Physical mapping of differences in chloroplast DNA of the five wild-type plastomes in Oenothera subsection Euoenothera

Abstract

1) DNA has been isolated from the five genetically distinguishable plastid types of Oenothera, subsection Euoenothera. DNA of plastomes I to IV was obtained from plants with identical nuclear backgrounds containing the genotype AA of Oenothera hookeri whereas the DNA of plastome V came from Oenothera argillicola (genotype CC). 2) The DNAs of the five basic Euoenothera wild-type plastomes can be distinguished by restriction endonuclease analysis with Sal I, Pst I, Kpn I, Eco RI and Bam HI. The fragment patterns exhibit distinct common features as well as some degree of variability. 3) Physical maps for the circular DNAs of plastome I, II, III and V could be constructed using the previously detailed map of plastome IV DNA (Gordon et al. 1981). This has been achieved by comparing the cleavage products generated by restriction endonucleases Sal I, Pst I and Kpn I which collectively result in 36 sites in each of the five plastome DNAs, and by hybridization of radioactively labelled chloroplast rRNA or chloroplast cRNA probes of spinach to Southern blots of appropriate restriction digests. The data show that the overall fragment order is the same for all five plastome DNAs. Each DNA molecule is segmentally organized into four regions represented by a large duplicated sequence in inverted orientation whose copies are separated by two single-copy segments. 4) The alterations in position of restriction sites among the Euoenothera plastome DNAs result primarily from insertions/deletions. Eleven size differences of individual fragments in the Sal I, Pst I and Kpn I patterns measuring 0.1-0.8 Md (150-1,200 bp) relative to plastome IV DNA have been located. Most changes were found in the larger of the two single-copy regions of the five plastomes. Changes in the duplication are always found in both copies. This suggests the existence of an editing mechanism that, in natural populations, equalizes or transposes any change in one copy of the repeat to the equivalent site of the other copy. 5) Detailed mapping of the two rDNA regions of the five plastomes, using the restriction endonucleases Eco RI and Bam HI which each recognize more than 60 cleavage sites per DNA molecule, disclosed a 0.3 Md deletion in plastome III DNA and a 0.1 Md insertion in plastome V DNA relative to DNA of plastome IV, I and II. These changes are most probably located in the spacer between the genes for 16S and 23S rRNA and are found in both rDNA units.