Renate Horn

Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene

SummaryThe organization and expression of the mitochondrial (mt) genome of fertile, male-sterile and restored lines of Helianthus annuus and of H. petiolaris were compared to identify alterations which might lead to cytoplasmic male sterility (CMS). The mtDNAs of fertile and male-sterile lines differ by an 11 kb inversion and a 5 kb insertion. The rearrangements seem to be the result of recombination events within an inverted repeat of 261 bp. Detectable alterations in the transcript pattern of the rearranged mtDNA regions are restricted to the atpA locus. The male-sterile line CMSBaso shows three additional transcripts of the atpA locus of about 2500, 1200 and 250 nucleotides which are not detectable in Baso. However, the coding sequences of the atpA gene are entirely identical in the fertile line Baso and the male-sterile line CMSBaso. But a new open reading frame (orfH522) of 522 nucleotides is co-transcribed with the atpA gene as an additional larger transcript of about 2500 nucleotides in CMSBaso. orfH522 is also included in a second additional transcript of about 1200 nucleotides. The predicted translation product of orfH522 might play a role in CMS in sunflower.

A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower

Cytoplasmic male-sterile lines CMS89 and CMSBaso of sunflower (Helianthus annuus) differ from the fertile lines HA89 and Baso in a mitochondrial DNA sequence in the vicinity of theatpA gene. In addition, the transcriptional pattern of theatpA gene is changed in male-sterile lines compared to fertile ones. Besides one main transcript in the fertile lines, the male-sterile lines additionally show larger transcripts. Investigation of Baso and CMSBaso revealed that the two fertility-restored lines of CMS89 have the same transcripts as CMSBaso or a combination of CMSBaso and CMS89. Comparing the mitochondrialin organello translation products we observed a unique 16 kDa protein, which is expressed in male-sterile lines carrying theH. petiolaris cytoplasm but is not detectable in fertile lines withH. annuus cytoplasm. The 16 kDa protein can also be observed in restored lines but not inH. petiolaris. As the expression of the 16 kDa polypeptide seems to be linked to the interspecific cross betweenH. petiolaris andH. annuus it may play a role in CMS. By different criteria such as molecular mass, isoelectric point and peptide fingerprinting the α subunit of the F1-ATPase of male-sterile and fertile lines is very similar if not identical.

The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower

In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3′-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3′-coding region of orfH522 to verify by immunological methods the identity of the protein in the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 is located in the 3′-flanking region of the atpA gene. Transcript analyses using atpA and orfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor.

High regeneration rates in anther culture of interspecific sunflower hybrids

Optimization of anther culture with regard to the induction of callus formation and direct embryogenesis was obtained for interspecific hybrids ofH. annuus withH. tuberosus, H. laetiflorus, andH. resinosus by investigating six different induction media and four regeneration media. One media combination (MS-13, MS-R3 and MS-R4) used under different culture conditions (30°C / 35°C and different dark treatments) gave up to 92.7% embryogenic anthers with an average of 8.5 embryos per anther. However, direct embryogenesis as well as callus formation showed a strong genotypec and treatment specific reaction. From 5,600 anthers of the four investigated genotypes more than 2,000 plants could be regenerated. Regenerants were characterized by morphological traits and isozyme analyses to prove their androgenetic origin.

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.

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.

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.

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.

MAPPING OF THE RESTORER GENE Rf1 IN SUNFLOWER (Helianthus annuus L.)

