2n=34) by successive backcrossing, resulted in progenies with indehiscent anthers containing white, rather than normal yellow, pollen. Further backcrossing by cv. Saturn failed to restore pollen shed, suggesting that the male sterility was cytoplasmic. In vivo germination tests of pollen from 23 such plants from eight BC₅ lines, indicated complete pollen sterility for 14 plants, but normal seed set, suggesting that female fertility was not affected. Meiosis in all plants examined was normal.Crosses between seven sources of pollen-fertility restorer, one collection of wild H. annuus, and an existing source of cytoplasmic male sterility, resulted in a high frequency of plants with normal pollen shed in all F₁ progenies. However, no normal pollen shed was evident in F₁ progenies for similar crosses between BC₅ male-steriles and three of the seven restorer sources, nor for the single wild H. annuus evaluated. The foregoing suggests that the backcross substitution lines are a new source of cytoplasmic male sterility. The inheritance of restoration of pollen shed was complex and not fully elucidated. Some data suggested that two independent, complementary, dominant genes were required, but others indicated two to three independent, dominant genes.     

Accumulation of soluble phenolic compounds in sunflower capitula correlates with resistance to Sclerotinia sclerotiorum

Disease symptoms and total soluble phenolics content have been analysed in four sunflower (Helianthus annuus L.)lines with different resistance levels(from highly susceptible to resistant) to head rot caused by Sclerotinia sclerotiorum (Lib.) de Bary. At the beginning of the flowering stage, capitula were inoculated by spraying with a water suspension of ascospores, and disease symptoms were evaluated from day 6 to day14 after inoculation. The most susceptible genotypes showed all their ovaries to be necrosed and abundant lesions in corollas, bracts and receptacle. In the resistant line, the ovary and corolla were only partially necrosed with no symptoms in the bracts or the receptacle. Total soluble phenolics were extracted and quantified from different parts of the capitulum in both inoculated and non-inoculated plants. The amount of phenolic compounds depended on the sunflower line, the time after inoculation, and the tissue. Higher constitutive and induced phenolic content as well as phenylalanine ammonia-lyase activity were present in the most resistant line, these differences correlated with the absence/presence of disease symptoms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of the high oleic acid content in cultivated sunflower (Helianthus annuus L.)

Summary Sunflower lines breeding true for very high oleic acid content in their oil (average levels higher than 85%) were crossed with standard sunflower lines with mean oleic acid levels of 30%. Analysis of the oil of F₁ seeds indicated dominance for high oleic levels and control of the genotype of the embryo. Segregating generations were obtained selfing heterozygous high oleic BC_nF₁ plants from several generations of a backcrossing program to incorporate the high oleic character to standard inbred lines and testcrossing these plants to low oleic material. Analysis of F₂ and testcrossed seeds showed three kind of segregations, in both F₂ and testcrossed populations, with different proportions of low, intermediate and high oleic types. Genetic analysis of these data supported the hypothesis, that the high oleic character is controlled by three dominant complementary genes OL₁, OL₂ and OL₃. Additional data showing F₁ seeds with intermediate oleic content and segregations for high oleic in progenies of intermediate types, suggest the presence of major factors modifying high oleic acid content.     

Path-coefficient analysis of some yield components of sunflower (Helianthus annuus L.), I

Summary Interactions of seven bio-morphological characteristics of sunflower (head diameter, number of flowers per head, number of seeds per head, 1,000-seed weight, volume weight, kernel content in seed, oil content) and their correlations with seed yield per plant were examined. The path-coefficient analysis provided information about direct and indirect effects of the examined characteristics on seed yield per plant. The correlation coefficients were positive and highly significant. The highest direct positive effect on seed yield per plant was exhibited by the weight of 1,000 seeds. Head diameter, volume weight and kernel content had a direct negative effect on seed yield per plant.     

Inheritance of resistance to two races of sunflower downy mildew (Plasmopara halstedii) in two Helianthus annuus L. lines

