Sevindik et al. 2024, Biologica Nyssana 15(2) 15 (2) December 2024: 61-68 DOI: 10.5281/zenodo.14258237 Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye Original Article Emre Sevindik Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Aydın Adnan Menderes University, Aydin, Türkiye ph.d-emre@hotmail.com (corresponding author) Filiz Yangılar Department of Nutrition and Dietetics, Faculty of Health Sciences, Erzincan Binali Yıldırım University, Erzincan, Türkiye Bayram Atasagun Department of Medical Services and Techniques, Vocational School of Health Services, Selcuk University, Konya, Türkiye Erengül Sofyalıoğlu Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Aydın Adnan Menderes University, Aydin, Türkiye Selçuk Alp Şimşek Bilkent University, Department of Molecular Biology and Genetics, Ankara, Türkiye Muhammed Ebrar Çayır Isparta Egirdir Fruit Research Institute, Isparta, Türkiye Martin Vivodík Slovak University of Agriculture, Faculty of Biotechnology and Food Sciences, Institute of Biotechnology, Nitra, Slovakia Received: August 13, 2024 Revised: November 13, 2024 Accepted: November 15, 2024 Abstract: In this study, ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars were collected from different locations in Erzincan province, Türkiye, and genetic diversity was determined using the Start Codon Targeted (SCoT) marker technique. The SCoT marker technique was chosen because its gene targeting, long primer, and high annealing temperature make it more effective than other marker techniques. Using ten SCoT primers, 60 bands were obtained, and 42 of them were polymorphic. The polymorphism rate was determined to be 70%. The UPGMA (Unweighted Pair Group Method with Arithmetic mean) dendrogram created using the PAUP 4.0b10 program consists of two clades. The genetic distance between apple cultivars varies between 0.13462 and 0.45614. Principal Component Analysis (PCA) results were compatible with the UPGMA dendrogram. With the SCoT marker technique, genetic diversity among apple cultivars can be determined in a shorter time and with more reliable results. Key words: apple, genetic diversity, SCoT, Türkiye Apstrakt: Analiza genetske raznovrsnosti i strukture sorti jabuka ‘‘Ak Sakı’’ i ‘‘Kara Sakı’’ koje rastu u Erzindžanu/Turska U ovoj studiji, sorte jabuka ‘Ak Sakı’ i ‘Kara Sakı’ prikupljene su sa različitih lokaliteta u provinciji Erzindžan, Turska, koristeći tehniku markera startnog kodona (SCoT) za određivanje genetske raznovrsnosti. Tehnika SCoT odabrana je zbog ciljanog delovanja na gene, dužine prajmera i visoke temperature vezivanja, što je čini efikasnijom od drugih tehnika markera. Korišćenjem deset SCoT prajmera dobijeno je 60 traka, od kojih su 42 bile polimorfne. Procenat polimorfizma utvrđen je na 70%. UPGMA dendrogram (Unweighted Pair Group Method with Arithmetic mean), kreiran korišćenjem programa PAUP 4.0b10, sastoji se od dve klade. Genetska udaljenost između sorti jabuka varira između 0.13462 i 0.45614. Rezultati analize glavnih komponenti (PCA) bili su kompatibilni sa UPGMA dendrogramom. Tehnikom SCoT može se u kraćem vremenskom periodu i sa pouzdanijim rezultatima odrediti genetska raznovrsnost među sortama jabuka. Ključne reči: jabuka, genetska raznovrsnost, SCoT,Turska Introduction The apple (Malus × domestica Borkh.), a member of the Rosaceae family, originates from the region between the Caspian Sea and the Black Sea. It is one of the most economically and culturally significant fruit species globally (Han et al., 2020; Geană et al., 2021; Chen et al., 2021; Fotirić Akšić et al., 2022). Due