Acta agriculturae Slovenica, 119/1, 1–7, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2787 Original research article / izvirni znanstveni članek Preliminary assessment of genetic diversity between Glebionis coronaria and G. discolor (Asteraceae) by AFLP markers Alessia GALLUCCI 1, Carmelo Maria MUSARELLA 2, 3, Ana CANO-ORTIZ 4, José Carlos PIÑAR FUENTES 5, Ricardo QUINTO CANAS 6, 7, Clizia VILLANO 1 Received July 25, 2022; accepted February 21, 2023. Delo je prispelo 25. julija 2022, sprejeto 21. februarja 2023 1 Department of Agricultural Sciences, University of Naples Federico II, Naples, Italy 2 Department of Agriculture, Mediterranean University of Reggio Calabria, Reggio Calabria, Italy 3 Corresponding author, e-mail: carmelo.musarella@unirc.it 4 Department of Didactics of Experimental Social Sciences and Mathematics, Section of Didactics of Experimental Sciences, Faculty of Education, Complutense Uni- versity of Madrid, Madrid, Spain 5 Department of Animal and Plant Biology and Ecology, Section of Botany, University of Jaen, Jaén, Spain 6 Faculty of Sciences and Technology, University of Algarve, Faro, Portugal 7 Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal Preliminary assessment of genetic diversity between Glebio- nis coronaria and G. discolor (Asteraceae) by AFLP markers Abstract: Glebionis coronaria is a valuable and medicinal herb native of Mediterranean region. Recently, G. coronaria var. discolor has been elevated to the rank of species as G. discolor (d’Urv.) Cano based on morphological characteristics, distin- guishing it from G. coronaria var. coronaria (=G. coronaria). To investigate the genetic basis of this diversity, AFLP mark- ers were applied to genotypes of G. discolor and G. coronaria sampled in three different Mediterranean regions (Italy, Spain, and Portugal). Our results showed that among 1347 fragments identified with five primer combinations 99.55  % were poly- morphic. The genetic distance and the Shannon Index values suggested that the two species can be genetically distinguished, but further studies are needed to confirm this hypothesis. Key words: biodiversity; Compositae; Chrysanthemum; daisy; garland chrysanthemum; taxonomy Preliminarno ovrednotenje genetske raznolikosti med vrsta- ma Glebionis coronaria and G. discolor (Asteraceae) z AFLP markerji Izvleček: Vrsta Glebionis coronaria je cenjeno zdravilno zelišče, samoniklo na območju Mediterana. V zadjem času je bila različica G. coronaria var. discolor dvignjena na rang vrste kot vrsta G. discolor (d’Urv.) Cano na osnovi morfoloških last- nosti, po katerih se razlikuje od vrste G. coronaria var. coronaria (=G. coronaria). Za preučitev genetske osnove te raznolikosti so bili uporabljeni AFLP markerji za analizo genotipov vrst G. di- scolor in G. coronaria vzorčenih na različnih območjih Medite- rana (Italija, Španija in Portugalska). Rezultati so pokazali, da je bilo med 1347 fragmenti, identificiranih s kombinacijami petih primerjev 99,55 % polimorfnih. Genetska razdalja in vrednosti Shannonovega indeksa nakazujejo, da sta vrsti genetsko ločeni, a so potrebne nadaljne raziskave za potrditev te hipoteze. Ključne besede: biodiversiteta; Compositae; Chrysanthe- mum; ivanjščica; užitna ivanjščica; taksonomija Acta agriculturae Slovenica, 119/1 – 20232 A. GALLUCCI et al. 1 INTRODUCTION Asteraceae is one of the worlds’ richest and the most diverse plant families in biological and ecological terms (Cano et al., 2020). This angiosperm family comprises 1,739 genera including 36,033 species (Hassler, 2021), taxonomically continuously updated. Several of them have various uses around the world, due to their chemical composition for medicinal and food purposes (Mezhoud et al., 2012; Saoud et al., 2019; Pace et al., 2020; Nkuimi Wandjou et al., 2020; Singh et al., 2020; Bhat et al., 2021; Sicari et al., 2021). Among all, Glebionis coronaria (L.) Cass. ex Spach (synonym: Chrysanthemum coronarium L.) is an annual plant, frequent in ruderal vegetation, in