TrnH and the intergenic region between trnH and psbA of the chloroplast genomes of alfalfa (Medicago sativa), Fabaceae, and petunia (Petunia hybrida), Solanaceae, were sequenced and compared to published sequences of that region from other members of those families. A striking feature of these comparisons is the occurrence of insertions/deletions between short, nearly perfect AT-rich direct repeats. The directionality of these mutations in the petunia, tobacco and Nicotiana debneyi lineages within the Solanaceae cannot be discerned. However, we present several alternative hypotheses that are consistent with Goodspeed's 1954 evolutionary treatment of the genus Nicotiana and family Solanaceae. Within the Fabaceae, the major size differences in the intergenic region between alfalfa, pea and soybean are due to insertions/deletions between direct repeats. The alfalfa intergenic region has an inverted repeat stem-loop structure of 210 bases directly 5' to trnH. This structure is an insert relative to the liver-wort. Marchantia polymorpha. Portions of the insert are found also in pea and soybean as well as in published sequences from other dicots representing diverse orders: petunia, tobacco, N. debneyi (Scrophulariales), spinach (Caryophyllales), and Brassica napus (Capparales). Some of the regions of the insert that are missing in these plants appear to have resulted from deletions of sequences between different imperfect direct repeats within, or 5' to and within the insert. Other deletions are not flanked by repeated sequences. A short insert flanked by imperfect direct repeats in B. napus occurs just within the longer alfalfa insert suggesting that both alfalfa and B. napus have remnants of an even longer insert relative to M. polymorpha. From these analyses we hypothesize the insertion of a stem-loop structure into an M. polymorpha-like ancestral land plant, followed by deletions of sequences, often between different imperfect direct repeats within and upstream of the insert, leading to the psbA-trnH intergenic sequences represented by the present-day plants examined.
Two orientations of the Chlamydomonas reinhardii chloroplast (ct) genome are shown to be produced by recombination within the inverted repeat (IR) sequences that separate the two single copy (SC) regions. SC region 1 is bounded on its two ends by EeoRI restriction endonuclease fragments of 3.2 and 4.7 kilobase pairs (kb) (Rochaix 1978). The 3.2 kb EeoRI fragment overlaps a 51.3 kb BglII fragment spanning one of the 19.7 kb IR sequences, and the 4.7 kb EcoRI fragment overlaps a 42.1 kb BglII fragment spanning the other 19.7 kb IR sequence. We have shown by hybridization analysis that the 3.2 kb fragment also overlaps a BgIII fragment with a predicted size of 52.3 kb, and that the 4.7 kb fragment also overlaps a BglII fragment of a predicted size of 41.1 kb. The second set of BglII fragments are isomers produced by recombination localized to the IR region. The two isomers are present in approximately equimolar ratio. Knowledge of the isomeric composition of the C. reinhardii ctDNA is essential for establishing a correlation between genetic and physical maps of the ct genome.
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