Chloroplast DNA evolution among legumes: Loss of a large inverted repeat occurred prior to other sequence rearrangements

Summary We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions.     

Chloroplast DNA from Adiantum capillus-veneris L., a fern species (Adiantaceae); clone bank,physical map and unusual gene localization in comparison with angiosperm chloroplast DNA

Summary We cloned all of Adiantum capillus-veneris chloroplast DNA PstI fragments longer than 1.0 kb, which cover 98% of the genome. These cloned fragments were used to construct a physical map for five restriction enzymes. The genome of A. capillus-veneris is approximately 153 kb long and contains a 24 kb inverted repeat. Mapping of 12 chloroplast DNA genes and heterologous hybridization, involving A. capillus-veneris chloroplast DNA and angiosperm chloroplast DNA probes, demonstrated that chloroplast DNA of A. capillus-veneris has a different gene order from typical angiosperm cpDNA (e.g., tobacco) in the inverted repeat region and the flanking segment of the large single copy region.     

The site of deletion of the inverted repeat in pea chloroplast DNA contains duplicated gene fragments

Summary Analysis of a nucleotide sequence from pea chloroplast DNA which spans the site of deletion of one copy of the large inverted repeat (IR) element suggests that the IR unit has been entirely deleted, but that the single-copy coding sequences which flanked it (trnH and ndh5) have been left intact. However, these flanking sequences have not simply been ligated together: between them there is instead a novel 200 by DNA sequence which includes two regions apparently derived by the duplication of fragments of the psbA and rbcL genes.     

Organization and sequence of five tRNA genes and of an unidentified reading frame in the wheat chloroplast genome: evidence for gene rearrangements during the evolution of chloroplast genomes

Summary The genes for the initiator tRNA_CAU ^Met. tRNA_UCC ^Gly, tRNA_GGU ^Thr, tRNA_UUC ^Glu and tRNA_GUA ^Tyr and an open reading frame of 62 codons have been identified by sequencing a 2,358 by BamHl and a 1,378 by BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 by intergenic region between the genes for the initiator tRNA^Met and the gene for tRNA_UCC ^Gly is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 by of the intergenic region and the first 28 by of the tRNA^Met gene, the second copy is found downstream of the tRNA^Met gene.     

Physical mapping of the plastid genome from the chlorophyll c-containing alga,Cryptomonas Φ

Summary A physical map of the circular plastid genome of Cryptomonas has been constructed using the enzymes SacI, BamHI, SmaI, SalI, PstI and XhoI. In addition, fine-structural mapping of the inverted repeat region has been performed using AvaI, BglII, EcoRI and XbaI. The inverted repeat is very small, encompassing no more than 6 kb and containing only genes for the rRNAs. It divides the plastid genome into a small singlecopy region of 12–13 kb which contains genes for phycoerythrin and the 32 kd photosystem II polypeptide, and a large single-copy region of 93–94 kb, giving a total size of 118 kb. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) and the beta subunit of ATP synthase CF₁ are encoded in the large single-copy region. The evolutionary significance of the organization of this plastid genome, the first presented from the chlorophyll c-, phycobiliprotein-containing group of algae, is discussed.     

Characterization of a large inversion in the spinach chloroplast genome relative to Marchantia: a possible transposon-mediated origin

Summary A 7,022 by BamHI-EcoRI fragment, located in the inverted repeat of spinach chloroplast, has been sequenced. It contains a 2131 codon open reading frame (ORF) homologous to both tobacco ORFs 581 and 1708, and to Marchantia ORF 2136. Relative to the Marchantia chloroplast genome, spinach ORF 2131 is located at the end of a large inversion; the other end point is close to trnL, the position of which is the same in Marchantia, tobacco and spinach. In Marchantia, two 8 by direct repeats flanking two 10 by indirect repeats are present near the end points of the inversion. These repeats may result from a transposon-mediated insertion which would have facilitated the subsequent inversion. From a comparison of the gene organization of the spinach, tobacco, and Marchantia genomes in this region, we propose a step-wise process to explain the expansion of the inverted repeat from a Marchantia-like genome to the spinach/tobacco genome.     

