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.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Inheritance of reduced plant height in the sunflower line Dw 89

The objective of the present research was to study the inheritance of reduced plant height in the sunflower line Dw 89. Plants of the cytoplasmic male sterile version of this line, cmsDw 89 (mean plant height of 47.4 cm) were crossed with plants of the restorer line RHA 271 (mean of 120.9 cm). F₁ plants averaged 120.4 cm, which indicated dominance of standard over reduced plant height. F₂ plants followed a segregation pattern of 1 : 15 (reduced : normal height), suggesting that reduced plant height in Dw 89 is controlled by alleles at two loci, designated Dw1 and Dw2. Class assignment in the F₂ was confirmed through the evaluation of the F₃ generation. Backcrosses to Dw 89 segregated with 1 : 3 (reduced : normal height) ratios, which confirmed the digenic inheritance of the trait. The evaluation of plant height distributions in F₃ families suggested possible genetic interaction between the Dw1 and Dw2 loci.     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

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

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Screening for resistance to broomrape (Orobanche cernua) in cultivated sunflower

Racial evolution of sunflower broomrape (Orobanche cernua) has been very rapid in Spain during recent years, in which resistance has been overcome several times and there has been an important increase in areas infested with this parasitic angiosperm. In order to find resistance to a highly virulent population of sunflower broomrape that could be used directly in breeding programmes, three different sets of cultivated plant material composed of 429 entries were tested by artificial inoculation. All evaluated inbred lines from Moden, Canada, were fully susceptible. Out of the 240 P.I. accessions tested, only 10 segregated for resistance to broomrape, the rest being susceptible. From the 160 USDA breeding lines evaluated, 5% were resistant and 19% segregated for resistance to O. cernua. These lines traced back mainly to crosses of RHA 274 and RHA 801 with Russian, Turkish and Romanian hybrids. The origin of P.I. accessions that segregated for resistance were primarily derived from the former USSR and from Romania.     

Optimal allocation of resources in evaluating current sunflower inbred lines for resistance to Sclerotinia

Sclerotinia sclerotiorum (Lib.) de Bary is a major limiting factor for sunflower (Helianthus annuus) production. This study was conducted to (1) evaluate a large number of current sunflower lines for their reaction to Sclerotinia, and (2) determine the optimum allocation of resources for their selection. A set of 85 inbred lines was screened under artificial leaf infection in three environments. The test revealed ample variation for all resistance traits in the current germplasm. A line selected from NDBLOS, an oilseed sunflower germplasm poo, and its derivatives were most resistant to Sclerotinia. Lines derived from interspecific crosses with the wild species Helianthus tuberosus and Helianthus argophyllus also showed satisfactory Sclerotinia resistance. The evaluation of five plants per plot in two replications and four environments appears to be a reasonable allocation of resources in order to optimize the selection response.     

Fertility restoration of new CMS sources in sunflower (Helianthus annum L.)

The development of commercial sunflower hybrids based on new CMS sources is of special interest for reducing the potential risk of vulnerability to pathogens and for increasing genetic diversity. From 263 test crosses involving nine new CMS sources, i.e. ANL1, ANL2, MAX1, PEF1, PET2, ANN1, ANN2, ANNS and ANN4, five lines were selected as potential restorers for PEF1, PET2 and ANN4. In test crosses between all nine CMS sources and these five restorer lines evaluated in 2 years, seven fully restored hybrids could be identified. These hybrids, based on ANL1, ANL2, MAX1, PEF1, PET2, and ANN4, showed good agronomic performance for plant height, days to flowering, maturity and oil content. Segregation analyses of the F2 populations indicate that a single dominant restorer gene was sufficient to restore pollen production of hybrids based on ANL2, PEF1 and PET2. For restoration of ANN4, two dominant complementary genes are required. In restoration of fertility in the crosses of ANL1 and MAX1 investigated, two dominant genes are involved each of which on its own allows the production of fertile plants.