Abstract

RNA editing sites were systematically examined for the transcripts of 74 known protein-coding genes in the chloroplasts of Phalaenopsis aphrodite . A total of 44 editing sites were identified in 24 transcripts, the highest reported in seed plants. In addition, 21 editing sites are unique to the Phalaenopsis orchid as compared with other seed plants. All editing is C-to-U conversion, and 42 editing sites bring about the changes in amino acids. One of the remaining two editing sites occurs in the transcripts of the ndhB pseudogene, and another in the 5′-untranslated region of psbH transcripts.

RNA editing is one of the post-transcriptional regulation mechanisms of gene expression in the chloroplast of land plants. Plastid RNA editing is generally found as C to U conversion in seed plants and was first documented in maize rpl2 transcripts for the creation of an initiation codon by ACG to AUG conversion, and since then instances of the generation and removal of translational stop codons have also been reported (Bock 2000 , Fiebig et al. 2004 ). However, editing sites usually reside at the internal positions in transcripts and most frequently affect the second codon positions. Sometimes they alter the first or third codon positions (Bock 2000 ). Typically, the codon changes resulting from RNA editing restore the identity of conserved amino acids in plant phylogeny. Although conserved cis -elements surrounding plastid editing sites are scarce, the regions about 20 nucleotides immediately upstream of the editing sites have been mapped by transplastomic and in vitro approaches (Bock 2000 , Hirose and Sugiura 2001 ). The cis -elements are recognized by nuclear-encoded trans -acting factors that are believed either to be site specific or to bind to small clusters of related sites (Hirose and Sugiura 2001 , Chateigner-Boutin and Hanson 2003 , Miyamoto et al. 2004 ). Recently, a pentatricopeptide repeat protein, CRR4, acting as a site-specific recognition factor, which is required for editing at the translational initiation codon in ndhD transcripts, was identified in Arabidopsis (Okuda et al. 2006 ).

To date, RNA editing has been systematically examined for the chloroplast protein-coding transcripts in the following plant species: dicot plants, Nicotiana tobacum (Hirose et al. 1999 , Sasaki et al. 2003 , Sasaki et al. 2006 ), Arabidopsis thaliana (Lutz and Maliga 2001 , Tillich et al. 2005 ), Atropa belladonna (Schmitz-Linneweber et al. 2002 ), Pisum sativum (Inada et al. 2004 ) and Solanum lycopersicum (Kahlau et al. 2006 ); monocot plants, Zea mays (Maier et al. 1995 ), Oryza sativa (Corneille et al. 2000 ) and Saccharum officinarum (Calsa Junior et al. 2004 ); the gymnosperm, Pinus thunbergii (Wakasugi et al. 1996 ); the fern, Adiantum capillus-veneris (Wolf et al. 2004 ); and the hornwort, Anthoceros formosae (Kugita et al. 2003 ). Genome-wide analysis of transcripts in the chloroplasts has revealed that plants have undergone dramatic changes in both the levels and patterns of editing, from hornworts (1.2% conversion of all nucleotides examined) and ferns (0.38%) to seed plants (<0.05%) (Kugita et al. 2003 , Wolf et al. 2004 ). In seed plants, a relatively constant number of editing sites, 21–37, were identified in plastids (Tsudzuki et al. 2001 , Sasaki et al. 2003 , Kahlau et al. 2006 , Sasaki et al. 2006 ). On comparing editing sites among a dicot (tobacco) and monocots (maize and rice), Tsudzuki et al. ( 2001 ) identified 12 common editing sites between tobacco and monocot plants, and 20 common sites between the two monocots (Tsudzuki et al. 2001 ). However, as expected, when looking at more closely related taxa, the number of shared sites increases. For instance, maize, rice and sugarcane in the poaceae family share at least 23 editing sites (Calsa Junior et al. 2004 ), and tobacco, tomato and Atropa in the solanaceae family share 30 (Schmitz-Linneweber et al. 2002 , Kahlau et al. 2006 ). Furthermore, a recent study revealed 31 conserved sites out of a total of 35 editing sites among three species of tobacco, N. tobacum, N. sylvestris and N. tomentosiformis (Sasaki et al. 2003 ). Moreover, at the subspecies level, three different ecotypes of A. thaliana have all 28 editing sites in common though the consequences of RNA editing differ at one position between the ecotypes (Tillich et al. 2005 ). These studies suggest that determining the distribution and pattern of editing sites across taxa and across the entire chloroplast genome is an important step in investigating the evolutionary process of RNA editing in angiosperms. However, these plants still represent a poor sample of major clades in the phylogeny of seed plants.

