Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum.

Flower color, which is determined by various chemical pigments, is a vital trait for ornamental plants, in which anthocyanin is a major component. However, the epigenetic regulation of anthocyanin biosynthesis remains poorly understood. During chrysanthemum cultivation, we found a heterochromatic chrysanthemum accession (YP) whose progeny generated by asexual reproduction contained both yellow-flowered (YP-Y) and pink-flowered (YP-P) plants. In this study, we aimed to elucidate the epigenetic mechanisms of different flower colors in the YP plant progeny. Metabolome and transcriptome analyses revealed that the difference in flower color between YP-Y and YP-P was caused by expression variation of the anthocyanin biosynthesis gene CmMYB6. Bisulfite sequencing revealed that methylation at the CmMYB6 promoter, especially in the CHH context, was higher in YP-Y than YP-P. After demethylation of the CmMYB6 promoter using the dCas9-TET1cd system, the flower color returned from yellow to pink. Furthermore, the methylation status of the CmMYB6 promoter was higher in YP-Y over three consecutive generations, indicating that this methylation status was heritable mitotically. Finally, investigation of other chrysanthemum cultivars showed that the methylation of CmMYB6 decreased gradually with the increase in anthocyanin content. These results lay an epigenetic foundation for the improvement of flower color in horticultural plants.

Optimization of CRISPR-Cas9 system in Eustoma grandiflorum.

The optimization of the CRISPR-Cas9 system for enhancing editing efficiency holds significant value in scientific research. In this study, we optimized single guide RNA and Cas9 promoters of the CRISPR-Cas9 vector and established an efficient protoplast isolation and transient transformation system in Eustoma grandiflorum, and we successfully applied the modified CRISPR-Cas9 system to detect editing efficiency of the EgPDS gene. The activity of the EgU6-2 promoter in E. grandiflorum protoplasts was approximately three times higher than that of the GmU6 promoter. This promoter, along with the EgUBQ10 promoter, was applied in the CRISPR-Cas9 cassette, the modified CRISPR-Cas9 vectors that pEgU6-2::sgRNA-2/pEgUBQ10::Cas9-2 editing efficiency was 37.7%, which was 30.3% higher than that of the control, and the types of mutation are base substitutions, small fragment deletions and insertions. Finally we obtained an efficient gene editing vector for E. grandiflorum. This project provides an important technical platform for the study of gene function in E. grandiflorum.