Theor Appl Genet (1995) 91:1 8 @ Springer-Verlag 1995 M. Nakazono 9 A. Kanno 9 N. Tsutsumi 9 A. Hirai Homologousrecombinationmediated by two palindromicrepeated sequences in the mitochondrialgenome of Oryza Received: 20 April 1994 / Accepted: 18 October 1994 Abstract Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evol- ution of Oryza. We first performed Southern hybridiz- ation of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization pat- terns revealed genome type-specific and/or species-speci- fic variations. We speculated that homologous recom- bination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after diver- gence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, there- fore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs. Key words Oryza 9 Mitochondrial DNA 9 Palindromic repeated sequences 9 Polymorphism 9 Homologous recombination Introduction The mitochondrial (mt.) genomes of higher plants are much larger and more complicated than those of other Communicated by G. S. Khush N. Nakazono 9N. Tsutsumi , A. Hirai ([]) Laboratory of Radiation Genetics, Faculty of Agriculture, The Uni- versity of Tokyo, 1-i-1 Yayoi, Bunkyo, Tokyo 113, Japan A. Kanno Institute of Genetic Ecology, Tohoku University, 2-1-1 Katahira, Aoba, Sendal 980, Japan eukaryotic organisms. Their complexity is mainly due to recombination via large repeated sequences and to the transfer of DNA fragments from the chloroplast or the nucleus (reviewed by Lonsdale et al. 1988; Schuster and Brennicke 1988). Inter- and/or intramolecular recom- bination via recombination repeats seems to create multipartite structures and to occur reversibly (Lon- sdale et al. 1984). In addition to such active recombina- tion repeats, mtDNAs of higher plants have a number of small repeated sequences (reviewed by Andr6 et al. 1992). These are not involved in the frequent recombina- tion events mentioned above. However, several analyses of recombination events have indicated that the small repeated sequences may be related to rearrangements of mtDNA that have been postulated to have occurred during evolution or that are seen in tissue culture (Brears et al. 1989; Small et al. 1989; Shirzadegan et al. 1991). Many chimeric genes, produced by such infre- quent recombination via the small repeated sequences, are found in the mtDNA of several plants (Dewey et al. 1986; Young and Hanson 1987; Kadowaki et al. 1990.) Some of the chimeric genes are associated with the male-sterile phenotype (Dewey et al. 1986; Young and Hanson 1987). Therefore, the small repeated sequences appear to have contributed to the variation in mitochondrial genomes and to the phenotypes of plants. We reported previously that small repeated se- quences of 60-66 basepairs (bp) are present in the mitochondrial genome of rice (Oryza sativa; Nakazono et al. 1994). These sequences are highly conserved with respect to one another and are widely distributed in at least ten regions of rice mtDNA. We designated these sequences PRSs (Palindromic Repeated Sequences) be- cause they are potentially capable of forming a stem- and-loop structure. We noted that some of the PRSs had been inserted into the intron of the gene for ribosomal protein $3 (rps3) and into the flanking sequence of the gene for chloroplast-like tRNA Ash (trnN) of the Oryza mtDNA during the course of evolution. In this study, we found that at least one PRS might have been used as a site of homologous recombination during the evolution