iPBS ve ISSR Markörleri Kullanarak In Vitro ve In Vivo Koşullarda Yetiştirilen Aronya Çeşitlerinin Genetik Karakterizasyonu

Öz

Aronya (Aronia spp.), yüksek antioksidan kapasitesi nedeniyle son yıllarda gıda ve sağlık sektörlerinde giderek artan bir öneme sahip, Rosaceae familyasına ait küçük bir meyve türüdür. Cins üç ana türle temsil edilir: siyah aronya (Aronia melanocarpa), kırmızı aronya (Aronia arbutifolia) ve bunların doğal melezi mor aronya (Aronia prunifolia). Bu çalışmada, dört ticari çeşidi ("Viking", "Nero", 'Aron' ve "Galicjanka") temsil eden sekiz genotip, ISSR ve iPBS moleküler belirteçleri kullanılarak hem in vivo hem de in vitro koşullar altında değerlendirilmiştir. Çeşit içi değişkenliği çeşitler arası değişkenlikten ayırma için, aynı çeşide ait in vitro ve in vivo örnekler tek bir örneklemde birleştirilmiştir (toplamda dört kesinti; sekiz genotip). ISSR primerleri monomorfik bant desenleri oluşturdu ve çeşitler arasındaki polimorfizmi tespit edilememiştir. Buna karşılık, beş iPBS primerinden ikisi polimorfik profiller üretti; iPBS2400 ve iPBS2378 primerleri sırasıyla %57,89 ve %62,50 polimorfizm oranları sergiledi. Bu sonuçlar, her iki primerin de analiz edilen aronya genotiplerindeki genetik varyasyonu tespit edebildiğini göstermektedir. Bununla birlikte, toplam amplifiye bant sayısı ve polimorfizm seviyeleri bakımından farklılık göstermişlerdir. iPBS2400 primeri için PIC, He, Ne ve Shannon çeşitlilik indeksi (I) değerleri sırasıyla 0,426, 0,487, 1,772 ve 0,614 olarak hesaplandı. iPBS2378 primeri için ise karşılık gelen değerler 0,431, 0,493, 1,809 ve 0,617 idi. Bu primerler için PIC değerleri 0,426 ile 0,431 arasında değişirken, He değerleri 0,487 ile 0,493 arasında ve Shannon bilgi indeksi (I) değerleri 0,614 ile 0,617 arasında değişmiştir. İncelenen popülasyonlar arasında en yüksek genetik çeşitlilik 'Aron' popülasyonunda gözlemlenirken, 'Viking' popülasyonu en düşük çeşitliliği göstermiştir. Jaccard benzerlik katsayısı kullanılarak oluşturulan dendrogram, 0,333 ile 1,000 arasında değişen genetik mesafeleri göstermiştir. Genel olarak, bulgular ISSR işaretleyicilerinin sınırlı ayırt edici güce sahip olduğunu, iPBS işaretleyicilerinin ise aronya çeşitleri arasındaki genetik değişkenliği tespit etmek için daha sağlam bir araç olduğunu göstermektedir. Ayrıca in vivo ve in vitro örnekler arasındaki yüksek benzerlik derecesi, doku kültürü kaynaklı materyalin genetik stabilitesini desteklemektedir.

Anahtar Kelimeler

• Aronya
• in vitro
• Genetik stabilite
• Moleküler belirteçler
• ISSR
• iPBS

Destekleyen Kurum

This research was carried out in Tekirdağ Namık Kemal University Agricultural Biotechnology Research and Production Unit (ZIRAATBIYOTEK) and Agricultural Biotechnology Department Molecular Genetics Laboratory

Proje Numarası

Proje numarası yoktur

Etik Beyan

There is no need to obtain permission from the ethics committee for this study.

Teşekkür

We want to thank Dr. Selen YATKIN and Ramazan ÇETİNKAYA for their support of this research.

