SummaryA single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. 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 pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf 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.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. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value # 10 230) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5# to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5# noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms .5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, smallto medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks.

This paper examines macro and micro-level patterns of genome size evolution in the Brassicaceae. A phylogeny of 25 relatives of Arabidopsis thaliana was reconstructed using four molecular markers under both parsimony and Bayesian methods. Reconstruction of genome size (C value) evolution as a discrete character and as a continuous character was also performed. In addition, size dynamics in small chromosomal regions were assessed by comparing genomic clones generated for Arabidopsis lyrata and for Boechera stricta to the fully sequenced genome of A. thaliana. The results reveal a sevenfold variation in genome size among the taxa investigated and that the small genome size of A. thaliana is derived. Our results also indicate that the genome is free to increase or decrease in size across these evolutionary lineages without a directional bias. These changes are accomplished by insertions and deletions at both large and small-scales occurring mostly in intergenic regions, with repetitive sequences and transposable elements implicated in genome size increases. The focus upon taxa relatively closely related to the model organism A. thaliana, and the combination of complementary approaches, allows for unique insights into the processes driving genome size changes.

The two forms of cytoplasmic male sterility (CMS) native to the oilseed rape or canola species Brassica napus, nap and pol, have novel features that may provide insight into the molecular mechanisms through which CMS/nuclear restorer systems evolve. One such feature is the finding that the distinct nuclear restorer genes for the two systems represent different alleles or haplotypes of the same nuclear locus. Improved understanding of how these systems have evolved will require molecular cloning and characterization of this novel locus. We have employed an approach that exploits the regional co-linearity between the Arabidopsis and Brassica genomes to construct a high-resolution genetic map of the nuclear restorer for the pol system, Rfp. Specifically, Arabidopsis-derived sequences have been used as a set of ordered RFLP probes to localize Rfp to a region of the B. napus genome equivalent to a 115 kb interval on Arabidopsis chromosome 1. Based on the known relationship of physical distances between orthologous segments of Arabidopsis and Brassica chromosomes, it is anticipated that the B. napus restorer locus is now mapped to sufficient resolution to permit its isolation and characterization.