Tree Genetics and Molecular Breeding 2024, Vol.14, No.4, 177-184 http://genbreedpublisher.com/index.php/tgmb 181 enzymes involved in the betalain biosynthesis pathway, such as polyphenol oxidase and DOPA dioxygenase, which are differentially expressed during the red pulp stage of Hylocereus polyrhizus (Tao et al., 2014). These findings underscore the complex regulatory networks governing betalain biosynthesis and highlight potential targets for genetic manipulation to enhance red pulp coloration. Understanding these molecular mechanisms is essential for developing breeding strategies that can optimize betalain content, thereby improving the nutritional and commercial value of dragon fruit. Figure 2 Gene distribution in the 11 longest scaffolds (pseudochromosomes) which account for 88.7% of the dragon fruit draft genome (Adopted from Zheng et al., 2021) Image caption: Protein-coding genes, noncoding RNA gene, and tRNA genes resided in these scaffolds account for 87.8%, 72.6%, and 58.0% of all these genes, respectively. A A photo of the whole plant of Hylocereus undatus cultivar “David Bowie” from the USDA-ARS Tropical Agriculture Research Station in Mayaquez, Puerto Rico. B Protein-coding gene density of dragon fruit in the 11 longest scaffolds/pseudochromosomes with a window size 100,000 bp, which is plotted by Rldeogram111. C Distribution of protein-coding genes (blue), noncoding RNA genes (including rRNAs, orange), and tRNA genes (green) on the 11 longest scaffolds. The Chr7 (Scaffold 33675) has the most (1478) noncoding RNAs, including 1125 5S rRNAs. The mapping of scaffolds and pseudochromosomes is as follows: Chr1:Scaffold 33678, Chr2:Scaffold 19641, Chr3:Scaffold 33676, Chr4:Scaffold 10417, Chr5:Scaffold 33679, Chr6:Scaffold 33677, Chr7:Scaffold 33675, Chr8:Scaffold 33673, Chr9:Scaffold 33680, Chr10:Scaffold 3410, Chr11:Scaffold 2055 (Adopted from Zheng et al., 2021) 4.3 Functional validation of candidate genes using CRISPR-Cas9 The CRISPR-Cas9 system has emerged as a revolutionary tool for functional genomics, enabling precise editing of specific genes to validate their roles in key traits of dragon fruit. This technology leverages the complementarity of guide RNA (gRNA) to target specific DNA sequences, allowing the Cas9 enzyme to introduce double-stranded breaks at precise locations in the genome (Muranty et al., 2015). By employing CRISPR-Cas9, researchers can create targeted knockouts or modifications in candidate genes, facilitating the study of their functions in traits such as disease resistance, fruit quality, and stress tolerance (Tao et al., 2014). The application of CRISPR-Cas9 in dragon fruit involves designing gRNAs specific to genes of interest, followed by the delivery of the Cas9-gRNA complex into plant cells, often using traditional methods or advanced techniques like CRISPR ribonucleoproteins (RNPs) (Silva et al., 2017). This approach not only accelerates the validation process but also enhances the precision of breeding programs by enabling the introduction of beneficial traits without altering other genetic characteristics. 4.4 Implications for breeding programs The integration of CRISPR-Cas9 technology into dragon fruit breeding programs holds significant promise for the rapid development of new cultivars with enhanced traits. By enabling precise gene editing, CRISPR-Cas9 allows
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