Sains Malaysiana 54(3)(2025): 797-814

http://doi.org/10.17576/jsm-2025-5403-15

Exploring the Diversity of Shan Tea in Vietnam through SSR Markers, Morphological Traits, and Biochemical Content

(Meneroka Kepelbagaian Teh Shan di Vietnam melalui Penanda SSR, Ciri Morfologi dan Kandungan Biokimia)

KHUYNH THE BUI¹, THAI HOANG DINH¹, NGOC-THANG VU¹, VAN THU DANG², THI VIET HA DO³, XINGHUI LI⁴ & XUAN HOANG TRAN^3,*

¹Faculty of Agronomy, Vietnam National University of Agriculture, Hanoi 131000, Vietnam

²The Vietnamese Tea Science – Technology Association, Phu Tho 293823, Vietnam

³Tea Research and Development Center, Northern Mountainous Agriculture and Forestry Science Institute, Phu Tho 293823, Vietnam

⁴Tea Research Institute, Nanjing Agricultural University, Nanjing 210095, China

Diserahkan: 7 Jun 2024/Diterima: 14 Disember 2024

Abstract

Shan tea (Camellia sinensis var. Shan), a variety native to the mountainous regions, used to prepare ‘Che Shan Tuyet’- a high quality standard tea product with cultural significance, is considered the most precious tea in Vietnam. However, little is known about its genetic diversity, composition, and variation in biochemical content across the central growing regions until now. Here, the use of 30 pairs of SSR primers selected based on their proven high utility in previous studies in tea with high polymorphisms showed that the Shan tea population exhibit rich genetic diversity, with gene diversity (H) varying from 0.47 to 0.82 and the polymorphic information content (PIC) ranging from 0.47 to 0.84. The cluster (UPGMA-based) analysis showed that 60 Shan tea accessions can be categorized into three groups with different origins. Biochemical profiles including tannin and catechins were observed to have high variation by harvest season of which the highest content was recorded during summer. Though the variation in biochemical profiles was not considerably significant among the three groups of origin, accessions from Suoi Giang (Yen Bai) significantly had lower content of tannin, EC, ECG, and EGC compared to Shan tea in Cao Bo (Ha Giang). In addition, morphology-based PCA also showed that it is practical to discriminate three groups of different origins, with the essential traits being leaf blade width, pericarp thickness, leaf area (PC1), fruit length, and fine pluck weight (PC2). The clustering of 60 Shan accessions based on morphological traits also showed consistent results with the genetic diversity analysis conducted using SSR, where accessions from Suoi Giang and Cao Bo had higher similarity levels than accessions from Tua Chua.

Keywords: Biochemical profile; genetic diversity; morphology; Shan Tea germplasm; SSR marker

Abstrak

Teh Shan (Camellia sinensis var. Shan), varieti asal di kawasan pergunungan, digunakan untuk menyediakan ‘Che Shan Tuyet’ - produk teh berkualiti tinggi dengan kepentingan budaya, dianggap sebagai teh paling berharga di Vietnam. Walau bagaimanapun, sedikit yang diketahui tentang kepelbagaian genetiknya, komposisi dan variasi kandungan biokimia di seluruh rantau kawasan tengah berkembang sehingga kini. Di sini, penggunaan 30 pasang primer SSR yang dipilih berdasarkan utiliti tinggi yang terbukti dalam kajian teh terdahulu dengan polimorfisme tinggi menunjukkan bahawa populasi teh Shan menunjukkan kepelbagaian genetik yang kaya dengan kepelbagaian gen (H) berbeza dari 0.47 hingga 0.82 dan kandungan maklumat polimorfik (PIC) antara 0.47 hingga 0.84. Analisis kelompok (berasaskan UPGMA) menunjukkan bahawa 60 aksesi teh Shan boleh dikategorikan kepada tiga kumpulan dengan asal usul yang berbeza. Profil biokimia termasuk tanin dan katekin diperhatikan mempunyai variasi yang tinggi mengikut musim menuai yang mana kandungan tertinggi direkodkan semasa musim panas. Walaupun variasi dalam profil biokimia tidak begitu ketara dalam kalangan tiga kumpulan asal, aksesi daripada Suoi Giang (Yen Bai) secara signifikan mempunyai kandungan tanin, EC, ECG dan EGC yang lebih rendah berbanding teh Shan di Cao Bo (Ha Giang). Di samping itu, PCA berasaskan morfologi juga menunjukkan bahawa adalah praktikal untuk mendiskriminasi tiga kumpulan asal yang berbeza, dengan ciri penting ialah lebar helai daun, ketebalan perikarpa, luas daun (PC1), panjang buah dan petik halus berat (PC2). Pengelompokan 60 aksesi Shan berdasarkan ciri morfologi juga menunjukkan hasil yang tekal dengan analisis kepelbagaian genetik yang dijalankan menggunakan SSR, dengan aksesi daripada Suoi Giang dan Cao Bo mempunyai tahap persamaan yang lebih tinggi daripada aksesi daripada Tua Chua.