Genetic Characterization of Aronia Varieties Cultivated In Vitro and In Vivo Conditions Using iPBS and ISSR Markers

Öz

Aronia (Aronia spp.) is a minor fruit species within the Rosaceae family that has gained increasing prominence in the food and health sectors in recent years, owing to its high antioxidant capacity. The genus is represented by three primary species: black chokeberry (Aronia melanocarpa), red chokeberry (Aronia arbutifolia), and their natural hybrid, purple chokeberry (Aronia prunifolia). In the present study, eight genotypes representing four commercial cultivars (‘Viking’, ‘Nero’, ‘Aron’, and ‘Galicjanka’) were evaluated for genetic stability under both in vivo and in vitro conditions using ISSR and iPBS molecular markers. To separate within-variety variability from between-variety variability, in vitro and in vivo samples of the same variety were combined into a single population (four populations in total; eight genotypes). ISSR primers generated monomorphic banding patterns and failed to detect polymorphism among the cultivars. In contrast, two of the five iPBS primers produced polymorphic profiles; the primers iPBS2400 and iPBS2378 exhibited polymorphism rates of 57.89% and 62.50%, respectively. These results demonstrate that both primers can detect genetic variation in the analyzed aronia genotypes. However, they differ in the total number of amplified bands and in the levels of polymorphism. For the iPBS2400 primer, the PIC, He, Ne, and Shannon’s diversity index (I) values were calculated as 0.426, 0.487, 1.772, and 0.614, respectively. For the iPBS2378 primer, the corresponding values were 0.431, 0.493, 1.809, and 0.617. The PIC values for these primers ranged from 0.426 to 0.431, while He values were between 0.487 and 0.493, and Shannon’s information index (I) values ranged from 0.614 to 0.617. Among the examined populations, the ‘Aron’ population showed the highest genetic diversity, whereas the ‘Viking’ population showed the lowest. The dendrogram generated using the Jaccard similarity coefficient indicated genetic distances ranging from 0.333 to 1.000. Overall, the findings demonstrate that ISSR markers exhibit limited discriminatory power, whereas iPBS markers are a more robust tool for detecting genetic variability among aronia cultivars. Additionally, the high degree of similarity between in vivo and in vitro samples supports the genetic stability of tissue culture-derived material.

Anahtar Kelimeler

• Aronia
• in vitro
• Genetic stability
• Molecular markers
• ISSR
• iPBS

Destekleyen Kurum

Bu araştırma Tekirdağ Namık Kemal Üniversitesi Tarımsal Biyoteknoloji Araştırma ve Geliştirme Merkezi'nde gerçekleştirilmiştir.

Proje Numarası

Proje numarası yoktur

Etik Beyan

Bu çalışma için etik kuruldan izin alınmasına gerek yoktur.

Teşekkür

Araştırmayı destekleyen Dr. Selen YATKIN ve Ramazan ÇETİNKAYA'ya teşekkür ederiz.

Kaynakça

1. Alan, F., Uzun, A. and Pinar, H. (2025). Genetic diversity and population structure of some blackberry genotypes from Türkiye using ISSR markers. Genetic Resources and Crop Evolution, 72: 9001-9019.
2. Aljuaid, B. S., Ismail, I.A., Attia, A. O. and Dessoky, E. D. S. (2022). Genetic stability of in vitro propagated grapevine (Vitis vinifera L.) cv. AlBayadi. Journal of Agriculture and Crops, 8(1): 12-19. https://doi.org/10.32861/jac.81.12.19
3. Asif, J. M. and Othman, R. Y. (2005). Characterization of Fusarium wilt–resistant and Fusarium wilt–susceptible somaclones of banana cultivar Rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers. Plant Molecular Biology Reporter, 23: 241-249.
4. Banach, M., Wiloch, M., Zawada, K., Cyplik, W. and Kujawski, W. (2020). Evaluation of antioxidant and anti-inflammatory activity of anthocyanin-rich water-soluble aronia dry extracts. Molecules, 25(18):4055. https://doi.org/10.3390/molecules25184055
5. Basturk, B. and Ozerson, Z. (2025). The role of functional foods in health policies: An evaluation through the case of Aronia. Journal of Original Studies, 6: e2832. https://doi.org/10.47243/jos.2832
6. Bednarek, P. T., Pachota, K. A., Dynkowska, W. M., Machczyńska, J. and Orłowska, R. (2021). Understanding in vitro tissue culture-induced variation phenomenon in microspore system. International Journal of Molecular Sciences, 14: 22 (14):7546. https://doi.org/10.3390/ijms22147546
7. Bilgen, B. B., Yuruk, B. and Nasirian, H. (2023). Brown rust resistance screening and molecular characterization of wheat cultivars by molecular markers. Turkish Journal of Agriculture and Forestry, 47(5): 787-800.
8. Cekic, C., Calis, O. and Erdem, O. (2018). Genetic diversity of wild raspberry genotypes (Rubus idaeus L.) in North Anatolia based on ISSR markers. Applied Ecology and Environmental Research, 16(5): 6835-6843.

