Journal of Applied Botany and Food Quality 89, 82 - 88 (2016), DOI:10.5073/JABFQ.2016.089.010 1Erciyes University Department of Horticulture, Melikgazi, Kayseri, Turkey 2 Fruit Research Institute, Egirdir, Isparta, Turkey 3Plant Protection Central Research Institute, Ankara, Turkey 4Ataturk University Department of Horticulture, Erzurum, Turkey Determination of genetic relatedness among Turkish apple germplasm based on ISSR markers Aydın Uzun1, Serif Ozongun2, Osman Gulsen1, Kadir Ugurtan Yılmaz1, Suat Kaymak3, Sezai Ercisli4* (Received October 4, 2015) * Corresponding author Summary Apple (Malus domestica Borkh.) is one of the most economically important pome fruits worldwide and Turkey is within origin cen- ter of apple. In this research, inter-simple sequence repeat (ISSR) markers were used to determine relationships among the Turkish apple accessions and some selected foreign cultivars and species. Fourteen ISSR primers produced a total of 111 fragments and 76 of them were polymorphic. The number of average polymorphic frag- ments per primer was 5.4. The mean polymorphism information con- tent (PIC) was 0.37. The unweighted pair group method arithmetic average (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.79 to 0.98. All accessions studied were discriminated and many subgroups were determined in the dendro- gram based on the UPGMA analysis. High level of variation among the Turkish apples existed. Foreign cultivars, M.baccata, M. pruni- folia and M. sylvestris accessions studied mix-clustered among the Turkish accessions. For sub-structuring Bayesian analysis, 71 loose- ly or uncorrelated markers with less than 10 % missing data were used. This indicated absence of subpopulations, meaning well and equal introgression of genetic backgrounds or species available among the accessions. It can be concluded that Turkey was rich in apple genetic diversity, which may provide opportunities for apple breedind programs. Introduction Fruit, which contains phytochemicals that are being studied for ad- ded health benefits, has been recognized as a good source of vitamins and minerals, and for their role in preventing vitamin C and vitamin A deficiencies (BACVONKRALJ et al., 2014; ROP et al., 2014). Among fruits, apple has special importance because it is one of the most produced fruit crops among the temperate fruits with over 75 million tons of production per year (FAO, 2012). Domesticated apples (Malus domestica Borkh.) have been cultivated since ancient times and are now produced in a range of area from Siberia with freezing temperatures during winter as low as -40 oC to some equa- torial locations with high temperatures (JANICK et al., 1996). Four origin centers were reported for apples including East Asia, Middle Asia, East Asia-Europe and North America. Turkey belongs to East Asia-Europe origin center and has considerable diversity (JANICK et al., 1996). In earlier time, breeding programs were based only on selections from naturally growing apple trees. Then, hybridization became a more preferable tool for obtaining economically important new cultivars. Origin of domesticated apples was probably based on M. sieversii known as wild apple in central Asia (HARRIS et al., 2002; COART et al., 2006 ). It was argued that M. sylvestris, the wild apple of Western Europe, might have contributed little or even nothing to the domesticated apple gene pool at least as maternal ancestor (ROBINSON et al., 2001). Similarly, M. sieversii was reported as the main contributor to the genome of the cultivated apple and M. syl- vestris, in particular, determined as the secondary contributor. Evo- lution of domesticated apples occurred over a long time period and involved more than one wild species (CORNILLE et al., 2012). But