Kata kunci: Germplasma Teh Shan; kepelbagaian genetik; morfologi; penanda SSR; profil biokimia

RUJUKAN

Ahmed, S., Griffin, T.S., Kraner, D., Schaffner, M.K., Sharma, D., Hazel, M., Leitch, A.R., Orians, C.M., Han, W., Stepp, J.R., Robbat, A., Matyas, C., Long, C., Xue, D., Houser, R.F. & Cash, S.B. 2019. Environmental factors variably impact tea secondary metabolites in the context of climate change. Front in Plant Sci. 10: 939.

Bali, S., Raina, S.N., Bhat, V., Aggarwal, R.K. & Goel, S. 2013. Development of a set of genomic microsatellite markers in tea (Camellia L.) (Camelliaceae). Molecular Breeding 32: 735-741.

Chen, L. & Yamaguchi, S. 2002. Genetic diversity and phylogeny of the tea plant (Camellia sinensis) and its related species and varieties in the section Thea genus Camellia determined by randomly amplified polymorphic DNA analysis. Journal of Horticultural Science and Biotechnology 77(6): 729-732. https://doi.org/10.1080/14620316.2002.11511564

Chinese National Standard. 2008. GB/T 8313-2008. Determination of total polyphenols and catechins content in tea.

Clarke, C., Richter, B.S. & Rathinasabapathi, B. 2023. Genetic and morphological characterization of United States tea (Camellia sinensis): Insights into crop history, breeding strategies, and regional adaptability. Front. in Plant Sci. 14: 1149682. https://doi.org/10.3389/fpls.2023.1149682

Deka, H., Barman, T., Dutta, J., Devi, A., Tamuly, P., Paul, R.K. & Karak, T. 2021. Catechin and caffeine content of tea (Camellia sinensis L.) leaf significantly differ with seasonal variation: A study on popular cultivars in North East India. Journal of Food Composition and Analysis 96: 103684. https://doi.org/10.1016/j.jfca.2020.103684

Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19(1): 11-15.

Fang, W., Cheng, H., Duan, Y., Jiang, X. & Li, X. 2011. Genetic diversity and relationship of clonal tea (Camellia sinensis) cultivars in China as revealed by SSR markers. Plant Syst. Evol. 298: 469-483. https://doi.org/10.1007/s00606-011-0559-3

Freeman, S., West, J., James, C., Lea, V. & Mayes, S. 2004. Isolation and characterization of highly polymorphic microsatellites in tea (Camellia sinensis). Molecular Ecology Notes 4: 324-326. https://doi.org/10.1111/j.1471-8286.2004.00682.x

Gong, A.D., Lian, S.B., Wu, N.N., Zhou, Y.J., Zhao, S.Q., Zhang, L.M., Cheng, L. & Yuan, H.Y. 2020. Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons. BMC Plant Biology 20: 294. https://doi.org/10.1186/s12870-020-02443-y

Guo, R., Xia, X., Chen, J., An, Y., Mi, X., Li, R., Zhang, C., Chen, M., Wei, C. & Liu, S. 2021. Genetic relationship analysis and molecular fingerprint identification of the tea germplasms from Guangxi Province, China. Breeding Science 71(5): 584-593.