The Orchidaceae, with approximately 30,000 species, is among the largest families of flowering plants, and Phalaenopsis aphrodite subsp. formosana is the only species in which the chloroplast genome has been completely determined (Chang et al. 2006 ). The chloroplast genome of the Phalaenopsis orchid contains 110 genes including four rRNA, 30 tRNA, 74 known protein-coding genes, and two conserved reading frames of unknown function (Chang et al. 2006 ). The ACG codon rather than an ATG codon at the translation initiation sites was observed in the plastid rpl2 and ndhD genes of P. aphrodite (Chang et al., 2006 ). Previously, we confirmed the presence of an RNA editing system in the chloroplast of P. aphrodite because a C to U conversion was verified in the initiation codon of rpl2 transcripts (Chang et al. 2006 ). Here, we further extend the study of the RNA editing pattern for the chloroplast transcripts of all known protein-coding genes in P. aphrodite and compare them with those of other seed plants.

To study the pattern of RNA editing, we sequenced 60,651 bp of cDNA representing 74 known chloroplast protein-coding transcripts of the Phalaenopsis orchid. A total of 44 editing sites were identified in the 24 transcripts of P. aphrodite chloroplast genes, which represented an average of 0.07% of the nucleotides examined ( Table 1 ). This is the highest number of RNA editing sites reported in seed plants so far. All the RNA editing sites were of the C-to-U conversion type. Of the 42 sites that involved codons, four (9.5%) were in the first position and 38 (90.5%) in the second position, and all resulted in the substitution of one amino acid for another ( Table 1 ; Supplementary Table S1). This result is consistent with previous reports regarding the patterns of genome-wide RNA editing across widely divergent taxa, which show a bias in favor of second codon position edits (Supplementary Table S2; Bock, 2000 ). The consequence of RNA editing in the codon position for the Phalaenopsis orchid is mostly to restore the conservation of amino acids with other seed plants. The most frequently edited codon was serine converted to leucine, followed by serine to phenylalanine, and proline to leucine ( Table 1 ). One of the remaining two editing sites occurred in the transcripts of the ndhB pseudogene, and another in the 5′-untranslated region (UTR) of psbH transcripts. Among 44 sites, seven partially edited sites were detected in the transcripts of atpA (site 2), clpP (site 2), ndhB, psbF, rpoA (site 1 and 3) and rps8 genes in the Phalaenopsis orchid as shown in Table 1 and Supplementary Fig. S1. This is not surprising, since RNA editing efficiency was reported to vary in different organs, developmental stages and environmental conditions (Ruf and Kossel 1997 , Chateigner-Boutin and Hanson 2003 ).

