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Journal of Food, Agriculture & Environment Vol.11 (1): 516-521. 2013

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Molecular characterization and genetic diversity analysis of mandarin genotypes by SSR and SRAP markers Yildiz Kacar 1*, Aydin Uzun 2, Ilknur Polat 3, Turgut Yesiloglu 1, Bilge Yilmaz 1, Osman Gulsen 2, Onder Tuzcu 1, Müge Kamiloglu 4, Senay Kurt 3 and Ubeyit Seday 5 Department of Horticulture, Faculty of Agriculture, Cukurova University, 01330 Balcali, Adana, Turkey. 2 Department of Horticulture, Faculty of Agriculture, Erciyes University, 38039 Melikgazi, Kayseri, Turkey. 3 Bati Akdeniz Agricultural Research Institute, 07100 Antalya, Turkey. 4 Department of Horticulture, Faculty of Agriculture, Mustafa Kemal University, 31034 Antakya, Hatay, Turkey. 5Alata Horticultural Research Institute, 33740 Erdemli, Mersin, Turkey. *e-mail: [email protected] 1

Received 20 November 2012, accepted 30 January 2013.

Abstract The aim of the present study was to characterize the mandarin germplasm conserved at the Tuzcu Citrus Variety Collection at Cukurova University in Turkey using simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) based molecular approaches. Genetic characterization of 65 mandarins was performed using 14 SSR markers and 21 SRAP markers. We originally tested 26 SSR markers, and 14 of these markers were selected due to their high polymorphism information content in the molecular assays. Thirty-eight alleles were detected at 14 loci. The number of alleles per SSR locus ranged from 2 to 4, with a mean of 2.7 alleles. The most informative loci were CAGG 9, CAT 01 and TAA 52 (4 alleles/primer). From the SRAP analysis, a total of 187 alleles were generated, and the polymorphism rate was 77%. The number of alleles detected by a single primer set ranged from 4 to 15, with an average of 8.90. The UPGMA dendrogram, defined by SSR and SRAP markers, revealed the genetic relatedness of the mandarin genotypes. These findings can be used to guide future breeding studies and germplasm management of these mandarin genotypes. Key words: Genetic diversity, mandarin, markers, SSR, SRAP.

Introduction Citrus is the most important fruit crop in the world, with a production of more than 120 million tons. Turkey is one of the important citrus producing countries in the Mediterranean, as well as in the world, and produced more than 3.5 million tons 1. Mandarins are the second most important group of citrus plants in the world, with the highest climatic adaptation among cultivated citrus 2.Turkey is considered the 4th largest mandarin producer, with an estimated annual production of 858,699 tons 1. Research on germplasm characterization, genetic variation and breeding in mandarin and other Citrus species has been hampered by characteristics related to the reproductive biology of these species, i.e., high interspecific fertility, apomictic reproduction, polyembryony, a long juvenile phase and a paucity of polymorphic DNA markers 3. There are many molecular marker systems available for plant scientists to characterize genetic resources and cultivars. These systems have advantages and disadvantages for each study that depend on several factors, such as the study’s objectives and the investigated crop 4. Genetic diversity and phylogenic studies have been performed in Citrus using various marker systems, including RFLP (Restriction Fragment Length Polymorphism), RAPD (Randomly Amplified Polymorphic DNA), SSR, ISSR (Inter Simple Sequence Repeat), AFLP (Amplified Fragment Length Polymorphism) and SRAP (Sequence-Related Amplified Polymorphism). Several studies focused on genetic diversity among mandarins have been performed using different molecular markers 2, 5-7. 516

SSR markers have several advantages over the previously studied molecular markers: They are abundant in most genomes and are co-dominant, so their information content is very high. SSRs are PCR-based, thus requiring little DNA for the amplification; every SSR locus is defined by a unique pair of primers, so that information exchange between laboratories is easy 8, 9. Thus, SSR markers are considered to be ideal markers for genetic and physical genome mapping, identification and discrimination of genotypes, studies of population genetics, inbred line characterization, forensic studies and medical genetics. In citrus, this marker has been used in studies related to phylogenetic analysis and linkage 10-13. SRAP technology is a newly developed method based on two-primer amplification that preferentially amplifies open reading frames (ORFs) 14. Due to its unique primer design, SRAP markers are more reproducible, more stable and less complex than other molecular marker techniques. SRAP markers have been demonstrated to be more powerful for revealing genetic diversity among closely related cultivars compared to SSR, ISSR and RAPD markers 15. In this study, SSR and SRAP molecular markers were used to characterize mandarin genotypes in the Tuzcu Citrus Germplasm Collection. Materials and Methods Sixty-five mandarin genotypes were used in this study (Table 1). For DNA extraction, leaf tissues of all accessions were obtained from the Tuzcu Citrus Collection at the University of Cukurova Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013