field margins, road verges, and urban wastelands, widely distributed in the Mediterranean basin, Western Africa and Asia (da Silva et al., 2005). In the last few years there has been a growing interest in this species due to its bio- logical activities, such as insecticidal, antifungal, cancer prevention, antioxidant and anti-inflammatory (Yildi- rim et al., 2018; Khareba et al., 2021). Historically, based on the different colour and morphology of the flowers, d’Urville (1822) distinguished two varieties of this spe- cies: Chrysanthemum coronarium var. concolor d’Urv. (= G. coronaria var. coronaria) and Chrysanthemum coro- narium var. discolor d’Urv. (= G. coronaria var. discolor (d’Urv.) Turland). Furthermore, a recent study demon- strated that G. coronaria var. discolor can be separated and elevated to the rank of species, based on the disposi- tion of the intercostal glands, the size of the disc cypsela wings and bioclimatological traits, as Glebionis discolor (d’Urv.) Cano, Musarella, Cano-Ortiz, Piñar Fuentes, Spamp. et Pinto Gomes (Cano et al., 2017). These authors documented that G. coronaria has totally yellow ray flo- rets and intercostal glands aligned, while G. discolor has white ray florets on a yellow base and intercostal glands arranged randomly. Another feature highlighted by Cano et al. (2017) concerns the distribution of these two spe- cies in the Mediterranean basin: indeed, G. coronaria is distributed mainly throughout the thermo-Mediterrane- an bioclimatic belt, while G. discolor is spread also in the meso-Mediterranean one. Few other authors phenotypi- cally recognize and/or report G. discolor, such as Cueto et al. (2018), Bartolucci et al. (2018), Portal to the Flora of Italy (2022) and POWO (2022). Among them, the last three sources endorse that G. discolor is a doubtful taxon or is a synonym of G. coronaria, contrary to Cano et al. (2017). In this contest, DNA-based markers (also named molecular markers) represent a powerful tool to fingerprint unequivocally the identity of these species. Indeed, in the last decades, they have been successfully used for investigation of interspecific and intraspecific genetic variability in various plants (Carputo et al., 2013; Villano et al., 2014, 2022, 2023). Among the plethora of molecular markers available, the best choice for Glebionis spp. can be represented by AFLP (Amplified Fragment Length Polymorphism) markers, due to the absence of a reference genome. Their main advantage is the employ- ment of a standard protocol in combination with differ- ent restriction endonucleases to achieve optimal finger- prints without prior knowledge of the organism’s genome sequence. These markers have been pioneered by Vos et al. (1995) and have been used in various species, such as Dioscorea spp. (Rivera-Jiménez et al., 2011) and Gyneri- um sagittatum (Aubl.) P.Beauv. (Rivera-Jiménez et al., 2008). This paper aims to analyse the genetic variability between G. coronaria and G. discolor using combinations of AFLP markers. 2 MATERIAL AND METHODS 2.1 PLANT MATERIAL AND DNA EXTRACTION Three samples of G. coronaria and three of G. dis- color (Figure 1) were collected in Italy, Spain, and Portu- gal in three biological replicates and stored at the herbar- ium of the Mediterranean University of Reggio Calabria (REGGIO) (acronym follows Thiers, 2023), as below de- tailed according to the original labels: Italy: 1) G. coronaria - SIC Fiumara di Melito P.S. (Reg- gio Calabria). 03/05/2018. Collectors: C.M.Musarella & G.Spampinato. 2) G. discolor - Spiaggia di Palizzi Marina, Palizzi (Reggio Calabria). 14/05/2018. Collectors: C.M.Musarel- la & G.Spampinato. Spain: 1) G. coronaria - Near Urbanización Salobreña (Gra- nada). Alt. 13 m asl. 30S0448140/4064980. 05/05/2018. Collectors: E.Cano, A.Cano-Ortiz & J.C.Piñar Fuentes. 2) G. discolor - Near Hotel Soto, Andujar (Jaén). Alt. 220 m asl. 30S0405061/4209759. 