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.     

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.     

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.     

Biomass partitioning and genetic analyses of salinity tolerance in sunflower (Helianthus annuus L.)

Biomass partitioning was studied in sunflower (Helianthus annuus L.) inbred lines and their hybrids differing in salinity resistance. Differential biomass partitioning was observed among resistant and susceptible lines as well as within resistant lines, which were grown in large pots. Traits such as number of dead leaves and total number of nodes may be used as dominant markers for understanding the mechanism of resistance to salinity. Multi-location trials differing for salinity levels showed low and non-significant heritabilities across the environment for biochemical traits showing their sensitivity to the environment and a significant G X E interaction. Thus selection could only possible within the salinity level for these traits. Contrastingly, morphological traits such as number of nodes showed significant heritabilities (narrow or broad sense) across the environment. These traits may be exploited by the selection of inbred lines across or with salinity levels. Among various salinity levels, 12 dSm^?1 was found to be feasible for screening and selection as it promoted the additive type of gene action.     

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.     

Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (Helianthus annuus L.)

Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred line HA89 over four environments. Pairwise comparisons of alloplasmic male fertile (MF) with euplasmic MF lines suggested that cytoplasm of perennial H. angustifolius increased lodging while perennial cytoplasms of H. mollis, H. grosseserratus, and H. divaricatus reduced head size and consequently reduced yield and will need to be used with caution. Lines having annual species, cytoplasms had no effects on agronomic traits. Comparisons of CMS HA 89 with 10 CMS lines suggested yield‐reducing cytoplasmic effects of perennial H. maximiliani and annual H. annuus PI 413178 and PI 413024. Cytoplasmic effects on oil percentage and fatty acid composition were minimal and should not be of concern for sunflower breeders. In general, most cytoplasms of wild annual Helianthus species accommodate cultivated nuclear genes without drastic adverse interactions, and are potential sources of cytoplasmic diversity for sunflower breeding.     

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

Quantitative resistance to sunflower downy mildew was studied on inbred lines and hybrids not carrying efficient major gene resistance, in field trials in one to four sites over 3 years. Hybrids from factorial crosses showed that inheritance is under additive control and comparison with reactions of parental inbred lines gave narrow sense heritabilities of 27–57%. Analysis of a polymorphic recombinant inbred line population without efficient major gene resistance indicated that two highly significant Quantitative Trait Loci (QTL) explained 42% of variation in field reaction to downy mildew. These QTL were mapped on linkage groups 8 and 10, and do not appear related to any of the known major resistance gene clusters. Possible bases of this type of resistance and its use in breeding are discussed.     

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     

The inheritance and biochemistry of resistance to Sclerotinia sclerotiorum leaf infections in sunflower (Helianthus annuus L.)

Summary Resistance of sunflower leaves to attack by Sclerotinia sclerotiorum was studied by infecting them, in the field, with agar disks containing Sclerotinia mycelium. Resistance levels were determined by the length of lesions after a given period. There were significant differences in reaction between both sunflower hybrids and inbred lines. Different Sclerotinia isolates gave the same classification of sunflower genotypes. The results of trials repeated in one year or different years were significantly correlated. The general combining ability variance/specific combining ability variance ratio was 1.35. Strict sense heritability was 0.61±0.03. The midparent-offspring correlation coefficient was significant, but the relation between per se values of the male parents used with the values of their hybrids was much closer than that for the female parents. Mean heterosis for resistance was 28.9%, compared with the midparent. The results of the leaf resistance test are frequently correlated with levels of resistance to root attack by Sclerotinia. Possible use of this test in breeding both directly for leaf resistance and indirectly for root resistance are discussed. H.P.L. Chromatography studies of the phenols present in healthy and infected leaves distinguished 19 compounds, all of the inhibitin type. There was a large increase in phenol content in leaves infected by Sclerotinia, for all genotypes. However, more especially in uninfected leaves, the contents of 3 chromatogram peaks, numbered 4, 6 and 9, showed a close relation with levels of Sclerotinia resistance. It is proposed that these compounds could be used as markers of certain types of resistance.     

Genetic control of in vitro-organogenesis in recombinant inbred lines of sunflower (Helianthus annuus L.)

Ninety-three recombinant inbred lines (F₈) of sunflower developed by the single-seed descent method from the cross ‘PAC-2 × RHA-266’ were used to screen their regenerability by organogenesis. The experiment was designed in randomized complete blocks with three replications. Each replication consisted of 10 Petri dishes with four explants. Cotyledons were excised from 2-day-old seedlings. Each cotyledon was divided into two pieces (four explants), which were incubated in solid regeneration medium consisting of full-strength Murashige and Skoog medium modified by adding hormones. A high genotypic variability for organogenesis parameters between genotypes was observed in this study. The difference between all recombinant F₈ lines and their parents was not significant, showing that the 93 inbred lines used in this experiment are representative of the total possible recombinant lines from the cross ‘PAC-2 × RHA-266′. These F₈ lines were not consciously selected for any trait; therefore, they represent a random set of lines segregating for the organogenesis parameters, as well as for other traits which could be important for breeding.     

