Bot. Bull. Acad. Sin. (1998) 39: 245_250

Chiang et al. Primers for atpB-rbcL spacer

Universal primers for amplification and sequencing a noncoding spacer between the atpB and rbcL genes of chloroplast DNA

Tzen-Yuh Chiang1,4, Barbara A. Schaal2 and Ching-I Peng3

1Department of Biology, National Cheng-Kung University, Tainan 700, Taiwan

2Department of Biology, Washington University, St. Louis, Missouri 63130, USA

3Institute of Botany, Academia Sinica, Nankang, Taipei 115, Taiwan

(Received January 14, 1998; Accepted April 23, 1998)

Abstract. Universal primers for amplifying and sequencing a noncoding spacer between the atpB and rbcL genes of the chloroplast DNA were constructed from the published sequences of Marchantia (a liverwort), tobacco, and rice. Our results indicate an evolutionary trend of increasing spacer size from liverworts, through mosses, to vascular plants. This atpB-rbcL spacer is AT-rich, consistent with other chloroplast noncoding spacers. Due to weak functional constraints, the spacer is evolving rapidly. A sequence identity of 92.2% was observed between spacers of two closely related moss species, Rhytidiadelphus loreus and R. triquestrus. Insertion/deletion events are common in the evolution of this spacer. A 23 bp deletion occurrs in R. loreus. Variation is found between two populations of Amorphophallus henryi (Araceae) and between individuals in a populations of Pasania formosana (Fagaceae). We suggest that this spacer will be useful for molecular systematics at the subspecific, specific, and generic levels and, in some species, for population genetics studies.

Keywords: Amorphophallus henryi; atpB-rbcL chloroplast spacer; PCR; Rhytidiadelphus sp.; Universal primers.


The chloroplast genome evolves slowly in general (Clegg et al., 1991), making it an ideal system to assess phylogenetic relationships among genera (e.g., Onagraceae, Conti et al., 1993; Apiaceae, Downie et al., 1996), families (e.g., Asteridae, Olmstead et al., 1992) or higher levels (e.g., seed plants, Chase et al., 1993; monocots, Davis, 1995; angiosperms, Nickrent and Soltis, 1995). Nevertheless, some recent studies have revealed genetic variation between populations based on RFLP's of the total chloroplast genome (see Schaal and Leverich, 1996) or on specific sequences, such as noncoding spacers between several tRNAs of chloroplast DNA (Demesure et al., 1995, 1996).

Several universal primers for amplifying noncoding spacers of the chloroplast genome have been reported (Taberlet et al., 1991; Demesure et al., 1995; Dumolin-Lapegue et al., 1997). Most of the primers were designed for amplifying spacers between tRNA genes, which have been proved variable among species or populations (Demesure et al., 1996). In this study we describe one pair of universal primers for another noncoding spacer region that lies between the large subunit of ribulose-1,5-bisphosphate-carboxyalse (rbcL) and the beta-subunit of the chloroplast ATP-synthase (atpB) genes. Compared to the primers designed for specific taxa (e.g., Rubiaceae,

Manen et al., 1994), the primers proposed here work for plants of a wider phylogenetic array from bryophytes to flowering plants. The spacer is useful for both phylogenetic assessment (Golenberg et al., 1993; Manen et al., 1994; Natali et al., 1995; Savolainen et al., 1994) and species level or population studies.

Materials and Methods

Material Collection

Species of mosses and vascular plants representing a phylogenetic array were collected for testing the primers designed in this study. Two mosses, Rhytidiadelphus triquetrus and R. loreus (Hylocomiaceae), one fern (Angiopteris), one gymnosperm [Cunninghamia lanceolata (Taxodiaceae)], three monocots [i.e., Imperata cylindrica, Miscanthus transmorrisonensis (Poaceae) and Amorphophallus henryi (Araceae)], and three dicots [i.e., Begonia aptera (Begoniaceae), Glycine soja (Fabaceae), and Pasania formosana (Fagaceae)] were collected in the field in Taiwan, China, Canada or USA (Table 1). Vouchers are deposited at HAST herbarium. Leaf tissue, fast dried in silica gel after collecting, was powdered in liquid nitrogen and kept in a -70C freezer.