to its ecological compatibility and high nutritional value, it is popular among both producers and consumers (Eberhardt et al., 2000; Boyer and Liu, 2004; Hyson, 2011; Sarkate et al., 2017). ‘Ak Sakı’ is a variety grown in the Erzincan province of Türkiye and is the most commonly grown and readily consumed apple. In the region ‘Ak Sakı’ and ‘Kara Sakı’ ecotypes are known. It was also registered under the name ‘Ak Sakı’ on 03.05.1990. (Öztürk et al., 2013). The ‘Kara Sakı’ apple is a native apple variety of Erzincan, resembling the Amasya apple, but with a slightly lighter and brighter color and a mildly tart taste (Doğan, 2001). Its aroma is the most important feature that makes it the consumer’s preference. To date, more than 300 volatile compounds have been detected in apples © 2024 Sevindik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 61 (Dixon and Hewett, 2000; Aprea et al., 2011; Yang et al., 2021). Also, apples provide high amounts of carbohydrates, vitamins and bioactive compounds, such as phytosterols, β-carotene and phenolic compounds (Wu et al., 2020; Feng et al., 2021). Various apple phytochemical compounds with antioxidant activity and anticancer properties reduce the risk of chronic and degenerative diseases (Boyer and Liu, 2004; Inroga et al., 2021). Molecular markers are widely utilized in many plant species and are one of the most essential methods for analyzing genetic diversity (Yang et al., 2023). SCoT markers are simple and reliable markers designed to produce of gene-targeted markers. Compared to many other marker systems, the SCoT system provides more information about the universality and biological properties of plants (Yilmaz and Ciftci, 2021). The SCoT marker approach is more effective than other random markers because of its long primer and high annealing temperatures (Alzahrani et al., 2023). SCoT markers have been used in genetic diversity and phylogenetic relationships analysis in many plant species (Guo et al., 2016; Jalilian et al., 2018; Etminan et al., 2018; Vivodík et al., 2019; Zarei and Erfani-Moghadam, 2021; Vivodík et al., 2023). In this study, the genetic diversity and structure of the ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars, distributed in Erzincan – Türkiye, were analyzed using ten SCoT markers. Materials and Methods Plant Materials, Genomic DNA Isolation and PCR ‘Ak Sakı’ and ‘Kara Sakı’ cultivars were collected from different localities of Erzincan-Türkiye. For both cultivars, the collection was made from approximately seven centers. Genomic DNA isolation was performed using green leaves with a commercial kit (GeneMark Catalog No: DP022). Selected ten SCoT primers (Collard and Mackill, 2009) for PCR amplifications, PCR components and the protocol used are given in Tab. 1. PCR products were run on 1.0% agarose gel. In gel imaging, GeneRuler (Cat#: GMM100) between 100 bp and 3000 bp was used. Gel images of PCR results using the SCoT 11 primer are shown in Fig. 1. Start Codon Targeted (SCoT) Analysis Following the SCoT analyses, the DNA bands were scored by giving the value “1” in the presence of DNA, “0” in the absence of DNA and “?” or “9” for the missing cases. The UPGMA phylogenetic tree was drawn using the program PAUP 4.0b10 (Swofford, 2001). Pairwise distance was created with the same program. The genetic distance matrix between apple cultivars was calculated and shown in Tab. 2. The JMP Statistical Software which presents the distances between individuals in a two-dimensional diagram, was used to perform the PCA analysis. In addition, major allele