Influence of Exogenous Glycine Betaine on Gas Exchange and Biomass Production in Sunflower (Helianthus annuus L.) under Water Limited Conditions

This study investigated whether the exogenous application of glycine betaine (GB) could alleviate the adverse effects of water deficit on sunflower. Two sunflower lines, Gulshan-98 and Suncross, were subjected to water deficit at the vegetative or the reproductive stages of plant growth. Three levels (0, 50 and 100 mmol l⁻¹) of GB were applied as a pre-sowing seed treatment or as a foliar spray at the time of initiation of water deficit at the vegetative or reproductive stages. Foliar application of GB at the time of initiation of water deficit treatments showed a marginal increase in shoot biomass in drought-stressed plants. Exogenously supplied GB as a foliar spray also showed a positive role in reducing the effects of water deficit on net CO₂ assimilation rate, transpiration rate and sub-stomatal CO₂ concentration in both sunflower lines. Pre-sowing seed treatment with GB had no effects on the above-mentioned physiological and growth attributes in both normally irrigated and drought-stressed plants. Taken together, foliar application of GB increased net CO₂ assimilation rate which in turn showed a slight increase in growth of water-stressed plants but this increase was not related to stomatal regulation.     

Characterization of a green non‐dormant sunflower (Helianthus annuus) mutant NDG

A sunflower (Helianthus annuus L.) mutant was observed in the progeny of a cross between the sunflower cultivar ‘HA 89’ and an amphiploid of a Helianthus divaricatus L. × P21 cross that exhibited loss of dormancy induction in the developing embryo. Seeds of this mutant frequently germinate on the head about 40 days after pollination. The cotyledons of this mutant remain green, whereas some other non‐dormant mutants exhibit loss of pigmentation. The objectives of this investigation were to compare levels and activities of abscisic acid (ABA), a plant hormone that induces dormancy in developing embryos, in the non‐dormant green mutant (NDG) and ‘HA 89’ from which NDG was derived. Immunoassays showed that abscisic acid was present in NDG and the levels were not significantly different from those in ‘HA 89’. Exposure of excised NDG mutant embryos to 40 μm abscisic acid failed to prevent germination, suggesting that non‐dormancy could result from impairment in ABA receptors or from a defect in other proteins participating in the subsequent signalling pathway that normally induces dormancy.     

Evaluation of different microsatellite motifs for analysing genetic relationships in cultivated sunflower (Helianthus annuus L.)

A total of 16 different simple sequence repeat motifs and an M13 repeat sequence were used as hybridization probes in order to examine the molecular relationships between two German inbred and eight North American sunflower lines of known pedigree, and to determine the suitability of the individual short tandem repeats for genetic analyses and their occurrence in the sunflower genome. Only the oligonucleotides (ACA)₆, (CAT)₆, (CATA)₅, (GACA)₄ and (GATA)₄ proved to be well suited for the generation of scorable fingerprinting patterns, while the other (microsatellite) sequences were either too abundant or too rare. Although different levels of polymorphism were present in hybridizations with the different simple sequence repeats (SSRs), a clear separation of the German material from the USDA Hnes was feasible not only by evaluating 20 probe-enzyme combinations, but also by combining every individual probe with four different restriction enzymes. In the American material, the expected relationships could be proved with the single exception of a line that did not group in accordance with its pedigree.     

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.     

Development of a new initial breeding material in sunflower (Helianthus annuus L.) using direct organogenesis and somatic embryogenesis

Summary Immature zygotic embryos from three inbred lines of sunflower (Helianthus annuus L.) were used as donor material for the induction of direct organogenesis and somatic embryogenesis. The scope of the spontaneously appearing somaclonal variation has been studied among the regenerants produced according to the method of Freyssinet & Freyssinet (1988), and was compared to the results obtained after gamma ray mutagenesis (7 and 10 Gy). Freshly excised immature zygotic embryos were used for irradiation treatment. Genetic changes occurring spontaneously during the regeneration procedure were observed for date of flowering, vegetation period, plant height, head diameter, 1000 seed weight, kernel oil content, and fatty acid composition. The mutagenic treatment had a marked stimulatory effect on the frequency of the regenerants produced and the degree of the observed changes. The 7 Gy treatment was most efficient for the majority of the characters studied. The type of changes was correlated with the genotype. The degree and type of somaclonal variation, with or without mutagenic treatment, should be sufficient for an application in the production of new breeding material.     

