Distinguishing the Protected Designation of Origin Apricot (Prunus armeniaca L. cv. Şalak) from Closely Related Cultivars by High Resolution Melting

Şalak kayısı çeşidi (Prunus armeniaca cv. Şalak) Aras Havzası’nda üretimi yapılan ve coğrafi işarete sahip bir kayısı çeşididir. Tescilli ismi Iğdır Kayısısı olarak belirlenmiştir. Özellikle gıda aldatmacasına maruz kalma potansiyeli olan özel gıda ürünlerinin DNA temelli yöntemler ile tanımlanması güvenilir ve görece ucuz maliyetlidir. Bu çalışmanın amacı Şalak kayısı çeşidinin, yakın ilişkili kayısı çeşitlerden Yüksek Çözünürlüklü Erime (HRM) yöntemi kullanarak ayırt edilmesi için bir protokol geliştirmektir. Çalışmada, daha önceden Prunus türleri için doğrulanmış 12 adet SSR belirtecinin, Türkiye’de üretimi yapılan Şalak (AS), Hasanbey (HB), Hacıhaliloğlu (HH), Kabaaşı (KB) ve Şekerpare (SK) kayısı çeşitlerini ayırt etme başarısı test edilmiştir. Çalışmada ayrıca HRM verilerinden Temel Bileşenler Analizi yapılabilmesi için R yazılımında kullanılabilecek bir komut dosyası oluşturulmuştur. Kılcal elektroforez ile doğrulanmış 12 SSR belirtecinden 11 tanesinin, her kayısı çeşidi için farklı fragmentler çoğalttığı tespit edilmiştir. Temel Bileşenler Analizi ile desteklenmiş HRM sonuçlarının Şalak kayısı çeşidini diğer çeşitlerden başarılı bir şekilde ayırt ettiği belirlenmiştir.

Anahtar Kelimeler:

Genotipleme, mikrosatelitler, genetik doğrulama, gıda aldatmacası, SSR-HRM

Distinguishing the Protected Designation of Origin Apricot (Prunus armeniaca L. cv. Şalak) from Closely Related Cultivars by High Resolution Melting

The apricot cultivar Prunus armeniaca cv. Şalak (registered as “Iğdır Kayısısı”) is a Protected Designation of Origin (PDO) apricot and produced in Aras Basin (Iğdır, Turkey) region. Authenticating the special products, which has adulteration potential, by DNA based methods is reliable and cost-effective. The aim of the current study is to distinguish the PDO apricot from closely related cultivars by High Resolution Melting. We tested 12 SSR markers previously validated for Prunus spp. by means of distinguishing the ability of five closely related apricot cultivars that are Şalak (AS), Hasanbey (HB), Hacıhaliloğlu (HH), Kabaaşı (KB), and Şekerpare (SK) produced in Turkey. Capillary electrophoresis validation showed 11 of 12 markers amplified unique fragments for the cultivars. HRM analysis combined with the Principal Component Analysis (PCA) successfully distinguished the PDO AS from closely related cultivars. HRM analysis combined with PCA can be a cost-effective and reliable authenticating method for PDO food products.

Keywords:

