# Unravelling the Molecular Regulation Mechanisms of Slow Ripening Trait in _Prunus persica_
## 2 Results
### RNA Sequencing and bioinformatic Analysis  
The selected samples and the sequencing information details are presented in Table S1. The number of reads sequenced for each sample was 46,229,240 reads, with a GC content of 45% on average and no overrepresented libraries. When the samples were filtered, between 0.6% and 0.9% of the reads were lost by quality on each library. The alignment process resulted in an average of 93.3% of the filtered reads correctly aligned against the reference genome. In summary, of the total reads sequenced for each library, 92.8% on average  
Figure 1: Slow ripening versus normal ripening fruit phenotypes. Fruit changes between normal and slow ripening phenotypes at different developmental stages. The evaluation stages T1, T2, T3, T4, T5, T6 and T7 represent different days after 1 September (DASF), corresponding to 37, 51, 65, 79, 99, 112 and 120 DASF, respectively. (**A**) Photographic developmental evaluation of fruit with normal (NR) and slow (SR) ripening phenotypes. (**B**) Expression profile of the candidate gene Prupe.4G186800 (_NAC072_) during normal phenotype development. Letters a-d represent significant expression differences. (**C**) Changes in soluble solid content, fruit weight and firmness between NR (continuous lines) and SR (dashed lines) phenotypes. Significant differences between NR and SR phenotypes are represented with asterisk (* \(p\) \(<\) 0.05; ** \(p\) \(<\) 0.01; *** \(p\) \(<\) 0.001). were correctly aligned with the reference genome, and no overrepresented libraries and contaminations were found. A principal component analysis (PCA) was performed to observe the biological replicate distribution using the expression levels of each transcript. Figure 2A shows that the replicates of each sample presented similar behaviors and grouped close to each other. Furthermore, the four development stages analyzed are clearly separated. Two significant differences were identified, PC1 (41.85%) separated the T1 samples from the other developmental stages, and PC2 (38.43%) separated the T7 samples of NR from the SR. The T2, T3, and T7 SR samples showed a similar trend among them, and could only be separated by PC3 (10,95%). Similar results are demonstrated in Figure 2B, which uses all differential expression data to construct a heatmap. In general, the differences between T1 and the other three developmental stages were evident, as was the difference between the NR and SR T7 samples. Furthermore, the number of differentially expressed genes (DEG) in each developmental stage were compared separately through the expression levels between NR and SR individuals in a Venn diagram (Figure 2C). A total of 157, 269, 976, and 5,224 DEG were identified between the normal and the mutant phenotypes for T1, T2, T3, and T7, respectively. The section in gray displays the 78 transcripts differentially expressed in all developmental stages. In this group of genes, the described candidate gene for SR phenotype _NAC072_ is included (Figure 2C). Of these 78 genes related to the SR phenotype, three stand out for their description: one WRKY transcription factor (_WRKY35_) and two auxin-related genes (SAUR family proteins). These three genes presented with considerably higher expression values in NR individuals. A gene ontology term enrichment analysis was carried out to understands the fruit transcriptomic differences between the normal and slow ripening phenotypes (Figure 2D). Regarding the development of normally ripening fruits, it was possible to identify high activity of cell wall remodeling enzymes in T3, accompanied by nucleotide-sugar biosynthesis. While in the T7 NR fruits, increases in the genes associated with caroten biosynthesis, auxin response, and oxidative stress were observed. When analyzing the transcriptomes of slow ripening fruits, neither cell wall remodeling nor caroten biosynthesis were observed.