Length polymorphism scanning is an efficient approach for revealing chloroplast DNA variation.
Phylogeographic and population genetic screens of chloroplast DNA (cpDNA) provide insights into seed-based gene flow in angiosperms, yet studies are frequently hampered by the low mutation rate of this genome. Detection methods for intraspecific variation can be either direct (DNA sequencing) or indirect (PCR-RFLP), although no single method incorporates the best features of both approaches. We show that screening universal chloroplast amp li cons for length polymorphism provides an accurate and efficient method for identifying cpDNA variation. By sequencing 4500 bp of cpDNA from 17 accessions of Purshia tridentata (bitterbrush), we detected 9 haplotypes, 8 of which were identifiable by unique multilocus length combinations resolvable by automated fragment analysis. In silico estimates of PCR-RFLP for these loci show that 5 haplotypes would be resolved by agarose electrophoresis. A survey of 4 intraspecific data sets from diverse angiosperms revealed that length variation in cpDNA amplicons is nearly ubiquitous, and 61 of 67 haplotypes identified by direct sequencing could be identified by screening length variation. Combined with automated fluorescent detection, length polymorphism screening of universal cpDNA regions offers a simple screen for intraspecific variation that can be used across angiosperms with minimal optimization, providing detection limits that rival direct sequencing at a fraction of the cost.
Horning
Matthew E
ME
USDA Forest Service, Pacific Northwest Research Station, Corvallis, OR 97331, USA.
Cronn
Richard C
RC
D002736:Chloroplasts / Q000378:metabolism; D018742:DNA, Chloroplast / Q000235:genetics*; D018744:DNA, Plant; D017343:Genes, Plant; D014644:Genetic Variation; D005828:Genetics, Population; D006239:Haplotypes; D008957:Models, Genetic; D010802:Phylogeny; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D027824:Rosaceae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D018742:DNA, Chloroplast; D018744:DNA, Plant