some findings regarding to this issue that three most frequent chlo- roplast haplotypes of M. domestica and M. sylvestris were nearly absent in the analysed M. sieversii accessions concluded complex origin of domesticated apples. Also high level of cpDNA diversity was detected among the genus Malus cultivars, which was explai- ned by the hypothesis of the complex hybrid origin of M. domestica (COART et al., 2006). Genetic diversity and phylogenetic studies on apples have been carried out using various marker systems. Simp- le sequence repeats (SSRs) markers were the most reliable system for this kind of studies (PATZAK et al., 2012; GARKAVA-GUSTAVSSON et al., 2013). In addition, amplified fragment length polymorphism (AFLP) (KENIS and KEULEMANS, 2005; NING et al., 2007); chloro- plast DNA (cpDNA) (COART et al., 2006) markers were used for estimating apple genetic diversity, relationships and constructing genetic maps of apples. Inter-simple sequence repeat (ISSR) markers were reported to have higher reproducible rates due to the use of longer primers (16-25- mers) than the RAPD primers (10-mers). This marker system is cost effective because multiple loci are amplified during PCR amplifi- cation (ZIETKIEWICZ et al., 1994). It was previously used for apple genetic studies (GOULAO and OLIVEIRA, 2001; HE et al., 2011). Turkey is both within origin center of apples and a major apple pro- ducer with 2.88 million tons of production (FAO, 2012). Hence there is considerable genetic diversity of apples in Turkey. In addition, introductions from foreign countries are available in apple genetic resources of Turkey. Conservation and characterization of this ge- netic pool are required for breeding programs and future utilization. In present study, genetic variation and relationships among the apple accessions collected from different parts of Turkey and some selec- ted foreign cultivars were investigated. Materials and methods Plant material and DNA isolation One hundred and fifty-eight apple accessions were used for this stu- dy including 152 M. domestica cultivars and local genotypes, three M. sylvestris accessions, two M. baccata and one M. prunifola ac- cession (Tab. 1). The rest included common apple cultivars such as Golden Delicious, Granny Smith and Red Chief. For DNA extrac- tions, leaf tissues of all accessions were obtained from the collection located Fruit Research Station in Egirdir of Isparta, Turkey. Total genomic DNA was extracted from young leaves by the CTAB me- thod as described by DOYLE and DOYLE (1990). DNA concentration was measured with a spectrophotometer (BioTek Instruments, Inc. Vinooski, United States) and 10 ng/mL DNA templates were made using TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0). Tab. 1: Name of apple accessions utilized in this study 1 Lodiearlygolden 41 180887(5.2) 81 E73 121 42.bs.5(Almila) 2 Yazelmasi(2484) 42 190887(1.4) 82 E2411 122 42.e.2(Ankaraguz.) 3 Yazelmasi(2482) 43 180887(4.4) 83 E65 123 42.c.5(Yazamasya) 4 Beyazelma(2575) 44 200887(1.2) 84 E5 124 42.e.6(Kabaelma) 5 Ferik 45 Blackjon 85 E392e 125 42.ko.(Yaylapinari) 6 Karanfil(2570) 46 Daldabir 86 E24 126 42.e.4(Mayhosy.k.) 7 Beyelmasi(2477) 47 220887(3.2) 87 Batum 127 42.e.3(Hanimteni) 8 Kiselmasi(2590) 48 230887(1.2) 88 E14 128 42.e.7(Yildizkiran) 9 Sahelmasi(2600) 49 250887(1.10) 89 E13 129 42.kp.3(Karapinar) 10 Gelinelmasi(2475) 50 Yaztavsanbasi 90 Sarigobek 130 42.c.3(Tatlitavsanb.) 