9. Celebi-Toprak, F. and Alan, A. R. (2020). A Successful Micropropagation Protocol for Three Aronia (Aronia melanocarpa) cultivars. ISHS Acta Horticulturae 1285: XXX. International Horticultural Congress IHC2018: II International Symposium on Micropropagation and In Vitro Techniques. https://doi.org/10.17660/ActaHortic.2020.1285.27
10. Cilesiz, Y. (2025). Genetic relationship among grape (Vitis vinifera L.) genotypes grown in inner Anatolia region Turkey analyzed by ISSR and RAPD markers. Scientific Reports, 15: 32842 (2025). https://doi.org/10.1038/s41598-025-18463-3
11. Coskun, S. and Dumanoglu, H. (2025). Current knowledge on aronia systematics and cultivation. Fruit Science, 12(1): 1-6. https://doi.org/10.51532/meyve.1550561
12. Courtial, B., Feuerbach, F., Eberhard, S., Rohmer, L., Chiapello, H., Camilleri, C. and Lucas, H. (2001). Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana, and transposed copies integrate into genes. Molecular Genetics and Genomics, 265: 32-42.
13. Debergh, G. F. and Debergh, J. (1994). Micropropagation and the production of high-quality plants. Plant Cell, Tissue and Organ Culture, 37: 1–13.
14. Doyle, J. J. and Doyle, J. L. (1987). A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19: 11-15.
15. Doyle, J. J. and Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12: 13-15.
16. Engin, S. P. and Boz, Y. (2019). The latest developments at berry cultivation of Turkey. International Journal of Anatolia Agricultural Engineering, 1(5): 108-115.
17. Engin, S. P. and Mert, C. (2024). Determination of phenological and morphological properties of some aronia cultivars in Yalova Conditions. Bahçe, 53(1): 27-33.
18. Eskimez, I. and Polat, M. (2023). Effects of plant growth regulators on micropropagation of aronia (Aronia melanocarpa (Michx.). Fruit Science, 10: 92-99. https://doi.org/10.51532/meyve.1189817
19. George, E. F., Hall, M. A. and De Klerk, G. J. (2008). Plant Propagation by Tissue Culture. Springer Nature. https://doi.org/10.1007/978-1-4020-5005-3
20. Guclu, F., Koyuncu, F. and San, B. (2010). The in vitro micropropagation of some clonal cherry rootstocks. Suleyman Demirel University Journal of Natural and Applied Sciences 14(2): 144-147.
21. Haliloglu, K., Turkoglu, A., Ozturk, H. I., Ozkan, G., Elkoca, E. and Poczai, P. (2022). iPBS-Retrotransposon markers in the analysis of genetic diversity among common bean (Phaseolus vulgaris L.) germplasm from Türkiye. Genes, 13(7): 1147.
22. Hardin, J. W. (1973). The enigmatic chokeberries (Aronia, Rosaceae). Bulletin of the Torrey Botanical Club, 100(3): 178-184.
23. Hasnain, A., Naqvi, S. A. H., Ayesha, S. I., Khalid, F., Ellahi, M., Iqbal, S., Hassan, M. Z., Abbas, A., Adamski, R., Markowska, D., Baazeem, A., Mustafa, G., Moustafa, M., Hasan, M. E. and Abdelhamid, M. M. A. (2022) Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches. Frontiers in Plant Science, 13:1009395. https://doi.org/10.3389/fpls.2022.1009395
24. Hirochika, H., Sugimoto, K., Otsuki, Y., Tsugawa, H. and Kanda, M. (1996) Retrotransposons of Rice Involved in Mutations Induced by Tissue Culture. Proceedings of the National Academy of Sciences, 7783-7788, U. S. A.
25. Jaccard, P. (1901). Étude comparative de la distribution florale dans une portion des Alpes et des Jura. Bulletin de la Société vaudoise des sciences naturelles, 37: 547-579.
26. Jeppsson, N. (2000). The effect of cultivar and cracking on fruit quality in black chokeberry (Aronia melanocarpa) and hybrids between chokeberry and rowan (Sorbus). Gartenbauwissenschaft, 65(2): 93-98.