Table 1

RNA editing sites in chloroplast transcripts of Phalaenopsis orchid

Gene Site Nucleotide position Codon position Edited codon Amino acid change 
accD  1 a 1,184 395 uCa S→L 
  2 a 1,412 471 cCa P→L 
  3 a 1,430 477 cCu P→L 
atpA 773 258 uCa S→L 
  2 b 1,148 383 uCa S→L 
atpB  1 a 1,184 395 uCa S→L 
atpF 92 31 cCa P→L 
atpI  1 a 428 143 cCu P→L 
  2 a 629 210 uCa S→L 
clpP  1 a 82 28 Cau H→Y 
  2 b 559 187 Cau H→Y 
matK  1 a 533 178 uCu S→F 
  2 a 718 240 Cau H→Y 
 1,066 356 Cac H→Y 
ndhBc  1 b 1,977  C→U  
petB 611 204 cCa P→L 
petL cCu P→L 
psaI  1 a 80 27 uCu S→F 
psbF  1 b 77 26 uCu S→F 
psbH  1 a –30   C→U   
rpl2 aCg T→M 
rpl23  1 a 71 24 uCu S→F 
rpoA  1 a,b 200 67 uCu S→F 
  2 a 368 123 uCa S→L 
  3 b 830 277 uCa S→L 
rpoB  1 a 92 31 uCc S→F 
 401 134 uCu S→F 
 536 179 uCg S→L 
 614 205 uCa S→L 
 629 210 uCg S→L 
 686 229 cCg P→L 
 2,489 830 uCa S→L 
rpoC1 62 21 uCa S→L 
  2 a 203 68 uCu S→F 
  3 a 509 170 uCa S→L 
  4 a 638 213 uCg S→L 
rpoC2  1 a 2,846 949 uCu S→F 
rps2 134 45 aCa T→I 
rps8  1 b 182 61 uCa S→L 
rps14 149 50 uCa S→L 
rps16  1 a 143 48 uCa S→L 
ycf3 44 15 uCu S→F 
 185 62 aCg T→M 
  3 a 191 64 cCa P→L 
Total 44    42 
Gene Site Nucleotide position Codon position Edited codon Amino acid change 
accD  1 a 1,184 395 uCa S→L 
  2 a 1,412 471 cCa P→L 
  3 a 1,430 477 cCu P→L 
atpA 773 258 uCa S→L 
  2 b 1,148 383 uCa S→L 
atpB  1 a 1,184 395 uCa S→L 
atpF 92 31 cCa P→L 
atpI  1 a 428 143 cCu P→L 
  2 a 629 210 uCa S→L 
clpP  1 a 82 28 Cau H→Y 
  2 b 559 187 Cau H→Y 
matK  1 a 533 178 uCu S→F 
  2 a 718 240 Cau H→Y 
 1,066 356 Cac H→Y 
ndhBc  1 b 1,977  C→U  
petB 611 204 cCa P→L 
petL cCu P→L 
psaI  1 a 80 27 uCu S→F 
psbF  1 b 77 26 uCu S→F 
psbH  1 a –30   C→U   
rpl2 aCg T→M 
rpl23  1 a 71 24 uCu S→F 
rpoA  1 a,b 200 67 uCu S→F 
  2 a 368 123 uCa S→L 
  3 b 830 277 uCa S→L 
rpoB  1 a 92 31 uCc S→F 
 401 134 uCu S→F 
 536 179 uCg S→L 
 614 205 uCa S→L 
 629 210 uCg S→L 
 686 229 cCg P→L 
 2,489 830 uCa S→L 
rpoC1 62 21 uCa S→L 
  2 a 203 68 uCu S→F 
  3 a 509 170 uCa S→L 
  4 a 638 213 uCg S→L 
rpoC2  1 a 2,846 949 uCu S→F 
rps2 134 45 aCa T→I 
rps8  1 b 182 61 uCa S→L 
rps14 149 50 uCa S→L 
rps16  1 a 143 48 uCa S→L 
ycf3 44 15 uCu S→F 
 185 62 aCg T→M 
  3 a 191 64 cCa P→L 
Total 44    42 

a Unique editing sites in the Phalaenopsis orchid.

b Partial editing.

c Pseudogene.

Supplementary Table S1 summarizes the editing sites (total 110 sites) identified so far in the chloroplast transcripts from orchid as well as in six other angiosperms and one gymnosperm. On comparing editing sites among the monocot plants orchid (44 sites), maize (27 sites), rice (26 sites) and sugarcane (24 sites), the dicot plants pea (27 sites), tobacco (38 sites) and Arabidopsis (29 sites), and the gymnosperm black pine (27 sites), 21 sites are unique in orchid while 13 sites overlap between the orchid and any monocots, 15 sites between the orchid and any dicots, 22 sites between orchid and any angiosperm, two sites between orchid and the gymnosperm, and four sites between any angiosperm and the gymnosperm.