11 Sekerelması(2551) 51 210887(1.1) 91 Candir 131 32E1 12 Tatlielma(2511) 52 E220887 92 E25 132 Yenice 13 Sarielma 53 E180887(2.1) 93 E11 133 Orak 14 Gobek(2455) 54 E210887(1.4) 94 Uzunyumra 134 Pancarlik 15 Sogutelma(2480) 55 E170887(2.5) 95 E32 135 Samsun 16 Susuzelma(2500) 56 E210887(2.1) 96 Cigit 136 Harim 17 Mektepelmasi(2565) 57 E130887(2.3) 97 E383e 137 Gelendost 18 Altinokelmasi(2490) 58 Reinettetardiva 98 Karpuz 138 Inebolu 19 Elma(2590) 59 E70 99 Portakal 139 Golden Delicious 20 Demir(2486) 60 E42 100 E33 140 Jonagold 21 Rizedemir 61 E71 101 Gurcu 141 Ozark Gold 22 Sandik 62 E40 102 E2 142 Royal Gala 23 Petek(2577) 63 E55 103 542E 143 Elstar 24 Oltuelmasi(2594) 64 E52 104 E1 144 Melrose 25 Cincik(2471) 65 E51 105 Cidagut 145 Idared 26 Mahsusaelmasi 66 E50 106 E6 146 Fuji 27 Elma(2523) 67 E49 107 Sinap 147 Red Chief 28 Petevrekelmasi 68 E82 108 384E 148 Gloster 29 Tavsanbasi(2531) 69 E57 109 E4 149 Granny Smith 30 Tatlielma(2492) 70 E78 110 Seker 150 E72 31 Pasaelmasi 71 E66 111 E35 151 Cooper43 32 Lazelmasi(2507) 72 E47 112 E10 152 E33 33 Hüryemez 73 E56 113 Piraziz 153 M. prunifolia 34 Kadirhatice 74 E37 114 Gümüshane 154 M. baccata 1 35 Güztavsanbasi 75 E63 115 Gemlik.3 155 M. baccata 2 36 180887(5.1) 76 E81 116 Tokat.1 156 M. sylvestris 1 37 Sivanorelmasi 77 E9 117 Tokat.3 157 M. sylvestris 2 38 Kalkandelen 78 E76 118 Tokat.4 158 M. sylvestris 3 39 Karasaki 79 E48 119 42.kp.1 40 Gurcu 80 E67 120 42.a.1 ISSR analysis Fourteen ISSR primers previously evaluated by FANG and ROOSE (1997) and GULSEN et al. (2010) were used for all apple accessi- ons (Tab. 2). PCR reaction components and PCR cycling parameters were performed as described by UZUN et al. (2009). PCR products were separated on 2 % agarose gel in 1 X TBE buffer (89 mM Tris, 89 mM Boric acid, 2 mM EDTA) at 115 V for 2.5-3 h. The fragment patterns were photographed under UV light for further analysis. A 100 bp standard DNA ladder (GeneRuler, Fermentas) was used for estimating size of ISSR fragments. Data analysis Each band was scored as present (1) or absent (0) and data were analyzed with the Numerical Taxonomy Multivariate Analysis Sys- tem (NTSYS-pc version 2.1) software package (ROHLF, 2000). A similarity matrix was constructed based on Dice’s coefficient (DICE, 1945), which considers only one to one matches between two taxa for similarity. The similarity matrix was used to construct a dendro- gram using the unweighted pair group method arithmetic average (UPGMA) to determine genetic relationships in the germplasm stu- died. Goodness of fit test called Mantel test was performed by using 84 A. Uzun, S. Ozongun, O. Gulsen, K.U. Yılmaz, S. Kaymak, S. Ercisli ultrametric distance matrix obtained from the dendrogram and simi- larity matrix (MANTEL, 1967). The result of this test is a cophenetic correlation coefficient, r, indicating how well the dendrogram re- presents similarity data. Polymorphic Information Content (PIC) for dominant markers was calculated as: PIC = 1− [f 2 + (1− f)2], where ‘f’ is the frequency of the marker in the data set. PIC for dominant markers is a maximum of 0.5 for ‘f’ = 0.5 (DE RIEK et al., 2001). PIC provides an estimate of the discriminatory power of a locus by taking into account not only the number of alleles that are ex- pressed but also the relative frequencies of those alleles (SMITH et al., 1997). A Principal Coordinate Analysis (PCoA) was perfor- med based on the variance covariance matrix calculated from ISSR data. PCoA is a computational alternative to Principle Coordinate Analysis (PCA). PCA is used for similarities and PCoA for dissimi- larities. The data matrix was used to calculate distance matrix, then the distance matrix was double-centered, the double-centered matrix was then factored and a plot was made (ROHLF, 2000). Results and discussion ISSR analysis yielded 111 fragments and 76 of them (68.5 %) were polymorphic. Number of bands scored per primer varied between 4 (HVH(CA)7T) and 14 (GACA4), with a mean of 7.9. The GenAlEx ver. 6.5 program was employed, to determine allele frequency (p and q), no of effective