The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands

Background  

The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC) remains uncertain. The five complete chloroplast DNA (cpDNA) sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR), have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage.     

The chloroplast genome sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features and new insights into the branching order of chlorophyte lineages

One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

The chloroplast genome arrangement ofLobelia thuliniana (Lobeliaceae): Expansion of the inverted repeat in an ancestor of theCampanulales

A clone-bank ofSac I restriction fragments was constructed from the chloroplast DNA (cpDNA) ofLobelia thuliniana E. B. Knox (Lobeliaceae). These cloned fragments and a set of 106 clones spanning the tobacco chloroplast genome were used as probes to determine the cpDNA restriction fragment arrangement forSac I and six other restriction enzymes (BamH I,EcoR V,Hind III,Nci I,Pst I, andXho I) and the chloroplast genome arrangement ofL. thuliniana relative to tobacco, which has been fully sequenced and is collinear with the hypothesized ancestral genome arrangement of angiosperms. The results confirm and refine our previous understanding of the chloroplast genome arrangement in the large single-copy region (LSC) and reveal (1) a roughly 11 kilobase (kb) expansion of the inverted repeat (IR) into the small single-copy region (SSC) and (2) apparent sequence divergence of the DNA segment inL. thuliniana that corresponds to ORF1901 in tobacco. The expansion of the IR into the SSC is present in all other examined members ofLobeliaceae, Cyphiaceae, andCampanulaceae, which indicates that the IR expansion was an early event in the cpDNA evolution of theCampanulales. The IR expansion into the SSC was not present inSphenoclea, which additionally supports exclusion of this genus from theCampanulaceae.     

Unidirectional gene conversions in the chloroplast of Chlamydomonas interspecific hybrids

Summary With the goal of studying directly the inheritance and recombination of physically mapped markers on the chloroplast genome, we have recently identified and localized physical differences between the chloroplast DNAs (cpDNAs) of the interfertile algae Chlamydomonas eugametos and C. moewusii. Here we report the inheritance patterns of 24 polymorphic loci mapping throughout the chloroplast genome in hybrids recovered from reciprocal crosses between the two algae. Most polymorphic loci were found to be inherited mainly from the mt ⁺ parent, with no apparent preference for one or the other parental alternatives in reciprocal crosses. Virtually all hybrids, however, inherited exclusively the long alleles of three loci; i.e. an intron in the large subunit ribosomal RNA gene of C. eugametos, a 21 kbp sequence addition in the inverted repeat of the C. moewusii cpDNA and a 5.8 kbp sequence addition in one of the single-copy regions of C. moewusii cpDNA. As these alleles are derived from opposite parental strains, their unidirectional inheritance in hybrids results necessarily from interspecific recombination of cpDNA molecules. We propose that gene conversion events led to the spreading of the long alleles of the three loci.     

Sequence analysis of the junctions between a large inverted repeat and single-copy regions in tobacco chloroplast DNA

Summary Tobacco chloroplast DNA contains a large inverted repeat sequence of 26 kilobase pairs (kbp). The inverted repeat is separated by 20 kbp small single-copy and 90 kbp large single-copy regions. We have cloned four DNA fragments containing each junction between the inverted repeat and the single-copy regions. The sequence analysis revealed the exact edges of the inverted repeat. A putative coding region for a ribosomal protein CS19 was found 4 base pairs (bp) away from the inverted repeat on the left margin of the large single-copy region. A sequence AGGAG, which is complementary to the 3 terminal sequence of tobacco chloroplast 16S rRNA, was found within the inverted repeat. A tRNA^His gene was found 5 bp away from the inverted repeat on the right-hand margin of the large single-copy region.     

The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales,Chlorophyta): compact genomes and genes of bacterial origin

Background

Species of Bryopsidales form ecologically important components of seaweed communities worldwide. These siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. Little is known about the diversity of chloroplast genomes (cpDNAs) in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. We present the complete cpDNAs of Bryopsis plumosa and Tydemania expeditiones, as well as a re-annotated cpDNA of B. hypnoides, which was shown to contain a higher number of genes than originally published. Chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the Chlorophyta, such as monophyly of the Ulvophyceae (the class in which the order Bryopsidales is currently classified).

