Tree Genetics and Molecular Breeding 2025, Vol.15, No.2, 80-88 http://genbreedpublisher.com/index.php/tgmb 84 indicating that Punica granatum varieties with stronger adaptability may be cultivated through selection and breeding. 6.2 Molecular breeding techniques 6.2.1 Marker-assisted selection (MAS) MAS is mainly used in Punica granatum breeding to improve important traits such as disease resistance and fruit quality. The application of SSR markers in genetic diversity analysis provides strong support for MAS in Punica granatum (Zarei and Sahraroo, 2018; Liu et al., 2020). These molecular markers enable breeders to select plants with ideal traits more quickly and accurately, saving a significant amount of time and resources required for traditional breeding (Holland and Bar-Ya’akov, 2018). 6.2.2 Genomic selection (GS) GS predicts the breeding value of each plant through genome-wide marker data. Tiwari et al. (2022) hold that GS has been extensively studied in crops such as tomatoes, and its application in Punica granatum should also be promising, with the potential to accelerate the breeding process and enhance the efficiency of genetic improvement. The development of high-throughput sequencing technology and bioinformatics tools has made it possible to apply GS in Punica granatum breeding, which is useful for improving complex traits more quickly (Tiwari et al., 2022). 6.2.3 CRISPR/Cas9 and gene editing CRISPR/Cas9 and other gene editing technologies have provided Punica granatum with new methods for precise gene improvement. Chang et al. (2019) demonstrated that CRISPR/Cas9 is highly effective in regulating the synthesis of important metabolites such as hydrolyzable tannins, and can also be used to precisely knockout or insert specific genes, helping to cultivate Punica granatum varieties with stronger disease resistance, higher fruit quality, and better environmental adaptability. 6.3 Field trials and validation Researchers will evaluate the agronomic traits of the selected varieties in the field during field trials. The combination of molecular breeding data and field trial results can provide a more comprehensive understanding of the genetic basis of these important traits and better guide future breeding work (Figure 1) (Holland and Bar-Ya’akov, 2018; Khadivi et al., 2018; Peerajade et al., 2020). Good field trials can ensure that newly bred Punica granatum varieties meet the expected standards and are suitable for large-scale commercial cultivation. Figure 1 Variability in fruit, aril and juice colour in Punica granatum genotypes (Adopted from Peerajade et al., 2020)
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