frequency, Nei’s H (gene diversity) and PIC (polymorphism 62 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye Table 1. SCoT primers name, sequences and PCR components used for PCR amplification conditions SCoT Primers DNA Sequences(5’-3’) Tm oC PCR components PCR Amplification (35 cycle) SCoT 1 CAACAATGGCTACCACCA 54 oC SCoT 2 CAACAATGGCTACCACCC 56 oC SCoT 3 CAACAATGGCTACCACCG 56 oC SCoT 4 CAACAATGGCTACCACCT 54 oC SCoT 5 CAACAATGGCTACCACGA 54 oC 1 μL genomic DNA 1 μL primer, 5 μL master mix (Cat. No: RP02-II-400, RP02-II-2000, 0.75 U of Taq DNA polymerase, reaction buffer, 2 mM MgCl2, 250 µM dNTPs and enzyme stabilizer) and 18 μL dH2O. SCoT 6 CAACAATGGCTACCACGC 56 oC 95 oC/1min. SCoT 8 54 oC 95 oC/30 sec CAACAATGGCTACCACGT 54-56 oC/30sec. SCoT 9 CAACAATGGCTACCAGCA 54 oC 72 oC/1 min. SCoT 10 CAACAATGGCTACCAGCC 56 oC 72 oC/5 min. SCoT 11 AAGCAATGGCTACCACCA 54 oC BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye 63 information content) values were calculated using the PowerMarker software package version 3.25 (Liu and Muse, 2005). Using the statistical program Structure 2.3.4, a structure test was utilized to classify members of various populations (Pritchard et al., 2000). Results and discussion For SCoT analysis, ten primers were used. A total of 60 bands were obtained. 42 of these bands were polymorphic and the polymorphism rate was 70%. The highest number of bands was obtained from the SCoT 2 primer and the lowest number of bands was obtained from the SCoT 8 primer (Tab. 2). “PIC”, “H” and “major allele frequency” values of each primer are given in Tab. 3. The mean PIC value was determined at 0.2878, while the average H value and the mean major allele frequency were determined at 0.3581 and 0.7413, respectively. Fig. 1. Gel image of bands amplifed with SCoT 11 primer: 1: ‘Ak Sakı’ (Elma village), 2: ‘Ak Sakı’ (Yaylabaşı), 3: ‘Kara Sakı’ (Çağlayan), 4: ‘Ak Sakı’ (Çağlayan), 5: ‘Ak Sakı’ (Karatuş), 6: ‘Kara Sakı’ (Elma village), 7: ‘Kara Sakı’ (Karakaya), 8: ‘Ak Sakı’ (Karakaya), 9: ‘Kara Sakı’ (Karatuş village), 10: ‘Ak Sakı’ (Pişkidağ), 11: ‘Ak Sakı’ (Üzümlü), 12: ‘Kara Sakı’ (Yaylabaşı), 13: ‘Kara Sakı’ (Üzümlü) Table 2. Monomorphic and polymorphic band numbers of SCoT primers Primers Total bands Monomorphic bands Polymorphic bands SCoT 1 8 6 2 SCoT 2 11 2 9 SCoT 3 7 1 6 SCoT 4 5 1 4 SCoT 5 5 1 4 SCoT 6 7 1 6 SCoT 8 1 1 0 SCoT 9 3 1 2 SCoT 10 4 1 3 SCoT 11 9 3 6 Total 60 18 42 Table 3. Major allele frequency, gene diversity (H) and polymorphism information content (PIC) values Primers Major Allele Frquency Gene Diversity (H) PIC SCoT 1 0.6643 0.446 0.3465 SCoT 2 0.6014 0.4794 0.3645 SCoT 3 0.6783 0.4364 0.3412 SCoT 4 0.7692 0.355 0.292 SCoT 5 0.8392 0.2699 0.2335 SCoT 6 0.6573 0.4505 0.349 SCoT 8 0.9091 0.1653 0.1516 SCoT 9 0.8601 0.2406 0.2117 SCoT 10 0.8531 0.2506 0.2192 SCoT 11 0.5804 0.4871 0.3684 Mean 0.7413 0.3581 0.2878 The UPGMA dendrogram was created and it consists of two main clades (Fig. 2). Clade A consists of ‘Ak Sakı’ (Elma village), ‘Ak Sakı’ (Karatuş village), ‘Kara Sakı’ (Elma village), ‘Kara Sakı’ (Üzümlü), ‘Kara Sakı’ (Karatuş village), ‘Kara Sakı’ (Yaylabaşı), ‘Kara Sakı’ (Karakaya) apple cultivars. Clade B consists of ‘Ak Sakı’ (Yaylabaşı), ‘Kara Sakı’ (Çağlayan), ‘Ak Sakı’ (Çağlayan), ‘Ak Sakı’ (Karakaya), ‘Ak Sakı’ (Üzümlü) and ‘Ak Sakı’ (Pişkidağ) apple cultivars. According to Tab. 4, the 64 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye Fig. 2. The UPGMA tree generated using ten SCoT markers lowest distance (0.13462) was found between the ‘Kara Sakı’ (Yaylabaşı) and ‘Kara