Firenze University Press Caryologia www.fupress.com/caryologia International Journal of Cytology, Cytosystematics and Cytogenetics The technique of Plant DNA Barcoding: potential application in floriculture Citation: A. Giovino, F. Martinelli, A. Perrone (2020) The technique of Plant DNA Barcoding: potential application in floriculture. Caryologia 73(2): 27-37. Antonio Giovino1,*, Federico Martinelli2,*, Anna Perrone3 doi: 10.13128/caryologia-730 1 Council for Agricultural Research and Economics (CREA), Research Centre for Plant Received: December 12, 2019 Protection and Certification (CREA-DC), Bagheria, Italy 2 Department of Biology, University of Florence, Sesto Fiorentino, Florence, 50019, Italy Accepted: April 1, 2020 3 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies Uni- Published: July 31, 2020 versity of Palermo, Viale delle Scienze, Palermo, 90128, Italy *Correspondonding authors. E-mail: [email protected]; federico.martinelli@ Copyright: © 2020 A. Giovino, F. unifi.it Martinelli, A. Perrone. This is an open access, peer-reviewed article pub- lished by Firenze University Press Abstract. The objective of this work was to assess the ability of the DNA barcoding (http://www.fupress.com/caryologia) approach to identify different taxonomic groups from two flowering plant collec- and distributed under the terms of the tions: 1) the most relevant commercial taxa (nursery production) and 2) Mediterra- Creative Commons Attribution License, nean plants with ornamental attitude (new emerging species). “Core markers”, rbcL which permits unrestricted use, distri- and matK, were adoptedthe identification step of 100 taxa belonging to 20 families. A bution, and reproduction in any medi- third marker, the intergenic spacer trnH-psbA, was also tested, on 74 taxa, when the um, provided the original author and source are credited. core markers were not able to discriminate well the analysed germplasm.DNA barcode fragments were recovered for all the total taxa investigated (100%). The rbcL showed Data Availability Statement: All rel- the best performances: the greatest amplification success, the best sequencing perfor- evant data are within the paper and its mance both in terms of the number of sequences obtained and in terms of quality Supporting Information files. of the sequences obtained. Despite having recorded greater amplification difficulties, according to numerous other studies, matK has shown a good success in sequencing Competing Interests: The Author(s) declare(s) no conflict of interest. and quality of the obtained sequences (de Vere et al. 2012), unlike what is indicated in some protocols that suggests for this region the need for further primers to be adopted for the sequencing phase (Hollingsworth et. al 2011). Results showed that sixty-one taxa overall (61%) were totally resolved at specific or subspecific level, by at least one of the three markers. The matK and rbcL locus respectively resolved 44% and 35% of the taxa. The core markers inmultilocus approach led to the discrimination of a total of 49% taxa. The trnH-psbA was able to discriminate 52% of taxa analysed and resulting determinant in the discrimination of 14 taxa. Four families, including the major num- ber of taxa (Arecaeae, Fabaceae, Euphorbiaceae, Asteraceae), were evaluated in terms of genetic distance (K2P% value). This work highlighted the potential of the barcoding approach for a rapid identification of plant species in order to solve taxonomic disputes and support commercial traceability of floreal products. Keywords: DNA barcoding, DNA fingerprinting, floriculture,genetic identification. 1 INTRODUCTION Genetic certification of plant material is, today more than ever, a funda- mental requirement to increase the competitiveness of plant nurseries, even Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 27-37, 2020 ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-730 28 Antonio Giovino, Federico Martinelli, Anna Perrone in the ornamental sector. This represents a unique and Hebert of the University of Guelph (Canada) (Hebert effective tool for unambiguous determination of nature et al 2003). In this work it was used for the identifica- of plant species. These improvements will enhancethe tion of species, a gene sequence located in the region floriculture sector through the successful obtainment of the mitochondrial gene COI, coding for the subu- of the following to objectives: 1) genetic identification nit I of the cytochrome-c oxidase (also known as War- (especially for native plants) and particularly to iden- burg’s respiratory fragment), therefore the variability of tify the link between genetic resources of ornamental a molecular marker for the identification of biological