Tree Genetics and Molecular Breeding 2025, Vol.15, No.2, 80-88 http://genbreedpublisher.com/index.php/tgmb 83 technology through transcriptome and real-time qPCR analysis, indicating that transcriptome analysis is very useful in the study of Punica granatum functional genes. 5.2 Gene function studies The application of CRISPR/Cas9 technology enables scientists to have a clearer understanding of the gene functions in certain key metabolic pathways. In 2019, Chang et al. double-edited the two genes PgUGT84A23 and PgUGT84A24 in the hairy roots of Punica granatum and found that this would lead to the accumulation of 3-O- and 4-O-glucosides of gallic acid in a special way, indicating that these two genes have overlapping functions in the synthesis of β -gluconic acid. Chang et al. (2019) also discovered the PgUGT72BD1 gene that specifically acts on the glycosyltransferase at the 4-O- site of gallic acid, demonstrating the potential of CRISPR/Cas9 in the study of Punica granatum gene function and metabolic improvement. The recent research by Chen et al. (2023) found that the U-box gene family in Punica granatum has different functional manifestations when facing abiotic stress, indicating that these genes may be crucial in adapting to the environment. 5.3 Regulation of gene expression The regulatory process of gene expression in Punica granatum is rather complex. Chen et al. (2023) found that the U-box genes in Punica granatum would be activated when exposed to abiotic stress, indicating that they play a certain role in the process of plants coping with stress. Yan et al. (2019) analyzed the chloroplast genomes of different Punica granatum varieties and found that their gene structures were very conformed, with 11 genes in a positive selection state, most of which were related to photosynthesis or the genetic system, indicating that the gene regulation mode of Punica granatum is closely related to their ability to adapt to the environment. Patil et al. (2020) developed a large number of SSR markers using whole-genome information, which are beneficial for studying gene expression regulation and genetic diversity, and can also be used for marker-assisted selection in breeding. 6 Trait Improvement in Punica granatum 6.1 Traits of interest 6.1.1 Disease resistance Bacterial blight caused by the bacteria Xanthomonas axonopodis pv. punicae can lead to a significant reduction in Punica granatum yield (Singh et al., 2020). Singh et al. (2020) found by comparing the transcriptomes of different varieties that the significant increase in the expression of some genes related to defense mechanisms in disease-resistant varieties provided a basis for the breeding of new disease-resistant varieties. Peerajade et al. (2020) demonstrated that Punica granatum have a high heritability and improvement potential for resistance to this disease, indicating that this trait can be enhanced through selective breeding. 6.1.2 Fruit quality The quality traits of fruits such as fruit weight, aril weight, and the amount of juice will affect consumers’ preferences and market sales. Khadivi et al. (2018) and Peerajade et al. (2020) both found that different Punica granatum varieties have significant differences in these aspects, indicating that there is a lot of room for improvement. Peerajade et al. (2020) also held that traits such as fruit weight and phenol content have relatively high heritability and show significant genetic progression, supporting the possibility of improving fruit quality through genetic means. Jeong et al. (2018) identified genetic markers related to fruit color and anthocyanin content, which is very beneficial for the breeding of Punica granatum varieties with good appearance and high nutritional value. 6.1.3 Environmental adaptability Environmental adaptability is a key factor for Punica granatum to be smoothly cultivated in different climates. Peerajade et al. found in their genetic research in 2020 that the mutual influence between genotype and environment can affect traits such as plant growth, fruit length and fruit peel thickness. It is crucial to select varieties that perform well in specific environments. Zarei and Sahraroo (2018), as well as Liu et al. (2020), discovered rich genetic diversity from Punica granatum germplasm resources collected in different regions,
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