枳DNA甲基化修饰酶基因的鉴定与表达特征

doi:10.16420/j.issn.0513-353x.2024-1034

摘要/Abstract

摘要：

为了解甲基化修饰酶基因在柑橘基因组中的特性，在全基因组水平对枳甲基化转移酶和去甲基化酶基因进行了鉴定与分析。结果表明枳基因组中存在11个DNA甲基转移酶基因和3个去甲基化酶基因。这些基因编码蛋白的氨基酸数在665 ~ 1 962 aa之间，均属于亲水蛋白，其中13个成员定位在细胞核中。此外，PtrDME、PtrROS1、PtrDML3、PtrCMT2和PtrMET1-6中发生了3个片段重复事件，与拟南芥和水稻中同源基因都存在共线性关系。进一步分析表明，枳DNA甲基化修饰酶基因启动子区域含有较多的光响应和激素响应元件，且PtrMET1在蛋白互作网络中是核心蛋白。分析发现，PtrMET1-2和PtrCMT1在叶、根、成熟胚珠和幼嫩胚珠中的表达水平较高，PtrCMT2和PtrCMT3在成熟胚珠和幼嫩胚珠中较高。DNA甲基化抑制剂5-氮杂胞苷处理可降低枳叶片整体DNA甲基化水平，并上调甲基化修饰酶基因的表达水平，但降低了PtrDRM在茎中的表达。

关键词: 枳, DNA甲基化, 生物信息分析, 基因表达

Abstract:

In order to understand the characteristics of DNA methylation-modifying enzyme genes in the citrus genome，the DNA methyltransferase and demethylase genes in Poncirus trifoliata were identified and analyzed at the genome-wide level in this study. The results showed that 11 DNA methyltransferase genes and 3 demethylase genes existed in the Poncirus trifoliata genome. The peptide sequences of those proteins contain 665-1 962 amino acids，and all of them were hydrophilic proteins，with 13 members located at the nucleus. In addition，three fragment duplication events occurred in the PtrDME，PtrROS1，PtrDML3，PtrCMT2，and PtrMET1-6 genes，which were covariant with both Arabidopsis thaliana and Oryza sative. Further analysis revealed that promoter regions of these DNA methylation-modifying genes contain numerous light-responsive and hormone-responsive elements and that the PtrMET1 was a core protein in the protein interaction network. Gene expression analysis demonstrated that PtrMET1-2 and PtrCMT1 were highly expressed in leaf，root，late-stage ovule，and early-stage ovule，while PtrCMT2 and PtrCMT3 were highly expressed in flesh of mature and young fruit and seed. The DNA methylation inhibitor 5-azacitidine treatment reduced the leaf DNA methylation level in Poncirus trifoliata and increased the expression of most DNA methylation-modifying enzyme genes with the exception of PtrRM gene，which showed down expression in the stem. The result indicted that the methylation inhibitor methylation inhibitors may alter the methylation levels of plants via regulating the methylation-related genes expression.

Key words: Poncirus trifoliata, DNA methylation, bioinformatics analysis, gene expression

陈梦如, 吴庆安, 何思佳, 蔡祥斌, 马巧利, 辜青青, 魏清江. 枳DNA甲基化修饰酶基因的鉴定与表达特征[J]. 园艺学报, 2025, 52(12): 3167-3179.

CHEN Mengru, WU Qing’an, HE Sijia, CAI Xiangbin, MA Qiaoli, GU Qingqing, WEI Qingjiang. Genomic-Wide Identification and Expression Analysis of the DNA Methylation-Modifying Enzyme Genes in Poncirus trifoliata[J]. Acta Horticulturae Sinica, 2025, 52(12): 3167-3179.