Transcripts of the plastid rpoA, rpoB, rpoC1 and rpoC2 genes are the most extensively edited (15 sites) among the functional gene groups in P. aphrodite . Seven editing sites are unique to the Phalaenopsis orchid. Two unique sites at Ser 67 (UCU) and Ser 123 (UCA) were identified in the rpoA transcripts with conversion to Phe 67 (UUU) and Leu 123 (UUA), respectively. Both editing events result in the restoration of codon conservation in plants. There are seven editing sites in rpoB transcripts, the most of any chloroplast transcripts in the Phalaenopsis orchid or in the rpoB transcripts of the other angiosperm chloroplasts studied so far. Interestingly, the first editing site with conversion of Ser 31 (UCC) to Phe 31 (UUC) is the only site unique to the Phalaenopsis orchid and results in a codon diversification from other angiosperms, but it is conserved with gymnosperm black pine (Supplementary Table S1). In the Phalaenopsis orchid, four of the seven editing sites (sites 3–6) in rpoB transcripts are the same as in maize, clustered in the region corresponding to the dispensable domain I of Escherichia coli RNA polymerase β subunit (Corneille et al. 2000 ). Three unique RNA editing sites were identified in the rpoC1 transcripts, and they converted Ser 68 (UCU) to Phe 68 (UUU), Ser 170 (UCA) to Leu 170 (UUA), and Ser 213 (UCG) to Leu 213 (UUG), respectively. One unique RNA editing site was identified in rpoC2 transcripts, and it converted Ser 949 (UCU) to Phe 949 (UUU). Conversion of the above codon position in the rpoC1 and rpoC2 transcripts generally leads to codon conservation among seed plants. The transcripts of six ribosomal protein-coding genes, rpl2, rpl23, rps2, rps8, rps14 and rps16 , are converted by RNA editing, with one codon for each transcript in the Phalaenopsis orchid. However, two editing sites are unique to this orchid. One occurred at the Ser 24 (UCU) codon in rpl23 transcripts, and the other occurred at the Ser 48 (UCA) codon in rps16 transcripts, and they were converted to Phe 24 (UUU) and Leu 48 (UUA), respectively. C to U conversion in those two sites restores codon conservation among seed plants. Interestingly, among the analyzed seed plants, the 103rd codon of rpl20 transcripts is leucine (UUA), either with or without a C-to-U conversion from serine (UCA), except that the corresponding codon of rpl20 transcripts in the Phalaenopsis orchid is serine, and no RNA editing was observed by directly sequencing the RT–PCR products (Supplementary Table S1). This suggests that independent loss of this editing site might have occurred in the Phalaenopsis orchid during the evolutionary process.

In P. aphrodite , for the genes encoding subunits in the complexes (or assembly of the complexes) of the photosynthetic electron transport chain, five of them, psaI, psbF, psbH, petB and petL , have transcripts with one RNA editing site, and ycf3 transcripts have three sites. However, three editing sites were unique to the Phalaenopsis orchid. The first was at the Ser 27 (UCU) codon in psaI transcripts, and the second was at the Pro 64 (CCA) codon in the ycf3 transcripts. These changed to Phe 27 (UUU) and Leu 64 (CUA) in their respective transcripts, leading to amino acid conservation in plants. The third unique editing site resided in the −30 nucleotide (C) position in the 5′UTR of psbH transcripts in the Phalaenopsis orchid, and this nucleotide position is located at the variable region when the 5′UTR nucleotide sequences of psbH transcripts from eight species of seed plants were aligned ( Fig. 1 A). Previously, a C to U conversion identified at the −10 nucleotide position of the ndhG 5′UTR in monocot plants was predicted to modify the RNA secondary (stem/loop) structure (Drescher et al. 2002 ). To check if C to U conversion in the 5′UTR of psbH transcripts in the Phalaenopsis orchid also affected the RNA secondary structure, the sequence extending 32 nucleotides to each side of the editing site (nucleotide −62 to +3) was analyzed in its edited and unedited form with the RNAshapes prediction program (Giegerich et al. 2004 ). Indeed, the edited RNA sequence can form an energetically less stable secondary structure ( Fig. 1 B). Therefore, it is possible that editing in the psbH 5′UTR might influence psbH expression. C to U conversions at the Ser 26 (UCU) to Phe 26 (UUU) and Pro 204 (CCA) to Leu 204 (CUA) codons were identified in the psbF and petB transcripts of the Phalaenopsis orchid, respectively. The functional importance of both corresponding editing events has been demonstrated in transplastomic tobacco and Chlamydomonas reinhardtii , respectively, in which the lack of RNA editing results in a severe mutant phenotype (Bock 2000 ). For the six plastid genes involved in the ATP synthase complex, four of them, atpA, atpB, atpF and atpI , have transcripts that are edited (six sites) in the Phalaenopsis orchid. However, three sites are unique to the orchid. One occurred at the Ser 395 (UCA) codon in atpB transcripts, the other two at the Pro 143 (CCU) and Ser 210 (UCA) codons in atpI transcripts, and they were converted to Leu 395 (UUA), Leu 143 (CUU) and Leu 210 (UUA), respectively. Conversion of C to U in those sites led to the restoration of codon conservation among seed plants.

