A PROOF OF CONCEPT FOR THE GENETIC IDENTIFICATION OF PLANT SPECIES: Erythroxylum spp., Cymbopogon spp., and Cannabis sativa L. AS STUDY MODELS
plants; identification; genetics; Erythroxylum
The immense botanical variety combined with the difficulties in accurately identifying plant species made Plant DNA barcoding a topic of singular importance. Such aspects are even more evident in the field of forensic sciences. Plants that are fragmented, pulverized, or present in such a way that there are difficulties for unambiguous morphological identification are everyday situations in criminalistics. In the present work, we mainly analyzed wood and plant samples of the genera Erythroxylum spp., Cymbopogon spp., and Cannabis sativa L. Such approaches bear particularities of notorious practical interest in the forensic field, in addition to a profound relevance also in the academic field: the first approach (wood samples) due to the natural challenges for extracting DNA from tissue forests and the serious issue of illegal deforestation. The second approach (genus Erythroxylum spp. and species Cannabis sativa L.) is due to the problem concerning coca-producing plants and the forensic interest in this last species. Cymbopogon spp., in addition to academic interest, also has implications for industrial quality control. Plant DNA barcoding uses specific genomic regions as a differential diagnosis tool between different species. For that, we used the sequencing of rbcL, a plant-exclusive gene located in the genome of chloroplasts (plastids) and responsible for producing ribulose-1,5- bisphosphate, or RuBisCO. It is a genomic fragment already widely standardized for this purpose. Subsequently, we sequenced a stretch of ITS2 sequences, part of the ITS (internal transcribed spacer) regions of ribosomal DNA (rDNA), which consist of genomic regions where the genes that encode ribosomal RNA (rRNA) are found. We alternatively employ an alternative approach. We explored genetic diversity through insertions and deletions that occur in intergenic regions by analyzing fragments amplified by PCR. Thus, as proof of concept of the SPInDel approach (Species Identification by Insertions / Deletions), we analyzed two genomic regions still unexplored through this conception: the ITS2 region of the nuclear genome and the trnL-trnF region of the plastidial genome. There were, therefore, two fundamental contributions introduced by the present work, mainly for the field of forensic sciences: the first consists of making available a practical, effective, and currently accessible method to forensic laboratories for the extraction of DNA from plant material, including wood; the second consisted of creating, to the limit of our knowledge, the first duplex kit for amplifying plant DNA employing PCR to the identification of botanical species using fragment analysis based on capillary electrophoresis technology: “a fast DNA barcode for land plants.