DNA Isolation

Genomic DNA was extracted by a CTAB method (Doyle and Doyle, 1987) from frozen tissue. Isolated DNA of mosses was purified on low melting agarose gels to remove secondary compounds and RNA (Chiang, 1994).

4Corresponding author. Tel: +886-6-2757575 ext. 65525; FAX: +886-6-2742583; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 39, 1998

Table 1. Materials collected for amplification of atpB-rbcL spacer of cpDNA with size variation.

Taxon Sources Size (bp)


Rhytidiadelphus loreus (Hylocomiaceae) Canada: British Columbia 524

R. triquestrus (Hylocomiaceae) USA: Smoky Mt. 549


Angiopteris lygodiifolia (Marattiaceae) Taiwan: Yangmingshan 629


Cunninghamia lanceolata (Taxodiaceae) China: Chekiang Prov. 1,000



Miscanthus transmorrisonensis (Poaceae) Taiwan: Hohuanshan 800

Imperata cylindrica (Poaceae) Taiwan: Tainan Co. 800

Amorphophallus henryi (clone 1) (Araceae) Taiwan: Tainan Co. 800

A. henryi (clone 2) (Araceae) Taiwan: Kaohsiung City 800


Glycine soja (Fabaceae) Taiwan: Taipei Hsien 900

Begonia aptera (Begoniaceae) Taiwan: Taipei Hsien 841

Pasania formosana (Fagaceae) Taiwan: Pingtung Hsien 900

Primer Design and PCR

One pair of universal primers were designed by comparing the sequences of Marchantia (a liverwort, Umesono et al., 1988), Angiopteris (a fern, EMBL X58429), tobacco (Shinozawa et al., 1986), and rice (Nishizawa and Hirai, 1987). Primers, atpB-1: 5'-ACATCKARTACKGGACC AATAA-3' and rbcL-1: 5'-AACACCAGCTTTRAATCCAA-3', were used for PCR and sequencing. PCR amplification conditions were 30 cycles of 94C denaturing for 45 s, 49C annealing for 1 min 15 s, and 72C extension for 1 min 15 s, followed by 72C extension for 10 min and 4C for storing. PCR products were electrophoresed in a 1% agarose gel.

Nucleotide Sequencing

PCR products were separated and eluted using agarose gel purification (BM) and ligated to a pT7 Blue T-vector (Novagen). Plasmid DNA was purified using Wizard Plus SV kit (Promega) and quantified for further sequencing. Cycle sequencing based on dideoxy-mediated chain-termination methodology (Sanger et al., 1977) with Taq polymerase was performed using the fmolTM Sequencing System (Promega). Double strands were sequenced from both ends, using primers for PCR amplification and additional primers in between as well, with overlapping of about 100 bp. Clones of mosses and Amorphophallus henryi were sequenced completely. DNA fragments from the other species were sequenced only with one of the primers, atpB-1, to confirm the nature of the amplified segments.

Results and Discussion

PCR products show size variation of the atpB-rbcL spacer among groups. Our results indicate an evolutionary trend of increasing atpB-rbcL spacer size, from liv

erworts (507 bp in Marchantia), through mosses (ca. 550 bp in Rhytidiadelphus), to vascular plants including ferns (629 bp in Angiopteris), conifers (ca. 1,000 bp in Cunninghamia lanceolata), monocots (ca. 800 bp) and dicots (ca. 850_950 bp) (Table 1).