Sunflower downy mildew caused by Plasmopara halstedii is an important disease of sunflower capable of causing losses of more than 80% of production. Races 100, 300, 310, 330, 710, 703, 730 and770 of the fungus have been identified in Spain. Race 703, of high virulence, has been identified frequently in the northeast, while race 310 seems to occur over the south, the main sunflower growing region of the country. Oil sunflower lines RHA-274 and DM4 were studied for their resistance to races 310(RHA-274 and DM4) and 703 (DM4). In each cross, only one plant of the resistant parent was crossed to the inbred susceptible line HA-89 (or cmsHA-89).Plants from F₂ and backcross(BC₁F₁ to susceptible parent)generations were evaluated for fungal sporulation on true leaves and/or cotyledons. The resistant-to-susceptible ratios obtained in the F₂ and BC₁F₁ progenies from the crosses cmsHA-89 × RHA-274 and HA-89 × DM4suggested that one major gene in each line is responsible for resistance to race 703.The segregations of the progenies of the cross HA-89 × DM4 inoculated with race 703also fitted the ratios 1:1 and 3:1 (for BC₁F₁ and F₂, respectively)corresponding to control of resistance by a single dominant gene. In RHA-274, the gene for resistance to race 310 was designated Pl ₉, whereas Pl _v is tentatively proposed to designate the gene in DM4 responsible for resistance to races310 and 703. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetical studies of hullability in comparison with other sunflower seed characteristics

Summary To study the genetic control of hullability in sunflower (Helianthus annuus L.), 36 hybrids, produced by a factorial cross of six male sterile and six restorer lines, were evaluated in two locations in Spain and one in France. Hullability was calculated as the ratio of the quantity of hull removed by a mechanical huller compared with total hull content. In dry conditions in Spain, hullability was twice (83.1%) that observed in France (41.5%). In all locations, male, fernale and interaction effects were significant. Estimates of narrow sense heritability varied between 0.78 and 0.82, values similar to those for other seed characters such as oil content. There were positive genetic and phenotypic correlations between hullability and 1000 seed weight (means 0.81 and 0.74, respectively) but negative correlations with seed oil content (–0.73 and –0.68, respectively). It is concluded that selection in early segregating generations of crosses involving good hulling material needs a specific strategy in order to maintain reasonable levels of oil and protein contents in the seed of the selected genotypes.     

Construction and characterization of a BAC library for sunflower (Helianthus annuus L.)

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.     

Somaclonal variants of sunflower with modified coumarin expression under stress

Summary Two coumarins, scopoletin and ayapin, have been shown to accumulate in sunflower following insect damage or fungal infection (Tal & Robeson, 1986a, 1986b & Olson, 1989). The technique of shoot regeneration from embryo-derived callus was used to produce somaclonal variants of sunflower with enhanced levels of coumarin expression after an applied stress. Five regenerates (R1–R5) of inbred lines RHA 297 produced viable seeds following self-pollination. One of the S1 families from these plants had significantly higher concentration of scopoletin (12.75 g/g fresh weight) compared to the parental inbred line (5.66 g/g). The increased levels of scopoletin in S1 plants were partially inherited after a second self-fertilization (S2 plants). The ploidy of the regenerates and their S1 and S2 progeny was not examined. No consistent correlation between higher levels of scopoletin in stimulated S1 and S2 plants, and deterrence to feeding of Heliothis virescens (Fab) larvae on unstimulated plants was shown after in vivo assays. From inbred line HA 300, three regenerates (H1–H3) were derived which produced S1 progeny with higher or lower concentrations of ayapin or scopoletin than the parental inbred. The inheritance of increased ayapin in the HA 300-derived lines was not assessed in the S2 generation because of poor seed germination. Feeding deterrence of H. virescens larvae was not consistently correlated with ayapin concentrations in S1 progeny of HA 300 regenerates. However, a correlation (-0.61) between ayapin concentration and daily feeding score was observed in one planting of these unstimulated S1 progeny. Sunflower germplasm was developed with enhanced levels of stress-induced coumarin expression through somaclonal variation and this germplasm may also show increased resistance to some leaf-feeding insects.     

Genetic variation and changes with ontogeny of osmotic adjustment in sunflower (Helianthus annuus L.)

Summary Intraspecific variation for osmotic adjustment in sunflower was examined using a collection of 33 genotypes of different origin which were exposed to water stress at the 8-leaf stage. Changes in osmotic adjustment with ontogeny were also evaluated in the pre- and post-anthesis phases using seven genotypes drawn from this collection. Estimates of osmotic adjustment were derived from measurements of leaf relative water content (RWC) and osmotic potential () during a period in which the soil was allowed to dry out gradually. The degree of osmotic adjustment, expressed as the value of RWC for a of –1.7 MPa (RWCe), was derived from the ln RWC/ln relationship. Both monophasic and biphasic ln RWC/ln relationships were found. Irrespective of the form of the relationship, all genotypes at the 8-leaf stage showed some degree of osmotic adjustment. This was also true for the cultivars included in the subset examined in pre- and post-anthesis phases. Significant differences (P=0.05) in RWCe were found between extreme genotypes in all three phases.Significant (P=0.05) linear relationships were found between RWCe measured in the 8-leaf stage and that measured in the pre- and post-anthesis phases, establishing the viability of measurements in the 8-leaf stage as a means of selection for osmotic adjustment in later developmental stages. Genotype rank order was stable (P=0.01) across the three ontogenetic phases examined.Abbreviations ETp potential evapotranspiration - osmotic potential - RWC relative water content - RWCe value of RWC for a of –1.7 MPa     