(Adana, Turkey) and the West Mediterranean Agricultural Research Institute (Antalya, Turkey). DNA extraction: For each genotype, young leaves were collected from a single tree, immediately frozen in liquid nitrogen and stored -80°C. Total genomic DNA was extracted from frozen leaves using the CTAB method, as described by Doyle and Doyle 16. DNA concentration was assessed using a NanoDrop ND 100 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), and DNA templates were diluted to 10 ng/µl using TE (10 mM Tris HCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0). SSR analysis: The 26 SSR primers used in this study (Table 2) were previously described and used for citrus germplasm characterization 12 . These primers were selected based on their dispersion on linkage groups. PCR amplifications were conducted as described by Barkley et al. 12, with some modifications. Each 10 µl reaction consisted of 1µl of template DNA (10 ng), 1 µM 10× PCR buffer, 200 µM of each dNTP, 2.5 mM MgCl2, 0.5 unit of Taq DNA polymerase, 1µM of forward and reverse primers, and 4.9 µl ddH2O. PCR conditions were those of Barkley et al. 12 with some modifications. A DNA thermal cycler (Biorad DNA-Engine Gradient Cycler, Hercules, CA, USA) was used, and the cycling parameters included 3 min of denaturing at 94°C; 35 cycles of 30 s of denaturing at 94°C, 30 s of annealing at 50°C, except for the three primers described below, and 1 min of elongation at 72°C; and one cycle at 72°C for 10 min. The annealing temperature was different for three primers; TAA52, TAA15 and CAGG9 were amplified using an annealing

temperature of 40°C. The PCR products were separated on 2% high-resolution agarose gels (Amresco SFR, OH, USA) in 1× TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA) at 115 V for 3.5 h, and the resulting bands were photographed under UV light for further analysis. A 100-bp DNA ladder was used as a molecular standard to confirm the appropriate SSR markers. SRAP analysis: All SRAP primer 17 combinations were initially screened using a group of samples. The 21 primers that produced scorable polymorphic bands were used to amplify the rest of the accessions (Table 3). The PCR reaction components and PCR cycling parameters were those described by Uzun et al. 17. The resulting PCR products were separated on 2% agarose gels in 1× Tris/Borate/EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA) at 115 V for 3–4 h. Data analysis: A similarity matrix was constructed using the similarity coefficient of simple matching for SSR and SRAP data based on the presence (1) or absence (0) of fragments for each primer. Cluster analysis was performed using the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) software package 18. The genetic similarity matrix and ultrametric distance matrix produced from the UPGMA-based dendrogram, with the COPH module nested in the same software, were compared using Mantel’s matrix correspondence test 19. The result of this test is a cophenetic correlation coefficient, r, which indicates how well the dendrogram represents the similarity data. Polymorphism information content (PIC) values were calculated for SSR and SRAP markers, as previously described 20.

Table 1. Plant materials, their cultivar or common name and Latin name. Common name Tardivo SRA 358 Palazelli SRA 430 Oroval SRA 360 Sidi Aissa Fuzhu SRA 599 Mency SRA 466 Suenkat Nucellar Swatow Nuseller Fallglo Campeona Dweet Tangor Murcott Tangor Ortanique Tangor Mandora Tangor Temple Tangor Ellendale Tangor Tankan Tangor Agma-I Batangas Bover Encore Fairchild Fortune Fremont 8076R Honey Kara 3450R Kinnow 10003R King (Kunembo) Clementine SRA 38 Clementine SRA 80 Clementine Nules Clementine Fino

Latin name Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco C. reticulata X C.sinensis C. reticulata X C.sinensis C. reticulata X C.sinensis C. reticulata X C.sinensis C. reticulata X C.sinensis C. reticulata X C.sinensis C. reticulata X C.sinensis Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus nobilis Lour. C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka

Common name Clementine Marisol Clementina Hernandina Algerian Tangerina Clementine 90 A Clementine Tuzcu 06 Clementine 22 D Lee Nova Robinson Osceola Parson Special Pixie Ponkan Satsuma SRA 12 Satsuma Hayashi Satsuma Mihowase Satsuma Zairai Satsuma Clausellina Satsuma Okitsu Satsuma Silverhille Satsuma 23 M Satsuma I4-2 Sampson Tangelo Seminello Tangelo Mapo Tangelo Thornton Tangelo Lake Tangelo Minneola Tangelo Orlando Tangelo Sunburst Wilking Yerli-Birecik Yerli 24-12

Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013

Latin name C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka C. clementina Hort. ex Tanaka Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. Citrus unshiu Marc. C. reticulata X C.paradisi C. reticulata X C.paradisi C. reticulata X C.paradisi C. reticulata X C.paradisi C. reticulata X C.paradisi C. reticulata X C.paradisi C. reticulata X C.paradisi Citrus reticulata Blanco Citrus reticulata Blanco Citrus deliciosa Tenore Citrus deliciosa Tenore

517

Results and Discussion The genetic diversity among mandarin genotypes was evaluated using SSR and SRAP markers. For SSR analysis, amplification was successful with 14 primer pairs, and 26 primers were used for SRAP analysis. A total of 38 alleles were generated using the fourteen SSR primers. The polymorphism rate was 83.0% among the 65 mandarin genotypes based on SSR data. The number of alleles detected by a single primer set ranged from two to four, with a mean number of alleles per primer pair of 2.70 (Table 2). An SSR analysis of Turkish lemon accessions revealed that the CAGG9 primer pair identified more fragments (5 alleles/primer) than any of the other primer pairs used. The second most polymorphic SSR identified in the Turkish lemon germplasm was CAT01, with 4 alleles scored 13. The PIC value for SSR primers in this study was between 0.15 (CAC19) and 1.0 (CCT01), with a

mean value of 0.57 (Table 2). SRAP analysis generated a total of 187 alleles, and the polymorphism rate was 77%. The number of amplified fragments was dependent on the individual genotypes. The number of fragments produced by each primer varied from 4 (em11 me1) to 17 (em2 me3), with an average of 8.90 (Table 3). The size of the DNA fragments obtained from SRAP analysis ranged from 80-1600 bp. The PIC value for SRAP primers in this study was between 0.21 (em1 me4) and 0.71 (em14 me1), with a mean of 0.47 (Table 3). The number of alleles detected by a single primer set ranged from two to four, with a mean number of alleles per primer pair of 2.70. This mean value is lower than the value reported for other plant species, such as Prunus cerasus and Actinidia chinensis2123 . This difference may be due to the narrow genetic base of mandarin accessions. The CAGG9, CAT01 and TAA52 primer pairs were the most informative, and they produced four fragments of Table 2. List of the SSR primers, their numbers of total and different sizes. polymorphic fragments, percentage of Das et al. 24 investigated the genetic diversity of 25 mandarin polymorphism, and polymorphism information genotypes using RAPD markers. They reported that the number contents used in this study. of alleles detected by a single primer ranged from 2 to 13, with an SSR Total Polymorphic Polymorphism average of 7.60. In another study, AFLP markers were used, and PIC primers fragments fragments (%) the polymorphism rate was 86% among mandarin genotypes 5. AC01 2 2 100 0.53 The difference between the average number fragments produced ATC09 2 2 100 0.51 by the primers was dependent on the marker system used. CAC19 2 1 50 0.15 Cluster analysis (UPGMA) employing SSR and SRAP CAC23 2 1 50 0.43 data produced a dendrogram with two main branches (Fig. 1). The CAC33 3 2 67 0.44 CAG01 3 3 100 0.71 Orlando Tangelo (C. reticulata x C.paradisi) genotype was found CAGG9 4 2 50 0.32 to be genetically divergent from all of the other genotypes by CAT01 4 4 100 0.56 22%. All genotypes, except for the Orlando Tangelo, were clustered CCT01 2 2 100 1.00 together in the second main branch of the dendrogram. Tangelos, CT21 2 1 50 0.50 TAA15 3 3 100 0.59 which are known to be a hybrid of grapefruit and mandarin, TAA33 2 2 100 0.82 clustered in the same subgroups of the dendrogram, with one TAA45 3 3 100 0.66 exception, the Orlando Tangelo was separated from tangelos. TAA52 4 4 100 0.82 Although most of the mandarin genotypes were separated from Total 38 32 8.04 Mean 2.7 2.3 83 0.57 each other, some mandarins were identical based on the SSR and SRAP data (Fig. 2). Table 3. List of the SRAP primers, their numbers of total and The matrix correlation coefficient obtained from the SSR polymorphic fragments, percentage of polymorphism, and polymorphism information contents used in this study. and SRAP analysis was calculated as r = 0.820 for the dendrogram. This value indicates that the similarity index is Fragment SRAP Total Polymorphic Polymorphism represented well by the dendrogram 25. Interpretation of the size ranges PIC primers fragments fragments (%) (bp) correlation coefficient matrix is as follows: r≥0.9 is very good, em1 me4 100-1270 8 5 63 0.21 0.8≤ r