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     

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.     

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.     

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.     

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.     

Precision phenotyping of imidazolinone-induced chlorosis in sunflower

Chlorosis level is a useful parameter to assess imidazolinone resistance in sunflower (Helianthus annuus L.). The aim of this study was to quantify chlorosis through two different methods in sunflower plantlets treated with imazapyr. The genotypes used in this study were two inbred lines reported to be different in their resistance to imidazolinones. Chlorosis was evaluated by spectrophotometrical quantification of photosynthetic leaf pigments and by a bioinformatics-based color analysis. A protocol for pigment extraction was presented which improved pigment stability. Chlorophyll amount decreased significantly when both genotypes were treated with 10 μM of imazapyr. Leaf color was characterized using Tomato Analyzer^® color test software. A significant positive correlation between color reduction and chlorophyll concentration was found. It suggests that leaf color measurement could be an accurate method to estimate chlorosis and infer chlorophyll levels in sunflower plants. These results highlight a strong relationship between imidazolinone-induced chlorosis and variations in leaf color and in chlorophyll concentration. Both methods are quantitative, rapid, simple, and reproducible. Thus, they could be useful tools for phenotyping and screening large number of plants when breeding for imidazolinone resistance in this species.     

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.     

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.     

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.     

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.     

Polymorphism and inheritance of methionine-rich 2S albumins in sunflower

2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell 1990) from seeds of 103 sunflower (Helianthus annuus L.) accessions and analysed by SDS-PAGE, IEF andRP-HPLC. Two methionine-rich albumins SFA7 and SFA8 showed nodifferences in mobility on SDS-PAGE gels but were readily separated byRP-HPLC. Their levels also varied widely between different genotypes, inrelation to each other and as proportions of the total albumin fraction. Avariant form of SFA8 was identified which differed from the normal SFA8in its pI (6.5 compared to 6.0) and mobility on SDS-PAGE. N-terminal sequences of both the variant form of SFA8 and the majorform of SFA7 were identical to that reported previously for the normalform of SFA8 from the cultivar Hysun (Kortt et al., 1991) indicating theirstructural relatedness. Analysis of segregation in the F₂ of the crossbetween lines VIR130 (variant SFA8) and VIR104 (normal SFA8) showedthat the normal and variant forms of SFA8 are encoded by alleles at asingle Mendelian locus. The levels of SFA7 and SFA8 in the seeds ofparental lines, F₁ hybrids and individual F₂ seeds classifiedfrom SDS-PAGE and IEF as homozygous for normal SFA8 (VIR104 type),homozygous for variant SFA8 (VIR130 type) and heterozygous (F₁type) were determined by RP-HPLC. Seeds of the parental line VIR130contained 3.7% SFA7 and 19.0% SFA8 whereas seeds of VIR104contained 9.9% SFA7 and 12.8% SFA8. The F₁ hybrid seedscontained a higher total amount of SFA7+8 proteins (32% comparingto 22% in each parent) which was largely accounted for by a highproportion of SFA7. The mean combined proportions of SFA7+8 in eachof the three phenotypic classes of F₂ seeds were about 18–19% ofthe total. However, the combined proportions of SFA7+8 varied in therange 10–20% among the individual seeds. The ratio of SFA7 to SFA8was highest in the VIR104-type and heterozygous seeds, with the amountof SFA7 exceeding that of SFA8 in six heterozygous seeds. Theproportions of SFA7 and SFA8 were inversely correlated among individualF₂ seeds. The results suggest that the amounts and proportions ofSFA7 and SFA8 are determined by genetic factors in addition toavailability of sulphur.     