Sakı’ (Üzümlü), ‘Kara Sakı’ (Elma village) and ‘Kara Sakı’ (Üzümlü) cultivars. The highest distance (0.45614) was found between the ‘Ak Sakı’ (Üzümlü) and ‘Kara Sakı’ (Yaylabaşı) cultivars. The graphical results and eigenvalues obtained on the two-dimensional plane according to PCA analysis are given in Fig. 3. The first three eigenvalues explained 64.755% of the total variance in the cultivars. As a result of the analysis, 13 samples were reduced to two dimensions. The first dimension consists of seven cultivars and the second one consists of six (eigen > 1). The clustering in the two-dimensional graph obtained from the PCA analysis partially showed parallelism with the dendrogram results. Çokran et al. (2019) determined the genetic diversity of 30 local ‘Misket’ apple genotypes using AFLP, SSR, and RAPD markers. In their study, AFLP, SSR and RAPD markers amplified a total of 423 bands and obtained 205 polymorphic bands, while 30 RAPD primers obtained 207 bands, 91 of which were polymorphic (40.1%), and 10 SSR primers obtained 33 bands and 26 polymorphic (78.78%) bands. Five AFLP combinations obtained 183 bands, 88 of which were polymorphic (48.08%). Sevindik et al. (2018) determined the genetic diversity of apple genotypes in Ardahan province with the ISSR-PCR technique. The authors found the polymorphism rate to be 60% in their study results. Kaya et al. (2015) used RAPD markers to perform molecular genotyping on apples collected from Van province, Türkiye. As a result of their study, they found the polymorphism rate to be 89.29%. Khachtib et al. (2024) determined the genetic diversity of 29 apple cultivars in different regions of Morocco using ISSR-PCR technique. They obtained 177 bands from 15 ISSR primers, 156 of which were polymorphic. Also, the mean values of PIC, Rp, I and H were determined as 0.46, 4.58, 0.43 and 0.28, respectively. In conclusion, they revealed that ISSR markers could be useful in detecting genetic diversity in this fruit crop. Najar et al. (2023) have collected samples from the North Kashmir region and screened 62 apple genotypes using ten SSR markers. In their study, they amplified a total of 77 alleles with an average polymorphism percentage of 87.5%, PIC of 0.71 and resolving power (RP) of 3.58. Dar et al. (2020) have determined the genetic diversity of 19 apple varieties from the Kashmir region using ten RAPD markers. In their study, they detected a total of 70 polymorphic bands with a polymorphism percentage of 83.33. The same study suggested that these results can be implemented in apple-related conservation and breeding programs. In our study, the cultivar structure of individuals was estimated using the Structure test. Structure analysis of 13 cultivars was performed using ten SCoT primers. Our results showed a clear peak point for ΔK at K = 3. At the first level of clustering (ΔK at K = 3), although ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars were divided into three subpopulations; all 13 cultivars were considered as mixtures. (Fig. 4). This suggests significant gene flow with high exchange rates and different allele combinations between populations, in agreement with the results reported by Najar et al. (2023) for Malus×domestica germplasm from North Kashmir, India. BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye 65 Ta bl e 4. P ai rw ise g en et ic di st an ce m at rix o bt ai ne d fro m te n SC oT p rim er s C ul tiv ar s 1 2 3 4 5 6 7 8 9 10 11 12 13 ‘A k Sa kı ’ (E lm a vi lla ge ) - 0. 25 0. 32 72 7 0. 36 66 7 0. 28 33 3 0. 31 57 9 0. 28 33 3 0. 4 0. 26 66 7 0. 