Dynamic Changes in DNA Methylome and Transcriptome Patterns During Papaya Fruit Ripening

doi:10.16420/j.issn.0513-353x.2020-1046

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (3): 519-532.doi: 10.16420/j.issn.0513-353x.2020-1046

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Dynamic Changes in DNA Methylome and Transcriptome Patterns During Papaya Fruit Ripening

ZHOU Chenping¹, YANG Min¹, GUO Jinju², KUANG Ruibin¹, YANG Hu¹, HUANG Bingxiong¹, WEI Yuerong¹^,^*()

¹Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs,Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research,Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China
²Institute of Facility Agriculture,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China

Received:2021-07-19 Revised:2021-08-20 Online:2022-03-25 Published:2022-03-25
Contact: WEI Yuerong E-mail:weid18@163.com

Abstract

Abstract:

The whole genome bisulfite sequencing（WGBS）and transcriptome sequencing（RNA-seq）techniques were used to sequence papaya fruit samples at four stages,namely green stage,color break stage,half yellow stage and full yellow stage. The average methylation level of C locus,CG,CHG,and CHH sequence contexts were 17.15%,49.33%,34.30%,and 8.28%,respectively. The level of genome-wide DNA methylation decreased with the ripening of fruit,accompanied by the increased number of differentially expressed genes（DEGs）. Combined analysis of genome-wide DNA methylation and transcriptome data showed that the gene expression presented an obvious positive correlation with promoter and genebody methylation. Four differentially expressed candidate genes possibly responsible for papaya fruit ripening,namely CpACO4,CpPDS,CpXTH30 and CpXTH31,were screened out. Their expression levels and DMRs methylation levels obtained by RT-qPCR and McrBC-PCR matched well with the results of the transcriptome and methylome,respectively. These genes might play an important role in papaya fruit ripening.

Key words: Carica papaya, fruit, ripening, whole genome, DNA methylation, gene, differentially expression

CLC Number:

S667.9

ZHOU Chenping, YANG Min, GUO Jinju, KUANG Ruibin, YANG Hu, HUANG Bingxiong, WEI Yuerong. Dynamic Changes in DNA Methylome and Transcriptome Patterns During Papaya Fruit Ripening[J]. Acta Horticulturae Sinica, 2022, 49(3): 519-532.

Figures/Tables 11

Fig. 1 Different maturation stages of papaya fruits

Table 1 Primers used for McrBC-PCR and qRT-PCR

用途 Application	基因名称 Gene name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
McrBC-PCR	CpXTH30	GGTTGGCTAATGATTGT	AGAAGGAAGGAGGGTGC
	CpPDS	CTAGTTTCTAACTGCGTAT	AACCATTATCATGTGGG
	CpACO4	GAAGACCATTAGCCACA	TTCGCTTAACTTAAACAC
	CpXTH31	ATGAAGTAGGGTGAGAA	ATGGTGGAATAAGTGTT
qRT-PCR	CpXTH30	GTTCCATCGGTATAGCAT	CTTTGTATCTTCCACCTGA
	CpPDS	AGTTGGCGTCCCTGTTA	GGATTGATTTGGGTTGTA
	CpACO4	CACGAGCAGCCAAACGA	TCACCCACCCATCCCAC
	CpXTH31	GGGAACCCTACACTTTG	CTTGGATACCTCCGAAT

Table 2 Summary of WGBS data of papaya fruits at different ripening stages

成熟期 Stage	序列总数量 Total reads	对比参考基因组获得的序列数量 Mapped reads	匹配率/% Mapping rate	重复序列占比/% Duplication rate	≥ Q30/%	测序深度 Sequencing depth	甲基化C位点数量Number of Methylated C site	平均甲基化水平/% Mean methylation level
成熟期 Stage	序列总数量 Total reads	对比参考基因组获得的序列数量 Mapped reads	匹配率/% Mapping rate	重复序列占比/% Duplication rate	≥ Q30/%	测序深度 Sequencing depth	甲基化C位点数量Number of Methylated C site	C	CG	CHG	CHH
绿熟期 Green	49 375 154	35 135 889	70.97	14.37	92.78	21.87	251 784 698	17.79	51.22	35.59	8.62
破色期 Color break	49 361 724	34 881 444	70.67	12.75	91.56	22.08	258 718 931	17.51	50.64	35.09	8.44
熟化期 Half yellow	49 461 069	34 774 513	70.36	12.62	92.57	21.95	232 054 629	17.04	48.60	33.76	8.26
腐熟期 Full yellow	48 863 321	34 071 375	70.61	10.79	92.19	22.54	233 578 504	16.26	46.88	32.77	7.81

Fig. 2 DNA methylation levels of genes and TEs（A）and C,CG,CHG,and CHH contexts（B）at different ripening stages of papaya fruits GS：Green stage;CB：Color break stage;HY：Half yellow stage;FY：Full yellow stage;-2K and 2K：2 kb upstream and downstream regions of genes;5′ and 3′：5′ and 3′ flanking regions of genes;rep1 and rep2：Two biological replicates. The same below.

Fig. 3 Number of DMCs（A）and DMRs（B）in CB vs. GS,HY vs. GS,and FY vs. GS

Fig. 4 Circos plot of twenty genome scaffold methylation density Number on outer rim：Number of the twenty genome scaffolds;Scales of 5mC/RNA,TE and Gene：The methylation levels of CG,CHG,CHH sequence context,RNA abundance,the ratio of transposon repeats and gene number were from low to high,respectively.

Fig. 5 Relationship between distribution of methylation levels in three contexts within the genebody,upstream,and downstream 2 kb rigions（A）and methylation levels of genebody and promoterwith the gene expression levels（B） -2K and 2K：2 kb upstream and downstream regions of genes;Expression：genes with FPKM value < 1 were considered as none（non-expressed）,expressed genes（FPKM value ≥ 1）were ranked from small to large and divided into three groups based on the upper and lower quartile;Methylation group：Methylation levels of genebodies or promoters were ranked from small to large and divided into quinitiles.

Fig. 6 Number of up-and down-regulated DEGs in different ripening stages

Fig. 7 Homologous alignment of DNA methylation genes（A）and their expression changes at different ripening stages in papaya fruits（B） Scale：From blue to red represent the gene expression levels was from low to high. 1-3：Three biological replicates.*：P < 0.05;-：No significant difference.

Fig. 8 Expression changes of carotenoid biosynthetic-related genes at different ripening stages of papaya fruits Scale：From blue to red represent the gene expression levels was from low to high;1-3：Three biological replicates.

Fig. 9 The DMRs methylation and expression levels of four candidate genes at different maturation stages of papaya fruits A：DMRs methylation levels of CpACO4,CpXTH30 and CpXTH31 promoters and CpPDS genebody;B：The McrBC-PCR validation（M：PCR products been digested by McrBC,Un：PCR products not been digested）;C：Fold change（FC）;D：qRT-PCR validation.

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Dynamic Changes in DNA Methylome and Transcriptome Patterns During Papaya Fruit Ripening

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