Design and evaluation of PCR primers for amplification of four chloroplast DNA regions in plants
The high genetic diversity of plants can be a problem when developing molecular methods that require conserved DNA sequences among species. Several chloroplast DNA (cpDNA) regions have been used for the identification of plant DNA from broad taxonomic groups, but many species fail to amplify due to genetic variation at primer-binding sites. Here, we evaluated the conservation degree of four chloroplast DNA (cpDNA) regions commonly used in plant investigations (atpF-atpH, psbA-trnH, trnL CD and trnL GH). We propose new conserved PCR primers for the study of the most common plant families, designed using consensus sequences obtained from 28 multiple sequences alignments with over 11,000 reference sequences. The new primers were able to amplify all target regions in representative samples from the seven families. The conserved genomic regions and PCR primers can be used in diverse areas of plant research, including DNA barcoding, molecular ecology, metagenomics or phylogeny.
KeywordsPlants Conserved genomic regions cpDNA PCR primers
The authors would like to thank Elisabete Couto and Luís Alves from ‘Cantinho das Aromáticas’ (http://www.cantinhodasaromaticas.pt), Paulo Almeida from Jardim Botânico da Universidade de Trás-os-Montes e Alto Douro (http://jb.utad.pt/) and Liliana Ramuge for providing samples. CS is supported by the “Programa Ciências Sem Fronteiras” from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil. FP is supported by the Portuguese Foundation for Science and Technology (FCT), ERDF and Programa Operacional Potencial Humano (IF/01356/2012). CIIMAR was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT and ERDF in the framework of the programme PT2020.
- Doyle JJ (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem bull 19:11–15Google Scholar