Analysis of mitochondrial DNA, chloroplast DNA, and double-stranded RNA in fertile and cytoplasmic male-sterile sunflower (Helianthus annuus) GREGORY G. BROWN, HOWARD BUSSEY, AND LEE J. DESROSIERS Department of Biology, McGill University, 1205 Dr. Penjield Avenue, Montre'al, P.Q., Canada H3A 1Bl Corresponding Editor: H. Bertrand Received August 12, 1985 Accepted October 7, 1985 BROWN, G. G., H. BUSSEY, and L. J. DESROSIERS. 1986. Analysis of mitochondrial DNA, chloroplast DNA, and double- stranded RNA in fertile and cytoplasmic male-sterile sunflower (Helianthus annuus). Can. J. Genet. Cytol. 28: 121 - 129. The extent of variation in the mitochondrial DNAs (mtDNAs), chloroplast DNAs (ctDNAs), and double-stranded RNAs (dsRNAs) of sunflower lines carrying fertile and male-sterility conferring cytoplasms was examined. To minimize nuclear gene effects, efforts were concentrated on two chromosomally isogenic lines, CM400 (fertile) and cmsCM400 (male sterile), which differ only in their cytogenes. A circular 1.45 kilobases (kb) plasmid DNA was found in the mitochondria of the four fertile lines examined, but was absent in the male-sterile line. Restriction enzyme analysis of mtDNAs of the fertile and male-sterile cytoplasms with BamHI, EcoRI, and Hind11 revealed no fragment mobility differences between them other than those which could be ascribed to the 1.45-kb circle. Similar restriction analysis of ctDNA showed no differences between fertile and male-sterile cytoplasms. Both CM400 and cmsCM400 contain dsRNA molecules. The number and sizes of these dsRNAs varied from preparation to preparation in both lines. Species of 3.3 and 1.5 kb, which were the only dsRNAs common to all preparations from CM400, were also the only species common to all preparations from cmsCM400. Thus, no consistent differences between the fertile and male-sterile cytoplasms were seen in these molecules. The specific association of the 1.45-kb plasmid with fertile cytoplasm together with the absence of variation in ctDNA and dsRNA, suggests the involvement of mtDNA in sunflower cytoplasmic male sterility. Key words: DNA (mitochondrial), sterility (male), sterility (cytoplasmic), Helianthus, sunflower, DNA chloroplast. BROWN, G. G., H. BUSSEY, et L. J. DESROSIERS. 1986. Analysis of mitochondrial DNA, chloroplast DNA, and double- stranded RNA in fertile and cytoplasmic male-sterile sunflower (Helianthus annuus). Can. J. Genet. Cytol. 28: 12 1 - 129. L'etendue de variation de I'ADN des mitochondries (ADNmt), de I'ADN des chloroplastes (ADNct) et de I'ARN a double brin (ARNdb) des lignkes de tournesol qui sont porteuses de cytoplasmes qui confkrent la stQilite mile ou la fertilite a Cte ktudiee. Afin de minimiser les effets des genes nucleaires, les travaux ont port6 sur deux lignees a chromosomes isogknes, CM400 (fertile) et cmsCM400 (mile-sterile), qui ne different que par leurs cytogknes. Un plasmide de forme circulaire avec ADN de 1,45 kb a kt6 trouvk chez les mitochondries de quatre lignees fertiles examinees, mais non chez la lignee sterile. L'analyse des enzymes de restriction de 1'ADNmt des cytoplasmes fertiles et steriles avec BamHI, EcoRI et Hind11 n'a rkv6l6 aucune difference de mobilite de fragments entre elles, sauf de celles qui pourraient etre reliees au 1,45 kb circulaire. Une analyse de restriction semblable des ADNct n'a revel6 aucune difference entre les cytoplasmes fertiles et les mile-stkriles. La CM400 et la cmsCM400 contiennent toutes deux des mol6cules d' ARNdb. Le nombre et la diminution de ces ARNdb ont varie de preparation en preparation chez les deux lignees. Des ARNdb a 3,3 et 1,5 kb, qui furent les seules espkces communes a toutes les preparations CM400, furent egalement les seules espkces communes a toutes les preparations de cms CM400. Donc, ces molCcules n'ont present6 aucune difference consequente entre les cytoplasmes fertiles et mile-stkriles. L'association spkcifique du plasmide a 1,5 kb avec le cytoplasme fertile et I'absence de variation des ADNct et ARNdb suggkrent l'implication de 1'ADNmt dans la stenlit6 mile cytoplasmique chez le tournesol. Mots cle's: ADN (mitochondial), sterilite (mile), sterilite (cytoplasmique), Helianthus, tournesol, ADN (chloroplastique). [Traduit par le journal] Introduction The maternally transmitted inability of higher plants to produce functional pollen is known as cytoplasmic male sterility (CMS). This trait is widespread in occur- rence and exists in different forms that can block pollen development at different stages (Edwardson 1970; Laughnan and Gabay-Laughnan 1983). For some instances of CMS, specific nuclear "restorer" genes can suppress the male-sterile phenotype and allow for nor- mal pollen production. CMS is useful in the large scale production of hybrids because it prevents self-pollina- tion of the seed parent. In principle, any cytoplasmic genetic element (mito- chondrial DNA (mtDNA), chloroplast DNA (ctDNA), a seed transmitted virus, or another yet undescribed genetic constituent of the cytoplasm) could carry CMS determinants. In maize, the mitochondrial genomes of fertile and male-sterile cytoplasms can be distinguished by restriction enzyme analysis (Pring and Levings 1978) and by their complement of low molecular weight DNA species (Pring et al. 1977; Kemble et al. 1980). The chloroplast DNAs (ctDNAs) of these cytoplasms cannot be distinguished so easily (Pring and Levings 1978). Mitochondria from the male-sterile maize cytoplasms Can. J. Genet. Cytol. 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