Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 108-116 http://genbreedpublisher.com/index.php/tgmb 112 were particularly closely related to the accumulation of valencene (an aroma component), suggesting that they might be the key to regulating the aroma. Some genes related to plant hormones have also been found to be associated with the formation of fragrance (Feng et al., 2021). 6 Metabolomics Approaches in Citrus Aroma Research 6.1 Analytical methods: GC-MS, LC-MS, and SPME techniques Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and solid-phase microextraction (SPME) are often used to study the aroma components of Citrus. LC-MS can well separate and analyze various polar and non-polar substances and is suitable for studying various aroma metabolites in Citrus. GC-MS is mainly used to detect volatile aroma components, while SPME “concentrates” these low-content volatiles during the sample preparation stage, making the analysis more sensitive (Miao et al., 2022; Wang et al., 2023). 6.2 Quantification and classification of aroma compounds Researchers can measure the content of each aroma substance in Citrus fruits by using metabolomics methods and also classify them. In 2022, Miao et al. used the derivatization -LC-MS method to identify 28 sugars and 18 carboxylic acids in limes, and classified 147 metabolites, among which 92 were discovered for the first time. The combined analysis of metabolomics and genomic data can also identify which genes control the synthesis of these aroma components (Wang et al., 2023). 6.3 Metabolite profiling in different Citrus species and tissues These techniques can also compare the fragrance substances in different varieties and different parts. Traband et al. (2025) recently analyzed data from over 200 Citrus leaves and fruits and found a clear metabolic connection between them, indicating that substances in different parts are flowing and mutually regulating. Metabolomics can also identify certain “local characteristic” metabolites when analyzing Citrus samples from different regions, which is very helpful for improving aroma quality and differentiating varieties (Miao et al., 2022; Wang et al., 2023). 7 Integration of Transcriptomic and Metabolomic Data 7.1 Correlation analysis between metabolites and gene expression By combining the transcriptome and metabolome data of different tissues or varieties of Citrus, researchers have discovered the relationship between some specific metabolites (such as aroma terpenes and flavonoids) and their related genes. For instance, in the leaves and flowers of Citrus, 31 major terpenes are highly present in wild or semi-wild varieties. Moreover, the accumulation of these compounds is highly consistent with the high expression of key genes in the two synthetic pathways of MVA and MEP, indicating that the higher the gene expression, the more of these flavor substances there are (Zhang et al., 2020). In addition, the antioxidant capacity in Citrus fruits is also related to the distribution of compounds such as flavonoids and coumarin, and the generation of these substances is closely related to the expression of genes such as CHS, CHI, and FNS (Liang et al., 2024). 7.2 Network-based multi-omics integration methods Network analysis methods like “WGCNA” can link genes and metabolites and draw their “circles of friends”. Liang et al. (2024) identified some gene modules related to the accumulation of flavonoids and coumarin in their research on different tissues of Citrus fruits, and also determined the OMT genes involved in coumarin synthesis and the related regulatory factors. Xiong et al. (2024) found in their study on the grafting of ‘Newhall’ fruit peels that transcription factors like AP2/ERF and MYB may have a significant impact on the synthesis of phenolic acids after combining KEGG and GO analyses. 7.3 Discovery of candidate genes through co-expression-metabolite mapping This method can also help us identify the key genes that regulate the aroma components. For example, in the study of Citrus flowers, scientists discovered a gene called sabinene synthase, whose expression level was consistent with the changes of a kind of aroma terpene (Zhang et al., 2020). Also, in the grafted peels of ‘Newhall’,
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