Structural alterations of the chloroplast genome tend to occur at "hot spots" on the physical map. To clarify the mechanism of mutation of chloroplast genome structure in higher plants, we determined the nucleotide sequence of the hot-spot region of chloroplast DNAs related to length mutations (deletions/insertions) in Triticum (wheat) and Aegiops. From a comparison of this region in wheat with the corresponding region of tobacco or liverwort, it is evident that one of the open reading frames in tobacco (0RF512) has been replaced in wheat by the rpl23 gene, which is a member of the ribosomal protein gene operon. In the deleted positions and in the original genome of Triicum and Aegilops, consensus sequences forming short direct repeats were found, indicating that these deletions were a result of intramolecular recombination mediated by these short direct-repeat sequences. By two independent recombination events in theAegilops crassa type of chloroplast genome, which is shared by Triticum monococcum, Ae. bicornis, Ae. sharonensis, Ae. comosa, and Ae. mutica, the novel chloroplast DNA sequences of T. aestivum and Ae. squarrosa were generated. This finding indicates the existence of illegitimate recombination in the chloroplast genome and presents a mechanism for producing genetic diversity of that genome.

We have characterized a so-called D genome specific repetitive DNA sequence (pAs1) of Aegilops squarrosa L. (2n = 14, genome DD) with respect to its DNA sequence and its distribution among Triticeae species. The clone consisted of three units of a repetitive DNA sequence of 336 or 337 base pairs, and was AT rich (65.2%). DNA analyses revealed the presence of the pAs1-like sequences in other genomes of Triticeae species, although the repetition was greatly (as much as 100-fold) variable among the genomes. The repetitive sequences from 10 diploid species were amplified using PCR with specific primers, and the sequential variability was analyzed by the digestion pattern obtained with five restriction enzymes. Since the AfaI site was the most conservatively present in the unit of the repetitive sequences, we named them "Afa family." The analysis clearly displayed the variation of the repetitive sequences regardless of the uniformity of the size of the amplified product. These results indicated that plural amplification events of these repetitive sequences happened independently in the genome evolution of Triticeae.

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