Huang, D.J., Wang, Y.P., Chen, X., Wu, J., Wang, H.J., Tan, R.R., Jiao, L. & Mao, Y.X. 2022. Application of tea-specific fertilizer combined with organic fertilizer improves aroma of green tea. Horticulturae 8(10): 950.

Jhou, H.C., Chen, C.I., Shen, J.Y., Wang, C.T. & Chang, F.C. 2025. Influence of natural light intensities on growth and content of catechins and caffeine in Camellia formosensis leaves and tea infusion. BioResources 20(1): 877-887.

Kottawa-Arachchi, J.D., Gunasekare, M.K. & Ranatunga, M.A. 2019. Biochemical diversity of global tea [Camellia sinensis (L.) O. Kuntze] germplasm and its exploitation: A review. Genetic Resources and Crop Evolution 66: 259-273. https://doi.org/10.1007/s10722-018-0698-2

Labate, J.A., Lamkey, K.R., Mitchell, S.E., Kresovich, S., Sullivan, H. & Smith, J.S.C. 2023. Molecular and historical aspects of Corn Belt dent diversity. Crop Science 43(1): 80-91. https://doi.org/10.2135/cropsci2003.8000

Le, T.L., Jang, D.C., Pham, L.A.M., Le, H.D., Nguyen, T.H., Nguyen, V.K., Nguyen, T-H., Hoang, T-N., Vu, D-T., Bui, T-K., Vu, T-T-H., Dinh, T-H., Tran, A-T., Phan, T-T-H., Tong, V-G., Le, T-T-C. & Vu, N-T. 2023. Genetic diversity and population structure of Canna edulis accessions in Vietnam revealed by ISSR markers. Vegetos 37: 1009-1018. https://doi.org/10.1007/s42535-023-00633-1

Liu, K.J. & Muse, S.V. 2005. PowerMarker: Integrated analysis environment for genetic marker data. Bioinformatics 21: 2121-2129. https://doi.org/10.1093/bioinformatics/bti282

Liu, S., Liu, H., Wu, A., Hou, Y., An, Y. & Wei, C. 2017. Construction of fingerprinting for tea plant (Camellia sinensis) accessions using new genomic SSR markers. Mol. Breeding 37(8): 93. https://doi.org/10.1007/s11032-017-0692-y

Ma, J.Q., Zhou, Y.H., Ma, C.L., Yao, M.Z., Jin, J.Q., Wang, X.C. & Chen, L. 2010. Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant, Camellia sinensis (Theaceae). American Journal of Botany 97(12): e153-e156. https://doi.org/10.3732/ajb.1000376

Mu, S.Q., Gu, X.F., Zhang, S.P., Wang, X.W. & Wang, Y. 2003. Genetic diversity of Cucumber (Cucumis sativus L.) germplasm by SSR.  Acta Horticulture Sinica 35(9): 1323-1330.

Nadeem, M.A., Nawaz, M.A., Shahid, M.Q., Doğan, Y., Comertpay, G., Yildiz, M., Hatipoğlu, R., Ahmad, F., Alsaleh, A., Labhane, N., Özkan, H., Chung, G. & Baloch, F.S. 2018. DNA molecular markers in plant breeding: Current status and recent advancements in genomic selection and genome editing. Biotechnology & Biotechnological Equipment 32(2): 261-285.

Ni, J., Colowit, PM. & Mackill, D.J. 2002. Evaluation of genetic diversity in rice subspecies using microsatellite markers. Crop Science 42(2): 601-607. https://doi.org/10.2135/cropsci2002.6010

Nguyen, K.O.T., Nguyen, P.L., Le, H.L. & Le, H.T. 2022. Discriminative chemical profiles of Shan Tuyet Tea (Camellia sinensis var. Shan) and Sinensis Tea (Camellia sinensis var. sinensis) collected in Ta Xua, Son La, Vietnam and their correlation with antioxidant activity. Natural Product Communications 17: 9. https://doi.org/10.1177/1934578X221128410

Paiva, L., Lima, E., Motta, M., Marcone, M. & Baptista, J. 2021. Influence of seasonal and yearly variation on phenolic profiles, caffeine, and antioxidant activities of green tea (Camellia sinensis (L.) Kuntze) from Azores. Applied Sciences 11(16): 7439.

