Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 98-107 http://genbreedpublisher.com/index.php/tgmb 98 Feature Review Open Access Cloning and Functional Analysis of Key Genes Involved in Anthocyanin Biosynthesis in Morella rubra Zhen Liu 1, Dandan Huang 2, Wenfang Wang 1 1 Institute of Life Sciences, Jiyang Colloge of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China 2 Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: wenfang.wang@jicat.org Tree Genetics and Molecular Breeding, 2025, Vol.15, No.3 doi: 10.5376/tgmb.2025.15.0012 Received: 17 Apr., 2025 Accepted: 19 May, 2025 Published: 30 May, 2025 Copyright © 2025 Liu et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Liu Z., Huang D.D., and Wang W.F., 2025, Cloning and functional analysis of key genes involved in anthocyanin biosynthesis in Morella rubra, Tree Genetics and Molecular Breeding, 15(3): 98-107 (doi: 10.5376/tgmb.2025.15.0012) Abstract This study reviews the research achievements made in recent years regarding the synthesis of anthocyanins in Morella rubra. Key genes such as CHS, CHI and F3H play an important role in the synthesis process of anthocyanins. In addition, some factors that regulate these genes, such as R2R3-MYB, bHLH and WD40, also play a role in the regulatory process. There is also a mutually cooperative relationship among them. To figure out how these genes work, researchers employed methods such as cloning genes, analyzing expression patterns, and also studied the epigenetic regulation of genes. This study also introduces the application prospects of molecular breeding, genetic engineering and CRISPR technology in improving the color and nutritional value of Morella rubra. It is hoped that these achievements can provide a scientific basis for improving the quality of Morella rubra and also bring practical methods for planting and breeding. Keywords Morella rubra; Anthocyanin biosynthesis; MYB transcription factor; Molecular breeding; Epigenetic regulation 1 Introduction Morella rubra is a common fruit tree, and its fruits come in a variety of colors, ranging from white, red to deep purplish red. These color differences are mainly due to the varying content of anthocyanins in the fruits. Anthocyanins not only make the fruit look good and improve its appearance, but also are beneficial to human health and can help prevent some diseases. They are very important nutrients in Morella rubra (Niu et al., 2010; Shi et al., 2021). Therefore, the amount of anthocyanins directly affects the quality and nutritional value of Morella rubra (Duan et al., 2015; Li et al., 2023). Anthocyanins are synthesized through a process called the “biosynthetic pathway”, which requires some structural genes, such as CHS, CHI, F3H, F3’H, DFR, ANS and UFGT. The expression of these genes is regulated by some transcription factors, especially the proteins of the MYB, bHLH and WD40 families (Liu et al., 2013b; Yan et al., 2021). In Morella rubra, some R2R3-MYB transcription factors such as MrMYB1 and MrMYB9 can promote anthocyanin synthesis, while MrMYB6 has the opposite effect and inhibits anthocyanin accumulation (Shi et al., 2021; Li et al., 2023). In addition, MrbHLH1 and MrWD40-1 can also cooperate with MYB protein to better promote the formation of anthocyanins (Liu et al., 2013a). The expression of these genes varies in different varieties and fruit development stages, which determines the depth of fruit color and the amount of anthocyanins (Niu et al., 2010; Duan et al., 2015). This study mainly aims to identify these key genes and investigate how they affect the color change of Morella rubra. Through the cloning research of some key genes, it explores their expression and role during the fruit development process. This study hopes that these results can provide assistance in improving the fruit color and nutritional value of Morella rubra, and also offer tools and theoretical support for future molecular breeding. 2 Biology and Distribution of Morella rubra 2.1 Botanical characteristics and genetic background Morella rubra is a common evergreen fruit tree in southern China. It is dioecious, that is to say, the male and female plants are separate, and only the female plants can bear fruit. Research has found that female plants have a slightly higher number of alleles and heterozygosity, but the difference is not significant compared to male plants.
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