GENETIC DIVERSITY OF CROCUS SATIVUS AND ITS CLOSE RELATIVE SPECIES ANALYZED BY iPBS-RETROTRANSPOSONS

Turk J Field Crops 2017, 22(2), 243-252 DOI: 10.17557/tjfc.357426 GENETIC DIVERSITY OF CROCUS SATIVUS AND ITS CLOSE RELATIVE SPECIES ANALYZED BY iPBS-RETROTRANSPOSONS Aysun GEDIK1*, Duygu ATES2*, Semih ERDOGMUS2, Gonul COMERTPAY3, Muhammed Bahattin TANYOLAC2** Hakan OZKAN1,4** 1 Cukurova University, Department of Biotechnology, Institute of Natural and Applied Sciences, Adana, TURKEY 2 Ege University, Department of Bioengineering, Izmir, TURKEY 3 Eastern Mediterranean Agricultural Research Institute, Adana, TURKEY 4 Cukurova University, Faculty of Agriculture Department of Field Crops, Adana, TURKEY *: These authors contributed equally to this article **Corresponding authors: , Received: 18.10.2017 ABSTRACT Saffron (Crocus L.) is a member of Crocoideae, the biggest of four subfamilies in the Iridaceae family. It has 2n = 3x = 24 chromosomes and is triploid; thus, it is sterile. In previous research, different molecular DNA markers were used but molecular characterization and genetic diversity of this complex genus have not yet been clarified. Therefore, current study aimed to determine the molecular characterization of saffron and its close relative species using inter-primer binding site (iPBS)-retrotransposon markers. Eighty-three iPBSretrotransposon primers were used in 28 C. sativus genotypes and 17 close relative species of saffron to identify their genetic diversity. Sixteen polymorphic iPBS-retrotransposon primers generated a total of 401 polymorphic scorable bands. The mean PIC value, Nei’s genetic diversity and Shannon’s information index (I) were calculated as 0.85, 0.16 and 0.29, respectively. The results of the Unweighted Pair Group Method with Arithmetic mean UPGMA dendrogram and Principal Coordinates Analysis PCoA analysis indicated a spatial representation of the relative genetic distances among 28 saffron samples and the 17 close relative species were categorized under two distinct groups. Saffron genotypes showed very limited genetic variation and according to the iPBS-retrotransposon data, its close relatives were C. cartwrightianus and C. pallasii subsp. pallasii. Keywords: Crocus sativus, genetic diversity, inter-primer binding site retrotransposons, saffron. INTRODUCTION Crocus L. is a member of Crocoideae, the biggest of presently known four subfamilies in the Iridaceae family (Busconi et al., 2015; Goldblatt et al., 2006). While this genus was originally considered to contain approximately 90 small species (Petersen et al., 2008), molecular, morphological and karyological studies undertaken in recent years (Kerndorff et al., 2012; Kerndorff et al., 2013; Harpke et al., 2013; Schneider et al., 2012) have shown that the number of Crocus species is presumably higher than 160 (Harpke et al., 2015) and these species are found in a wide range of habitats, including meadows, scrub and woodland (Rubio-Moraga et al., 2009). The majority of Crocus species and subspecies are naturally grown in the Balkan Peninsula, Greek, and Turkey (Petersen et al., 2008; Kerndorff and Pasche, 2011; Kerndorff et al., 2012; Peruzzi and Carta, 2011). Among all Crocus diversity in the world, Turkey’s flora includes a total of 132 Crocus species (108 endemic) with different flowering times (Mathew, 1982; Ozhatay, 2002; Kerndorff and Pasche, 2004; Arslan et al., 2007; AlaviKia et al., 2008; Yuzbasioglu et al., 2015). Crocus L. is primarily known for the commercially cultivated species C. sativus, generally called “saffron”, which is one of the most consumed spices in the world (Negbi, 2003). Saffron and its efficacious components have many applications in many industries such as textile (Mortazavi et al., 2012), perfume (Mousavi and Bathaie, 2011), food (Babaei et al., 2014) and pharmaceuticals (Hadizadeh et al., 2010). In addition, saffron is used as an enhancer of learning ability (Pitsikas and Sakellaridis, 2006), antidepressant (Basti et al., 2007), anti-cancer agent (Abdullaev, 2002; Fernández, 2006), and antioxidant component (Verma and Bordia, 1998). The retail price of saffron can reach 20.000 Euro/kg; thus making it one of the most expensive spices in the world 243 (Busconi et al., 2015). Due to its great commercial importance, the exploration of the genetic diversity of saffron and relationships between its accessions are significant for germplasm conservation and breeding strategies. Saffron has 2n=3x=24 chromosomes and is triploid; thus, it is sterile (Gribbon et al., 1999). However, the sterility of this species has limited the application of conventional genetic diversity strategies (Busconi et al., 2015). In the last four decades, classical strategies have been complemented with morphological markers to determine the genetic diversity of saffron; however, DNA markers have the potential to provide more reliable results than morphological markers in genetic diversity research since they represent the variation at the DNA level and are not affected by different environmental conditions (Mondini et al., 2009; Weising et al., 2005; Kumar, 1999; Kumar et al., 2009). Despite this potential, in the literature, there are only few articles reporting on the determination and classification of the systematic and genetic diversity of saffron (Frello and Heslop-Harrison, 2000; Nørbæk et al., 2002; Castillo et al., 2005; Frizzi et al., 2007; Petersen et al., 2008; Seberg and Petersen, 2009; Moraga et al., 2010). These studies have employed different molecular DNA markers such as inter simple sequence repeat (ISSR) (Rubio-Moraga et al., 2009), random amplified polymorphic DNA (RAPD) (RubioMoraga et al., 2009), sequence-related amplified polymorphism (SRAP) (Babaei et al., 2014), amplified fragment length polymorphism (AFLP) (Busconi et al., 2015), simple sequence repeats (SSR) (Mir et al., 2015) and inter-retroelement amplified polymorphism (IRAP) (Alsayied et al., 2015; Alavi-Kia et al., 2008) markers but have not fully elucidated the molecular characterization and genetic diversity of this complex genus. Alsayied et al. (2015) reported that according to the IRAP data, C. sativus had minimum genetic variation and its ancestors were C. cartwrightianus and C. pallasii subsp. pallasii. Retrotransposons are mobile genetic elements that generally constitute 50% of plant genome (may reach as high as 90%) and play a significant role in the formation of genetic diversity (SanMiguel et al., 1996; Pearce et al., 1996; Schulman et al., 2012). In addition, retrotransposons are very useful for the development of new markers because of their high prevalence and genome-specific distribution (Schulman et al., 2012). To date, several types of retrotransposons such as retrotransposon-microsatellite and amplified polymorphism (REMAP), IRAP and retrotransposon-based insertion polymorphism (RBIP) have been used for research on plant characterization (Kalendar et al., 2011; Schulman et al., 2012; Alavi-Kia et al., 2008; Alsayied et al., 2015). However, the process of developing retrotran (...truncated)