Additional file 2 of Increased DNA methylation contributes to the early ripening of pear fruits during domestication and improvement
Creators: name: Song, Bobo
name: Yu, Jinshan
name: Li, Xiaolong
name: Li, Jiaming
name: Fan, Jing
name: Liu, Hainan
name: Wei, Weilin
name: Zhang, Lingchao
name: Gu, Kaidi
name: Liu, Dongliang
name: Zhao, Kejiao
name: Wu, Jun
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 2: Fig. S1. The computational pipeline utilized to examine the WGBS (whole-genome bisulfite sequencing) data in our study. Fig. S2. Read mapping rates for the WGBS data across the 41 pear accessions using biological duplicates. The mapping rates (a) and the number of methylated cytosine (mC) sites (b) with and without using pseudo reference genomes across all 41 accessions. Fig. S3. The Pearson correlation coefficients between DNA methylation levels and gene/TE densities. DNA methylation levels were positively associated with TE density but negatively correlated with gene density. Fig. S4. Distribution of DNA methylation levels across the upstream 2 kb, gene body, and downstream 2 kb regions of different TEs. Fig. S5. DNA methylation changes during pear domestication and improvement process. (a) Kernel density distribution of DNA methylation changes between wild and landrace pear populations. (b) Kernel density distribution of DNA methylation changes across landrace and improved pear population. Fig. S6. Phylogenetic examination of DNA demethylases (a) and methyltransferases (b) from Arabidopsis thaliana and Pyrus pyrifolia. Fig. S7. The expression levels of 14 methylation genes in wild, landrace, and improved pear populations. Fig. S8. Expression levels of nine demethylation genes in wild, landrace, and improved pear populations. Fig. S9. Selective sweep examination for three Demeter-like1 (DML1) genes from P. pyrifolia during pear domestication and improvement. Comparisons of π (nucleotide diversity) in the three pear populations on two chromosomes and one contig. The red line represents the wild population, the blue line represents the landrace population, and the orange line represents the improved population. The grey region depicts the genomic locations of the DML genes. Fig. S10. Methylated cytosine site frequency spectrum (mC reads/total reads) for CG, CHG, and CHH contexts in the wild, landrace, and improved pear populations. The x-axes are composed of 10 different degree windows (from 0-10% to 90%-100%) for DNA methylation levels. The y-axes depict frequency distribution. The CG and CHG sites are highly methylated. However, the distribution is highly skewed towards unmethylation for the CHH sites. Fig. S11. The distribution of DMRs in the upstream and downstream regions of various genes. Fig. S12. Selection sweep analysis throughout pear domestication and improvement processes. (a) The identification of selective sweeps during pear domestication. Each point represents a 10-kb sliding window. If one sliding window with π ratio &gt; 1.509 and FST &gt; 0.075 is defined as a selective sweep region during domestication. (b) The identification of selective sweeps during pear improvement. Each point represents a 10-kb sliding window. If one sliding window with π ratio &gt; 1.918 and FST &gt; 0.157 is defined as selective sweep regions during domestication. (c) Overlap of selective sweep region in the wild vs. landrace (domestication process) and landrace vs. improved (improvement process) comparisons. Fig. S13. Correlation analyses between methylation levels of the CG and CHG contexts in the u_CG_DMRs (a) and the u_CG_DMRs (b) during pear domestication. Correlation analysis between the methylation levels of the CG and CHG contexts in the u_CG_DMRs (c) and the u_CG_DMRs (d) throughout pear improvement. Fig. S14. Genetic diversity comparisons between DMRs, DSRs, and NSRs across all pears and the wild, landrace, and improved populations (*P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001, two-tailed paired Student’s t-test). Fig. S15. Genetic diversity differences in various genomic backgrounds for the wild, landrace, and improved populations across all pears. The four different genomic backgrounds exhibited varying degrees of diversity, and the introns had the highest levels of genetic diversity. Fig. S16. KEGG enrichment analysis of genes linked to“Pure-CG-DMRs” (a) and “Pure-CHG-DMRs” (b). Fig. S17. The number of genes identified near DMRs in wild vs. landrace, landrace vs. improved, and wild vs. improved comparisons. Fig. S18. Correlation analysis between DNA methylation level of DMRs (Chr13:26073542-26073668) and the expression level of CAMTA2. Fig. S19. 5’-Aza, and ddH2O (mock) were injected into pear fruits. (a) Comparison of fruit firmness between 5’-Aza treated samples and mock. (b) Comparison of CG methylation level (mCG) gene expression levels in candidate DMR (Chr13: 26,073,542-26,073,668) between 5’-Aza treated samples and mock. (c) Comparison of gene expression levels of CAMTA2 between 5’-Aza treated samples and mock (*P &lt; 0.05, two-tailed paired Student’s t-test). Fig. S20. Expression levels of CAMTA2 in callus tissues of wild-type (WT) and three CAMTA2overexpression lines (*P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; NS = not significant, two-tailed paired Student’s t-test). Fig. S21. Expression level of the ACO gene in wild-type (WT) and CAMTA2-overexpressing pear callus tissue (*P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; NS = not significant, two-tailed paired Student’st-test). Fig. S22. Identification of CAMTA2 in transgenic T1-generation tomato plants. (a) Detection of transgenic plants carrying CAMTA2 through PCR amplification. WT represents wild-type plants (the cultivar‘Micro-Tom’; the negative control), P denotes the plasmid used for transformation (the positive control), and Lines 1-6 are transgenic tomato plants. (b) Quantitative RT-PCR was employed to determine the relative expression of CAMTA2 in plants of the three transgenic lines (*P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; NS = not significant, two-tailed paired Student’s t-test). Fig. S23. Relative expression levels of the SlACO gene in wild-type (WT; the cultivar ‘Micro-Tom’) and transgenic CAMTA2-overexpressing (CAMTA2-OE) tomato plants *P&lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; NS = not significant, two-tailed paired Student’s t-test). Fig. S24. Expression level of PpyDML1.1, PpyDML1.2, and PpyDML1.3 genes in the leaves of three wild, three landrace, and three improved pears. Fig. S25. The cis-acting element analysis and haplotype analysis of promoter sequences of PpyDML1.1 genes. Fig. S26. The cis-acting elements analysis and haplotype analysis of promoter sequences of PpyDML1.2 genes. Fig. S27. The cis-acting elements analysis and haplotype analysis of promoter sequences of PpyDML1.3 genes.