06/05/2018. Collectors: E.Cano & A.Cano-Ortiz. Portugal: 1) G. coronaria - Faro. 20/05/2018. Collector: R. Quinto Canas 2) G. discolor - Tavira, Pedras del Rey. 15/06/2018. Collector: R. Quinto Canas. The samples were processed using the DNeasy Plant Mini Kit (Qiagen) previously described by Tengel et al. (2001). The quantity and quality of the isolated gDNA were measured using the NanoDrop ND-1000 spectro- photometer (Thermo Scientific, Wilmington, DE) and Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA). Acta agriculturae Slovenica, 119/1 – 2023 3 Preliminary assessment of genetic diversity between Glebionis coronaria and G. discolor (Asteraceae) by AFLP markers 2.2 AFLP ANALYSIS The analysis was performed using the method de- scribed by Vos et al. (1995) and the commercially avail- able AFLP kit and protocol (Gibco-BRL AFLP analysis System I, Life Technologies, Gaithersburg, MD), which employs EcoRI and MseI as restriction enzymes. For se- lective amplification, five combinations of primers were used (E-AGG + M-CAG; E-AGC + M-CAC; E-AGG + M-CTT; E-AGC + M-CAC; E-ACT + M-CAG) with the E primer labelled with FAM or HEX. Amplicons were separated with the ABI PRISM® 3130 DNA Analyzer sys- tem (Life Technologies, Carlsbad, California, USA). Size calibration was performed with the molecular weight ladder GenScan® 500 ROXTM Size Standard (Life Tech- nologies, Carlsbad, California, USA). AFLP fragments were detected and scored using Peak Scanner® software (Applied Biosystems, Foster) and combined into a binary matrix. Three technical and three biological replicates were considered. 2.3 DATA AND PHYLOGENETIC ANALYSIS The statistical software Genalex 6.5 (Peakall & Smouse, 2006) was used for data analysis. The input file was created considering each band as one diallelic locus (1 means presence of band, 0 means absence of band). Per each species were calculated the observed number of alleles (Na), the number of total bands and number of bands unique, the effective number of alleles (Ne), the Shannon’s information index (I) and the percentage of polymorphic loci (P %). To visualize interspecies and in- dividuals’ relationships, a principal coordinates analysis (PCoA) was performed (Nei & Li, 1979). 3 RESULTS AND DISCUSSION Five selective AFLP primer combinations generated a total of 1347 fragments, distributed between 50 and 500 bp, of which 1341 (99.55  %) were polymorphic (Table 1). The combinations E-AGG/M-CAG and E-AGG/M- CTT were the most informative ones with 248 and 274 polymorphic bands, respectively (Table 1). Two-hundred seventy-two species-specific bands were identified in G. discolor and 224 in G. coronaria (Table 1). The genetic variation was measured within the two species considering the number of effective and differ- ent alleles (named Ne and Na, respectively), the Shan- non’s information index (I), the percentage of polymor- phic loci (P %) and the number of species-specific bands, called private bands (Table 2). Among all, the Shannon’s indexes in G. coronaria and G. discolor species were 0.369 and 0.353, respectively. The Na values were 1.398 in G. coronaria and 1.341 in G. discolor while the Ne values were 1.414 in G. coronaria and 1.398 in G. discolor. The percentage of polymorphic loci showed that the most Figure 1: Capitula of (left) Glebionis coronaria (L.) Cass. ex Spach (Pentidattilo, Reggio Calabria, Italy – April 18, 2022) and (right) G. discolor (d’Urv.) Cano, Musarella, Cano-Ortiz, Piñar Fuentes, Spamp. et Pinto Gomes (Sevilla, Spain – March 28, 2013). Ph. C.M.Musarella Acta agriculturae Slovenica, 119/1 – 20234 A. GALLUCCI et al. polymorphic species was G. coronaria, with 67.12 % of polymorphic loci and 359 private bands. To investigate the genetic distance of the analysed species, the Nei’s value was calculated. Our results showed that the highest variation was found between G. coronaria from Spain and G. discolor from Portugal (0.642), and between G. coronaria from Italy and G. dis- color from Spain (0.642) (Table 3). In order to obtain further information on the grouping of the two species, we carried out PCoA, us- ing