Tree Genetics and Molecular Breeding 2025, Vol.15, No.5, 192-201 http://genbreedpublisher.com/index.php/tgmb 196 affects metabolites or disease resistance (Fister et al., 2016). At present, there are not many studies directly verifying theobromine synthesis genes using CRISPR or RNAi. However, these techniques have been applied in some defense genes (such as chitinase), and their functions have been confirmed, laying the foundation for subsequent studies of theobrosin-related genes (Fister et al., 2016). 5.3 Integration of transcriptomics with metabolomic data Combining transcriptome and metabolome data for analysis can provide a clearer view of the relationship between gene expression and metabolites. Research has found that the accumulation of theobromine at different developmental stages is closely related to the expression of related synthetic genes. Meanwhile, genotype differences among different populations can affect the efficiency of alkaloid metabolic pathways (Koyama et al., 2003; Zheng et al., 2004). Genomic selection signal analysis also revealed that during domestication, theobromine synthesis-related genes were strongly selected, further indicating that these genes are important in Theobroma cacao quality formation (Cornejo et al., 2018). 6 Case Study: Metabolomic and Genomic Analysis in Cacao Cultivars 6.1 Selection of cultivars with contrasting theobromine content The varieties of Theobroma cacao vary greatly in the content of theobromine. Traditionally, the main cultivation types are Criollo, Forastero and Trinitario. Modern genomic research has further classified Theobroma cacao germplasm into ten genetic populations, including Amelonado, Contamana, Criollo, Curaray, Guianna, Iquitos, Marañon, Nacional, Nanay and Purús. These groups showed significant differences in genetic structure and metabolite accumulation, providing a basis for comparing high and low theobromine varieties (Zheng et al., 2004; Cornejo et al., 2018). 6.2 Methodology: integrated metabolomic- genomic analysis pipeline The research process is generally divided into several steps. First, collect leaf or fruit samples from different varieties. Secondly, whole-genome resequencing was conducted using high-throughput sequencing technologies (such as the Illumina platform) to obtain a large amount of SNP and gene annotation information (Argout et al., 2011; Cornejo et al., 2018). Thirdly, the contents of theobromine and related metabolites were determined by metabolomics methods such as HPLC-MS/MS (Zheng et al., 2004). Finally, through GWAS and expression profiling analysis, the associations between metabolite levels and gene variations or gene expression patterns were identified (Zheng et al., 2004; Cornejo et al., 2018). 6.3 Findings: key genes, metabolite variations, and breeding implications The results indicated that domesticated varieties such as Criollo underwent intense selection in genes related to theobromine metabolism. Some genes are closely related to the synthesis of flavor substances such as theobromine and anthocyanins (Cornejo et al., 2018). Theobromine is mainly synthesized in young fruits and cotylodes. The pathway is that AMP/IMP is first converted to 7-methylxanthine nucleoside, and then theobromine is generated through multiple steps of reaction (Koyama et al., 2003). There are significant differences in the ability of theobromine synthesis and degradation among different varieties and tissues. Moreover, most theobromine is directly synthesized in the seeds rather than transported from the pericarp (Zheng et al., 2004). Genomic studies have also found that beneficial genes related to flavors such as theobromine were selected during domestication, but some harmful mutations were also accumulated at the same time, which may affect the adaptability and yield of plants (Cornejo et al., 2018). These achievements provide important genetic resources for molecular breeding and contribute to the cultivation of new varieties with high theobromine content, excellent flavor and stress resistance (Argout et al., 2011; Cornejo et al., 2018). 7 Applications for Breeding and Industry 7.1 Marker-assisted breeding for flavor and health-enhancing alkaloid content Studies have shown that the biosynthetic pathway and genetic basis of theobromine are relatively clear, which provides strong support for molecular marker-assisted breeding (MAS). The results of population genomics show that during the domestication of Theobroma cacao, theobromine metabolism-related genes were strongly selected, especially in the Criollo variety, where the variations of related genes were closely related to flavor and functional
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