Chloroplast DNA from the fern Osmunda cinnamomea: physical organization,gene localization and comparison to angiosperm

Summary Chloroplast DNA from the fern Osmunda einnamomea was isolated by a sucrose gradient procedure utilizing PEG to stabilize chloroplasts. Analysis with the restriction endonucleases PvuII, Sacl and BstEII indicates a chloroplast genome size of 144 kb. A physical map of the fragments produced by these three enzymes was constructed by filter hybridizations using purified PvuII fragments as hybridization probes. The Osmunda chloroplast genome is circular and contains an inverted repeat 8–13 kb in size.Gene probes from tobacco, corn and spinach were used to map the positions of six genes on the Osmunda chloroplast chromosome. The 16S and 23S ribosomal RNAs are encoded by duplicate genes which lie within the inverted repeat. Genes for the large subunit of ribulose-1,5-bisphosphate carboxylase, a photosystem II polypeptide, and the alpha and beta subunits of chloroplast coupling factor are located in three different segments of the large single copy region.The Osmunda chloroplast genome is remarkably similar in size, conformation, physical organization, and map positions of known genes, to chloroplast DNA from a number of angiosperms. The major difference between chloroplast DNA from this fern and angiosperms is that the inverted repeat is smaller in Osmunda (8–13 kb) than in angiosperms (22–25 kb).Abbreviations PEG polyethylene glycol 4000 - kb kilobase pairs - bp base pairs - rRNA ribosomal RNA - LS large subunit of ribulose-1,5-bisphosphate carboxylase - PII 32,000 dalton photosystem II polypeptide (Mattoo et al. 1981) - CF alpha subunit of chloroplast coupling factor - CF beta subunit of chloroplast coupling factor     

The ribosomal RNA repeats are non-identical and directly oriented in the chloroplast genome of the red alga Porphyra purpurea

A detailed restriction map of the chloroplast genome of the red alga Porphyra purpurea has been constructed. Southern hybridization experiments with cloned or gel-purified restriction fragments and PCR products indicate that the P. purpurea chloroplast genome is approximately 188 kb in size. This circular molecule contains two rRNA-encoding repeats (approximately 4.9 kb) that separate the genome into single-copy regions of 34 kb and 144 kb. Interestingly, these repeats are arranged in a direct orientation. In addition, DNA sequencing of the ends of both repeats revealed that the two rRNA repeats are not identical. No intramolecular recombination between the repeats can be detected. We discuss the possibility that the chloroplast genome of P. purpurea is organized like that of the ancestral chloroplast.     

The chloroplast genome of the gymnosperm Pinus contorta: a physical map and a complete collection of overlapping clones

Summary Overlapping restriction fragments of chloroplast DNA from the conifer Pinus contorta were cloned. Out of a total of 49 clones, 33 comprise the minimum set required to represent the entire genome. Using the purified inserts of these clones as probes in filter hybridizations, all sites for the three restriction enzymes KpnI, HapI and SacI in the P. contorta chloroplast genome were mapped. Heterologous filter hybridizations and sequence analysis of some of the P. contorta clones were used to determine the position of 15 genes on the restriction map. The size of the genome, which lacks an inverted repeat organization, was found to be approximately 121 kilobase pairs (kbp). Unusual features of this genome are a duplication of the psbA gene and the presence of two genes, gidA and frxC, which are not found in angiosperms. The genome appeared essentially colinear with that of Pinus radiata, for which a map has previously been published. Two different restriction fragment length polymorphisms were found to be produced by variable numbers of copies of 124 bp-and 150 bp-long, tandemly repeated elements.Communicated by H. Kössel     

The endpoints of an inversion in wheat chloroplast DNA are associated with short repeated sequences containing homology toatt-lambda

Summary The physical mapping of Aval, BstEII and EcoR1 restriction sites on the chloroplast genome of the green alga Chlamydomonas eugametos is presented. The circular map, with a size of 243 kilobase pairs, is the largest yet reported for a chloroplast genome. It features a large inverted repeat sequence, part of which encodes the 16S and 23S ribosomal RNAs (rRNAs), the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and the 32-kdodalton thylakoid membrane protein (psbA). Such an rRNA-encoding inverted repeat sequence is also found in the chloroplast genomes of Chlamydomonas reinhardtii and most land plants. These genomes, however, differ from that of C. eugametos by the absence of the rbcL gene from the inverted repeat sequence of C. reinhardtii and by the absence of both the rbcL and psbA genes from the inverted repeat sequence of land plants. Possible evolutionary implications of these differences are discussed.Abbrevations cpDNA chloroplast DNA - kbp kilobase pairs - psbA 32 kilodalton thylakoid membrane protein gene - rbcL ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit gene - rRNA ribosomal RNA     

Organisation of the chloroplast genome of kiwifruit (Actinidia deliciosa)

Summary A restriction map of the chloroplast genome has been determined for kiwifruit, Actinidia deliciosa. Single and multiple enzyme digests of kiwifruit chloroplast DNA were hybridised to a set of Brassica chloroplast probes, and the kiwifruit bands aligned with the known Brassica map. The chloroplast DNA of kiwifruit is typical of the majority of angiosperm chloroplast genomes; it is 160 kb in size, contains a 15–34 kb inverted repeat, and its gene content and gene order are similar to those of the Brassica chloroplast genome.     