Pandolfi, C., Mugnai, S., Azzarello, E., Bergamasco, S., Masi, E. & Mancuso, S. 2009. Artificial neural networks as a tool for plant identification: A case study on Vietnamese tea accessions. Euphytica 166: 411-421. https://doi.org/10.1007/s10681-008-9828-9

Parmar, R., Seth, R. & Sharma, R.K. 2022. Genome-wide identification and characterization of functionally relevant microsatellite markers from transcription factor genes of Tea (Camellia sinensis (L.) O. Kuntze). Scientific Reports 12(1): 201.

Phong, N.H., Pongnak, W., Soytong, K., Poeaim, S. & Poeaim, A. 2016. Diversity of Tea (Camellia sinensis) grown in Vietnam based on morphological characteristics and Inter-primer Binding Sites (iPBS) Marker. International Journal of Agriculture & Biology 18(2): 385-392.

Powell, W., Machray, G.C. & Provan, J. 1996. Polymorphism revealed by simple sequence repeats. Trends in Plant Science 1(7): 215-222. https://doi.org/10.1016/1360-1385(96)86898-1

Rajkumar, S., Karthigeyan, S., Sud, R.K., Rajkumar, R., Muraleedaran, N., Das, S.C., Hazarika, M. & Ahuja, P.S. 2010. Genetic diversity of Indian tea (Camellia sinensis (L.) Kuntze) germplasm detected using morphological characteristics. Journal Cell and Plant Science 1(1): 13-22.

Rohlf, F.J. 2000. Statistical power comparisons among alternative morphometric methods.  Am. J. Phys. Anthropol. 111: 463-478.

Saravanan, M., Maria, J.K.M., Raj Kumar, R., Pius, P.K. & Sasikumar, R. 2005. Genetic diversity of UPASI tea cones (Camellia sinensis (L.) O. Kuntze) on the basis of total catechins and their fractions. Phytochemistry 66: 561-565. https://doi.org/10.1016/j.phytochem.2004.06.024

Sealy, J.B. 1958. A Revision of the Genus Camellia. London: Royal Horticultural Society. p. 239.

Sharma, R.K., Bhardwaj, P., Negi, R., Mohapatra, T. & Ahuja, P.S. 2009. Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.). BMC Plant Biology 9: 1-24.

Sharma, V., Joshi, R. & Gulati, A. 2011. Seasonal clonal variations and effects of stresses on quality chemicals and prephenate dehydratase enzyme activity in tea (Camellia sinensis). European Food Research and Technology 232: 307-317.

Takeda, Y. 2000. History and development in Japanese tea breeding. In: Tea Culture, Tea Food Industry and Tea Breeding in Korea, China and Japan. The Korea Tea Society, Korea Park Y.G. and Shin D.I. (eds). pp. 139-158.

Tan, L.Q., Wang, L.Y., Wei, K., Zhang, C.C., Wu, L.Y., Qi, G.N. & Liang, J.B. 2013. Floral transcriptome sequencing for SSR marker development and linkage map construction in the tea plant (Camellia sinensis). PLoS ONE 8(11): e81611.

Taniguchi, F., Furukawa, K., Ota-Metoku, S., Yamaguchi, N., Ujihara, T., Kono, I. & Tanaka, J. 2012. Construction of a high-density reference linkage map of tea (Camellia sinensis). Breeding Science 62(3): 263-273.

Tao, L.L., Ting, Y.J., Chen, H.R., Wen, H.L., Hui, X., Luo, L.Y., Huang, K.L., Zhu, J.Y., Liu, S.R. & Wei, C.L. 2023. Core collection construction of tea plant germplasm in Anhui Province based on genetic diversity analysis using simple sequence repeat markers. Journal of Integrative Agriculture 22(9): 2719-2728. https://doi.org/10.1016/j.jia.2023.07.020

Tian, X., Chen, S., Zhong, Q., Wang, J., Chen, J., Chen, L., Moon, D. & Ma, J. 2024. Widely targeted metabolomics analysis reveals the effect of cultivation altitude on tea metabolites. Agronomy 14(4): 812. https://doi.org/10.3390/agronomy14040812

Tounekti, T., Joubert, E., Hernández, I. & Munné-Bosch, S. 2013. Improving the polyphenol content of tea. Critical Reviews in Plant Sciences 32(3): 192-215.