27. Kafkas, S., Ozkan, H. and Sutyemez, M. (2005). DNA polymorphism and assessment of genetic relationships in walnut genotypes based on AFLP and SAMPL markers. Journal of the American Society for Horticultural Science, 130(4): 585-590.
28. Kalendar, R. and Schulman, A. H. (2014). Transposon-Based Tagging: IRAP, REMAP, and iPBS. In: Molecular Plant Taxonomy: Methods and Protocols (Vol. 1115), Ed(s): Besse, P., pp. 233-255. Totowa, NJ: Humana Press, U. S. A.
29. Kalendar, R., Antonius, K., Smýkal, P. and Schulman, A. H. (2010). iPBS: a universal method for DNA fingerprinting and retrotransposon isolation. Theoretical and Applied Genetics, 121(8): 1419-1430.
30. Kaya, Ç. (2025). The biochemical composition, health benefits, and potential use of the aronia (Aronia melanocarpa L.) in the food industry. Nevşehir Journal of Science and Technology, 14(1): 1-15.
31. Larkin, P. J. and Scowcroft, W. R. (1981). Somaclonal variation A novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics, 60(4): 197-214.
32. Leonard, P. J., Brand, M. H., Connolly, B. A. and Obae, S. G. (2013). Investigation of the origin of Aronia mitschurinii using amplified fragment length polymorphism analysis. Horticulture Science, 48(5): 520-524.
33. Metiner, E. E. and Ersus, S. (2023). The effects of different drying methods on the quality of Aronia (Aronia melanocarpa) fruit and its dried powder. Journal of Agriculture Faculty of Ege University, 60(2): 353. https://doi.org/10.17100/nevbiltek.1626095
34. Miguel, C. and Marum, L. (2011). An epigenetic view of plant cells cultured in vitro: Somaclonal variation and beyond. Journal of Experimental Botany, 62(11): 3713-3725.
35. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15(3): 473-497.
36. Obae, S. G., Brand, M. H., Connolly, B. A., Beasley, R. R. and Lance, S. L. (2017). Microsatellite markers for Aronia melanocarpa (Black Chokeberry) and their transferability to other aronia species. Horticulture science, 52(1): 20-23.
37. Oszmianski, J. and Wojdylo, A. (2005). Aronia melanocarpa phenolics and their antioxidant activity. European Food Research and Technology, 221: 809–813. https://doi.org/10.1007/s00217-005-0002-5
38. Ozbucak, T. and Gumus, A. F. (2024). Determination of ecological and phytochemical variations of aronia fruit. Turkish Journal of Agricultural and Natural Sciences, 11(4): 1035-1045. https://doi.org/10.30910/turkjans.1499475
39. Ozkan, S. and Bilgen, B. B. (2019). Genetic characterization of sainfoin (Onobrychis viciifolia) varieties and populations using microsatellite markers. Journal of Tekirdag Agricultural Faculty, 16(1), 45-54.
40. Parab, A. R., Lynn, C. B. and Subramaniam, S. (2021). Assessment of genetic stability on in vitro and ex vitro plants of Ficus carica var. black jack using ISSR and DAMD markers. Molecular Biology Reports, 48(11): 7223-7231.
41. Peakall, R. and Smouse, P. E. (2012). GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research an update. Bioinformatics, 28(19): 2537-2539.
42. Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V., Vanderplas, J., Passos, A., Cournapeau, D., Brucher, M., Perrot, M. and Duchesnay, É. (2011). Scikit-learn: Machine learning in Python. The Journal of Machine Learning Research, 12: 2825-2830.
43. Petrova, M., Bonchev, G., Dimitrova, L., Dimitrova, M. and Vassilevska-Ivanova, R. (2023). In vitro cultivation of saffron (Crocus sativus L.) and assessment of genetic homogeneity using iPBS markers. Comptes rendus de l’Acad ́emie bulgare des Sciences, 76(11): 1670–1678. https://doi.org/10.7546/CRABS.2023.11.04.