Variation of hullability and other seed characteristics among sunflower lines and hybrids

Summary The hullabilities of 40 inbred lines (20 male sterility maintainers and 20 male fertility restorers) and 36 hybrids were studied in several environments in France. Thousand seed weight, oil and hull contents and the quantity of hulls remaining with the kernel after hulling were also measured. Principal component analyses and a hierarchical clustering method were used to described the variation of both inbreds and hybrids. Hullability varied from 11 to 66% for inbreds and 16 to 60% for hybrids. This variation is sufficient to permit breeding programmes for this character. Hullability is correlated positively with hull content (r=0.35^* to 0.69^*) but negatively with oil content (r=–0.34^*to–0.71^**). Large seeds generally hull better than small seed (r=0.21ns to –0.66^**). The value of the different classes of genotypes is discussed. Inbreds and hybrids producing achenes with a quite small hull content, a high oil content but a good hullability are the most promising for improvement of the quality of sunflower seed meal. Such genotypes are rare so that a recurrent selection programme is recommended to increase the frequency of favorable genes.     

Genetic mapping,marker assisted selection and allelic relationships for the Pu6 gene conferring rust resistance in sunflower

Rust resistance in the sunflower line P386 is controlled by Pu₆, a gene which was reported to segregate independently from other rust resistant genes, such as R₄. The objectives of this work were to map Pu₆, to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu₆ and R₄. Genetic mapping of Pu₆ with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu₆ was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu₆ and R₄ indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R_adv/R₄/R₁₁/ R_13a/R_13b/Pu₆ cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.     

Diversity among sources of cytoplasmic male sterility in sunflower (Helianthus annuus L.)

Summary Ten cytoplasmic male sterile (CMS) sunflower (Helianthus annuus L.) lines were crossed with nine maintainer or male fertility restorer lines in a diallel crossing scheme. Based on fertility restoration of the F₁ generation, CMS lines were divided into four groups. At least two new sources of CMS, CMS PET2 and CMS GIG1, were found to be potentially useful for commercial production of hybrids. Environment had an influence on fertility restoration of one CMS line, CMS MAX1. Effective restoration of male fertility for CMS RIG1, CMS ANN2, and CMS ANN3 was not found.     

Inheritance of fertility restoration in sunflower (helianthus annuus L.)

Summary Fertility restoration in the cross between a cytoplasmic male sterile line, 2 cm 183, and the restorer line, BCZ 111, (both obtained from France) was dominant in F₁ and segregated in a 9:7 ratio in the F₂ generation and thus suggested the action of two independent, complementary dominant genes controlling restoration. The behaviour of F₃ families broadly confirmed the F₂ ratio. The reasons underlying this pattern of inheritance has been discussed and the genetic symbols rf ₁ rf ₁ rf₂ rf₂and Rf ₁ Rf ₁ Rf ₂ Rf ₂ have been suggested for the male sterile and the restorer parents respectively.     

The effect of nitrate nutrition on iron chlorosis and leaf growth in sunflower (Helianthus annuus L.)

The objective of this work was to investigate the effects of nutrient solution pH, nitrogen form (NO₃, NH₄NO₃), bicarbonate and different Fe concentrations in the nutrient solution on the Fe concentration in roots and on the development of Fe deficiency symptoms in sunflower plants (Helianthus annuus L.). High pH in the nutrient solution induced by nitrate supply or by a pH-stat device led to increased Fe concentrations in roots and low leaf Fe concentrations associated with a significant decrease in leaf chlorophyll concentration manifested by yellow leaves. Plants of the nitrate fed treatments with 1 μM Fe in the nutrient solution were also characterized by reduced leaf growth and by the suppression of new leaf formation. The reduced leaf growth and the suppression of new leaves only occurred with nitrate and not with NH₄NO₃ in all treatments with 1 μM Fe in the nutrient solution. All symptoms were removed by a high Fe concentration in the nutrient solution (100 μM) at low external pH proving that suppression of leaf formation, reduced leaf growth and low chlorophyll concentration were caused by Fe deficiency. In the nitrate treatment with a low Fe supply (1 μM Fe) and pH 4 in the nutrient solution leaf chlorophyll concentrations similar to the controls were found. In comparison to control plants (NH₄NO₃, 1 μM Fe), leaf growth was still significantly reduced, and new leaf formation was suppressed. The chlorophyll concentration and CO₂ assimilation rate did not differ from those of the control plants. These results show that Fe deficiency is also characterized by small green leaves and the suppression of leaf formation. At the onset of leaf development, leaf growth and new leaf formation may respond more sensitively to poor Fe efficiency than chlorophyll concentration. In experiments with NO₃ plus HCO₃, simulating soil solution conditions prevailing in calcareous soils, the Fe efficiency of the youngest leaves was poor, showing retarded leaf growth and low chlorophyll concentration.     

Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.)

Summary The Pl6 locus in the inbred sunflower (Helianthus annuus L.) line HA335 giving resistance to French races of downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni. was localized by molecular techniques. A bulked segregant analysis was made on the F2 progeny from a cross between this line and H52, a downy mildew susceptible line. The resistance gene in HA335 was found to have the same linked RFLP marker loci as those determined for Pl1 (resistance to race 1 in the line RHA266) on linkage group 1 of the consensus RFLP map of the cultivated sunflower. Pl1 and Pl6 thus appear either to be allelic or closely linked. The implications for sunflower breeding are discussed.     

Resistance of sunflower inbred lines to various forms of attack by Sclerotinia sclerotiorum and relations with some morphological characters

Summary Twenty sunflower inbred lines were studied for their reactions to 7 Sclerotinia sclerotiorum tests on different plant parts. A principal component analysis (P.C.A.) and t-tests on the means of resistant and susceptible groups indicated that reactions of inbred lines to infection of capitula by ascospores are independent from those to ascospore infections of terminal buds. They are even more contrasted with the results of any test measuring mycelial extension. However, for the last, there is a close association between the reactions of roots, leaves and capitula. Of the inbred lines, some showed good levels of resistance to most forms of attack, others were generally susceptible and there were some with good resistance to one particular form of attack. A P.C.A. of 18 morphological and field characters showed no general association between these characters and Sclerotinia sclerotiorum test results, although it was found that lines resistant to mycelial extension on capitula were generally earlier than those that were more susceptible. It is proposed that breeding programmes for general resistance to Sclerotinia sclerotiorum should include a combination of two or three tests.     

The genetics of resistance in sunflower capitula to Sclerotinia sclerotiorum measured by mycelium infections combined with ascospore tests

To enhance efficiency of breeding programmes for Sclerotinia sclerotiorum resistance in sunflower capitula, two separate resistance tests have been recommended. However, the time necessary to develop genotypes with two types of resistance makes this impractical. A strategy to combine the two tests was consequently proposed to reduce the number of seasons per selection cycle, but genetic studies were necessary to determine if it could be applied in breeding programmes. This was the objective of this work. Data from two genetically different sunflower inbred lines and their F1, F2 and backcross generations were analysed in two years to determine the genetics of resistance to S. sclerotiorum in capitula measured by application on the same plants of a mycelium test combined with an ascospore test. Effects of maternal origin were detected when the reciprocal generations were evaluated for the mycelium test. This suggests the importance of choice of the inbred line used as female in population formation by hybridisation. Progeny subjected to the mycelium test did not show any change in relative reactions to two S. sclerotiorum isolates suggesting that ranking genotypes according to the results of combined S. sclerotiorum tests is repeatable across Sclerotinia isolates and experimental seasons. Moderate narrow sense heritability indicated that selection of the best F2 plants should be effective. Genetic gain from selection is possible because a reduction of lesion areas produced by the mycelium test can be expected. Additive gene effects contribute significantly to reduction in lesion area. Consequences of results in population improvement for S. sclerotiorum resistance in capitula are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Use of immature seed germination technique as an alternative to in vitro culture of sunflower (Helianthus annuus L.) embryos

Summary The importance of developing new techniques that would simplify the complexity and operative costs of genetic breeding plans has been widely recognized. In that sense, germination from immature seeds in sunflower is a simpler and cheaper alternative to that of in vitro embryo culture, as it is produced in non-sterile conditions. Different techniques to maximize germination percentage were tested using 15 day old seeds. The results indicate that it was important: 1) to scarify seeds and 2) to soak them in 100 ppm of gibberellic acid for 1 hour. Through the application of this technique more than 94% of the seeds germinated.