Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 108-116 http://genbreedpublisher.com/index.php/tgmb 113 important MYB and ERF transcription factors that may regulate phenolic acid synthesis were identified (Xiong et al., 2024). This method of WGCNA can also discover which structural genes and regulatory factors are related to the accumulation of coumarin, which is very helpful for further research (Liang et al., 2024). 8 Case Study: Aroma Regulation in ‘Newhall’ Navel Orange vs. Wild Relatives 8.1 Experimental design and sampling strategy This study selected the ‘Newhall’ navel orange (Citrus sinensis ‘Newhall’) and its wild relatives as research materials, and conducted a comparative analysis of the accumulation of aroma substances and gene expression during their fruit development. A combined transcriptomic and metabolomic analysis method was adopted, and samples were taken at multiple stages before and after fruit ripening to comprehensively capture the changes in aroma. Gene expression information was obtained by high-throughput sequencing, and aroma components were detected by gas chromatography-mass spectrometry (GC-MS) technology. Combined with bioinformatics analysis, the main differential metabolites and the key genes that may be involved in regulation were screened out (Alquézar et al., 2017; Wang et al., 2024a). 8.2 Key differential metabolites and candidate genes identified The results show that there are significant differences in the accumulation of some terpene aroma substances (such as valencene, β -caryophyllene and β -humulene) between ‘Newhall’ navel oranges and their wild relatives. Further analysis revealed that some terpene synthase (TPS) genes related to terpene synthesis were expressed more highly in ‘Newhall’, such as Cstps1, β -caryophyllene synthase and β -humulene synthase. These genes may directly determine its aroma characteristics (Alquézar et al., 2017). Meanwhile, transcription factors such as LMI1, DRNL and MYC5 were also identified, which may be related to the development of oil glands and the accumulation of aroma precursors (Wang et al., 2024a). 8.3 Functional verification of a key terpene synthase gene In vitro expression experiments and enzyme activity tests indicated that Cstps1 (valencene synthase) could efficiently convert farnesyl diphosphate (FPP) into valencene, a compound that is the main component of the aroma of ‘Newhall’ fruit. The expression of Cstps1 significantly increased in the later stage of fruit ripening, which was consistent with the increasing trend of valencene. Moreover, the expression of this gene is also regulated by ethylene, suggesting that it may play an important role in the process of fruit ripening and aroma formation (Sharon-Asa et al., 2003). These findings provide a useful theoretical basis for improving the aroma quality of Citrus through breeding or genetic engineering in the future. 9 Breeding Applications and Biotechnology 9.1 Marker-assisted selection based on aroma-related loci The aroma of Citrus fruits is related to the genetic inheritance of the variety. Researchers have conducted extensive explorations on the genetic basis of Citrus aroma using methods such as QTL mapping and molecular markers. They identified multiple SNP markers and QTLS related to aroma in the hybrid offspring of 'Fortune' and ‘Murcott’, which are closely associated with aroma substances such as monoterpenes and sesquiterpenes. These candidate genes such as QTL and terpene synthase provide a basis for molecular marker-assisted breeding, which is beneficial for breeders to select varieties with better aroma more quickly (Yu et al., 2017; Gill et al., 2022). Yu et al. (2018) demonstrated that the method of GC-MS analysis combined with molecular markers has been able to distinguish different Citrus varieties well and can support breeding decisions. 9.2 Genome editing tools for aroma trait improvement Nowadays, gene editing technologies such as CRISPR-Cas9 have also begun to be used to regulate the aroma of Citrus fruits. By this method, some key genes, such as terpene synthase or aromatic synthase, can be precisely knocked out or altered to affect the composition of aroma (Mansoor and Kim, 2024). Although gene editing is still mostly used to improve disease resistance at present, with the increasing understanding of aroma synthesis genes, it is very likely that such technologies will be used in the future to enhance the aroma performance of Citrus (Salonia et al., 2020; Conti et al., 2021). Moreover, gene editing can also be combined with molecular marker breeding to improve the efficiency and accuracy of breeding (Gill et al., 2022).
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