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Journal of Integrative Agriculture  2026, Vol. 25 Issue (4): 1519-1530    DOI: 10.1016/j.jia.2025.06.026
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Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study

Kaixin Rao1, Yuting Ouyang1, Yanjun Chen1, Xiaojing Wang1, 2, Ting Liu3, Qinfei Song1, Shaojuan Zhang1, 2, Biao Xiong1, Suzhen Niu1, 2#

1 College of Tea Science/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, China

2 The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in the Mountainous Region (Ministry of Education), Guiyang 550025, China

3 Zhengzhou Foreign Language School, Zhengzhou 450006, China

 Highlights 

GWAS identified 19 SNPs and a key gene (CsAK) that are significantly associated with caffeine (CAF) content in tea accessions.
The CsAK gene is involved in CAF biosynthesis by catalyzing L-aspartate to generate the key intermediate-L-methionine.

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摘要  咖啡碱(CAF)作为茶叶主要风味成分,是茶饮料广受欢迎的核心元素之一。作为茶树重要的次级代谢产物,不同茶树种质资源间CAF含量呈现显著差异。然而,CAF生物合成的遗传调控机制尚未明晰。在这项研究中,基于贵州高原359份茶树种质资源开展全基因组关联研究(GWAS)以确定与CAF含量相关的遗传变异。共鉴定出19个显著关联的单核苷酸多态性(SNPs)及CAF生物合成关键基因(CsAK)。亚细胞定位分析表明CsAK-GFP融合蛋白位于细胞膜上。通过反义寡核苷酸(AsODN)靶向沉默茶树芽叶CsAK基因,结果显示AsODN处理的茶树中CsAK基因的表达水平显著下调,且CAF含量降低。在真核细胞中过表达CsAK基因可促进CAF生物合成过程中关键中间产物(L-蛋氨酸)的积累。该研究为后续培育具有高或低CAF水平的优良种质资源的茶树育种计划提供了理论依据。




Abstract  

Caffeine (CAF), a primary flavor component in tea, is one of the main reasons for the popularity of tea beverages.  As an important secondary metabolite in tea plants, the CAF content varied greatly among different tea accessions.  However, the genetic mechanisms underlying the CAF biosynthesis were still unclear.  In this study, we performed a genome-wide association study (GWAS) on 359 tea accessions in the Guizhou Plateau to identify genetic variation associated with CAF content.  A total of 19 significant single nucleotide polymorphisms (SNPs) and key gene (CsAK) involved in CAF biosynthesis were identified.  Subcellular localization revealed that the CsAK-GFP fusion protein was located on the cell membrane.  Antisense oligodeoxynucleotide (AsODN) targeting the CsAK gene to the buds and leaves revealed that the expression levels of the CsAK gene were significantly reduced, and the corresponding CAF content was also decreased in AsODN-treated tea plants.  Overexpression of the CsAK gene in eukaryotic cells resulted in the accumulation of key intermediate product (L-methionine) during CAF biosynthesis process.  These findings offered a theoretical foundation for future tea breeding programs aimed at cultivating excellent germplasm with high or low levels of CAF.

Keywords:  tea accessions       caffeine (CAF)       single nucleotide polymorphism (SNP)       genome-wide association study (GWAS)       Guizhou Plateau  
Received: 08 January 2025   Accepted: 20 May 2025 Online: 30 June 2025  
Fund: 

This study was supported by the National Natural Science Foundation of China (32060700), the National Guidance Foundation for Local Science and Technology Development of China [(2023) 009], the Guizhou Provincial Basic Research Program (Natural Sciences) - Youth Initiative Project, China([2024] Youth 157), the Guiyang Science and Technology Plan Project (Construction Technology Contract, China [2023] 48-21), the Post-funding Project of the National Key R&D Program of China (2021YFD1100307), the Guizhou Provincial Science and Technology Program, China ([2023] General 481), the Qianxinan Prefecture Science and Technology Program, China (2022-1-52), the Youth Science and Technology Talent Project of Guizhou Provincial Department of Education, China (KY [2022]153), and the Basic Research Project of Guizhou University, China (2023-38).

About author:  Kaixin Rao, E-mail: 1778424029@qq.com; #Correspondence Suzhen Niu, E-mail: niusuzhen@163.com

Cite this article: 

Kaixin Rao, Yuting Ouyang, Yanjun Chen, Xiaojing Wang, Ting Liu, Qinfei Song, Shaojuan Zhang, Biao Xiong, Suzhen Niu. 2026. Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study. Journal of Integrative Agriculture, 25(4): 1519-1530.

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