Cytoplasmic male sterility (CMS) in plants is associated with mitochondrial dysfunction. We have proposed in this study that the mitochondrial-encoded chimeric peptide thought to be responsible for cytoplasmic male sterility in Polima system could function as a dominant male sterility inducer when expressed in the nucleus and targeted to the mitochondria. Transgenic plants expressing such mitochondrial targeting constructs exhibited reduction of pollen production that was characterized in fertile Westar (nap ) and restored fertile Westar (pol) plants by homeotic transformation of floral organs and in male-sterile Westar (pol) plants by a reduction in pollen production with shortening of the stamens. Genetic and molecular analysis has shown that the phenotypic changes were correlated with the effective genetic transmission of the inserted transgene through female gametes. Most significantly, we have found that differences in floral morphology induced by transgene expression between pol CMS and fertile Westar plants might be related to differences in transcriptional activity of the APETALA3 MADS box gene. We suggest that the alterations in floral morphology that accompany CMS in several plant species might be due to effects of mitochondria on transcriptional activity of floral organ identity genes.

DNA markers tightly-linked to nuclear fertility restorer genes for cytoplasmic male sterility (CMS) are valuable tools for breeders and researchers working with these genes. Two different targeting approaches were used to identify markers linked to the Rfp1 restorer gene for the pol CMS of canola (Brassica napus L.): nearly isogenic line (NIL) comparison and bulked segregant analysis. These methods were equally efficient in identifying markers linked to Rfp1; combining them allowed a targeting efficiency of 100% to be achieved. The efficiency of bulked segregant analysis was found to be limited by the inadvertent occurrence of shared homozygosity at specific chromosomal regions in the bulks, in contrast with the efficiency of NIL comparison which was limited by the occurrence of residual DNA from the donor cultivar at scattered sites around the genome of the NILs. Eleven DNA markers linked to the Rfp1 gene were identified, one of which perfectly co-segregates with Rfp1. The linkage group on which Rfp1 is localized contains 17 DNA markers. Two restorer genes of the pol CMS, Rfp1 and Rfp2, and a Rfn restorer gene of the nap CMS were found to be at least tightly linked to one another and may all reside at the same locus. A fourth restorer gene, the Rfo restorer for the ogu CMS, was, however, found to be unlinked to the other restorer genes. Different restorer genes for the nap CMS were found in the lines 'Westar-Rf and 'Karat'. A linkage map of the B. napus genome containing 146 markers organized into 23 linkage groups covering a total length of 850.2 cM was constructed from a BC$ sb1$ population. This map contains 63 loci previously localized on the B. napus genome through analysis of an F$ sb2$ population. Comparative analysis indicates that the total length of the BC$ sb1$-derived map is smaller than that of the F$ sb2$-derived map, which suggests that a reduction in recombination frequency is occurring in male gametes. The preferential use of two or three probe-

In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai

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Genetic variation exists in <i>Raphanus</i> that could be of use to <i>Brassica</i> breeders. Of particular interest is the Ogura system of cytoplasmic male sterility (CMS) which has been worked on extensively in a <i>Brassica napus</i> background. Problems have been experienced in <i>B. napus</i>restorer lines due to the inheritance of a large segment of <i>Raphanus</i> chromosome containing the fertility restoring locus. This restorer introgression is located on the <i>Brassica</i> C genome making it only of use for <i>B. napus</i> and not for <i>B. rapa</i> or <i>B. juncea</i>. This thesis describes the development of the materials necessary for the introgression into the <i>Brassica</i> A genome of a defined segment of <i>Raphanus</i> chromosome containing a restorer locus. Defined genetic stocks of <i>Raphanus</i> were developed that contained specific loci controlling restoration of Ogura CMS. This material was used to develop populations segregating for specific restorer loci. Extensive RFLP maps of three <i>Raphanus</i> populations were developed and aligned, resulting in a robust consensus map of the entire <i>Raphanus</i> genome. Three restorer loci were accurately mapped on three separate linkage groups. The segment of <i>Raphanus</i> that is implicated in the restoration of Ogura CMS in a <i>B. napus</i> restorer line developed by INRA was identified and it did not correspond to any of the regions containing the three mapped restorer loci, suggesting the presence of more restorer loci in <i>Raphanus</i>. Comparative mapping between the <i>Raphanus</i> genome map and previously generated <i>Brassica</i> A genome RFLP maps demonstrated large regions of collinearity between segments of chromosomes of the two species. Preliminary examination of the two genome maps suggest they contain essentially the same overall genetic content but with large segments of the genomes rearranged with respect to each other. Likely sites of <i>Raphanus</i> restorer introgression into the <i>Brassica</i> A genome were predicted. Trigenomic tetraploids were developed in which pairing and recombination between homoeologous segments of <i>Raphanus</i> and <i>Brassica</i> A chromosomes should result. Progeny of these individuals will allow an assessment of the pattern and extent of recombination that occurs between the chromosomes of the <i>Raphanus</i> and <i>Brassica</i> A genomes and should lead to the development of 'B. napus' lines carrying Ogura CMS restorer alleles from <i>Raphanus</i>.

Previous studies have shown that the mitochondrial orf224/ atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus, and that the effects of nuclear fertility restoration on orf224/ atp6 transcripts co-segregate with the pol restorer gene Rfp in genetic crosses. Results presented in this thesis indicate that the pol CMS restorer gene Rfp acts in an organ-specific manner to promote the processing of primary, dicistronic orf224/atp6 transcripts into new restorer-specific, monocistronic transcripts. The single 1.1 kb atp6 transcript of nap cytoplasm and some orfB transcripts of nap and pol cytoplasm B. napus mitochondria are shown to arise by removal of sequences from the 5' end of a longer precursor. Specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may thus represent a common mechanism for tailoring transcripts in plant mitochondria. Northern analysis of a segregating F2 population indicates that the recessive rfp allele at the restorer gene locus, or a very tightly linked gene, acts as a dominant gene designated Mmt (modifier of mitochondrial transcripts). Mmt controls the presence of additional, smaller transcripts of the nad4 and ccl1-like (ccl1-l) genes. These results suggest that Rfp/Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes. One of these genes, ccl1-l, is split in the B. napus mitochondrial genome into two widely separated and independently transcribed parts, contrasting with the situation in other plants where this gene is present as an uninterrupted ORF. Although transcripts of both parts are edited, no "trans-spliced" transcripts spanning both parts of the split ORF, that could be translated into a full sized protein product, were detected. Moreover, an antiserum directed against the product of t

Cytoplasmic male sterility (CMS) is a widespread, maternally inherited trait that results in an inability of plants to produce functional pollen. The Ogura CMS system originated in radish, but has since been transferred to, and confers male sterility on, plants in the related genus Brassica . A gene which restores male fertility is needed for the Ogura CMS system to be exploited commercially for hybrid seed production in oilseed species such as Brassica napus. The restorer gene Rfo is a dominant radish nuclear gene that restores the male fertility to plants with Ogura cytoplasm. This gene has been transferred into Brassica napus through intergeneric crosses; however the introgressed segment of radish DNA contains an unknown number of genes, some of which confer undesirable traits, such as an elevated content of seed glucosinolates, antinutritive compounds that render the seed meal unusable as animal feed. A fine scale linkage map of the region in radish containing Rfo was constructed, and a map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687 amino acid protein with a predicted mitochondrial targeting presequence was found to confer male fertility upon transformation into Ogura CMS B. napus . This gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. A transcript representing a non-functional allele (rfo) was detected in sterile radish plants. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis , although a smaller and related PPR gene is found about 40 kb from this site.