alleles (Ne), Shannon’s information index (I), expected (He) and unbiased expected heterozygo- sity (uHe) (PEAKALL and SMOUSE, 2012). The PIC values for the 21 primer combinations ranged from 0.02 (HVH(CA)7T and DBDA(CA7) to 0.27 (GACA4) with a mean of 0.15 (Tab. 2). Cophenetic correlation between ultrametric similarities of tree and similarity matrix was found to be relatively high (r = 0.76, P < 0.01). Values for effective alleles (Ne) ranged from 1.02 ((TCC)5RY) to 1.48 ((AG)7YC) (average 1.28), for Shannon’s information index from 0.04 ((TCC)5RY) to 0.43 ((GT)8YA) (average 0.28), for ex- pected heterozygosity (He) and unbiased expected heterozygosity (uHe) from 0.02 ((TCC)5RY) to 0.28 ((GT)8YA) (average 0.18) (Tab. 3). A dendrogram was constructed by using the UPGMA ana- lysis based on 111 ISSR markers. The apple accessions studied had similarity values ranging from 0.79 to 0.98 indicating a high level of variation (Fig. 1). Similarly, GOULAO and OLIVEIRA (2001) found similarity levels of ~ 0.75-1.00 among 41 apples according to ISSR data. On the other hand, GULSEN et al. (2010) determined higher va- riation among 192 apple accessions using POGP (peroxidase gene- based polymorphism) markers. In the present study, all accessions were distinguished. The dendrogram consisted of many subgroups. Foreign cultivars and three apple species studied (M. baccata, M. prunifolia and M. sylvestris) nested mixed with Turkish accessions. PCA was performed based on the genetic distance matrix to better Tab. 2: Results on ISSR primers used for apple accessions Primers Total Fragments Polymorphic Fragments Polymorphism (%) PIC Resolving Power (AG)7YC 8 6 75 0.18 12.7 (AGC)6G 12 8 67 0.14 16.5 (CA)8R 6 3 50 0.06 10.1 (CAA)6 10 10 100 0.26 8.5 (CAC)3GC 6 4 67 0.26 8.4 (CT)8TG 7 6 86 0.24 5.7 (GA)8YG 5 3 60 0.04 9.8 (GACA)4 14 12 86 0.27 11.1 (GT)6GG 10 8 80 0.22 10.1 (GT)8YA 9 8 89 0.26 9.9 HVH(TCC)7 6 3 50 0.04 10.1 (TCC)5RY 8 2 25 0.03 12.3 DBDA(CA)7 6 2 33 0.02 11.9 HVH(CA)7T 4 1 25 0.02 7.9 Mean 7,9 5,4 68,5 0.15 10.3 Total 111 76 - - - Tab. 3: ISSR primers studied, their estimated allele frequency (p & q), no of effective alleles (Ne), Shannon’s information index (I), expected (He) and unbiased expected heterozygosity (uHe). Primers P q Ne I He uHe (AG)7YC 0.66 0.34 1.48 0.39 0.27 0.27 (AGC)6G 0.60 0.40 1.26 0.27 0.17 0.17 (CA)8R 0.80 0.20 1.13 0.14 0.09 0.09 (CAA)6 0.30 0.70 1.40 0.41 0.26 0.26 (CAC)3GC 0.57 0.43 1.42 0.37 0.25 0.25 (CT)8TG 0.39 0.61 1.37 0.37 0.23 0.23 (GA)8YG 0.90 0.10 1.24 0.26 0.16 0.16 (GACA)4 0.29 0.71 1.34 0.33 0.21 0.21 (GT)6GG 0.38 0.62 1.38 0.38 0.24 0.24 (GT)8YA 0.41 0.59 1.47 0.43 0.28 0.28 HVH(TCC)7 0.79 0.21 1.13 0.17 0.10 0.10 (TCC)5RY 0.76 0.24 1.02 0.04 0.02 0.02 DBDA(CA)7 0.94 0.06 1.14 0.15 0.10 0.10 HVH(CA)7T 0.93 0.07 1.15 0.19 0.11 0.11 Mean 0.62 0.38 1.28 0.28 0.18 0.18 Diversity analysis of Turkish apple germplasm 85 Fig 1: Dendrogram of 158 apple accessions based on the ISSR markers and the UPGMA method. understand genetic relationships. Fig. 2 presents the distribution of different genotypes according to two principal axes of variation using PCoA, which revealed the variation among the accessions si- milar to the UPGMA analysis. Among the apples studied ‘Sandik’ and ‘Demir (2486)’ were the most distinct accessions with similarity value of 0.79. These two apples were collected from Northeast Anatolia and East Black Sea region of Turkey (CETINER, 1981). In another study, ‘Demir (2486)’ was clearly separated from the other apples (GULSEN et al., 2010). ‘Reinette Tardiva’ was also apart from other apples with similarity 86 A. Uzun, S. Ozongun, O. Gulsen, K.U. Yılmaz, S. Kaymak, S. Ercisli of 0.84. ‘Cigit’, ‘Daldabir’, ‘Tatlielma (2492)’, ‘Beyazelma (2575)’ were nested in the same