Molecular analysis of a new cytoplasmic male sterile genotype in sunflower.

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S + 5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mitochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.     

Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus.

Comparison of the physical maps of male fertile (cam) and male sterile (pol) mitochondrial genomes of Brassica napus indicates that structural differences between the two mtDNAs are confined to a region immediately upstream of the atp6 gene. Relative to cam mtDNA, pol mtDNA possesses a 4.5 kb segment at this locus that includes a chimeric gene that is cotranscribed with atp6 and lacks an approximately 1kb region located upstream of the cam atp6 gene. The 4.5 kb pol segment is present and similarly organized in the mitochondrial genome of the common nap B.napus cytoplasm; however, the nap and pol DNA regions flanking this segment are different and the nap sequences are not expressed. The 4.5 kb CMS-associated pol segment has thus apparently undergone transposition during the evolution of the nap and pol cytoplasms and has been lost in the cam genome subsequent to the pol-cam divergence. This 4.5 kb segment comprises the single DNA region that is expressed differently in fertile, pol CMS and fertility restored pol cytoplasm plants. The finding that this locus is part of the single mtDNA region organized differently in the fertile and male sterile mitochondrial genomes provides strong support for the view that it specifies the pol CMS trait.     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.     

Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower

Background

More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.

Results

Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.

Conclusion

The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997