Molecular mapping of the fertility restoration locus Rf1 in sunflower and development of diagnostic markers for the restorer gene

Fertility restoration by dominant nuclear genes is essential for hybrid breeding based on cytoplasmic male sterility (CMS) to obtain heterotic effects and high seed yields. In sunflower, only the PET1 sterility inducing cytoplasm has been used in commercial hybrid breeding until now. This particular male sterility was derived from an interspecific hybrid Helianthus petiolaris × H. annuus. For the recent work we used the segregating population RHA325(CMS) × HA342, based on the PET1 cytoplasm. Molecular markers were mapped within 1.1 cM around the restoration locus Rf1. At the distal side, the marker OP-K13_454 mapped at a distance of 0.9 cM and E32M36-155R at 0.7 cM from Rf1. At the proximal side the markers E44M70-275A, E42M76-125A and E33M61-136R were mapped at 0.1, 0.2, and 0.3 cM from the restorer locus, respectively. These markers provide an excellent basis for a map based cloning approach and for marker-assisted sunflower breeding.     

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.     

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.     

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.     

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.     

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     

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 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.     

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.     

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.     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.     

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.     

Photosynthesis as Influenced by Assimilate Level in Sunflower (Helianthus annuus L.)

The regulation of photosynthesis by its assimilates was investigated in sunflower by altering the assimilate level in leaves by girdling, detopping, shading and defoliation. The level of assimilates like sucrose accumulated in leaves did not inhibit photosynthesis or Rubisco activity. Starch levels also did not have an influence on the photosynthetic rate.     

Inheritance and molecular mapping of restorer‐of‐fertility (Rf) gene in A2 hybrid system in pigeonpea (Cajanus cajan)

Inheritance of fertility restorer gene in pigeonpea was studied using F₂ and BC₁F₁ populations derived from cross AL103A × IC245273. It was found to be controlled by single dominant gene. Out of 228 SSR primer pairs, 33 primer pairs showed parental polymorphism, while four primers were found polymorphic in bulk segregant analysis (BSA). These four primers viz., CcM 1615, CcM 0710, CcM 0765 and CcM 1522 were used for genotyping of F₂ population and were found to be placed at 3.1, 5.1, 28.1 and 45.8 cM, respectively. Two of them, CcM 1615 and CcM 0710, evinced clear and unambiguous bands for fertility restoration in F₂ population. The Rf gene was mapped on linkage group 6 between the SSR markers CcM 1615 and CcM 0710 with the distances of 3.1 and 5.1 cM, respectively. The accuracy of the CcM 1615 was validated in 18 restorers and six maintainer lines. The marker CcM 1615 amplified in majority of male restorer lines with a selection accuracy of 91.66%.     

Fine mapping of Rf2, a major locus controlling pollen fertility restoration in sorghum A1 cytoplasm,encodes a PPR gene and its validation through expression analysis

Sorghum is one of the pioneering cereal crops where cytoplasmic male sterility (CMS) was successfully exploited for mass production of F₁ hybrid seed. Mapping genes for fertility restoration (Rf) is an important aspect of understanding the molecular basis of fertility restoration in crop plants. In this study, we fine‐mapped a fertility restoration locus, Rf2 of sorghum reported earlier (Jordan, Mace, Henzell, Klein, & Klein, 2010 ), involving two F₂ populations (296A × RS29 and 296A × DSV1) and newly developed SSR markers delimited Rf2 locus to 10.32‐kb region on chromosome 2. The Rf2 locus was tightly linked with two new SSRs, MS‐SB02‐3460 (0.14 cM) and MS‐SB02‐3466 (0.75 cM) on both sides, and hosted only one gene (Sobic.002G057050) of PPR gene family. Another new SSR marker developed in the study, MS‐SB02‐37912, forms the part of PPR gene and could act as a perfect marker in marker‐assisted breeding for fertility restoration involving Rf2 in sorghum breeding. The strong involvement of Sobic.002G057050 gene in fertility restoration was supported through RNA expression analysis.     

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.     

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.     

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.