图/表 8

表1 qRT-PCR引物序列

Table 1 The sequences of qRT-PCR primers

基因 Gene	正向引物序列（5′-3′） Forward primer	反向引物序列（5′-3′） Reverse primer
PtrActin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC
Pt5g021700	AGGTTGT·GGTGGCTTATCTGATGG	TTCTCCTGCTGGTTGCTCATACTC
Pt7g014310	TGATACCTTGGTGCCTGCCTAATAC	TGCCTTCCCAATCAAGCCTTCC
PtUn014750	AGATTTAATACCCTGGTGCTTACC	AAAGTTTCCTTCCCAGTCCAA
PtUn014790	TTATTTCCGCCCCAGGTTT	CTCAGGTGATGCTGCCCAT
PtUn014840	CTTCTGCGAACATTTATACGATCC	TCCTGCTGGTTGCTCATACTCT
PtUn030910	TGCCGCCTCACATCAAGA	CCATCAGATAAGCCACCACAA
Pt2g001260	AAGTAAAGAAGCAGAAGCGAAGA	TCCCATCAACTGCCTCAAAC
Pt5g018630	AGACGGTTCCTACAACAGCTACTTC	CGAGGCATAATCCAGTTGACATTCC
Pt5g023400	AGCGAAGAAGAAGAAGAGGAGGAAG	TTCGGATCACCATAGCAGACAGC
Pt9g000560	GTTGTCAGTCTTCAGCGGAATTGG	CCCACCACCTCTTGAGAATCCTTC
Pt1g006140	GACATAGACCAGGAAGGCAAGGAG	ATGACAATCACCGCAAACACGAAG
Pt6g006730	TGCGACTTCTAACACTCCACCATC	GCACCCAGCCGAGTCTTACAG
Pt8g006600	GACTGTTGCCAGAACTGAGTATTGC	TGATCGCCTGTGCCTGTCTTG
Pt4g018910	ACAACCGATGCTAGTGCGAGAC	TCTCTGCCTGCTCACCAATTACTTC

表2 枳DNA甲基化修饰酶蛋白理化性质

Table 2 Physicochemical properties of DNA methylation-modifying enzyme genes in Poncirus trifoliata

基因名称 Gene name	基因号 Gene ID	编码序列长度/bp CDS Length	氨基酸数 No. of amino acids	分子量/D Molecular weight	等电点 pI	脂溶性指数 Aliphatic index	不稳定系数 Instability index (II)	亲水性 Gravy	亚细胞定位 Subcellular localization
PtrMET1-1	Pt5g021700.1	5 247	1 748	196 887.65	6.18	78.07	40.18	-0.439	细胞核 Nucleus
PtrMET1-2	Pt7g014310.1	4 677	1 558	175 472.98	5.85	74.85	43.70	-0.502	细胞核 Nucleus
PtrMET1-3	PtUn014750.1	2 829	942	105 994.39	6.27	77.09	35.16	-0.433	细胞质 Cytosol
PtrMET1-4	PtUn014790.1	4 488	1 495	168 753.73	6.27	77.00	42.11	-0.469	细胞核 Nucleus
PtrMET1-5	PtUn014840.1	4 305	434	161 602.75	5.84	77.97	37.77	-0.392	细胞核 Nucleus
PtrMET1-6	PtUn030910.1	5 088	1 695	190 981.84	6.21	77.70	41.75	-0.434	细胞核 Nucleus
PtrCMT1	Pt2g001260.2	2 616	871	99 098.07	5.06	77.07	45.44	-0.569	细胞核 Nucleus
PtrCMT2	Pt5g018630.1	1 998	665	75 200.79	8.04	77.05	43.81	-0.437	细胞核 Nucleus
PtrCMT3	Pt5g023400.1	2 493	830	93 924.59	5.60	78.57	42.02	-0.484	细胞核 Nucleus
PtrDRM	Pt9g000560.2	2 094	697	78 513.03	5.26	75.35	50.29	-0.546	细胞核 Nucleus
PtrDNMT2	Pt1g006140.1	3 543	1 180	132 606.24	5.80	70.49	39.30	-0.580	细胞核 Nucleus
PtrROS1	Pt6g006730.1	5 889	1 962	218 463.80	6.91	68.59	48.65	-0.733	细胞核 Nucleus
PtrDML3	Pt8g006600.1	5 250	1 749	196 115.50	8.25	73.01	54.29	-0.689	细胞核 Nucleus
PtrDME	Pt4g018910.1	5 874	1 957	219 765.43	6.49	66.69	55.85	-0.763	细胞核 Nucleus