Results

Both DNAs are circular and lack a large inverted repeat. The cpDNA of B. plumosa is 106,859 bp long and contains 115 unique genes. A 13 kb region was identified with several freestanding open reading frames (ORFs) of putative bacterial origin, including a large ORF (>8 kb) closely related to bacterial rhs-family genes. The cpDNA of T. expeditiones is 105,200 bp long and contains 125 unique genes. As in B. plumosa, several regions were identified with ORFs of possible bacterial origin, including genes involved in mobile functions (transposases, integrases, phage/plasmid DNA primases), and ORFs showing close similarity with bacterial DNA methyltransferases. The cpDNA of B. hypnoides differs from that of B. plumosa mainly in the presence of long intergenic spacers, and a large tRNA region. Chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the Ulvophyceae, and the relationship of the Bryopsidales within the Chlorophyta.

Conclusions

The cpDNAs of B. plumosa and T. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core Chlorophyta. The presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1418-3) contains supplementary material, which is available to authorized users.     

The complete chloroplast genome and characteristics analysis of Musa basjoo Siebold

Background

An ornamental plant often seen in gardens and farmhouses, Musa basjoo Siebold can also be used as Chinese herbal medicine. Its pseudostem and leaves are diuretic; its root can be decocted together with ginger and licorice to cure gonorrhea and diabetes; the decoct soup of its pseudostem can help relieve heat, and the decoct soup of its dried flower can treat cerebral hemorrhage. There have not been many chloroplast genome studies on M. basjoo Siebold.

Methods and results

We characterized its complete chloroplast genome using Novaseq 6000 sequencing. This paper shows that the length of the chloroplast genome M. basjoo Siebold is 172,322 bp, with 36.45% GC content. M. basjoo Siebold includes a large single-copy region of 90,160 bp, a small single-copy region of 11,668 bp, and a pair of inverted repeats of 35,247 bp. Comparing the genomic structure and sequence data of closely related species, we have revealed the conserved gene order of the IR and LSC/SSC regions, which has provided a very inspiring discovery for future phylogenetic research.

Conclusions

Overall, this study has constructed an evolutionary tree of the genus Musa species with the complete chloroplast genome sequence for the first time. As can be seen, there is no obvious multi-branching in the genus, and M. basjoo Siebold and Musa itinerans are the closest relatives.

     

The complete chloroplast DNA sequences of the charophycean green algae <Emphasis Type="Italic">Staurastrum</Emphasis> and <Emphasis Type="Italic">Zygnema</Emphasis> reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

Background  

The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum.     

Chloroplast DNA variation in Populus. III. Novel chloroplast DNA variants in natural Populus x canadensis hybrids

A rare phenomenon of the occurrence of novel non-parental chloroplast DNA (cpDNA) variants in natural sexual interspecific hybrids between Populus deltoides var deltoides and P. nigra, P. x canadensis is described. Restriction fragment variation of cpDNA in 17 P. x canadensis cultivars was examined and compared with that of representative samples of P. deltoides and P. nigra using 83 combinations of 16 restriction enzymes and six Petunia hybrida cpDNA probes. Twelve cultivars had one to five novel non-parental cpDNA fragments in the chloroplast genome region homologous to the 9.0-kb PstI cpDNA fragment of Petunia from the large single-copy region.     

Gene rearrangements in Chlamydomonas chloroplast DNAs are accounted for by inversions and by the expansion/contraction of the inverted repeat

