Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct an F₁ population by crossing ‘Zhongcha 302’ (theacrine-free) and a tea germplasm ‘Ruyuan Kucha’ (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negatively correlated with caffeine (R2 > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicated that the theacrine synthase (TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, an in vitro enzyme activity experiment revealed that the 241st amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker that is co-segregate with high-theacrine individuals in RY’s offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

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