图1 DNA甲基化修饰酶基因进化、染色体定位和共线性分析 A：枳（Poncirus trifoliata，Ptr）、拟南芥（Arabidopsis thaliana，At）、水稻（Oryza sativa，Os）DNA甲基化修饰酶基因蛋白统进化分析；B：枳DNA甲基化修饰酶基因染色体定位和物种内的共线性分析；C：枳、拟南芥、水稻DNA甲基化修饰酶基因共线性分析

Fig. 1 Phylogenetic analysis，chromosome localization，and syntenic analysis of DNA methylation-modifying enzyme genes A：Phylogenetic analysis of DNA methylation-modifying enzyme genes from Poncirus trifoliata（Ptr），Arabidopsis thaliana（At），and Oryza sativa（Os）；B：Chromosome localization and syntenic analysis of DNA methylation-modifying enzyme genes in Poncirus trifoliata；C：Syntenic analysis of DNA methylation-modifying enzyme genes from Poncirus trifoliata，Arabidopsis thaliana，and Oryza sativa

图2 枳DNA甲基化修饰酶基因基序（A）和保守结构域（B）

Fig. 2 Conserved motif（A）and domain（B）of DNA methylation-modifying enzyme genes in Poncirus trifoliata

图3 枳DNA甲基化修饰酶基因启动子顺式作用元件种类（A）、数量（B）和蛋白互作分析（C）

Fig. 3 The type（A）and number（B）of cis-acting elements in the promoter region，and protein interaction network（C）of DNA methylation-modifying enzyme genes in Poncirus trifoliata

图4 枳DNA甲基化修饰酶基因组织特异性表达数据显示值为log2FPKM

Fig. 4 Expression heatmap of DNA methylation-modifying enzyme genes in different tissues of Poncirus trifoliata The data in the heatmap shows the log2FPKM value

图5 5-azaC处理后枳叶片DNA甲基化水平平均值差异采用t检测，*代表处理和对照之间差异显著（P < 0.05）

Fig. 5 DNA methylation level in leaf of Poncirus trifoliata after 5-azaC treatment Differences between the means were evaluated using Student’s t-test，and the * indicates significant difference between the control and 5-azaC treatment（P < 0.05）

图6 5-azaC处理后枳根、茎、叶中DNA甲基化修饰酶基因的表达平均值差异采用t检测，*，**和***分别代表在P < 0.05，P < 0.01和P < 0.001水平差异显著

Fig. 6 Expression of DNA methylation-modifying enzyme genes in root，stem，and leaf of Poncirus trifoliata after 5-azaC treatment Differences between the means were evaluated using Student’s t-test，and the *，**，and *** indicate significant difference between the CK and 5-azaC treatment at P < 0.05，P < 0.01，and P < 0.001，respectively