Fig. 1

RNA editing site of psbH transcripts in the chloroplasts of the Phalaenopsis orchid. (A) The nucleotide sequences in the 5′-untranslated region (UTR) of psbH transcripts from eight species of seed plants were aligned by GeneDoc. The position of the translational start site, A, is indicated as +1. RNA editing at position −30 converts C to U in orchid as indicated by the arrow. Orchid, Phalaenopsis aphrodite ; maize, Zea mays ; rice, Oryza sativa ; sugarcane, Saccharum officinarum ; pea, Pisum sativum ; tobacco, Nicotiana tabacum ; Arabidopsis, Arabidopsis thaliana ; black pine, Pinus thunbergii . (B) Predicted RNA secondary structures formed by the unedited and edited psbH 5′UTR using the RNAshapes algorithm. The edited and unedited nucleotides are indicated by arrows. The psbH translational start codon is boxed.

Fig. 1

RNA editing site of psbH transcripts in the chloroplasts of the Phalaenopsis orchid. (A) The nucleotide sequences in the 5′-untranslated region (UTR) of psbH transcripts from eight species of seed plants were aligned by GeneDoc. The position of the translational start site, A, is indicated as +1. RNA editing at position −30 converts C to U in orchid as indicated by the arrow. Orchid, Phalaenopsis aphrodite ; maize, Zea mays ; rice, Oryza sativa ; sugarcane, Saccharum officinarum ; pea, Pisum sativum ; tobacco, Nicotiana tabacum ; Arabidopsis, Arabidopsis thaliana ; black pine, Pinus thunbergii . (B) Predicted RNA secondary structures formed by the unedited and edited psbH 5′UTR using the RNAshapes algorithm. The edited and unedited nucleotides are indicated by arrows. The psbH translational start codon is boxed.

RNA editing with conversion of Ser 267 to Leu 267 was identified in accD transcripts of pea, soybean, canola and Arabidopsis, and the corresponding codon was converted from proline to leucine in black pine and resulted in amino acid conservation among seed plants (Sasaki et al. 2001 ). The RNA editing at this codon position has been demonstrated to be required for the functional acetyl-CoA carboxylase in vitro in pea chloroplasts (Sasaki et al. 2001 ). However, in Phalaenopsis , the corresponding codon is Phe 266 , and RNA editing is not apparent (Supplementary Table S1). Considering the similarities in the hydrophobic properties of phenylalanine and leucine, the accD gene is probably still functional in Phalaenopsis chloroplasts. However, experimental verification is still needed. On the other hand, three unique editing sites which converted Ser 395 (UCA) to Leu 395 (UUA), Pro 471 (CCA) to Leu 471 (CUA), and Pro 477 (CCU) to Leu 477 (CUU), respectively, were observed in the accD transcripts of Phalaenopsis chloroplasts. Furthermore, three other unique editing sites were present in clpP and matK transcripts in the Phalaenopsis orchid. The former occurred at His 28 (CAU) with conversion to Tyr 28 (UAU), and the latter occurred at Ser 178 (UCU) with conversion to Phe 178 (UUU), and His 240 (CAU) changed to Tyr 240 (UAU). All of the above editing events tended to restore the codon conservation in seed plants.

