Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 108-116 http://genbreedpublisher.com/index.php/tgmb 111 5 Transcriptome Analysis in Citrus Aroma Studies 5.1 RNA-seq approaches to identify differentially expressed genes RNA-Seq is now the most commonly used method for studying the expression of genes related to Citrus fragrance. By conducting high-throughput sequencing on different varieties, tissues or developmental stages of Citrus fruits, scientists can identify which genes are related to the accumulation of aroma substances. Zhang et al. (2020) combined transcriptomic and metabolomic studies and found that 31 terpenoids accumulated in large quantities in the leaves of wild or semi-wild Citrus, which was closely related to the high expression levels of some key genes in the MVA and MEP pathways. Some terpene synthase genes have also been verified to indeed affect the synthesis of specific fragrance components (Hu et al., 2024). 5.2 Tissue-specific and developmental stage expression patterns The structure of Citrus fruits is complex. Different tissues, such as the peel, flesh and vascular bundles, as well as different developmental stages, all have different gene expressions. High-resolution transcriptome analysis revealed that these differences were important causes of aroma changes (Figure 2) (Feng et al., 2021). For instance, some genes involved in the synthesis of terpene and aldehyde aromas have increased expression levels in specific stages and tissues of fruit development (Hu et al., 2024). Recent studies have also used laser microdissection to separate different cells of Citrus peels and then conducted RNA sequencing, which can more clearly observe the expression differences among different cell types (Mei et al., 2024). Figure 2 Fruit tissue collection and global analysis of the fruit transcriptomes (Adopted from Feng et al., 2021) Image caption: (a) Six stages of C. sinensis fruit development and four fruit tissue types, epicarp, albedo, segment membrane and juice sac, collected for RNA-seq. (b) Cluster dendrogram showing the global relationships between biological replicates and among different stages and tissues. The y-axis is the degree of variance. In the following figures, samples are named ‘Tissue_stage_replicate’ or ‘Tissue_stage’; ‘EP_2_1’ represent ‘Epicarp_Stage 2_Replicate 1’. (c) Number of genes expressed in each tissue with an average FPKM higher than 0.3. (d) Venn diagram showing the number of shared and uniquely expressed genes among the four fruit tissues (Adopted from Feng et al., 2021) 5.3 Gene co-expression network analysis for aroma-related modules Co-expression network analysis (such as WGCNA) can help scientists identify a group of genes closely related to aroma synthesis and also identify key regulatory factors (Feng et al., 2021). Hu et al. (2024) found in their comparison of ‘Zong Cheng’ navel oranges with common varieties that Cs5g12900 and six transcription factors
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