Sequence analysis of the junction of the large single copy region and the large inverted repeat in the petunia chloroplast genome

Summary We have determined the nucleotide sequence at the junction of the large single copy (LSC) region and the right and left members of the large inverted repeat, IRA and IRB, respectively, of the petunia chloroplast (cp) genome. As in Nicotiana debneyi and spinach (Zurawski et al. 1984), coding sequences of rps19 of petunia overlap the junction of IRB and LSC. Immediately into the LSC region upstream of IRA in the petunia cp genome are two small insertions relative to N. debneyi that occur at sites just inside IRA of N. debneyi. We discuss how these additions in one copy of the large inverted repeat of an N. debneyi like ancestor to petunia resulted in shortening of the petunia large inverted repeat by 8 bases and in the resultant slight movement of rps19 farther into LSC. On a larger scale, the large inverted repeat in the tobacco, N. debneyi and petunia lineage relative to a spinach-like ancestor may have sustained several contractions due to deletions between short direct repeats found within IRA and the IRA/LSC junction. We also show how the large inverted repeat of N. debneyi instead may have been expanded relative to a tobacco-like ancestor by insertion into the large inverted repeat of bases between short inverted repeat sequences in LSC and the LSC/IRB junction.     

A restriction endonuclease map of the chloroplast genome of pearl millet

Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127–138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86–93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the -coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.     

Recombination within the inverted repeat sequences of the Chlamydomonas reinhardii chloroplast genome produces two orientation isomers

Summary 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.     

Physical mapping of differences between the chloroplast DNAs of the interfertile algae Chlamydomonas eugametos and Chlamydomonas moewusii

Summary The chloroplast genomes from the interfertile green algae Chlamydomonas eugametos and C. moewusii have been compared in their overall sequence organization. Physical mapping of Aval, BstEII and EcoRI restriction sites on the C. moewusii chloroplast genome revealed that this 292 kilobase-pair (kbp) genome is 49 kbp larger than the C. eugametos genome. Heterologous fragment hybridizations indicated the same order of common sequence elements on the two algal genomes. Almost all of the 49 kbp size difference is accounted for by the presence of two large extra sequences in C. moewusii: a 21 kbp sequence in the inverted repeat and a 5.8 kbp sequence in the single copy-region bordering the 16S ribosomal RNA (rRNA) genes. In addition to these two major deletion/addition differences, 42 restriction site and fragment length differences (ranging from 100 to 500 base pairs) were mapped on the two algal genomes. Surprisingly, the greatest density of these differences was found to be confined within the inverted repeat, one of the most conserved regions of land plant chloroplast genomes.     

Dispersed repetitive sequences in the chloroplast genome of Douglas-fir

Summary Restriction mapping and DNA sequencing were used to characterize dispersed repetitive DNA in the chloroplast genome of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. To map repeat families, chloroplast DNA (cpDNA) clones were hybridized at high stringency to one another and to cpDNA cut with restriction enzymes. Repeats are clustered in four regions of the genome and comprise at least six families. Sequence analysis of one repeat family shared among three XbaI fragments indicated the presence of a 633 by inverted repeat which contains a complete tRNA-Serine (GCU) gene and a highly conserved open reading frame (ORF 3.6). Both ends of this 633 bp dispersed repeat have a transposon-like combination of short direct and inverted repeats. One copy of the repeat flanks one of the endpoints of a major inversion which differentiates Douglas-fir from tobacco cpDNA. Dispersion of repetitive DNA by transposition, coupled with loss of the large inverted repeat, appears to have predisposed conifer cpDNA to a number of inversions. An 8 by (CATCTTTT) direct repeat in tobacco is located between two inverted sections in Douglas-fir; it may be a target sequence for homologous recombination.     

Expansion of the IR in the chloroplast genomes of buckwheat species is due to incorporation of an SSC sequence that could be mediated by an inversion

The chloroplast genomes in buckwheat species contain large inverted repeats which are at least 4 kbp longer than the majority of those in land plants. The length of the buckwheat inverted repeats was attributable to an additional region located adjacent to the borders of the small single-copy region. We have cloned and sequenced a 5.2-kbp SmaI fragment corresponding to this extra region in the inverted repeats. A homology search revealed that the sequence of the SmaI fragment is highly homologous to one side of the small single-copy region of the inverted repeats in dicot chloroplast DNAs such as tobacco and beechdrops. Interestingly, a 3.7-kbp segment in the middle of the SmaI fragment is inserted in the opposite orientation relative to those of the other dicot species, and 17-bp direct repeats are found located at both the ends of the additional region. These results suggest that expansion of the inverted repeats in buckwheat chloroplast DNA might have been associated with an inversion. Received: 6 May / 1 November 1996