ndh genes that encode the subunits of the NADH dehydrogenase complex are involved in the cyclic electron flow of PSI and chlororespiration in tobacco, and deletion of ndh genes from the tobacco plastid genome has no significant phenotypic effects under normal growth conditions (Burrows et al. 1998 ). All 11 subunits of ndh genes are present in the chloroplast genomes of photosynthetic vascular plants sequenced so far, with the exception of black pine and Phalaenopsis (Wakasugi et al. 1994 , Chang et al. 2006 ). As shown in Supplementary Table S1, RNA editing occurs frequently in the ndh transcripts, which contain >40% of the editing sites in the chloroplast transcripts of higher plants, except in the Phalaenopsis orchid and black pine. In particular, the ndhB transcripts of barley, tobacco and Arabidopsis differ from the corresponding genomic sequence at nine sites, the highest number of editing events for a single chloroplast mRNA reported so far (Tsudzuki et al. 2001 , Tillich et al. 2005 , Kahlau et al. 2006 ). The relative neutrality hypothesis provides an explanation for why editing sites evolve more readily in those genes (or domains of genes) in which a transitory loss of function can be tolerated (Fiebig et al. 2004 ). However, in Phalaenopsis , the ndhA, ndhF and ndhH genes are completely absent from the chloroplast genome. The ndhB, ndhC and ndhJ genes have small regions of nucleotide insertion/deletion, and the ndhD, ndhE, ndhG, ndhI and ndhK genes are truncated with large deletions, ranging from 26 to 60%, as compared with tobacco, and they are all frameshifted (Chang et al. 2006 ). In addition, the plastid ndhD genes of Phalaenopsis have an ACG codon rather than an ATG codon at their translation initiation sites (Chang et al. 2006 ). C to U conversions at the translation initiation sites of ndhD transcripts were previously reported in dicot plants (Tsudzuki et al. 2001 ). Therefore, we hypothesized that RNA editing may be required to repair internal stop codons and/or initiation sites and thus restore function for at least some ndh genes in the Phalaenopsis orchid. However, in our RT–PCR assays, we did not detect any RNA editing sites for the ndhC, ndhD, ndhE, ndhG, ndhI, ndhJ or ndhK transcripts converting C to U, either in the creation of an initiation codon or in repairing the internal stop codon in their transcripts. Only one partial editing site corresponding to the maize codon 494 was identified in the ndhB transcripts of the Phalaenopsis orchid, but it did not repair the internal stop codon of frameshifted ndhB transcripts. Therefore, we further confirmed that all the ndh genes are pseudogenes in the chloroplast genome of P. aphrodite . The remaining one partial editing site is probably an evolutionary remnant from before complete loss of chloroplast RNA editing sites for non-functional ndh genes in the Phalaenopsis orchid.

Materials and Methods

Leaves of P. aphrodite subsp. formosana were obtained from seedlings at the two-leaf stage of development, from which total cellular RNA was isolated according to the method described by Gehrig et al. ( 2000 ). The RNA samples were then treated with DNase I (Promega, Madison, WI, USA) for 30 min at 37°C to eliminate DNA contamination. To demonstrate the absence of DNA in the RNA preparation after DNase I digestion, the RNA quality was further checked by PCR with at least three pairs of primers from chloroplast genes of the Phalaenopsis orchid. Primers were designed based on 74 known protein-coding genes encoded by the chloroplast genome of P. aphrodite (AY916449) using Vector NTI Suite software (InforMax, Rockville, MD, USA). Reverse transcriptase (Promega) was used to synthesize cDNA from total RNA using a reverse primer for each gene at 42°C for 1 h. We multiplexed primers for up to 11 genes within a single reverse transcription reaction, and at least two independent reverse transcription reactions were performed. The PCR was then applied to amplify cDNA with a primer pair of both forward and reverse primers for each gene separately. Genomic DNA was used as a template in a positive control to ensure the primer pairs were effective for PCR. The PCR contained a final concentration of 200 nM gene-specific primers, 200 nM of each dNTP, 3 U of Taq DNA polymerase and 5 μl of 10× Taq DNA polymerase buffer in a 50 μl reaction mixture. The amplification was started with one 2 min cycle at 94°C, followed by 35–40 cycles of 1.5 min at 94°C, 2 min at 60°C and 3 min at 72°C, and this was followed by one 5 min cycle at 72°C. Each PCR sample was electrophoresed on a 0.8% agarose gel and visualized by staining with ethidium bromide. PCR products were sliced from the gel and purified with a gel extraction kit (Viogene, Taipei, Taiwan). The cDNA fragments were either directly sequenced or first cloned into pGEM-T vector (Promega), and then the propagated plasmid DNA was sequenced. The sequencing reaction was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the protocol recommended by the manufacturer. The DNA sequencer was an Applied Biosystems ABI 3700. To determine editing sites, the cDNA sequences were then aligned with that of genomic DNA.

Supplementary material

Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org .

Acknowledgments

We thank Dr. John Gray for providing the complete, unpublished chloroplast DNA sequence of pea. This work was financially supported in part by grants from the National Science Council of Taiwan.

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Abbreviations:

    Abbreviations:
  • UTR

    untranslated region