This is also the first report of variation of cpDNA sequences between closely related species of bryophytes. 553 nucleotide bases of the atpB-rbcL noncoding spacer were aligned (Figure 1). Clones of Rhytidiadelphus loreus (EMBL no. L21722) and R. triquestrus (EMBL no. L23410) shared 92.2% identity. Substitutions occured with no bias in transitions (5 times) versus transversions (6 times). Insertion/deletion is a common phenomenon in this spacer. Several length mutations were observed between the two taxa. Rhytidiadelphus loreus lacked a three bases (TAG) and a 13 bases (positions 381_403) sequences in contrast to R. triquestrus. Additionally, R. triquestrus lacked a three bases sequence (TTC) in a different portion of the sequence. The variability of this spacer among species is close to that of the internal transcribed spacer (ITS region) of nuclear ribosomal DNA, in which 87.6_93.7% identity between Raillardella species (family Asteraceae, Baldwin, 1992) has been reported.

Sequences (Figure 2) of the spacer in Amorphophallus henryi showed variation between two populations. In 810 aligned bases 11 sites (1.4%) were variable. Two indels occured at positions 169 and 718. Nine substitutions were observed with more transitions (6 sites) than transversions (3 sites), which is consistent with reports for other genes (cf. Li, 1997). The atpB-rbcL spacer is AT rich. In the sequence of A. henryi, 29.9% of the bases (242 out of 810) were either C or G. Most noncoding spacers and pseudogenes are AT rich due to low functional constraints (Li, 1997). Lack of functional constraints also results in a faster rate of evolution for most spacer regions. Our preliminary data

Chiang et al. Primers for atpB-rbcL spacer

Figure 1. Alignment of sequences of atpB-rbcL spacer of Rhytidiadelphus loreus (R-l) and R. triquestrus (R-t), with identical sequences () and deletions (_).

spacers of nuclear ribosomal DNA in plants: an example from the Compositae. Mol. Phylogen. Evol. 1: 3_16.

Chase, M.W., D.E. Soltis, R.G. Olmstead, D. Morgan, D.H. Les, B.D. Mishler, M.R. Duvall, R.A. Price, H.G. Hills, and Y.L. Qiu. 1993. Phylogenies of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Missouri Bot. Gard. 80: 528_580.

Chiang, T.Y. 1994. Phylogenetics and evolution of the Hylocomiaceae (Mosses, Hypnales) based on sequences of nrDNA ITS and cp DNA atpB-rbcL spacer. Ph.D. Dissertation, Department of Biology, Washington University, St. Louis, USA.

Clegg, M.T., G.H. Learn, and E.M. Golenberg. 1991. Molecular evolution of chloroplast DNA. In R. K. Selander, A. G. Clark and T. S. Whittam (eds.), Evolution at the Molecular Level. Sinauer Publishers, Sunderland, pp. 135_149.

Conti, E., A. Fischbach, and K.J. Sytsma. 1993. Tribal relationships in Onagraceae: implications from rbcL sequence data. Ann. Missouri Bot. Gard. 80: 672_685.

Davis, J.I. 1995. A phylogenetic structure of the monocotyledons,

on Pasania formosana shows much higher diversity (data not shown) in the nucleotide sequence of the atpB-rbcL spacer than has been detected in any other plant groups reported previously, such as the taxa of Rubiacaeae (Manen et al., 1994; Natali et al., 1995). Here we find variation among individuals within populations of Pasania. Therefore, we suggest that this spacer will be useful for molecular systematics at the subspecific, specific, and generic levels and, in some cases, for population genetic studies.

Acknowledgments. We are indebted to T. F. Chiang for lab assistance. This research was granted by National Science Council, ROC to CIP and TYC and by National Science Foundation, USA to BS.

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Botanical Bulletin of Academia Sinica, Vol. 39, 1998

Figure 2. Nucleotide sequences of atpB-rbcL chloroplast DNA spacer of clones (Hen 1 and Hen 2) of Amophorphalus henryi, with identical sequences () and deletions (_).

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