Turkish Journal of Agriculture and Forestry Turk J Agric For (2017) 41: 142-153 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1606-124 Evaluation of the genetic diversity of pomegranate accessions from Turkey using new microsatellite markers 1, 1 2 3 Oğuzhan ÇALIŞKAN *, Safder BAYAZIT , Mert ÖKTEM , Ali ERGÜL 1 Department of Horticulture, Faculty of Agriculture, Mustafa Kemal University, Antakya, Hatay, Turkey 2 Department of Biotechnology, Institute of Science, Mersin University, Mersin, Turkey 3 Biotechnology Institute, Ankara University, Ankara, Turkey Received: 27.06.2016 Accepted/Published Online: 08.04.2017 Final Version: 03.05.2017 Abstract: Turkey has many valuable genetic resources for pomegranate; however, there have not been many studies on the identification and characterization of these important genetic resources. New microsatellite markers were used to characterize a set of 78 pomegranate accessions from Turkey. Using six SSR primers, a total of 41 alleles were characterized with an average of 4.6 alleles per locus and mean probability of identity (PI) value of 0.366. These data indicated a high level of polymorphism in pomegranate germplasm. Five synonymous groups could be detected among 30 accessions. This microsatellite-based key is a first step towards a database for marker- assisted identification of pomegranate accessions in Turkey. The present study provides essential information to devise a pomegranate core germplasm collection without duplication of plant material, to sustainably manage pomegranate breeding programs, and to establish conservation strategies for preserving local pomegranate genetic resources. Key words: Genetic diversity, new microsatellites, pomegranate, synonymous group 1. Introduction al., 2006) and the phytochemical and medicinal properties Horticultural plants have been an indispensable part of its juice (Mena et al., 2011; Caliskan and Bayazit, 2012). of human life for ages. Since ancient times, their fruits, Precise identification of genotypes and determination of seeds, and even roots and branches have been used to the genetic relationships among them will be necessary meet personal and social needs such as food, medicine, for conserving its genetic diversity. Such data will also and decoration (Ercisli, 2009; Erturk et al., 2010; Hricova facilitate improved selection of genotypes with traits et al., 2016; Sarıdaş et al., 2016; Yazıcı and Şahin, 2016). preferred by consumers. Pomegranate (Punica granatum L.; 2n = 2x = 18) is a diploid, Turkey is one of the main pomegranate producers perennial, woody plant and belongs to the monogeneric among the Mediterranean countries. The majority of its family Lythraceae, which possesses two species, Punica pomegranate orchards are located in the Mediterranean, Aegean, and southeastern regions of the country. Annual granatum L. and Punica protopunica Balf. The genome size pomegranate production in Turkey was about 445.75 t in of pomegranate has been ascertained to be 704 Mbp, about 2015 and production has rapidly increased from year to six times the size of Arabidopsis thaliana (http://data.kew. year. Fresh pomegranate exports from Turkey were 3.591 t org/cvalues/). in 2000 and 151.174 t in 2015 (http://www.ffv.org.tr/home. Pomegranate (Punica granatum L.) is one of the oldest aspx). The Mediterranean region of Turkey has the most edible fruits and has been used in the folk medicine of suitable ecological conditions for pomegranate cultivation various countries (Holland et al., 2009). The center of and includes 60% of the country’s total pomegranate origin of pomegranate is considered to be Central Asia, production. This region also has rich pomegranate genetic especially parts of modern-day Iran, Turkey, Azerbaijan, diversity useful for breeding programs. In recent years, and Georgia in the Transcaucasia-Caspian region, from pomegranate resources have suffered genetic erosion due where it has spread to the rest of the world (Levin, 2006). to vulnerability to biotic and abiotic stresses and loss of Recently, pomegranate has garnered increasing interest, agricultural land to intensive urbanization in the country especially because of its high nutritional value (Seeram et (Caliskan and Bayazit, 2013). * Correspondence: [email protected] 142 ÇALIŞKAN et al. / Turk J Agric For Local pomegranate accessions are numerous and many fruit crop species (Ergül et al., 2002; Sánchez-Pérez well adapted to different ecological regions of Turkey. et al., 2005; Boz et al., 2011; Caliskan et al., 2012). Diverse These accessions have been cultivated using traditional SSR primer pairs have been published for pomegranate methods since ancient times and can be useful for their (Curró et al., 2010; Hasnaoui et al., 2010; Soriano