SUMMARY In sunflower, commercial hybrid breeding is based on a single CMSinducing cytoplasm, the so-called PET1 cytoplasm. The introduction of one dominant, nuclear-encoded restorer gene (Rf1) is in most cases sufficient for fertility restoration. Little has been learned so far about the mode of action of the restorer gene Rf1. For map-based cloning of the restorer gene Rf1, an F2 population of the cross RHA325 (cms) x HA342 has been used. The χ²-test confirmed segregation for one dominant gene which corresponds to Rf1. For the AFLP analyses 256 EcoRI/MseI primer combinations have been used so far. In addition, RAPD analyses were performed using 1,200 decamer primers. Twenty-three primers had polymorphic amplification products, differentiating the bulks, and could therefore be mapped. The hybridization of the marker HP4 against a BAC library resulted in three positive clones. The overlapping end of the smallest clone was used to get a new hybridization against the BAC library. RESUMEN El mejoramiento comercial por hibridación en girasol, está basado en una inducción de citoplasma CMS simple, llamado citoplasma PET1. La introducción de un gen restaurador dominante (Rf1) con codificación nuclear, es en la mayoría de los casos suficiente para restaurar la fertilidad. Hasta ahora se conoce poco acerca del modo de acción de este gen restaurador Rf1. Para la clonación basada en marcadores del gen restaurador Rf1 se uso la población F2 proveniente del cruzamiento de las lineas RHA325 (CMS) x HA342. La prueba χ² confirmó la segregación de un gen dominante, el cual corresponde a Rf1. Para el análisis de AFLP se usaron 256 combinaciones de primer EcoR1/MseI. Para el análisis de RAPD se usaron 1200 decamer primers. Veintitres primers presentaron productos de amplificación polimorficos en los bulks, permitiendo su mapeo. De la hibridación del marcador HP4 con la BAC-genoteca resultaron tres clones positivos. El sobrecruzamiento final del clon más pequeno, se uso para obtener una nueva hibridación con la BAC-genoteca. RÉSUMÉ Chez le tournesol, l’amélioration des hybrides commerciaux est basée sue l’induction unique CMS du cytoplasme, ce dernier étant appelé cytoplasme PET1. L’introgression du gène Rf1 dans le génome est dans la plupart des cas suffisante pour la restauration de la fertilité. Le mode d’action du gène Rf1 restaurateur de fertilité est peu connu. Afin de réaliser un clonage positionnel de ce gène, une population F2 dérivée du croisement RHA325 (CMS) x HA342 a été untilisée. Le test χ² a permis la confirmation de la ségrégation pour un gène dominant qui correspond á Rf1. L’analyse AFLP a été menée à terme au moyen de 256 combinaisons d’amorces EcoRI/MseI. Par ailleurs, des analyses RAPD ont été effectuées en utilisant 1200 amorces décamères. Vingt trois amorces ont engendré des produits d’amplification polymorphiques différenciant les bulks. Leur cartographie a été ensuite mise en oeuvre. L’hybridation du marqueur HP4 contre la banque d’ADN type BAC a donné trois clones positifs. La terminaison chevauchante du clone le plus court a été utilisée pou l’obtention d’une nouvelle hybridation contre la banque BAC.

Nuclear and cytoplasmic differences in the mitochondrial respiration and protein expression of CMS and maintainer lines of sunflower

In sunflower, the patterns of mitochondrially encoded proteins were compared in five cytoplasmic male sterile lines and the corresponding maintainer lines. The line RHA265 with the original fertile cytoplasm (N) showed a unique protein of 53 kDa that was not present in the male sterile isonuclear lines with the CMS-inducing cytoplasms GIG1, MAX1 and PET2. GIG1 and PET2 expressed an additional 12.4 kDa protein. In dependence of the nuclear background i.e. RHA265 or HA89, respectively, a nuclear encoded 24 kDa protein was present or absent in the mitochondrial protein patterns of GIG1, MAX1 and PET2.Nuclear and cytoplasmic differences in the total respiration of isolated mitochondria were detected using NADH, malate and succinate as substrates. For succinate oxidation in dependence of the nuclear background ANL1 and its maintainer RHA266 showed higher respiration rates than RHA265, ANL2, GIG1, MAX1 and PET2. For NADH total respiration of ANL2, GIG1 and PET2 was more than twice as high than for the isonuclear maintainer line RHA265. Also MAX1 showed an increased oxygen uptake even though not as high. The results demonstrated that considerable differences in the total respiration are possible without obvious relevance to the production of vital pollen. Regarding the engagement of the cytochrome oxidase and alternative pathway no differences were observed between CMS and maintainer lines.