Vo, T.D. 2007. Assessing genetic diversity in Vietnam tea [Camellia sinensis (L.) O. Kuntze] using morphology, inter-simple sequence repeat (ISSR) and microsatellite (SSR) markers. PhD Dissertation. Georg-August University Goettingen (Unpublished).

Wambulwa, M.C., Meegahakumbura, M.K., Kamunya, S. & Wachira, F.N. 2021. From the wild to the cup: Tracking footprints of the tea species in time and space. Frontiers in Nutrition 8: 706770. https://doi.org/10.3389/fnut.2021.706770

Wang, R.J., Gao, X.F., Kong, X.R. & Yang, J. 2016. An efficient identification strategy of clonal tea cultivars using long-core motif SSR markers. SpringerPlus 5(1): 1152.

Wight, W. 1962. Tea classification revised. Curr. Sci. 31: 298-299.

Yang, Y., Liu, Z., Zhao, Y., Liang, G.Q. & Zhao, X. 2009. Genetic diversity and relationship of Huangjincha cultivar based on EST-SSR markers. J. Tea Sci. 29: 236-242 (in Chinese with English abstract).

Yao, L., Caffin, N., D'Arcy, B., Jiang, Y., Shi, J., Singanusong, R., Liu, X., Datta, N., Kakuda, Y. & Xu, Y. 2005. Seasonal variations of phenolic compounds in Australia-grown tea (Camellia sinensis). J. Agric. Food Chem. 53(16): 6477-6483.

Yao, M.Z., Chen, L., Wang, X.C., Zhao, L.P. & Yang, Y.J. 2007. Genetic diversity and relationship of clonal tea cultivars in China revealed by ISSR markers. Acta Agron. Sin. 33: 598-604 (in Chinese with English abstract).

Ye, F., Guo, X., Li, B., Chen, H. & Qiao, X. 2022. Characterization of effects of different tea harvesting seasons on quality components, color and sensory quality of “Yinghong 9” and “Huangyu” large-leaf-variety black tea. Molecules 27(24): 8720. https://doi.org/10.3390/molecules27248720

Zagoskina, N.V., Alyavina, A.K., Gladyshko, T.O., Lapshin, P.V., Egorova, E.A. & Bukhov, N.G. 2005. Ultraviolet rays promote development of photosystem II photochemical activity and accumulation of phenolic compounds in the tea callus culture (Camellia sinensis). Russian Journal of Plant Physiology 52: 731-739.

Zhang, Q., Zhang, Y., Xie, J., Ye, J., Pang, X. & Jia, X. 2022. Differences in the analysis of the quality indexes and characteristic amino acids of the different grades of Wuyi Shuixian (Camellia sinensis) tea. Food Science and Technology 42: e66122.

Zhao, Z., Song, Q., Bai, D., Niu, S., He, Y., Qiao, D., Chen, Z., Li, C., Luo, J. & Li, F. 2022. Population structure analysis to explore genetic diversity and geographical distribution characteristics of cultivated-type tea plant in Guizhou Plateau. BMC Plant Biology 22(1): 55.

Zheng, X.Q., Jin, J., Chen, H., Du, Y.Y., Ye, J.H., Lu, J.L., Lin, C., Dong, J.J., Sun, Q.L., Wu, L.Y. & Liang, Y.R. 2008. Effect of ultraviolet B irradiation on accumulation of catechins in tea (Camellia sinensis (L) O. Kuntze. African Journal of Biotechnology 7(18): 3283-3287.

Zhou, Q., Li, H., Hoang, T.X., Ruan, X., Zhang, Y., Arkorful, E., Chen, X., Sun, K. & Li, X. 2019.  Genetic diversity and relationship of Dongting Biluochun Tea gemplasm in Suzhou revealed by SSR markers. Pak. J. Bot. 51(3): 895-902. DOI: 10.30848/PJB2019-3(31)

*Pengarang untuk surat-menyurat; email: hoangxuantrannomafsi@gmail.com