et al., adapted characteristics such as resistance to diseases, 2011). However, little is currently known about the level pests, cold, drought, and other biotic and abiotic stresses. of molecular genetic diversity available in pomegranate Some morphopomological studies have been performed germplasm in Turkey. (Drogoudi et al., 2005; Ferrara et al., 2011; Zaouay In the present study, a number of novel SSR markers and Mars, 2011; Caliskan and Bayazit, 2013), as have have been developed to investigate the potential of such studies of phytochemical characteristics (Tehranifar et markers for assessing the genetic diversity of pomegranate al., 2010; Mena et al., 2011; Caliskan and Bayazit, 2012) accessions from Turkey. This is also the first study using in pomegranate germplasm. Characterization based on microsatellite markers to evaluate the genetic diversity of morphological traits has commonly been used to resolve pomegranate accessions from the eastern Mediterranean duplication problems within pomegranate germplasm region of Turkey. These results will improve our (Zaouay and Mars, 2011). Although morphological understanding of the level of diversity of pomegranate parameters are generally significantly influenced by germplasm in Turkey and will be helpful for devising an environmental conditions and agronomic practices, effective strategy for the conservation, management, and their characterization is a highly recommended first use of these genetic resources in pomegranate breeding step for germplasm evaluation (Berinyuy et al., 2002). programs. Nevertheless, morphological differences are often too limited to allow closely related accessions to be 2. Materials and methods distinguished. Furthermore, the expression of these 2.1. Plant material and DNA extraction characteristics is not always consistent, so morphological The present study included 78 Turkish pomegranate information alone is inadequate for evaluating the genetic accessions and control cultivars listed in Table 1. Young diversity of collections. leaves were collected from each accession in Hatay Due to the limitations of morphological markers, Province, which is located in the eastern Mediterranean DNA markers are increasingly being used to evaluate region of Turkey. The standard cultivars Hicaznar and germplasm diversity (Soriano et al., 2011). DNA markers Katırbaşı were used as the control group. A pomegranate are independent of environmental conditions, are descriptor was used for evaluation of some fruit quality potentially unlimited in number, and can show a high level characteristics (Bellini and Giordani, 1998). Fruit size was of polymorphism. Therefore, they are invaluable tools for evaluated using a scale ranging from very small (<100 g) to determining genetic relationships, evaluating diversity, very large (>375 g). A color scale ranging from greenish- performing selection during plant breeding, and genome yellow to dark purplish-red was used to evaluate peel mapping (Currὸ et al., 2010; Zaouay and Mars, 2011). In color. In addition, fruit taste was investigated as sweet, recent years, RAPD (Sarkhosh et al., 2006; Zamani et al., sweet-sour, or sour. 2013), SRAP (Ranade et al., 2009; Soleimani et al., 2012), Genomic DNA was extracted from leaves using the ISSR (Zhao, 2011), AFLP (Moslemi et al., 2010; Ercisli procedure described by Lefort et al. (1998). DNA quality et al., 2011b; Nemati et al., 2012), and SNP (Ophir et al., and quantity were assessed on a 1% (w/v) agarose gel 2014) markers have been used to evaluate the diversity stained with ethidium bromide (Caliskan et al., 2012). of pomegranate genetic resources. However, despite The concentration and purity of the extracted DNA were the popularity of these markers, they can suffer from analyzed using a NanoDrop ND-1000 spectrophotometer poor reproducibility and can be insufficiently accurate (NanoDrop Products, Wilmington, DE, USA). for genotype identification. To overcome the potential 2.2. Primers and SSR analysis drawbacks of certain molecular markers, other markers Detection of microsatellite polymorphisms was performed based on nonspecific primers, microsatellites, or simple using six SSR markers, among which the PgAERB3 locus sequence repeats (SSR) including specific primers, which was described by Ergül and Bakır (2013). Five of them can anneal the flanking region of SSR repeat units, can be were novel SSR markers named PgAER121, PgAER138, advantageous due to their abundance, high polymorphism, PgAER154, PgAER194, and PgAERB7 loci (A Ergül, reproducibility, codominant pattern of inheritance, personal communication), and three SSR markers were and extensive genome coverage (Varshney et al., 2005; previously characterized by Pirseyedi et al. (2010). Six SSR Hasnaoui