Rearrangements in the chloroplast genomes of mung bean and pea

Abstract

We have mapped all the cleavage sites for the restriction endonucleases BstEII, Kpn I, Pst I, Pvu H, Sac I, Sal I, Sma I, and Xho I on the circular chloroplast chromosomes from mung bean and pea. The mung bean chloroplast genome measures 150 kilobase pairs (kb) in length; it includes two identical sequences of 23 kb that contain the ribosomal genes and are arranged as an inverted repeat separated by single-copy regions of 21 and 83 kb. The pea chloroplast genome is only 120 kb in size, has only one set of ribosomal genes, and does not possess any detectable repeated sequences. The mung bean inverted repeat structure is common to all other nonleguminous higher plant chloroplast genomes studied, whereas the pea structure has been found only in the closely related legume Viciafaba. We conclude from these data that loss ofone copy of the inverted repeat sequence has occurred only rarely during the evolution of the Angiosperms, and in the case of the legumes after the divergence of the mung bean line from the pea-Vicia line. We present hybridization data indicating that rearrangements that change the linear order of homologous sequences within the chloroplast genome have been quite frequent during the course of legume evolution.The great majority of higher plant chloroplast genomes studied consist of a circular molecule 134 kilobase pairs (kb) to 150 kb in length, which contains a large inverted repeat sequence of 20-25 kb. This pattern has been demonstrated in corn, spinach, and lettuce by intramolecular homoduplex formation (1) and in corn, wheat, spinach, Oenothera, tobacco, petunia, and Spirodela by restriction endonuclease mapping (2-7). The only exceptions to this pattern so far reported are pea and broad bean (Viciafaba), both members ofthe legume family. Pea chloroplast DNA lacks the inverted repeat structure as judged by electron microscopy (1) but is reported to possess two copies of the ribosomal RNA genes (8, 9), which may be repeated in tandem (10). Electron microscopy and restriction mapping reveal the absence of an inverted repeat in broad bean, as well as the presence of only one copy of the ribosomal RNA genes (11).We have constructed detailed restriction maps of the chloroplast genomes of mung bean and pea. The mung bean genome, unlike that ofpea and broad bean, possesses the inverted repeat structure common to all other higher plant chloroplast genomes. We confirm that the pea genome lacks an inverted repeat and demonstrate that it contains only one set ofribosomal genes and no detectable repeated sequences. From these data we conclude that deletion of one segment of the highly conserved inverted repeat has occurred during the evolution ofthe legumes. In addition, we have mapped a number of rearrangements of sequences common to the mung bean and pea chloroplast genomes. MATERIALS AND METHODSChloroplast DNA was isolated as described by Kolodner and Tewari (12). Recombinant plasmids containing mung bean and pea chloroplast DNA restriction fragments (13) (16) and hybridized to the filters in...

Localization of replication origins in pea chloroplast DNA.

“…When ctDNA was digested to completion with the restriction enzyme Sail, there were 10 identifiable ctDNA fragments generated, as determined by gel electrophoresis (14). These fragments were as follows: (i) the 44-kbp SalI-A; (ii) the 22.8-kbp SalI-B; (iii) the 16-kbp SalI-C; (iv) the 12.4-kbp SalI-D; (v) the 10.3-kbp SalI-E; (vi) the 10-kbp Sall-F; (vii) the 2.3-kbp Sall-G; and (viii) three fragments of approximately 1 kbp in size, all of which were clustered between the Sall C and Sall A fragments (Fig.…”

Cytogenomic Analyses Reveal the Structural Plasticity of the Chloroplast Genome in Higher Plants

“…Restriction enzyme site mapping across a plethora of plants demonstrated that the cpDNA has a circular plasmidlike structure, with the only significant difference being the number of IR regions (reviewed in Palmer, 1992). Restriction enzyme site mapping has the advantage over EM and reassociation kinetics in that smaller DNA fragments can be analyzed, resulting in less error, and multiple restriction enzymes can be used to map the same molecule (Palmer and Thompson, 1981). A more recently used technique was PFGE of chloroplasts embedded in agarose (Deng et al, 1989;Backert et al, 1995), which eliminated the need to purify cpDNA and reduced shearing.…”

“…Pea is a member of the Fabaceae and possesses a smaller chloroplast genome ( ‫ف‬ 120 kb) with only a single copy of the sequence corresponding to the IRs (Palmer and Thompson, 1981). Extended cpDNA fibers of pea were hybridized with both the LSC/SSC probes and the 12.2-kb IR probe.…”

DNA Strand-Transfer Activity in Pea (Pisum sativum L.) Chloroplasts

“…The 32 PssDNA was a 450-nucleotide segment internal to the plastid 16S rRNA gene (Cerutti and Jagendorf, 1991), and the complementary substrate was the 120-kb polyploid chloroplast genome (Palmer and Thompson, 1981;Palmer, 1985). After incubation, the DNA was purified and digested with Pstl, under conditions that minimize branch migration.…”