AFLP band pattern as raw data. The PCoA (Figure 2) clearly reflected the relationships among and between the genotypes analysed. The first and second component could explain 22.6 % and 20.7 % of the variation, respec- tively. G. discolor from Portugal have been classified apart from G. coronaria genotypes along the two axes. Further- more, the first axis separated two G. coronaria genotypes (Portugal and Italy) from two G. discolor ones (Spain and Italy). The obtained results showed that the G. coronaria and G. discolor can be distinguished using these markers, but further studies with a higher number of molecular markers are needed to confirm it. The genetic difference between these species has been always investigated using phenotypic attributes; only Ata et al. (2017) investigated the relationships of 12 species belonging to Asteraceae, including G. coronaria and G. discolor, using ITS se- quence barcoding. They affirm that the analysed species could be distinctly separated on the genetic basis. This claim is in line with our results. Indeed, the high num- ber of polymorphic bands, the Shannon index and Nei’s gene diversity values suggest that the two groups of gen- otypes are sharing only part of the analysed fragments, and so can be considered as distinct genotypes. The level of polymorphism obtained here in terms of percentage of polymorphic bands with AFLP markers and the ge- netic diversity expressed as Nei’s gene diversity as well as Shannon’s information index values is higher than that AFLP combinations Total bands Polymorphic bands, n° Polymorphic bands, % Specie-specific bands, n° G. coronaria G. discolor E-AGG/M-CAG 249 248 99.60 61 53 E-AGC/M-CAC 310 308 99.35 21 45 E-AGG/M-CTT 275 274 99.64 51 54 E-AGC/M-CAC 327 325 99.39 30 74 E-ACT/M-CAG 186 186 100.00 61 46 Table 1: Results of AFLP analyses used to differentiate Glebionis coronaria (L.) Cass. ex Spach and G. discolor (d’Urv.) Cano, Musarella, Cano-Ortiz, Piñar Fuentes, Spamp. et Pinto Gomes genotypes Species Na Ne I P % No. Private Bands G. coronaria 1.398 1.414 0.369 67.12% 359 G. discolor 1.341 1.398 0.353 63.90% 315 Table 2: Statistical analysis of Glebionis coronaria (L.) Cass. ex Spach and G. discolor (d’Urv.) Cano, Musarella, Cano-Ortiz, Piñar Fuentes, Spamp. et Pinto Gomes. Na = No. of different alleles; Ne = No. of effective alleles; I = Shannon’s information index; P % = percentage of polymorphic loci; No. Private Bands = No. of bands unique to a single species G. discolor P G. discolor I G. coronaria I G. coronaria S G. discolor S G. coronaria P G. discolor P 0 G. discolor I 0.574 0 G. coronaria I 0.580 0.599 0 G. coronaria S 0.642 0.527 0.556 0 G. discolor S 0.595 0.546 0.642 0.583 0 G. coronaria P 0.415 0.514 0.576 0.483 0.522 0 Table 3: Nei’s Original Measures of genetic distance among samples of Glebionis coronaria (L.) Cass. ex Spach and G. discolor (d’Urv.) Cano, Musarella, Cano-Ortiz, Piñar Fuentes, Spamp. et Pinto Gomes from: P = Portugal; S = Spain; I = Italy. The highest values are reported in bold Acta agriculturae Slovenica, 119/1 – 2023 5 Preliminary assessment of genetic diversity between Glebionis coronaria and G. discolor (Asteraceae) by AFLP markers reported for other Asteraceae (Nguyen et al., 2013; Kropf et al., 2017; Wu et al., 2019), confirming the effectiveness of the markers used to distinguish the population stud- ied. Furthermore, the PCoA analysis clearly separated G. coronaria apart from G. discolor along both axes. The dif- ferent distribution of G. coronaria from Portugal and Ita- ly vs genotypes from Spain and of G. discolor from Spain and Italy vs genotypes from Portugal could be related to the area of origin. 4 CONCLUSIONS In the present study, the genetic diversity of two Glebionis species has been investigated through an AFLP analysis. The genotypes considered came from G. coro- naria and G. discolor samples collected in three different countries (Spain, Portugal and Italy). 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