Tree Genetics and Molecular Breeding 2025, Vol.15, No.5, 192-201 http://genbreedpublisher.com/index.php/tgmb 195 4 Genomic Insights into Theobromine Biosynthesis 4.1 Genome sequencing and annotation of Theobroma cacao High-quality genomic sequencing and annotation of Theobroma cacao have provided a basis for studying the synthetic mechanisms of metabolites such as theobromine. Current research has covered approximately 80% of Theobroma cacao genome, and most genes are anchored on 10 chromosomes. The results showed that some gene families of Theobroma cacao had specific expansions, especially those related to secondary metabolites, such as flavonoids and methylxanthines (Argout et al., 2011). Meanwhile, the study also identified and annotated over 7 million SNPs. These data provided rich resources for functional gene localization and trait improvement (Cornejo et al., 2018). These genomic achievements not only contribute to the discovery of genes related to theobromine synthesis, but also lay a solid foundation for molecular breeding and functional gene research (Allègre et al., 2011). 4.2 Identification of candidate genes and regulatory networks Genome-wide annotation and functional analysis have identified a number of candidate genes and regulatory networks closely related to theobromine synthesis. Theobromine is a purine alkaloid, and its synthesis depends on multiple methyltransferase and other enzyme genes. Population genome studies have found that during domestication, theobromine metabolism-related genes were subjected to significant selective pressure (Cornejo et al., 2018). In addition, some gene families (such as GASA, BAHD, MADS-box, etc.) have been systematically identified in Theobroma cacao. Expression profiling analysis indicated that these genes were involved in seed development, disease resistance and metabolic regulation (Abdullah et al., 2021a; Abdullah et al., 2021b; Zhang et al., 2021). High-density genetic mapping and QTL mapping also helped achieve fine mapping of candidate genes related to theobromine content (Allègre et al., 2011). 4.3 Comparative genomics with other Theobromine/caffeine-producing species Comparative genomics shows that there are significant differences in genomic structure and functional genes between Theobroma cacao and other species that produce methyl xanthine, such as coffee and tea. Theobroma cacao mainly accumulates theobromine rather than caffeine, which is related to the expression and regulation mode of its methylase gene (Zheng et al., 2004; Agyirifo et al., 2019). Comparisons with the closely related species Theobroma grandiflorum also revealed differences in genomic structure, genetic content and evolutionary pathways between the two, providing evidence for understanding the evolutionary basis of theobromine synthesis (Kuhn et al., 2010). In addition, the comparison of chloroplast and mitochondrial genomes also provides new ideas for understanding the phylogenetic and metabolic diversity of Theobroma cacao and related species (De Abreu et al., 2022; Tineo et al., 2025). 5 Transcriptomic and Functional Genomics Approaches 5.1 Expression profiling of alkaloid biosynthesis genes Through high-throughput technologies such as RNA sequencing (RNA-seq), researchers can analyze the expression of alkaloid synthesis genes in Theobroma cacao. For instance, co-expression analysis has identified some gene modules related to protein modification, flowering and water transport. These modules showed obvious polygenic differentiation in different Theobroma cacao populations, providing clues for understanding the regulation of alkaloid synthesis genes (Hämäläet al., 2019). In addition, genome-wide analyses of some specific gene families (such as BAHD, GASA, etc.) revealed that some of their members were upregulated in expression during embryonic development and stress conditions (such as fungal infection), suggesting that they might be related to alkaloid metabolism and stress resistance (Abdullah et al., 2021a; Abdullah et al., 2021b; Nguyen et al., 2025). 5.2 Gene editing and functional validation In functional gene research, transient expression systems and gene editing techniques (such as CRISPR and RNAi) are often used to verify the functions of candidate genes. For instance, Agrobacterium-mediated transient transformation can enable the expression of exogenous genes in Theobroma cacao leaves within a short period of time. Researchers can detect the expression changes of the target gene through qRT-PCR and observe whether it
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