Genotyping, Microsatellites, Authenticity, Food adulteration, SSR-HRM,

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Aydın, A., Ince, A. G., Uygur Gocer, E., & Karaca, M. (2018). Single cotton seed DNA extraction without the use of enzymes and liquid nitrogen. Fresenius Environmental Bulletin, 27(10), 6722-6726.
Chou, L., Huang, S.J., Hsieh, C., Lu, M.T., Song, C.W., & Hsu, F.C. (2020). A High Resolution Melting Analysis-Based Genotyping Toolkit for the Peach (Prunus persica) Chilling Requirement. International journal of molecular sciences, 21(4), 1543. https://doi.org/10.3390/ijms21041543
Cipriani, G., Lot, G., Huang, W.G., Marrazzo, M.T., Peterlunger, E., & Testolin, R. (1999). AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theoretical and Applied Genetics, 99(1), 65-72. https://doi.org/10.1007/s001220051209
Downey, S.L., & Iezzoni, A.F. (2000). Polymorphic DNA markers in black cherry (Prunus serotina) are identified using sequences from sweet cherry, peach, and sour cherry. Journal of the American Society for Horticultural Science, 125(1), 76-80. https://doi.org/10.21273/JASHS.125.1.76
Druml, B., & Cichna-Markl, M. (2014). High resolution melting (HRM) analysis of DNA–Its role and potential in food analysis. Food chemistry, 158, 245-254.
Ercişli, S. (2004). A short review of the fruit germplasm resources of Turkey. Genetic Resources and Crop Evolution, 51(4), 419-435. https://doi.org/10.1023/B:GRES.0000023458.60138.79
FAOSTAT. (2020). Retrieved from http://www.fao.org/state-of-food-security-nutrition
Ganopoulos, I., Argiriou, A., & Tsaftaris, A. (2011). Microsatellite high resolution melting (SSR-HRM) analysis for authenticity testing of protected designation of origin (PDO) sweet cherry products. Food Control, 22(3-4), 532-541. https://doi.org/10.1016/j.foodcont.2010.09.040
Hollingsworth, P.M., Graham, S.W., & Little, D.P. (2011). Choosing and using a plant DNA barcode. PloS one, 6(5), e19254.
Lahaye, R., Van der Bank, M., Bogarin, D., Warner, J., Pupulin, F., Gigot, G., ... & Savolainen, V. (2008). DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences, 105(8), 2923-2928.
Li, J., Xiong, C., He, X., Lu, Z., Zhang, X., Chen, X., & Sun, W. (2018). Using SSR-HRM to identify closely related species in herbal medicine products: A case study on licorice. Frontiers in pharmacology, 9, 407. https://doi.org/10.3389/fphar.2018.00407
Osathanunkul, M., & Madesis, P. (2019). Bar-HRM: a reliable and fast method for species identification of ginseng (Panax ginseng, Panax notoginseng, Talinum paniculatum and Phytolacca americana). PeerJ, 7, e7660. https://doi.org/10.7717/peerj.7660
Passaro, M., Geuna, F., Bassi, D., & Cirilli, M. (2017). Development of a high-resolution melting approach for reliable and cost-effective genotyping of PPVres locus in apricot (P. armeniaca). Molecular Breeding, 37(6), 74. https://doi.org/10.1007/s11032-017-0666-0
Pentinsaari, M., Salmela, H., Mutanen, M., & Roslin, T. (2016). Molecular evolution of a widely-adopted taxonomic marker (COI) across the animal tree of life. Scientific reports, 6(1), 1-12.
RStudio, Team. (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA. http://www.rstudio.com/
Scrucca, L., Fop, M., Murphy, T.B., & Raftery, A.E. (2016). mclust 5: clustering, classification and density estimation using Gaussian finite mixture models. The R journal, 8(1), 289.
Sosinski, B., Gannavarapu, M., Hager, L.D., Beck, L.E., King, G.J., Ryder, C. D., ... & Abbott, A.G. (2000). Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]. Theoretical and Applied Genetics, 101(3), 421-428. https://doi.org/10.1007/s001220051499
Testolin, R., Marrazzo, T., Cipriani, G., Quarta, R., Verde, I., Dettori, M.T., ... & Sansavini, S. (2000). Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome, 43(3), 512-520. https://doi.org/10.1139/g00-010
Tindall, E.A., Petersen, D.C., Woodbridge, P., Schipany, K., & Hayes, V.M. (2009). Assessing high‐resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments. Human mutation, 30(6), 876-883.
Tuler, A.C., Carrijo, T.T., Nóia, L.R., Ferreira, A., Peixoto, A.L., & da Silva Ferreira, M.F. (2015). SSR markers: a tool for species identification in Psidium (Myrtaceae). Molecular Biology Reports, 42(11), 1501-1513.
TÜİK. (2020). Retrieved from http://www.turkstat.gov.tr
Turkish Apricot Research Institute (2019). Retrieved from https://arastirma.tarimorman.gov.tr/kayisi/Menus/47/Registered-Cultivars
Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org
Wittwer, C.T. (2009). High‐resolution DNA melting analysis: advancements and limitations. Human mutation, 30(6), 857-859.