subgroup while ‘Mektep’ apple, ‘Rizedemir’ and ‘E55’ apples were in the same cluster. ‘M. baccata 1’ was clus- tered with ‘Altinok’ and ‘Samsun’ accessions. Two M. baccata accessions studied in present research were distin- guished from each other and clustered with the Turkish local acces- sions. ‘Portakal’ and ‘542E’, two little acidulated accessions were in the same subgroup. ‘Mahsusa elmasi’, ‘E47’, ‘130887’, ‘180887’, ‘E66’, ‘E52’ and ‘E71’ were clustered closely. Another subgroup consisted of ‘42E6 Kabaelma’, ‘42A1 Yazelması’ sampled from Konya province, ‘32E1’, ‘Gelendost’ sampled from Isparta province and ‘Tokat1’, ‘Tokat3’. The UPGMA clustering grouped the geno- types into meaningful clusters. Three apples ‘Kalkandelen’, ‘Sogu- telma (2486)’ and ‘Beyelmasi (2477)’ having similar fruit charac- teristics with sourish flavour were clustered together. In the den- drogram describing the accessions above generated many different groups (A). In the dendrogram, the rest of 127 apple accessions were grouped into two main groups, B and C. Group B had more accessions and were separated into two subgroups. Two foreign red skin culti- vars (‘Gloster’ and ‘Red Chief’) and five local Turkish accessions were clustered in the smaller group of B. Foreign cultivars were not grouped apart from the Turkish accessions. They were mixed clustered with local apples, indicating similar genetic backgrounds. Accordingly, SONMEZOGLU and KUTUK (2014) found that 23 local apple genotypes collected from Karaman, Turkey and three foreign cultivars were divided into two major groups and numerous sub- groups, revealing a rich variation among the apple genotypes. On the other hand, PEREIRA-LORENZO et al. (2008) found genetic diffe- rence between Spanish apple accessions and non-native cultivars. Similarly, GASI et al. (2010) found that traditional Bosnia and Her- zegovina cultivars were differentiated quite clearly from foreign cul- tivars, except for few genotypes. Differences between the present study and others may be because of genetic backround of local apple accessions studied. Large subgroup of group B divided into eight subclusters. One of them consisted of sourish flavour accessions including ‘Sarielma’, ‘Sarigobek’, ‘Karpuz’, ‘Hanimteni (42E3)’, ‘Karapinar (42KP3)’. Last two apples also found in the same cluster in previous study based on POGP markers (GULSEN et al., 2010). These two accessions were collected from the same region of Turkey. One of the foreign cultivars, ‘Golden Delicious’ was apart from the other common cultivars and clustered with three local apples. In group B, the largest subcluster consisted of 30 apple accessions. Similarity of these accessions was between 0.89 and 0.98. In this subcluster, three foreign cultivars, ‘Granny Smith’, ‘Cooper’ and ‘Fuji’, three M. sylvestris accessions and many local accessions were nested. Some of the local apples, ‘Gelin’, ‘42KO1 Yaylapinari’ and ‘42KP1 Mayhostavsanbasi’ shared the same fruit characteristics such as soury flavor. ‘Granny Smith’ and ‘Fuji’ were closely related based on ISSR data. Similarly, these two cultivars were grouped closely according to SSR (GASI et al., 2010) and POGP markers (GULSEN et al., 2010). On the other hand, GOULAO and OLIVEIRA (2001) found that ‘Granny Smith’ and ‘Fuji’ were distinct based on ISSR data. Three M. sylvestris accessions studied nested in this subcluster and were nearly identical. They were closely related to the Turkish local apple accessions belong to M. domestica. COART at al. (2006) found high levels of haplotype sharing between M. sylvestris and M. do- mestica and assumed an interspecific gene flow, which is probably bidirectional and brought about by the use of (local) wild Malus ge- notypes for the (local) cultivation