Gms2019,10,465 8of14 es,mCG levels in TE regions differed by no more than 2%across the three developmental stages in both species. To test for a possible relationship between expression of TEs and their mCG levels,well-annotated TEswere classified into five categories:Gypsy,Copin,other long-terminal retrotransposons(LTR-others) long interspersed nuclearelements(LINEs)and DNA transposons.Asshown in Figure 3B,composition nd ee and 2i which nted -62%of total d the least p sy and 1.1%of Copia in Pt:13% of Gupsy and 2%i Pe)while relatively higher proportions of expressed TEs were found for the other three categories of TEs (96%of LTR-others,98%of LINEs and 96%DNA transposons in Pt;7.8%of LTR-others,10.1%of LINEs and 4.3%DNA transposons in Pe).Notably,the fruit body stage showed the largest proportions of expressed TEs of all categories in Pt,wher reas this hen nenon was see n only for Gypsy,LIF and DNA transposons in Pe As shown in Figure 3L els er type . i-Howell 1 of TEs,mCG levels For Pe all TE s showed virtually identical methylation levels in all three developmental stages (Figure S4C) Similarly,generally low expression levels occurred in different classes of TEs in all three developmental stages (Figure 3E and Figure S4D).Nevertheless,when focusing on TEs with TPM>0(average levels mong stages),a sigr ificant negative correlation between expression levels and mCGs was detected (Figure 3F;Figure S4E ated gh ge stages,sm all nu methyla etnEsFig n mu as the ed d ring do ts that at least bew adjacent developmental stages,the occurrence of minor changes of mCGs in TEs did not affect their generally low expression levels. 3.4.Correlation of CG methylation and expression in protein-Coding genes We investigated potential changes in mCG levels across the developmental stages for the expressed protein-coding genes(expressed at least at one stage).In both mushrooms,the average mCG levels in the body region of protein-coding genes(gene body)and their flanking regions were generally ery lo developmental stages(<3% ces were n een t one or more stag Pe 1 1以 nd Fi S6A)To explore the ntial correlatio n hetween mccs and ex ion of tein-coding we dissected genic regions into genes containing methylated promoters (MPs)and genes containing methylated gene bodies(MGBs)in each mushroom(Materials and Methods).More genes with MPs (722)and MGBs(365)were identified in Pe than in Pt(MPs and MGBs were 405 and 163,respectively). Strong correlations of mCG levels between adjacent developmental stages were detected for both groups of genes with or MGBs in both mushrc ting mCGs in protei ci cons ved during de ms,sug。 ent (e 1:0 ver in MP: ith ition 1 (14.8%v :4.7%in Ptan nd180 us 6.8%in Pe ectively):and (the numbers of nes with hype t-MPs were significantlyGenes 2019, 10, 465 8 of 14 differences, mCG levels in TE regions differed by no more than 2% across the three developmental stages in both species. To test for a possible relationship between expression of TEs and their mCG levels, well-annotated TEs were classified into five categories: Gypsy, Copia, other long-terminal retrotransposons (LTR-others), long interspersed nuclear elements (LINEs) and DNA transposons. As shown in Figure 3B, composition and content of TEs were very similar between the two mushroom species. In general, only small proportions of TEs were expressed during the three development stages (2.0%, 1.2% and 2.5% in Pt and 2.0%, 1.2% and 2.1% in Pe; Figure 3C and Figure S4B). The Gypsy and Copia retrotransposons, which represented ~62% of total repeat sequences in both species, showed the least proportions of expressed TEs among the different stages (on average, 1.3% of Gypsy and 1.1% of Copia in Pt; 1.3% of Gypsy and 2.2% of Copia in Pe), while relatively higher proportions of expressed TEs were found for the other three categories of TEs (9.6% of LTR-others, 9.8% of LINEs and 9.6% DNA transposons in Pt; 7.8% of LTR-others, 10.1% of LINEs and 4.3% DNA transposons in Pe). Notably, the fruit body stage showed the largest proportions of expressed TEs of all categories in Pt, whereas this phenomenon was seen only for Gypsy, LTR-others and DNA transposons in Pe. As shown in Figure 3D, the different categories of TEs showed minor difference of mCG levels in Pt: Gypsy was the highest methylated, whereas the other types of TEs showed similar mCG levels except for the comparison between Copia and DNA transposons (Games-Howell post-hoc test, p-value < 0.05). For each class of TEs, mCG levels were highly conserved across the three developmental stages. For Pe, all TE classes showed virtually identical methylation levels in all three developmental stages (Figure S4C). Similarly, generally low expression levels occurred in different classes of TEs in all three developmental stages (Figure 3E and Figure S4D). Nevertheless, when focusing on TEs with TPM > 0 (average levels among stages), a significant negative correlation between expression levels and mCGs was detected (Figure 3F; Figure S4E). Although generally conserved mCGs across the different stages, small numbers of differential￾methylated TEs (dmTEs) between adjacent stages did exist in both mushrooms. Relative to Pt, Pe possessed higher numbers of dmTEs (Figure S5A,C; Table S4). However, differential mCG levels in these TEs contributed little to their expression, as they remained largely repressed during development (Figure S5B,D; Mann-Whitney-Wilcoxon test, p-value > 0.05). This result suggests that, at least between adjacent developmental stages, the occurrence of minor changes of mCGs in TEs did not affect their generally low expression levels. 3.4. Correlation of CG Methylation and Expression in Protein-Coding Genes We investigated potential changes in mCG levels across the developmental stages for the expressed protein-coding genes (expressed at least at one stage). In both mushrooms, the average mCG levels in the body region of protein-coding genes (gene body) and their flanking regions were generally very low across the three developmental stages (<3%). However, moderate differences were noted between the two mushroom species at one or more stages. Specifically, Pt showed similar methylation levels among the three developmental stages, whereas Pe showed relatively higher methylation levels in fruit body, although the between-stage differences remained less than 1% (Figure 4A and Figure S6A). To explore the potential correlation between mCGs and expression of protein-coding genes, we dissected genic regions into genes containing methylated promoters (MPs) and genes containing methylated gene bodies (MGBs) in each mushroom (Materials and Methods). More genes with MPs (722) and MGBs (365) were identified in Pe than in Pt (MPs and MGBs were 405 and 163, respectively). Strong correlations of mCG levels between adjacent developmental stages were detected for both groups of genes with MPs or MGBs in both mushrooms, suggesting mCGs in protein-coding genes were largely conserved during development (correlation coefficient > 0.9; Figure 4B and Figure S6B). However, clear differences of mCG levels were detected in each mushroom: (i) mCG level difference in MPs was elevated in transition 2 compared with transition 1 (14.8% versus 4.7% in Pt and 18.0% versus 6.8% in Pe, respectively); and (ii) the numbers of genes with hyper-MPs were significantly