interest and the relative territory of origin, thus pro- identities is exploited.Over the years, COI has been suc- moting the products in harmony with the territory and cessfully used in various animal taxa, including birds with sustainability criteria, 2) traceability (native plants (Hebert et al., 2004b), arthropods (Barrett and Hebert, and imported plants) through the characterization of 2005), fish (Wardet al., 2005) and Lepidoptera (Hebert autochthonous products, plant material of supply chain et al., 2004a). In vegetables, COI has not proved to be and non-native incoming material. Therefore, the genet- an excellent marker for phylogenetic studies, due to the ic certification of plant material is an extremely impor- low evolutionary rate of the mitochondrial genome. In tant aspect for the resolution of related problems:1) order to overcome this problem, other markers for DNA taxonomic controversies (synonymy/homonymy) and barcoding of plants have been identified in recent years. species of difficult identification, 2) newly introduced These are DNA sequences present in some sections of programs, genetic improvement, 3) early identification the chloroplast genome, such as the trnH-psbA inter- of species with very long phenological cycles, 4)corre- genic region, the matK gene or the rbcL gene, which spondence checks of vegetable species entering the mar- have characteristics similar tocoxI useful for species kets, 5) protection of biodiversity, of native or endan- identification. There are several requirements for a gered species. Recently, DNA barcoding has emergedas marker to be considered appropriate for DNA barcod- a new molecular tool for taxonomists (Hebert, Ratnas- ing. First of all it is advisable that the marker has a wide ingham & deWaard, 2003). A DNA barcodeis a univer- taxonomic coverage (also called universality), which sally accepted short DNA sequence normally employed would allow the applicability of the gene chosen as bar- for the identification of species (Savolainen et al., 2005), code markern to the largest possible number of taxa promoted for a variety of biological applications (Holl- and have a high success rate of PCR and sequencing. A ingsworth, Graham & Little, 2011), including the identi- high resolution capacity of the gene is also important, fication of cryptic species, species discovery (Bickford et i.e the ability of a given barcode to differentiate species. al., 2007) and taxonomic revisions (Simeone et al 2013). This is typically based on the amount of interspecific The genotype is nothing but the set of all the genes differences between DNA sequences (Polymorphism). that make up the DNA of an organism. Thus DNA- Another fundamental assumption is that the molecular based taxonomy has proved to be a valuable support marker chosen as a barcode should show a higher inter- to the classical taxonomy allowing to face the growing specific variability than intraspecific variability. Inter- need for accurate and accessible taxonomic information and intra-specific variability are separated by a certain (Tautz et al., 2003). In particular, the advent of molecu- distance (discontinuity between intra and interspecific lar markers has marked a remarkable turning point in variability) called “barcoding gap” (Meyer and Paulay, the world of plant genetics allowing the construction 2005).The ideal marker therefore consists of a highly of association genetic maps and the identification of variable region, which provides for species discrimina- genes responsible for agronomic characters (Giovino et tion, flanked by highly conserved regions for which ade- al. 2015a). In taxonomic studies, markers are important quate primers can be designed (Saunders and Kucera, for botanical classifications and the analysis of phyloge- 2010). Therefore, for the plants, the Barcoding proto- netic relationships (Varshney et al., 2005). Among the cols refer to the indications of the Plant working Group, molecular techniques, a new approach to the study of which suggests the use of a multi-locus approach (Hol- biodiversity has become widespread, with all the prob- lingsworth et al., 2011; Domina et al. 2017). The gen- lems related to this study: the DNA barcoding, literally eral objective of the research was to use the technique “DNA barcode”. The name of this approach refers to the of DNA barcoding to help nursery production thanks identification method by which a scanner distinguishes to the easy identification of new products, ornamental various commercial products using linear bar codes or plants and ornamental-food valueto respond to new and “UPC” (Universal Product Code).This molecular inves- growing market needs. tigation approach was first proposed to the scientific