33 33 3 0. 41 66 7 0. 24 56 1 0. 26 96 3 ‘A k Sa kı ’ (Y ay la ba şı ) 15 - 0. 16 36 4 0. 25 0. 26 66 7 0. 31 57 9 0. 26 66 7 0. 31 66 7 0. 31 66 7 0. 31 66 7 0. 3 0. 42 10 5 0. 38 46 2 ‘K ar a Sa kı ’ (Ç ağ la ya n) 18 9 - 0. 14 54 5 0. 21 81 8 0. 26 92 3 0. 30 90 9 0. 2 0. 27 27 3 0. 30 90 9 0. 21 81 8 0. 38 46 2 0. 32 69 2 ‘A k Sa kı ’ (Ç ağ la ya n) 22 15 8 - 0. 25 0. 21 05 3 0. 28 33 3 0. 23 33 3 0. 3 0. 36 66 7 0. 28 33 3 0. 35 08 8 0. 26 92 3 ‘A k Sa kı ’ (K ar at uş ) 17 16 12 15 - 0. 17 54 4 0. 3 0. 25 0. 25 0. 28 33 3 0. 3 0. 28 07 0. 17 30 8 ‘K ar a Sa kı ’ (E lm a vi lla ge ) 18 18 14 12 10 - 0. 26 31 6 0. 33 33 3 0. 21 05 3 0. 19 29 8 0. 17 54 4 0. 28 07 0. 13 46 2 ‘K ar a Sa kı ’ (K ar ak ay a) 17 16 17 17 18 15 - 0. 25 0. 25 0. 31 66 7 0. 3 0. 36 84 2 0. 25 ‘A k Sa kı ’ (K ar ak ay a) 24 19 11 14 15 19 15 - 0. 4 0. 3 0. 18 33 3 0. 43 86 0. 30 76 9 ‘K ar a Sa kı ’ (K ar at uş v ill ag e) 16 19 15 18 15 12 15 24 - 0. 23 33 3 0. 31 66 7 0. 21 05 3 0. 17 30 8 ‘A k Sa kı ’ (P iş ki da ğ) 20 19 17 22 17 11 19 18 14 - 0. 18 33 3 0. 33 33 3 0. 26 92 3 ‘A k Sa kı ’ (Ü zü m lü ) 15 18 12 17 18 10 18 11 19 11 - 0. 45 61 4 0. 28 84 6 ‘K ar a Sa kı ’ (Y ay la ba şı ) 14 24 20 20 16 16 21 25 12 19 26 - 0. 13 46 2 ‘K ar a Sa kı ’ (Ü zü m lü ) 14 20 17 14 9 7 13 16 2 14 15 7 - BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye Fig. 3. Two dimensional graph and eigen values created as a result of Principal Component analysis with SCoT marker Fig. 4. Population structure analysis of apples Conclusion The study revealed a 70% polymorphism using ten SCoT primers, demonstrating the genetic diversity of the ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars. The PCA analysis results were consistent with the UPGMA dendrogram, and the cluster analysis of genetic structure indicated that the apple cultivars are best represented by three genetic groups (ΔK=3). In addition, the results obtained will enable the cultivars to be used more effectively in future breeding programs. References Alzahrani, O.R., Alshehri, M.A., Alasmari, A., Ibrahim, S.D., Oyouni, A.A., & Siddiqui, Z.H. (2023). Evaluation of genetic diversity among Saudi Arabian and Egyptian cultivars of alfalfa (Medicago sativa L.) using ISSR and SCoT markers. Journal of Taibah University for Science, 17(1), 2194187. https://doi.org/10.1080/16583655.2023.2194187 Aprea, E., Gika, H., Carlin, S., Theodoridis, G., Vrhovsek, U., & Mattivi, F. (2011). Metabolite profiling on apple volatile content based on solid phase microextraction and gas-chromatography time of flight mass spectrometry. Journal of Chromatography A, 1218(28), 4517–4524. https:// doi.org/10.1016/j.chroma.2011.05.019 Boyer, J., & Liu, R. (2004). Apple phytochemicals and their health benefits. Nutrition Journal, 3(1), 5. Chen, Z., Yu, L., Liu, W., Zhang, J., Wang, N., & Chen, X. (2021). Research progress of fruit color development in apple (Malus domestica Borkh.). Plant Physiology and Biochemistry, 162, 267–279. https://doi.org/10.1016/j.plaphy.2021.02.033 66 Çokran, B.D., Karadeniz, T., & İkten, H. (2019). Analysis of genetic diversity among ‘Misket’ apple clones using AFLP, SSR, and RAPD markers. Erwerbs-Obstbau, 61(3), 293–302. https://doi. org/10.1007/s10341-019-00430-8 Collard, B.C., & Mackill, D.J. (2009). Start codon targeted (SCoT) polymorphism: A simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Molecular Biology Reporter, 27, 86–93. https://doi.org/10.1007/s11105-008- 0060-5 Dar, J.A., Wani, A.A., & Dhar, M.K. (2020). Assessment of apple (Malus × domestica Borkh.) germplasm of Kashmir