High regeneration potential in vitro of sunflower (Helianthus annuus L.) lines derived from interspecific hybridization

Successful selection of interspecific hybrid progenies with superior ability to regenerate shoots from apical meristems was performed in sunflower which now allows for the development of lines for improved biotechnological applications. Early generations of interspecific hybrids originating from crosses between the two H. annuus CMS lines ‘HA89’ and ‘Baso’, and 9 wild species were screened for their ability to regenerate in vitro. Evaluation of 36 progenies allowed to identify seven progenies from crosses involving H. mollis, H. giganteus, H. strumosus, and H. decapetalus which showed a significantly higher regeneration potential than the commercial hybrid ‘Albena’ regarding the number of shoots per explant. Among these progenies, 47.2 to 62.4% of explants produced shoots with an average of 2.3 to 3.5 shoots per cultured explant. Regeneration in vitro was significantly determined by the genotype. More than half of the investigated interspecific hybrids performed better than the inbred ‘HA89’ demonstrating that the high regeneration potential available in the wild species can be efficiently transferred to cultivated sunflower. The seven progenies with high regeneration potential in vitro were characterised by agronomic performance in the field. Two of the interspecific hybrids derived from H. strumosus and H. decapetalus not only showed a superior regeneration potential but also proved to be competitive to commercial hybrids with regard to important agronomic traits, e.g. fat content and TGW. This revised version was published online in July 2006 with corrections to the Cover Date.     

Association between sporophytic reaction to Alternaria helianthi and gametophytic tolerance to pathogen culture filtrate in sunflower (Helianthus annuus L.)

Seven sunflower genotypes comprising of populations and hybrids showing differential sporophytic reaction to Alternaria leaf and stem blight were studied for their gametophytic reaction to pathogen culture filtrate. The sunflower pollen grains germinate well in the liquid medium and give good pollen tube growth in the absence of the culture filtrate. The addition of increasing concentrations of culture filtrate to medium significantly reduced the pollen germination and tube growth in all the genotypes. The reduction in pollen germination and tube growth in vitro due to culture filtrate was more in highly susceptible genotypes L-101 and Morden than the moderately resistant genotypes Acc. nos. 1229, 180 and ISFH-306. Pretreating the stigma and style with the culture filtrate before pollination reduced the number of pollen grains germinating compared to untreated control suggesting toxin stress can be created on the stigmatic surface before pollination. There was correspondence between pollen germination on stressed stigma (in vitro) and sporophytic reaction of the genotype suggesting pollen grain having resistance would germinate on the stressed stigma and fertilise the ovule achieving selective fertilisation. The correlation analysis indicated that there is a negative relation between sporophytic per cent disease index value and gametophytic parameters such as in vitro and in vivo pollen germination, culture filtrate required to inhibit 50% pollen germination and pollen tube growth. The association between pollen and the sporophytic reaction to the disease indicate the possibility of rapid screening of a large number of genotypes by means of pollen assay as an alternate technique with regard to sporophytic disease index in sunflower. The study also indicate the possibility of pollen selection before fertilisation to achieve rapid improvement in disease resistance. This revised version was published online in August 2006 with corrections to the Cover Date.     

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 (σ_f) were significant for seeds per head, seed weight, head sterile center, days to blooming and oil content. Female × male interactions (σ² _fm) 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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sporophytic and gametophytic recurrent selection for improvement of partial resistance to Alternaria leaf blight in sunflower (Helianthus annuus L.)

Alternaria leaf blight, caused by Alternaria helianthi Hanf., is one of the most important diseases of sunflower causing significant yield losses in several tropical countries. Yet, so far, only partial resistance for the disease has been discovered in the germplasm through conventional sporophytic selection. Therefore, the main objective of the present investigation was to compare sporophytic and gametophytic recurrent selection with the aim to enhance the level of resistance to Alternaria leaf blight. The base population was synthesized by random mating three populations- two interspecific derivatives involving different species of Helianthus and one germplasm accession based on their partial resistance to disease incidence. The base population was subjected to 1-2 cycles of both sporophytic and gametophytic selection. The gametophytic selection was practiced by applying pathogen culture filtrate to the stigma and style one hour before pollination. The selection response was measured by scoring the percent disease index at flowering, 15 days after flowering, and at physiological maturity and by quantifying economic yield gain. A significant reduction in mean per cent disease index values and a gain in seed yield were observed for both the types of selection cycles, but more so for gametophytic selection. The populations improved through gametophytic selection appear to be more promising as the pollen selection allowed the selection of rare favorable allelic combinations that would hardly be detected at the sporophytic level. A combination of gametophytic selection and conventional sporophytic selection should be considered as an effective tool in population improvement programs to achieve higher levels of resistance in relatively short time.