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.
Introduction
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 -70°C 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: tychiang@mail.ncku.edu.tw
