Identification of Species in the Aromatic Spice Family Apiaceae Using DNA Mini-barcodes

Abstract

The species of the aromatic plant family Apiaceae are mainly used as spices and foods, but the family also includes medicinal and some poisonous plant species. Due to the similar chemical compounds or aroma and morphology, the poisonous species are often mistaken for the edible aromatic species. It is therefore imperative to correctly identify the species present at the initial raw stage samples to ensure product safety and efficacy. At the molecular level, plant species can be identified using DNA loci either from nuclear or plastid genome with easily available universal oligonucleotides, a technique called DNA barcoding. However, this is possible when single-species plant material is present but may not work on a mixture of plants species. Another disadvantage is that using universal oligonucleotides is of limited help, especially if the adulterating material is present in low quantities. On the other hand, if using the species-specific oligonucleotides, only single specific adulterating plant material could be detected and, consequently, the unexpected adulterants may go undetected. Therefore, in the current work, four degenerated oligonucleotides from ITS1 and ITS2 regions of the nuclear genome were designed that can bind to a variety of Apiaceae genera only and not to other genera belonging to different plant families. These family-specific oligonucleotides were able to amplify a diagnostic PCR product from 16 Apiaceae species that, upon sequencing, revealed the identity of the plant it was derived from. The size of these products is around 140 bp for ITS1 and approximately 80 bp for the ITS2 region. The size range of the amplified products falls in the category of a desired mini-barcode size to be used for damaged/fragmented DNA and next generation sequencing.

Key words

Adulteration - Apiaceae - aromatic species - DNA mini-barcodes - family-specific oligonucleotides - universal oligonucleotides

• References

• 1 Pimenov MG, Leonov M. The Genera of the Umbelliferae: a Nomenclator. London: Royal Botanic Gardens, Kew and Botanical Garden of Moscow University, Russia; 1993
• 2 Downie SR, Spalik K, Katz-Downie DS, Reduron JP. Major clades within Apiaceae subfamily Apioideae as inferred by phylogenetic analysis of nrDNA ITS sequences. Plant Div Evol 2010; 128: 111-136
• 3 Liu J, Shi L, Han J, Li G, Lu H, Hou J, Zhou X, Meng F, Downies SR. Identification of species in the angiosperm family Apiaceae using DNA barcodes. Mol Ecol Res 2014; 14: 1231-1238
• 4 China Plant BOL Group. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci U S A 2011; 108: 19641-19646
• 5 White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct Sequencing of fungal ribosomal RNA Genes for Phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. eds. PCR Protocols: a Guide to Methods and Applications. London: Academic Press Inc.; 1990: 315-322
• 6 He Y, Hou P, Fan G, Song Z, Arain S, Shu H, Tang C, Yue Q, Zhang Y. Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes. Mitochondrial DNA 2012; 23: 100-105
• 7 Bell CE. Poisonous plants in the Apiaceae. Proc Calif Weed Sci Soc 2004; 56: 111-113
• 8 Altschul SF, Gish W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403-410
• 9 Little DP. Authentication of Ginkgo biloba herbal dietary supplements using DNA barcoding. Genome 2014; 57: 513-516
• 10 Parveen I, Gafner S, Techen N, Murch SJ, Khan IA. DNA barcoding for the identification of botanicals in herbal medicine and dietary supplements: strengths and limitations. Planta Med 2016; 82: 1225-1235
• 11 McCormack JE, Hird SM, Zellmer AJ, Carstens BC, Burnfield RT. Applications of next-generation sequencing to phylogeography and phylogenetics. Mol Phylogene Evol 2013; 66: 526-538
• 12 Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins D. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011; 11: 539
• 13 Nicholas KB, Nicholas HBJ. GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author 1997. Available at: http://www.psc.edu/biomed/genedoc Accessed February 21, 2018
• 14 Yoon H, Leitner T. PrimerDesign-M: a multiple-alignment based multiple-primer design tool for walking across variable genomes. Bioinformatics 2014; 17: 1472-1474
• 15 Brodin J, Krishnamoorthy M, Athreya G, Fischer W, Hraber P, Gleasner C, Green L, Korber B, Leitner T. A multiple-alignment based primer design algorithm for genetically variable DNA targets. BMC Bioinformatics 2013; 14: 255