Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 264 5 Case Study: Epigenetic Mechanisms in High-Lycopene Tomato Cultivars 5.1 Selection of contrasting tomato cultivars for analysis When studying the epigenetic regulation of lycopene accumulation, comparison varieties with high lycopene and low lycopene are usually selected. These varieties have significant differences in lycopene content during the fruit ripening process and are very suitable for studying the differences in epigenetic regulation. The commonly used varieties include ‘MicroTom’, ‘Ailsa Craig’ and their mutants. The lycopene content and the expression of related genes of these varieties show significant differences at the fruit development and ripening stages (Ming et al., 2023; He et al., 2024). 5.2 Experimental methods: methylation mapping, transcriptome profiling To systematically study epigenetic mechanisms, researchers usually employ high-throughput technologies such as whole-genome DNA methylation sequencing (WGBS) and transcriptome sequencing (RNA-seq). Methylation profiling analysis can determine the methylation status of the promoter regions of genes related to lycopene synthesis and reveal the regulatory role of DNA methylation on gene expression. Transcriptome analysis is used to compare the expression levels of key genes in the lycopene synthesis pathway under different varieties or different treatment conditions. Histone modifications and the expression of non-coding RNAs were also detected by methods such as ChIP-seq and small RNA sequencing (Ming et al., 2023; He et al., 2024). 5.3 Key findings on epigenetic marks correlating with lycopene levels Research has found that in high-lycopene varieties, the DNA methylation levels in the promoter regions of key lycopene synthesis genes (such as PSY1, PDS, etc.) are significantly reduced. This will enable these genes to be expressed at high levels, thereby promoting the accumulation of lycopene. In addition, histone deacetylases (such as SlHDA1 and SlHDA3) and histone variants (such as Sl_H2A.Z) also play significant roles in the expression of related genes. Their expression or modification state changes are closely related to the content of lycopene (Ming et al., 2023). Members of the AP2 family of transcription factors (such as SlAP2c and SlAP2a) regulate histone acetylation levels and directly affect the expression of lycopene synthesis genes, thereby controlling lycopene accumulation in fruits (He et al., 2024). In addition, environmental stress, such as exposure to nanoplastics, can also indirectly affect lycopene content by altering DNA methylation and histone modification (Nazari et al., 2025). 6 Biotechnological and Breeding Applications 6.1 Epigenome editing tools: CRISPR/dCas9-based epigenetic regulation The CRISPR/dCas9 system can link inactivated Cas9 (dCas9) to epigenetic modification enzymes, such as DNA methyltransferases, demethylases or histone acetyltransferases. In this way, DNA methylation, demethylation or histone modification can be achieved at specific locations, thereby precisely controlling the expression of target genes (Figure 2) (Pan et al., 2021; Cai et al., 2023). For instance, dCas9 can bind to transcriptional activators (such as VP64, p300) or inhibitors (such as KRAB, DNMT3A), respectively activating the gene (CRISPRa) or silencing it (CRISPRi) This method can be used to regulate the key genes related to lycopene synthesis (Moradpour and Abdulah, 2019; Jogam et al., 2022). Furthermore, the CRISPR/dCas9 platform can simultaneously regulate multiple genes and even alter chromatin structure, providing a powerful tool for precisely controlling the lycopene metabolic pathway (Moradpour and Abdulah, 2019). 6.2 Marker-assisted and epigenetic breeding strategies for lycopene enhancement The combination of molecular marker-assisted selection (MAS) and epigenetic markers can accelerate the genetic improvement of traits related to lycopene content. Tiwari et al. (2023) found that through genome-wide association study (GWAS) and epigenomic sequencing, epigenetic variations closely related to lycopene accumulation could be identified, and then efficient molecular markers could be developed for breeding screening. Genome editing technologies such as CRISPR/Cas9 and CRISPR/dCas9 can directly act on the key genes in the lycopene synthesis pathway to achieve rapid and precise trait improvement (Chandrasekaran et al., 2021; Tan et al., 2024).
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