参考文献 34

[35]	Vijay G, Harsha S, Tinku G, Paramjit K. 2022. Identification and characterization of DNA demethylase genes and their association with thermal stress in wheat(Triticum aestivum L.). Frontiers in Genetics, 13:894020.
[36]	Xu Jidi. 2015. Genome-wide analysis of DNA methylation and its regulation in citrus[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	徐记迪. 2015. 柑橘全基因组DNA甲基化分析及调控作用研究[博士论文]. 武汉: 华中农业大学.
[37]	Xiong Y, Liu X, You Q, Han L, Shi J, Yang J, Cui W, Zhang H, Chao Q, Zhu Y, Duan Y, Xue T, Xue J. 2022. Analysis of DNA methylation in potato tuber in response to light exposure during storage. Plant Physiology and Biochemistry, 170:218-224.
[38]	Yang Huidong, Peng Zhenyu, Peng Langzhi, Yuan Meng, Chun Changpin, Yuan Zhigao. 2023. Effect of growth and physiological indexes of navel orange budlings grafted on seven citrus rootstock cultivars. Acta Agriculturae Universitatis Jiangxiensis, 45(3):575-583. (in Chinese)
	杨惠栋, 彭震宇, 彭良志, 袁梦, 淳长品, 袁高鹏. 2023. 不同砧木对纽荷尔脐橙嫁接苗生长和生理指标的影响. 江西农业大学学报, 45(3):575-583.
[39]	Yu Z, Zhang G, Jaime A T S, Li M, Zhao C, He C, Si C, Zhang M, Duan J. 2021. Genome-wide identification and analysis of DNA methyltransferase and demethylase gene families in Dendrobium officinale reveal their potential functions in polysaccharide accumulation. BMC Plant Biology, 21:21.
[40]	Yang Zhining, Lu Xuke, Fan Yapeng, Sun Yuping, Yu Xin, Wang Liang, Gao Yongjian, Habuli G, Nayisi G, Abudu W, Huang Hui, Zhang Menghao, Wang Lidong, Chen Xiao, Xiao Lei, Zhang Xinlei, Wang Shuai, Chen Xiugui, Wang Junjuan, Guo Lixue, Gao Wenwei, Ye Wuwei. 2025. Response mechanism of cotton GhDMT7 gene to 5-azacytidine. Journal of Agricultural Science and Technology, 27(8):28-35. (in Chinese)
	杨志宁, 陆许可, 范亚朋, 孙玉平, 喻欣, 王亮, 高永健, 古丽加依那什·哈不力, 古丽沙西·拿斯依, 吾木尔·阿不都, 黄慧, 张梦豪, 王立东, 陈筱, 肖磊, 张鑫蕊, 王帅, 陈修贵, 王俊娟, 郭丽雪, 高文伟, 叶武威. 2025. 棉花GhDMT7基因对5-氮杂胞嘧啶核苷的响应机制. 中国农业科技导报, 27(8):28-35.
[41]	Zhang H, Lang Z, Zhu J K. 2018. Dynamics and function of DNA methylation in plants. Nature reviews. Molecular Cell Biology, 19(8):489-506.
[1]	Ahmad F, Huang X, Lan H X, Huma T, Bao Y M, Huang J, Zhang H S. 2014. Comprehensive gene expression analysis of the DNA(cytosine-5)methyltransferase family in rice(Oryza sativa L.). Genetics and Molecular Research, 13(3):5159-5172.
[2]	Akemi O, Katsushi Y, Sachiko F, Rie T, Toshiaki M, Shigeru I. 2012. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny. The Plant Journal, 71(4):564-574.
[3]	Ai Y, Jing S, Cheng Z, Song B, Xie C, Liu J, Zhou J. 2021. DNA methylation affects photoperiodic tuberization in potato(Solanum tuberosum L.)by mediating the expression of genes related to the photoperiod and GA pathways. Horticulture Research, 8(1):181-181.
[4]	Bossdorf O, Arcuri D, Richards L C, Pigliucci M. 2010. Experimental alteration of DNA methylation affects the phenotypic plasticity of ecologically relevant traits in Arabidopsis thaliana. Evolutionary Ecology, 24(3):541-553.
[5]	Cao D, Ju Z, Gao C, Mei X, Fu D, Zhu H, Luo Y, Zhu B. 2014. Genome-wide identification of cytosine-5 DNA methyltransferases and demethylases in Solanum lycopersicum. Gene, 550(2):230-237.