To gain insight into the mutational events responsible for the extensive variation of chloroplast DNA (cpDNA) within the green algal genus Chlamydomonas, we have investigated the chloroplast gene organization of Chlamydomonas pitschmannii, a close relative of the interfertile species C. eugametos and C. moewusii whose cpDNAs have been well characterized. At 187 kb, the circular cpDNA of C. pitschmannii is the smallest Chlamydomonas cpDNA yet reported; it is 56 and 105 kb smaller than those of its C. eugametos and C. moewusii counterparts, respectively. Despite this substantial size difference, the arrangement of 77 genes on the C. pitschmannii cpDNA displays only three noticeable differences from the organization of the corresponding genes on the collinear C. eugametos and C. moewusii cpDNAs. These changes in gene order are accounted for by the expansion/contraction of the inverted repeat and one or two inversions in a single-copy region. In land plant cpDNAs, these kinds of events are also responsible for gene rearrangements. The large size difference between the C. pitschmannii and C. eugametos/C. moewusii cpDNAs is mainly attributed to multiple events of deletions/additions as opposed to the usually observed expansion/contraction of the inverted repeat in land plant cpDNAs. We also found that the mitochondrial genome of C. pitschmannii is a circular DNA molecule of 16.5 kb which is 5.5 and 7.5 kb smaller than its C. moewusii and C. eugametos counterparts, respectively.     

Distinctive architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium helveticum

The chloroplast genome has experienced many architectural changes during the evolution of chlorophyte green algae, with the class Chlorophyceae displaying the lowest degree of ancestral traits. We have previously shown that the completely sequenced chloroplast DNAs (cpDNAs) of Chamydomonas reinhardtii (Chlamydomonadales) and Scenedesmus obliquus (Sphaeropleales) are highly scrambled in gene order relative to one another. Here, we report the complete cpDNA sequence of Stigeoclonium helveticum (Chaetophorales), a member of a third chlorophycean lineage. This genome, which encodes 97 genes and contains 21 introns (including four putatively trans-spliced group II introns inserted at novel sites), is remarkably rich in derived features and extremely rearranged relative to its chlorophycean counterparts. At 223,902 bp, Stigeoclonium cpDNA is the largest chloroplast genome sequenced thus far, and in contrast to those of Chlamydomonas and Scenedesmus, features no large inverted repeat. Interestingly, the pattern of gene distribution between the DNA strands and the bias in base composition along each strand suggest that the Stigeoclonium genome replicates bidirectionally from a single origin. Unlike most known trans-spliced group II introns, those of Stigeoclonium exhibit breaks in domains I and II. By placing our comparative genome analyses in a phylogenetic framework, we inferred an evolutionary scenario of the mutational events that led to changes in genome architecture in the Chlorophyceae.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Nucleotide sequence data reported are available in the GenBank database under the accession number DQ630521.     

The first report describes features of the chloroplast genome of Withania frutescens

Genomic studies not only help researcher not only to identify genomic features in organisms, but also facilitate understanding of evolutionary relationships. Species in the Withania genus have medicinal benefits, and one of them is Withania frutescens, which is used to treat various diseases. This report investigates the nucleotides and genic features of chloroplast genome of Withania frutescens and trying to clarify the evolutionary relationship with Withania sp and family Solanaceae. We found that the total size of Withania frutescens chloroplast genome was 153.771 kb (the smallest chloroplast genome in genus Withania). A large single-copy region (91.285 kb), a small single-copy region (18.373 kb) form the genomic region, and are distinct from each other by a large inverted repeat (22.056 kb). 137 chloroplast genes are found including 4 rRNAs, 38 tRNAs and 83 protein-coding genes. The Withania frutescens chloroplast genome as well as four closest relatives was compared for features such as structure, nucleotide composition, simple sequence repeats (SSRs) and codon bias. Compared to other Withania species, Withania frutescens has unique characteristics. It has the smallest chloroplast genome of any Withania species, isoleucine is the major amino acid, and tryptophan is the minor, In addition, there are no ycf3 and ycf4 genes, fourth, there are only fifteen replicative genes, while in most other species there are more. Using fast minimum evolution and neighbor joining, we have reconstructed the trees to confirm the relationship with other Solanacaea species. The Withania frutescens chloroplast genome is submitted under accession no. ON153173.     

