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.     

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.     

Comparison of evaluation methods for selection of resistance to Fusarium head blight in a recurrent selection programme in wheat (Triticum aestivum L.)

The objectives of this study were to compare efficiency of evaluation for resistance to Fusarium head blight (FHB) under two inoculation methods in a recurrent selection programme. Fifty selected homozygous F₅ fertile lines, from each of five cycles (C0, C1, C2, C3 and C4) of recurrent selection, and two control cultivars were evaluated in a split-plot design in 1995 and 1996 under the soil-surface inoculation with Fusarium graminearum-colonized kernels and the single-floret inoculation with ascospore suspension. Comparison of the two inoculation methods using means, ranges, coefficients of variation, heritabilities and correlations among infected spikelet rate (ISR), reaction index (RI) and disease index (DI) indicated that FHB resistance could be evaluated with similar accuracy and precision using either of the two inoculation methods. Regressions of disease scores in the soil-surface inoculation on disease scores in the single-surface inoculation were positive and highly significant, showing a strong relationship between both inoculation methods for FHB resistance. The percentage of lines with similar performance for FHB disease scores in both inoculation methods was high. The soil-surface inoculation and single-floret inoculation appear to be useful techniques for evaluating numerous individuals of segregating population and screening advanced homozygous lines for FHB resistance in a recurrent selection programme in wheat, respectively.     

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.     

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.     

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.     

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.     

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     

Identification and marker‐assisted introgression of QTL conferring resistance to bacterial leaf blight in cowpea (Vigna unguiculata (L.) Walp.)

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is widespread in major cowpea [Vigna unguiculata (L.) Walp.] growing regions of the world. Considering the resource poor nature of cowpea farmers, development and introduction of cultivars resistant to the disease is the best option. Identification of DNA markers and marker‐assisted selection will increase precision of breeding for resistance to diseases like bacterial leaf blight. Hence, an attempt was made to detect QTL for resistance to BLB using 194 F_2 : 3 progeny derived from the cross ‘C‐152’ (susceptible parent) × ‘V‐16’ (resistant parent). These progeny were screened for resistance to bacterial blight by the leaf inoculation method. Platykurtic distribution of per cent disease index scores indicated quantitative inheritance of resistance to bacterial leaf blight. A genetic map with 96 markers (79 SSR and 17 CISP) constructed from the 194 F₂ individuals was used to perform QTL analysis. Out of three major QTL identified, one was on LG 8 (qtlblb‐1) and two on LG 11 (qtlblb‐2 and qtlblb‐3). The PCR product generated by the primer VuMt337 encoded for RIN2‐like mRNA that positively regulate RPM1‐ and RPS2‐dependent hypersensitive response. The QTL qtlblb‐1 explained 30.58% phenotypic variation followed by qtlblb‐2 and qtlblb‐3 with 10.77% and 10.63%, respectively. The major QTL region on LG 8 was introgressed from cultivar V‐16 into the bacterial leaf blight susceptible variety C‐152 through marker‐assisted backcrossing (MABC).     

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.     

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.     

Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers

The Rf1 gene in sunflower can effectively restore the pollen fertility of PET1 cytoplasm in male-sterile lines and has been widely used in commercial hybrid production. Identifying molecular markers tightly linked to this gene will be useful in marker-assisted selection to develop maintainer and restorer lines. Rf1 has been mapped to Linkage Group (LG) 13 of the public sunflower simple sequence repeat (SSR) map by aligning maps constructed from different populations and only one SSR marker was reported to be loosely linked to Rf1 . This paper reports the result of applying target region amplification polymorphism (TRAP) and SSR markers to map and develop a sequence-tagged site (STS) marker tightly linked to Rf1 using two populations derived from a cross between two U.S. public sunflower lines, RHA439 and cmsHA441. An SSR marker, ORS511, was 3.7 cM from the Rf1 gene and a TRAP marker, K11F05Sa12-160, was linked to Rf1 at a distance of 0.4 cM. This TRAP marker was converted to an STS marker for using in 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.     

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.     

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.     

Growth and Yields of Intercropped Sorghum and Sunflower (Helianthus annuus L.) in Semi-arid Nigeria

Effect of five planting patterns on the growth, yield and yield components of intercropped sunflower and sorghum was studied during 1989–90 planting seasons at University of Maiduguri, Nigeria. Generally, intercropping depressed the performance of sorghum more than sunflower. Sorghum plants grown in alternate hills with sunflower had the shortest stems, the least dry matter and total seed yields per hectare, while the highest dry matter and seed yields were obtained from sorghum planted in five alternating rows with sunflower. Similarly, in sunflower, plants grown in five alternating rows with sorghum had the highest yields compared with other planting patterns, but there were no significant differences in the dry matter and total seed yields of sorghum and sunflower intercropped in three and five alternating rows. Light transmission, leaf area index and yields of both crops followed similar trends under the various planting patterns. Interplanting in five alternating rows that allowed the highest leaf area also allowed the lowest light transmission and produced the highest yields. Land use efficiency was highly improved under three and five rows interplanting by 52 and 74 % respectively.     

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.