[6]	Chan S W L, Henderson I R, Jacobsen S E. 2005. Gardening the genome:DNA methylation in Arabidopsis thaliana. Nature Reviews Genetics, 6(5):351-360.
[7]	Chang Y N, Zhu C, Jiang J, Zhang H, Zhu J K, Duan C G. 2020. Epigenetic regulation in plant abiotic stress responses. Journal of Integrative Plant Biology, 62(5):563-580.
[8]	Chen R, Chen X, Huo W, Zheng S, Lin Y, Lai Z. 2021. Transcriptome analysis of azacitidine(5-AzaC)-treatment affecting the development of early somatic embryogenesis in longan. The Journal of Horticultural Science and Biotechnology, 96(3):311-323.
[9]	Chen Tingxin, Fu Min, Li Na, Yang Leilei, Li Lingfei, Zhong Chunmei. 2024. Identification and expression analysis of DNA methyltransferase in Begonia masoniana. Chinese Bulletin of Botany, 59(5):726-737. (in Chinese)
	陈婷欣, 符敏, 李娜, 杨蕾蕾, 李凌飞, 钟春梅. 2024. 铁甲秋海棠DNA甲基转移酶全基因组鉴定及表达分析. 植物学报, 59(5):726-737.
[10]	Cui D, Meng J, Ren X, Yue J, Fu H, Huang M, Zhang Q, Gao S. 2020. Genome-wide identification and characterization of DCL,AGO and RDR gene families in Saccharum spontaneu. Scientific Reports, 10(1):13202.
[11]	Deng Xiuxin. 2022. A review and perspective for citrus breeding in China during the last six decades. Acta Horticulturae Sinica, 49(10):2063-2074. (in Chinese)
	邓秀新. 2022. 中国柑橘育种60年回顾与展望. 园艺学报, 49(10):2063-2074.
[12]	Feng S, Jacobsen E S. 2010. Epigenetic modifications in plants:an evolutionary perspective. Current Opinion in Plant Biology, 14(2):179-186.
[13]	Finnegan E J, Peacock W J, Dennis E S. 1996. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proceedings of the National Academy of Sciences, 93(16):8449-8454.
[14]	Gahlaut V, Samtani H, Khurana P. 2020. Genome-wide identification and expression profiling of cytosine-5 DNA methyltransferases during drought and heat stress in wheat(Triticum aestivum). Genomics, 112(6):4796-4807.
[15]	Gong Z, Morales-Ruiz T, Ariza R R, Roldán-Arjona T, David L, Zhu J K. 2002. ROS1,a repressor of transcriptional gene silencing in Arabidopsis,encodes a DNA glycosylase/lyase. Cell, 111(6):803-814.
[16]	Grzybkowska D, Morończyk J, Wójcikowska B, Gaj M D. 2018. Azacitidine(5-AzaC)-treatment and mutations in DNA methylase genes affect embryogenic response and expression of the genes that are involved in somatic embryogenesis in Arabidopsis. Plant Growth Regulation, 85(2):243-256.
[17]	Guo D, Li Q, Zhao H, Wang Z, Zhang G, Yu Y. 2019. The variation of berry development and DNA methylation after treatment with 5-azaC on ‘Kyoho’grape. Scientia Horticulturae, 246:265-271.
[18]	Hao X, Horvath P D, Chao S W, Yang Y, Wang X, Xiao B. 2014. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant[Camellia sinensis(L.)O. Kuntze]. International Journal of Molecular Sciences, 15(12):22155-22172.
[19]	He X J, Chen T, Zhu J K. 2011. Regulation and function of DNA methylation in plants and animals. Cell Research, 21(3):442-465.
[20]	Henderson I R, Jacobsen S E. 2007. Epigenetic inheritance in plants. Nature, 447:418-424.
[21]	Jia H, Su Z, Ren Y, Pei D, Yu K, Dong T, Zhang Y, Jia H. 2024. Exploration of 5-azacytidine and trichostatin a in the modulation of postharvest quality of‘Shine Muscat’grape berries. Postharvest Biology and Technology, 209:112719.