process of apple. Four local apples, ‘Candir’, ‘Güztavsanbasi’, ‘Tavsanbasi’, ‘Seker’ and ‘Cincik’ were grouped closely in the dendrogram. Two of them ‘Güztavsanbasi’ and ‘Tavsanbasi’ were collected from the same province of Turkey (CETINER, 1981). Most of foreign cultivars studied in the present study (‘Idared’, ‘El- star’, ‘Royal Gala’, ‘Melrose’, ‘Ozark Gold’ and ‘Jonagold’) were grouped together. Five local Turkish accessions were also nested in this group. ‘Elstar’, ‘Jonagold’ and ‘Gala (Galaxy)’ were clus- tered based on SSR data (GASI et al., 2010). In another study, ‘Ro- yal Gala’, ‘Jonagold’ and ‘Ozark Gold’ were clustered closely but ‘Idared’ and ‘Melrose’ nested in the same group and slightly distinct from these three cultivars (GOULAO and OLIVEIRA, 2001). Two apple species (M. baccata (No:1) and M. prunifolia) were closely rela- ted. They were clustered with several Turkish accessions such as ‘Huryemez’, ‘Karasaki’, ‘Gurcu’, ‘Cidagut’, ‘Harim’, ‘Pancarlik’ and ‘Ferik’. Two M. baccata accessions studied were apart from each other. The similar results reported by HOKANSON et al. (2001) and YAO et al. (2010) indicated variation among the M. baccata samples. Only one M. prunifolia accession was used in this study and it was highly similar to M. baccata (No:1) but not identical to Fig. 2: Principal coordinate analysis diagram showing the relationships among 158 apple accessions (numbers of accessions were identifed in Tab. 1). Diversity analysis of Turkish apple germplasm 87 M. baccata (No:2). Malus prunifolia and M. baccata were found in diverse clusters in a previous study (FORTE et al. 2002). Similar- ly, HARRIS et al. (2002) concluded that these two species were apart from each other according to nuclear ribosomal internal transcribed spacer gene. In addition, ZHOU and LI (2000) assumed that M. pru- nifolia originated from hybridization between M. sieversii and M. baccata. The last subcluster of large subgroup of group B consis- ted of 14 apple accessions including ‘Petevrekelmasi’, ‘Sivanora’, ‘Lazelmasi’, ‘Pasaelmasi’, ‘Sekerelmasi’, ‘Oltuelmasi’, ‘Susuzelma’, ‘Petek’, ‘Karanfil’, ‘Yaztavsanbasi’, ‘Yazelmasi’, ‘E82’, ‘E220887’ and ‘210887 (1.1)’. In the dendrogram, 16 Turkish accessions and two foreign cultivars (‘Blackjon’ and ‘Lodi Early Golden’) were clustered in group C. ‘Lodi Early Golden’ was very similar to ‘Ya- zelmasi (2484)’ and both were early maturing cultivars. Conclusions Several significant results were obtained from this study. ISSR mar- kers confirmed efficiency for characterization of apple germplasm and cultivar identification. All of 158 apple accessions were distin- guished from each other. The Turkish apple accessions were closely related to the other known species, M. sylvestris, M. prunifolia and M. baccata. These three species were clearly separated from each other and they were mixed grouped with the Turkish accessions. This verified gene flow among apple species and local apple genotypes. Turkey has considerable morphological and molecular diversity in its apple genetic resources. Turkey is located in East Asia-Europe origin center for apple and the middle of three important continents (Asia, Africa and Europe). This region including Turkey and Iran was important in apple domestication and their transfer from Central Asia to the western countries (GHARGANI et al., 2009). The acces- sions studied in present study are maintained in the germplasm plots and are being investigated for important agronomic characters to ex- ploit potential interest. Acknowledgements The authors thank Scientific Research Projects Unit of Erciyes Uni- versity for funding and supporting the project with the project num- ber of FBA-10-3299. 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