44. Pour, A. H., Ozkan, G., Nalci, O. and Haliloglu, K. (2019). Estimation of genomic instability and DNA methylation due to aluminum (Al) stress in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS analyses. Turkish Journal of Botany, 43(1): 27-37.
45. Rohela, G. K., Jogam, P., Saini, P., Sandhya, D., Peddaboina, V. and Shekhawat, M. S. (2022). Assessing the Genetic Stability of In Vitro Raised Plants. In: Commercial Scale Tissue Culture for Horticulture and Plantation Crops. Ed(s): Gupta, S. and Chaturvedi, P. Springer, Singapore. https://doi.org/10.1007/978-981-19-0055-6_11
46. Roldán-Ruiz, I., Dendauw, J., Van Bockstaele, E., Depicker, A. and De Loose, M. (2000). AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.). Molecular Breeding, 6(2): 125-134.
47. Saha, S., Adhikari, S., Dey, T. and Ghosh, P. (2016). RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1. Meta Gene, 7, 7-15.
48. Sahin, A. and Erdogan, Ü. (2022). Aronia (Aronia melanocarpa Michx Elliot) production and evaluation methods in the world and Turkey. Turkish Journal of Agriculture - Food Science and Technology, 10(1), 81-85.
49. Sanlier, N. and Cinar, E. N. (2025). Aronia berry (Aronia melanocarpa L.) as a bioactive treasure: Insights into its health-promoting effects. Journal of Functional Foods,135, 107116. https://doi.org/10.1016/j.jff.2025.107116 Shahin, L., Mohamed, M. H. and El-Mahrouk, M. E. (2019). Aronia (Chokeberry): an underutilized, highly nutraceutical plant. Journal of Applied Botany and Food Quality, 92: 204-213.
50. Siringam, T. and Vanijajiva, O. (2023). The effect of plant growth regulators on micropropagation of Melientha suavis Pierre. and assessment of genetic fidelity of regenerants based on iPBS and SRAP markers. Biodiversitas Journal of Biological Diversity, 24(9): 5325-5334.
51. Smolik, M., Ochmian, I. D. and Smolik, B. (2011). RAPD and ISSR methods used for fingerprinting selected, closely related cultivars of Aronia melanocarpa. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 39(2): 276-284.
52. TAGEM (2022). T.C. Ministry of Agriculture and Forestry, General Directorate of Plant Production, Aronia Feasibility Report and Investor Guide, Education and Publication Department, Ed: Toprak, E., Türktemel, E. Ankara, Türkiye. (In Turkish)
53. Tikendra, L., Dey, A., Jamir, I., Sahoo, M. R. and Nongdam, P. (2022). Cytokinin influence on in vitro shoot induction and genetic stability assessment of Dendrocalamus latiflorus Munro: A commercially important bamboo in Manipur, North-East India. Vegetos, 35(4): 1085-1095.
54. Wessler, S.R., Bureau, T.E. and White, S.E. (1995) LTR-retrotransposons and MITES important players in the evolution of plant genomes. Current Opinion in Genetics and Development, 5: 814-821.
55. Xu, J., Li, F., Zheng, M., Sheng, L., Shi, D. and Song, K. A. (2024). Comprehensive Review of the Functional Potential and Sustainable Applications of Aronia melanocarpa in the Food Industry. Plants (Basel), 20:13(24):3557. https://doi.org/10.3390/plants13243557
56. Yilmaz, E. C., Bilgen, B. B. and Tuna, M. (2023). Evaluation of the genetic structure of some accessions belonging to Onobrychis spp. using microsatellite DNA markers. Journal of Tekirdag Agricultural Faculty, 20(2): 399-409.
57. Yin, W. Y., Cui, X. Q., Zhao, Q. J., Deng, J. L., Huang, C. Y., Zhang, Z. B. and Li, J. W. (2025). Genetic relationship analysis and fingerprint construction of some species in the genus Vanda (Orchidaceae) by interprimer binding site (iPBS) markers. Genetic Resources and Crop Evolution, 72(1): 935-946.
58. Zietkiewicz, E., Rafalski, A. and Labuda, D. (1994). Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 20(2): 176-183.