Field evaluation of somaclonal variation in sunflower (Helianthus annuus L) and its application for crop improvement

Immature zygotic embryos from the American fertility restorer line RHA-857 were used as donor material for induction of direct organogenesis in sunflower (Helianthusannuus L.). The range of spontaneous somaclonal variation among the progenies of regenerants was studied. The genetic modifications observed in regenerants included agronomic traits such as oil content in seed, 1000 seed weight, plant height, leaf width, leaf length, petiole length, internode length, head diameter, number of branches, length of branches, number of ray florets, seed width, seed length, and seed thickness. RAPD molecular analysis carried out on sunflower materials in the R-11 generation showed the absence of a specific 358 bp band in somaclonal line 11/2/51 R. This line showed a modified architecture, full resistance to Phomopsis helianthiand higher oil content in seed in comparison to the standard RHA-857. Line31/3/53 R was with modified architecture and higher 1000 seed weight. Hybrid No. 144 produced with the participation of somaclonal line 20/5/52 R demonstrated high production capacity, shorter vegetation period and reduced height. The combination of these favourable changes is desirable in breeding work on sunflower. Somaclonal variation through director ganogenesis has facilitated the creation of genetically heritable variation in sunflower, which can be used with great success for hybrid seed production of highly productive hybrids. 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.     

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.     

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.     

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.     

Indicators of resistance of sunflower plant to basal stalk rot (Sclerotinia sclerotiorum): Symptomatological,biochemical, anatomical,and morphological characters of the host

Summary The object of this work was a study of the relationship between the field reactions of different sunflower genotypes to basal stalk rot (in terms of severe (dead plants) and incipient wilting, and lesion length) and some biochemical (phenol concentration), morphological (plant height, and stem and flower-bud diameters) and anatomical (xylem and cortical indexes) characters of the host. Plants from 8 inbred lines at closed flower-bud stage were artificially inoculated with mycelium at the base of the stem. The percentage of dead plants for each inbred line and the lesion length and wilting range for individual plants after 7 days were recorded.A positive and highly significant correlation coefficient between the percentage of dead plants and lesion length was found for the three years of the study (r=0.83; P<0.01). A highly significant association between lesion length and wilting range for individual plants was always found (P=0.00). Postinfectional phenol content exhibited a strong negative correlation with lesion length and the percentage of dead plants in all the experiments (P=0.05). Association between postinfectional phenol content and wilting range for individual plants was significant for all the years studied (P<0.05). No correlation between phenol levels in healthy plants of the different sunflower genotypes and their susceptibility was found. Morphological characters positively correlated with lesion length but only plant height exhibited significant values for the three years. Associations between wilting range and morphological characters for individual plants were significant for one of the two years analyzed (P<0.05). Xylem index showed a negative correlation with lesion length which was significant one of the two years studied.The lesion length measure seems to be a simple and direct method for resistance screening before the flowering period. Although strong relations with postinfectional phenol levels were found, their determinations would be too much time consuming and not completely reliable. The relationships between other characters measured and disease resistance would indicate that physiological mechanisms could be related to resistance.     

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.     

The genetic map position of the locus encoding the 2S albumin seed storage proteins in cultivated sunflower (Helianthus annuus L.)

Sunflower contains two classes of seed proteins: the 11S globulins (helianthinins) and the 2S albumins. Polymorphisms have been detected within both classes and therefore they have been proposed as genetic markers. The objectives of the present study were to identify sunflower seed protein polymorphisms and locate them on an RFLP linkage map. Seed proteins of 68 inbred lines and four Argentinian open-pollinated populations were studied by SDS-PAGE. The helianthinins were separated into ten bands arranged in fourgroups of M_r of 22.5, 25.5, 32 and 38 kDa and all lines showed identical banding patterns. The albumins were separated into eight to tenbands in the M_r range 10 and 18 kDa. The lines showed either a band of M_r of 14.5 or of 15.5 kDa. HAR1, HAR2, ZENB8 and ZENB12 had the 15.5 kDa, whereas the remaining lines had the 14.5 kDa. The 14.5 and 15.5 kDa polypeptides were also segregating in the Impira INTA population. One hundred and fifty F_2:3 families derived from ZENB8 × HA89 were used as the mapping population. The electrophoretic patterns of the families were either those of the two parental lines or presented both the 14.5 and 15.5 kDa polypeptides. No family showed absence of both bands, suggesting that they are allelic variants. The 2S albumin locus was located in the interval between the markers ZVG0051 and ZVG0052, at approximately 9 cM from ZVG0051 and at 6 cM from ZVG0052, on linkage group 11 of the public sunflower RFLP map. This revised version was published online in August 2006 with corrections to the Cover Date.