Complete sequence and organisation of the <Emphasis Type="Italic">Jatropha curcas</Emphasis> (Euphorbiaceae) chloroplast genome

Jatropha curcas is an important non-edible oil seed tree species and is considered a promising source of biodiesel. The complete nucleotide sequence of J. curcas chloroplast genome (cpDNA) was determined by pyrosequencing and gaps filled by Sanger sequencing. The cpDNA is a circular molecule of 163,856 bp in length and codes for 110 distinct genes (78 protein coding, four rRNA and 28 distinct tRNA). Genome organisation and arrangement are similar to the reported angiosperm chloroplast genome. However, in Jatropha, the infA and the rps16 genes are non-functional. The inverted repeat (IR) boundary is within the rpl2 gene, and the 13 nucleotides at the ends of the two duplicate genes are different. Repeat analysis suggests the presence of 72 repeat regions (>30 bp) apart from the IR; of these, 48 were direct and 24 were palindromic repeats. Phylogenetic analysis of 81 protein coding chloroplast genes from 65 taxa by maximum parsimony, maximum likelihood and minimum evolution analyses at 100 bootstraps provide strong support for the placement of inaperturate crotonoids of which Jatropha is a member as sister to articulated crotonoids of which Manihot is a member.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Complete Sequence of the Duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis>) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.     

The Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L. cv. ‘Aseel’)

Date palm (Phoenix dactylifera L.) is an economically important and widely cultivated palm of the family Arecaceae. We sequenced the complete date palm chloroplast genome (cpDNA) from Pakistani cv. ??Aseel??, using a combination of Sanger-based and next-generation sequencing technologies. Being very similar to a sequence from a Saudi Arabian date palm cultivar ??Khalas?? published recently, the size of the genome was 158,458?bp with a pair of inverted repeat (IR) regions of 27,276?bp that were separated by a large single-copy (LSC) region of 86,195?bp and a small single-copy (SSC) region of 17,711?bp. Genome annotation demonstrated a total of 138 genes, of which 89 were protein coding, 39 were tRNA, and eight were rRNA genes. Comparison of cpDNA sequences of cultivars ??Aseel?? and ??Khalas?? showed following intervarietal variations in the LSC region; (a) two SNPs in intergenic spacers and one SNP in the rpoc1 gene, (b) polymorphism in two mono-nucleotide simple sequence repeats (SSR), and (c) a 4-bp indel in the accD-psaI intergenic spacer. The SSC region has a polymorphic site in the mono-nucleotide SSR located at position 120,710. We also compared cv. ??Aseel?? cpDNA sequence with partial P. dactylifera cpDNA sequence entries deposited in Genbank and identified a number of potentially useful polymorphisms in this species. Analysis of date palm cpDNA sequences revealed a close relationship with Typha latifolia. Occurrence of small numbers of forward and inverted repeats in date palm cpDNA indicated conserved genome arrangement.     

The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae)

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8× depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373 bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,657 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperzia chloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophyte chloroplast genome data also enable a better reconstruction of the basal tracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, inferred amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.     

A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii

Summary Our recent physical mapping of chloroplast DNA (cpDNA) from Chlamydomonas moewusii, a unicellular green alga which is interfertile with Chlamydomonas eugametos, has revealed a two-fold size difference between the inverted repeat sequences of these algae. With a size of 42 kbp, the inverted repeat of C. moewusii is the largest yet identified in any chloroplast genome. Here we have compared the arrangement of conserved sequences within the two algal inverted repeats by hybridizing cloned restriction fragments representing over 90% of these repeats to Southern blots of cpDNA digests from the two algae. We found that the size difference between the two algal inverted repeats is due to the presence of an extra DNA segment of 21 kilobase pairs (kbp) in C. moewusii. Except for this sequence, the C. moewusii inverted repeat is highly homologous to the entire C. eugametos repeat and the arrangement of conserved sequences in the two repeats is identical. Southern hybridizations with specific gene probes revealed that the conserved sequences include the rDNA region and the genes coding for the large subunit of ribulose 1,5 bisphosphate carboxylase-oxygenase (rbcL) and for the 32 kilodalton thylakoid membrane protein (psbA). With respect to the conserved sequences, the extra 21 kbp DNA segment of C. moewusii lies in the region of psbA, most probably slightly downstream from this gene.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.