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 Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns''own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns.     

DEEP DIVISION IN THE CHLOROPHYCEAE (CHLOROPHYTA) REVEALED BY CHLOROPLAST PHYLOGENOMIC ANALYSES1

The Chlorophyceae (sensu Mattox and Stewart) is a morphologically diverse class of the Chlorophyta displaying biflagellate and quadriflagellate motile cells with varying configurations of the flagellar apparatus. Phylogenetic analyses of 18S rDNA data and combined 18S and 26S rDNA data from a broad range of chlorophycean taxa uncovered five major monophyletic groups (Chlamydomonadales, Sphaeropleales, Oedogoniales, Chaetophorales, and Chaetopeltidales) but could not resolve their branching order. To gain insight into the interrelationships of these groups, we analyzed multiple genes encoded by the chloroplast genomes of Chlamydomonas reinhardtii P. A. Dang. and Chlamydomonas moewusii Gerloff (Chlamydomonadales), Scenedesmus obliquus (Turpin) Kütz. (Sphaeropleales), Oedogonium cardiacum Wittr. (Oedogoniales), Stigeoclonium helveticum Vischer (Chaetophorales), and Floydiella terrestris (Groover et Hofstetter) Friedl et O’Kelly (Chaetopeltidales). The C. moewusii, Oedogonium, and Floydiella chloroplast DNAs were partly sequenced using a random strategy. Trees were reconstructed from nucleotide and amino acid data sets derived from 44 protein‐coding genes of 11 chlorophytes and nine streptophytes as well as from 57 protein‐coding genes of the six chlorophycean taxa. All best trees identified two robustly supported major lineages within the Chlorophyceae: a clade uniting the Chlamydomonadales and Sphaeropleales, and a clade uniting the Oedogoniales, Chaetophorales, and Chaetopeltidales (OCC clade). This dichotomy is independently supported by molecular signatures in chloroplast genes, such as insertions/deletions and the distribution of trans‐spliced group II introns. Within the OCC clade, the sister relationship observed for the Chaetophorales and Chaetopeltidales is also strengthened by independent data. Character state reconstruction of basal body orientation allowed us to refine hypotheses regarding the evolution of the flagellar apparatus.     

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

Background  

The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA) sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae), in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR) featuring an inverted rRNA operon and a small single-copy (SSC) region containing 14 genes normally found in the large single-copy (LSC) region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage.     

The complete chloroplast genome of the green algae <Emphasis Type="Italic">Hariotina reticulata</Emphasis> (Scenedesmaceae,Sphaeropleales, Chlorophyta)

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.     

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.     

Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.     

A Possible Role for Short Introns in the Acquisition of Stroma-Targeting Peptides in the Flagellate Euglena gracilis

The chloroplasts of Euglena gracilis bounded by three membranes arose via secondary endosymbiosis of a green alga in a heterotrophic euglenozoan host. Many genes were transferred from symbiont to the host nucleus. A subset of Euglena nuclear genes of predominately symbiont, but also host, or other origin have obtained complex presequences required for chloroplast targeting. This study has revealed the presence of short introns (41–93 bp) either in the second half of presequence-encoding regions or shortly downstream of them in nine nucleus-encoded E. gracilis genes for chloroplast proteins (Eno29, GapA, PetA, PetF, PetJ, PsaF, PsbM, PsbO, and PsbW). In addition, the E. gracilis Pbgd gene contains two introns in the second half of presequence-encoding region and one at the border of presequence-mature peptide-encoding region. Ten of 12 introns present within presequence-encoding regions or shortly downstream of them identified in this study have typical eukaryotic GT/AG borders, are T-rich, 45–50 bp long, and pairwise sequence identities range from 27 to 61%. Thus single recombination events might have been mediated via these cis-spliced introns. A double crossing over between these cis-spliced introns and trans-spliced introns present in 5′-UTRs of Euglena nuclear genes is also likely to have occurred. Thus introns and exon-shuffling could have had an important role in the acquisition of chloroplast targeting signals in E. gracilis. The results are consistent with a late origin of photosynthetic euglenids.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

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.     

Low levels of intraspecific genetic variation at a rapidly evolving chloroplast dna locus in North American duckweeds (Lemnaceae)

Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Regular Spliceosomal Introns Are Invasive in Chlamydomonas reinhardtii: 15 Introns in the Recently Relocated Mitochondrial cox2 and cox3 Genes

In the unicellular green alga, Chlamydomonas reinhardtii, cytochrome oxidase subunit 2 (cox2) and 3 (cox3) genes are missing from the mitochondrial genome. We isolated and sequenced a BAC clone that carries the whole cox3 gene and its corresponding cDNA. Almost the entire cox2 gene and its cDNA were also determined. Comparison of the genomic and the corresponding cDNA sequences revealed that the cox3 gene contains as many as nine spliceosomal introns and that cox2 bears six introns. Putative mitochondria targeting signals were predicted at each N terminal of the cox genes. These spliceosomal introns were typical GT–AG-type introns, which are very common not only in Chlamydomonas nuclear genes but also in diverse eukaryotic taxa. We found no particular distinguishing features in the cox introns. Comparative analysis of these genes with the various mitochondrial genes showed that 8 of the 15 introns were interrupting the conserved mature protein coding segments, while the other 7 introns were located in the N-terminal target peptide regions. Phylogenetic analysis of the evolutionary position of C. reinhardtii in Chlorophyta was carried out and the existence of the cox2 and cox3 genes in the mitochondrial genome was superimposed in the tree. This analysis clearly shows that these cox genes were relocated during the evolution of Chlorophyceae. It is apparent that long before the estimated period of relocation of these mitochondrial genes, the cytosol had lost the splicing ability for group II introns. Therefore, at least eight introns located in the mature protein coding region cannot be the direct descendant of group II introns. Here, we conclude that the presence of these introns is due to the invasion of spliceosomal introns, which occurred during the evolution of Chlorophyceae. This finding provides concrete evidence supporting the ``intron-late' model, which rests largely on the mobility of spliceosomal introns. Received: 22 August 2000 / Accepted: 28 February 2001     

Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly-labeled 32-kd protein of photosystem II, also identified as as herbicide-binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in-frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5'-and 3' -flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.     

INTRON REVEALED BY NUCLEOTIDE SEQUENCE OF LARGE SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM CODIUM FRAGILE (CHLOROPHYTA): PHYLOGENETIC ANALYSIS1

The coding sequence for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Codium fragile (Suringar) Hariot chloroplast DNA is 1428 bp in length and contains a 1813-bp group II intron. The only other organisms in which introns have been found in the rbcL gene are Euglena and Astasia. The Codium intron likely had a separate origin from the Euglena and Astasia introns, based on comparisons of intron sizes and sequences. Phylogenetic analyses of rbcL nucleotide and amino acid sequences place Codium between Chlorella and two Chlamydomonas spp., indicating that the Chlorophyceae may be polyphyletic.     

The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence

Background

Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga.

Principal Findings

A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support.

Conclusion

All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.     

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.     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.