using RAPD markers. International Journal of Fruit Science, 20(3), 635– 645. https://doi.org/10.1080/15538362.2019.16395 83 Dixon, J., & Hewett, E.W. (2000). Factors affecting apple aroma/flavour volatile concentration: A review. New Zealand Journal of Crop and Horticultural Science, 28(3), 155–173. https://doi.or g/10.1080/01140671.2000.9514136 Doğan, A. (2001). Erzincan ilinde yetiştiriciliği yapılan sakı elma çeşitlerinin klon seleksiyonu yoluyla islahı. Yüksek Lisans Tezi. Atatürk Üniversitesi Fen Bilimleri Enstitüsü, Bahçe Bitkileri Anabilim Dalı, Erzurum, 75 s. Eberhardt, M.V., Lee, C.Y., & Liu, R.H. (2000). Antioxidant activity of fresh apples. Nature, 405, 903–904. Etminan, A., Pour-Aboughadareh, A., Noori, A., Ahmadi-Rad, A., Shooshtari, L., Mahdavian, Z., & Yousefiazar-Khanian, M. (2018). Genetic relationships and diversity among wild Salvia accessions revealed by ISSR and SCoT markers. Biotechnology & Biotechnological Equipment, 32(3), 610–617. https://doi.org/10.1080/13102818. 2018.1447397 Feng, S., Yi, J., Li, X., Wu, X., Zhao, Y., Ma, Y., & Bi, J. (2021). Systematic review of phenolic compounds in apple fruits: Compositions, distribution, absorption, metabolism, and processing stability. Journal of Agricultural and Food Chemistry, 69(1), 7–27. Fotirić Akšić, M., Dabić Zagorac, D., Gašić, U., Tosti, T., Natić, M., & Meland, M. (2022). Analysis of apple fruit (Malus × domestica Borkh.) quality attributes obtained from organic and integrated production systems. Sustainability, 14(9), 5300. https://doi.org/10.3390/su14095300 Geană, E.I., Ciucure, C.T., Ionete, R.E., Ciocârlan, A., Aricu, A., Ficai, A., & Andronescu, E. (2021). Profiling of phenolic compounds and triterpene acids of twelve apple (Malus domestica Borkh.) cultivars. Foods, 10(2), 267. Guo, J., Yu, X., Yin, H., Liu, G., Li, A., Wang, H., & Kong, L. (2016). Phylogenetic relationships of Thinopyrum and Triticum species revealed by SCoT and CDDP markers. Plant Systematics and Evolution, 302, 1301–1309. https://doi.org/10.1007/ s00606-016-1332-4 Han, Q., Liu, F., Hao, Y., & Ni, Y. (2020). Characterization of membrane-bound polyphenol oxidase from Granny Smith apple (Malus × domestica Borkh.). International Journal of Biological Macromolecules, 158, 977–984. https:// doi.org/10.1016/j.ijbiomac.2020.04.225 Hyson, D. A. (2011). A comprehensive review of apples and apple components and their relationship to human health. Advances in Nutrition, 2(5), 408– 420. https://doi.org/10.3945/an.111.000513 Inroga, M.M.A.S., da Silva, M.M., Cantillano, R.F.F., Paese, K., Guterres, S.S., Flôres, S.H., & de Oliveira Rios, A. (2021). Apples (Malus domestica Borkh.) minimally processed biofortified with nanoencapsulated β-carotene. Journal of Culinary Science & Technology, 21(3), 356–370. https://doi.org/10.1080/15428052.2021.1948479 Jalilian, H., Zarei, A., & Erfani-Moghadam, J. (2018). Phylogeny relationship among commercial and wild pear species based on morphological characteristics and SCoT molecular markers. Scientia Horticulturae, 235, 323–333. https://doi. org/10.1016/j.scienta.2018.03.020 Kaya, T., Balta, F., & Şensoy, S. (2015). Fruit quality parameters and molecular analysis of apple germplasm resources from Van Lake Basin, Turkey. Turkish Journal of Agriculture and Forestry, 39(6), 864–875. https://doi.org/10.3906/tar-1406-24 Khachtib, Y., Bouda, S., Ait Bella, Y., Zinelabidine, L.H., & Haddioui, A. (2024). Use of ISSR markers for assessing genetic diversity of apple (Malus × domestica) cultivars growing in Morocco. Vegetos, 37, 1619–1626. https://doi.org/10.1007/ s42535-023-00712-3 Liu, K., & Muse, S. (2005). PowerMarker: An integrated analysis environment for genetic marker analysis. Bioinformatics, 21(9), 2128–2129. Najar, Z.H., Zargar, S.A., Kashtwari, M., & Wani, A.A. (2023). Genetic diversity and population structure analysis of apple (Malus × domestica Borkh.) germplasm collected from North Kashmir, India, using SSR markers. Erwerbs-Obstbau, 65, 2207–2218. https://doi.org/10.1007/s10341-023- BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye 67 00974-w Öztürk, B., Keskin, S., Yıldız, K., Kaya, Ö., Kılıç, K., & Uçar, M. (2013). The effects of pre-harvest napthalene acetic acid and aminoethoxyvinylglycine treatments on storage performance of ‘Ak Sakı’ apple cultivar grown in Erzincan conditions. Journal of Agricultural Faculty of Gaziosmanpasa University, 30(1), 52–60. https://doi.org/10.13002/jafag185 Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155, 945–959. Sarkate, A., Banerjee, S., Mir, J.I., Roy, P., & Sircar, D. (2017). Antioxidant and cytotoxic activity of bioactive phenolic metabolites isolated from the yeast-extract treated cell culture of apple. Plant Cell, Tissue and Organ Culture, 130, 641–649. https:// doi.org/10.1007/s11240-017-1253-0 Sevindik, E., Uysal, H., & Murathan, Z.T. (2018). Genetic diversity based on ISSR markers of apple genotypes in Ardahan/Turkey. Notulae Scientia Biologicae, 10(4), 554–558. https://doi. org/10.15835/nsb10410347 Swofford, D. L. (2001). PAUP: Phylogenetic Analysis Using Parsimony (and other methods). Version 4.0b10 for 32-bit Microsoft Windows. Sinauer Associates, Sunderland, MA. Vivodík, M., Balážová, Ž., Chňapek, M., Hromadová, Z., Mikolášová, L., & Gálová, Z. (2023). Genetic relationship of soybean (Glycine max L.) genotypes using SCoT markers. Journal of Microbiology, Biotechnology and Food Sciences, 13(1), e9961. https://doi.org/10.55251/jmbfs.9961 Vivodík, M., Balážová, Ž., Gálová, Z., & Petrovičová, L. (2019). Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated castor (Ricinus communis L.) genotypes. Genetika, 51(1), 137–146. https://doi.org/10.2298/GENSR1901137V Wu, C., Li, T., Qi, J., Jiang, T., Xu, H., & Lei, H. (2020). Effects of lactic acid fermentation-based biotransformation on phenolic profiles, antioxidant capacity and flavor volatiles of apple juice. LWT, 122, 109064. https://doi.org/10.1016/j.lwt.2020.109064 Yang, S., Hao, N., Meng, Z., Li, Y., & Zhao, Z. (2021). Identification, comparison, and classification of volatile compounds in peels of 40 apple cultivars by HS–SPME with GC–MS. Foods, 10(5), 1051. https://doi.org/10.3390/foods10051051 Yang, T., Zhang, X., Huang, S., Gao, M., Li, T., & Zhang, S. (2023). Evaluating the genetic diversity of Erythropalum scandens using inter-simple sequence repeat markers. Genetic Resources and Crop Evolution, 70, 2377–2390. https://doi.org/10.1007/ s10722-023-01567-y Yilmaz, A., & Ciftci, V. (2021). Genetic relationships and diversity analysis in Turkish laurel (Laurus nobilis L.) germplasm using ISSR and SCoT markers. Molecular Biology Reports, 48(5), 4537– 4547. https://doi.org/10.1007/s11033-021-06474-y Zarei, A., & Erfani-Moghadam, J. (2021). SCoT markers provide insight into the genetic diversity, population structure and phylogenetic relationships among three Pistacia species of Iran. Genetic Resources and Crop Evolution, 68(4), 1625–1643. https://doi.org/10.1007/s10722-020-01091-3 BIOLOGICA NYSSANA ● 15 (2) December 2024: Sevindik et al. ● Genetic diversity and structure analysis of ‘Ak Sakı’ and ‘Kara Sakı’ apple cultivars growing in Erzincan/Türkiye 68