~ Current Genetics 9 Springer-Verlag 1993 Comparative analysis of a recombining-repeat-sequenee family in the mitochondrial genomes of wheat (Triticum aestivum L.) and rye (Secale cereale L.) Michael B. Coulthart, David E Spencer, and Michael W. Gray Program in EvolutionaryBiology,Canadian Institute for Advanced Research, Department of Biochemistry,Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada Received May 29, 1992/Accepted July 23, 1992 Summary. The mitochondrial genomes of wheat and rye each contain a three-member family of recombining re- peat sequences (the "18S/5S repeat") that encode genes for 18S and 5S rRNAs (rrnl8 and rrn5) and tRNA fMet (trnfM). Here we present, for wheat and rye, the sequence and boundaries of the "common sequence unit" (CSU) that is shared between all three repeat copies in each species. The wheat CSU is 4,429 base-pairs long and con- tains (in addition to trnfM, rrnl8 and rrn5) a putative promoter, three tRNA-like elements ("t-elements"), and part of a pseudogene ("OatpAc") that is homologous to chloroplast atpA, which encodes the c~ subunit of chloro- plast F1 ATPase. The rye CSU is somewhat smaller (2,855 base pairs) but contains much the same genic and other sequence elements as its wheat counterpart, except that two of the three t-elements as well as OatpA c are found in only one of the three downstream flanks of the 18S/5S repeat, outside the CSU boundaries. In interpret- ing the sequence data in terms of the evolutionary history of the 18S/5S-repeat family of wheat and rye, we con- clude that (1) the wheat-rye form of the 18S/5S repeat most likely originated between 3 and 14 million years ago, in a lineage that gave rise to wheat and rye but not to barley, oats, rice or maize; (2) the close linkage (1-bp apart) between trnfM and rrnl8 is similarly limited in its taxonomic distribution to the wheat/rye lineage; (3) the trnfM-rrnl8 pair arose via a single mutation that inserted a sequence block containing trnfM immediately up- stream of rrnl8; and (4) the presence of a putative pro- moter upstream of rrnl8 in all wheat and rye repeats is consistent with all three repeat copies being transcrip- tionally active. We discuss these conclusions in the light of the possible functional significance of recombining-re- peats in plant mitochondrial genomes. Key words: Triticum aestivum - SecaIe cereale - Riboso- mal RNA genes - Mitochondrial DNA - Recombining- repeats - Evolution Correspondence to." M. W. Gray Introduction A conspicuous feature of most chloroplast and some mi- tochondrial genomes is the presence of families of repeat- ed sequences that recombine to generate structural heterogeneity in these DNAs. In chloroplast genomes (Palmer 1985, 1991) the repeats are in inverted orienta- tion on the circular chromosome, so that products of reciprocal exchange differ only by inversion of the single- copy regions between the repeats. In plant (angiosperm) mitochondrial DNA (mtDNA), the situation is consider- ably more complex. Complete physical maps of the mtDNAs of turnip (Palmer and Shields 1984), maize (Lonsdale et al. 1984), wheat (Quetier et al. 1985), and sunflower (Siculella and Palmer 1988), are consistent with the presence of a single circular "master chromo- some" that accounts for the entire sequence complexity of these mitochondral genomes [but see Folkerts and Hanson (1989) and Yamato et al. (1992) for counter- examples]. However, plant mtDNAs also typically con- tain one or more families of direct repeats that generate subgenomic molecules by recombination. This has sug- gested a "multicircular" structural model for the plant mitochondrial genome, wherein a collection of recombi- nationally generated chromosomal subcircles constitutes a substantial fraction of the mass of the mtDNA (Lons- dale et al. 1984; Palmer and Shields 1984). For plant mtDNAs with higher numbers of repeat families (e.g., maize and wheat - see Lonsdale et al. 1984; Quetier et al. 1985), the potential multicircular complexity is very great. It is unclear at present how much of this complex- ity is realized in vivo within populations of mitochondria, cells, or plants (Bendich 1985; Bendich and Smith 1990; Andr6 et al. 1992). Moreover, the evolutionary origins and dynamics of recombining-repeats, as well as their specific implications for plant mtDNA heredity, gene ex- pression and evolution, are largely unknown. Comparative studies of the structure and sequence of recombining-repeats will constitute an essential phase in understanding their function. Unfortunately, very little information of this type is presently available. Detailed