[22]	Kumar V, Staden V J. 2017. New insights into plant somatic embryogenesis:an epigenetic view. Acta Physiologiae Plantarum, 39:194.
[23]	Law J A, Jacobsen S E. 2010. Establishing,maintaining and modifying DNA methylation patterns in plants and animals. Nature Reviews Genetics, 11(3):204-220.
[24]	Li Z, Hua X, Zhong W, Yuan Y, Wang Y, Wang Z, Ming R, Zhang J. 2020. Genome-wide identification and expression profile analysis of WRKY family genes in the autopolyploid Saccharum spontaneum. Plant & Cell Physiology, 61(3):616-630.
[25]	Liu H, Wang X, Liu S, Huang Y, Guo Y, Xie W, Liu H, Tahir U Q M, Xu Q, Chen L. 2022. Citrus pan-genome to breeding database(CPBD):a comprehensive genome database for citrus breeding. Molecular Plant, 15(10):1503-1505.
[26]	Madhushree D, Vidhi R, Vijay G, Akhil K, Paras S, Vipasha V, Kishor G V, Salej S, Gaurav Z. 2022. The interplay of DNA methyltransferases and demethylases with tuberization genes in potato(Solanum tuberosum L.)genotypes under high temperature. Frontiers in Plant Science, 13:933740.
[27]	Markus S, Davison T S, Henz S R, Pape U J, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann J U. 2005. A gene expression map of Arabidopsis thaliana development. Nature Genetics, 37(5):501-506.
[28]	Mary G, Hoe J H, Tzung-Fu H, Jon P, Yeonhee C, Harada J J, Goldberg R B, Fischer R L. 2006. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell, 124(3):495-506.
[42]	Zhang Pan, Yu Yongxu, Cao Linggai, Zhu Guangbing, Wu Wei, Guo Yushuang, Yin Guoying, Jia Meng’ao. 2023. Research progress of m⁶A methylation modification response to plant biotic and abiotic stresses. Acta Horticulturae Sinica, 50(9):1841-1853. (in Chinese)
	张盼, 余永旭, 曹领改, 朱广兵, 吴伟, 郭玉双, 尹国英, 贾蒙骜. 2023. m6A甲基化修饰响应植物生物胁迫和非生物胁迫的研究进展. 园艺学报, 50(9):1841-1853.
[43]	Zhang Y, He X, Zhao H, Xu W, Deng H, Wang H, Wang S, Su D, Zheng Z, Yang B, Grierson D, Wu J, Liu M. 2020a. Genome-wide identification of DNA methylases and demethylases in kiwifruit(Actinidia chinensis). Frontiers in Plant Science, 11:514993.
[44]	Zhang Y, Si F, Wang Y, Liu C, Zhang T, Yuan Y, Gai S. 2020b. Application of 5-azacytidine induces DNA hypomethylation and accelerates dormancy release in buds of tree peony. Plant Physiology and Biochemistry, 147:91-100.
[45]	Zhu Shiping, Wang Fusheng, Chen Jiao, Yu Qin, Yu Hong, Luo Guotao, Hu Zhou, Feng Jinying, Zhao Xiaochun, Hong Qibin. 2020. Seed traits and seedling performances of different types of citrus rootstock. Scientia Agricultura Sinica, 53(3):585-599. (in Chinese)
	朱世平, 王福生, 陈娇, 余歆, 余洪, 罗国涛, 胡洲, 冯锦英, 赵晓春, 洪棋斌. 2020. 柑橘不同类型砧木的种子和苗期性状. 中国农业科学, 53(3):585-599.
[46]	Zhuang Zeyue. 2023. Identification of the DRM gene family and functional study of CaDRM1 gene in flower organ development of pepper(Capsicum annuum L.)[M. D. Dissertation]. Guangzhou: Zhongkai University of Agriculture and Engineering. (in Chinese)
	庄泽岳. 2023. 辣椒DRM基因家族的分析及CaDRM1在花器官发育中的功能研究[硕士论文]. 广州: 仲恺农业工程学院.
[29]	Ma Qingling, Liang Beibei, Yang Li, Li Yong, Liu Dechun. 2022. Genome-wide identification and expression analysis of WOX gene family in Trifoliate orange. Journal of Fruit Science, 39(5):712-729. (in Chinese)
	马青龄, 梁贝贝, 杨莉, 胡威, 刘勇, 刘德春. 2022. 枳WOX基因家族全基因组鉴定及表达分析. 果树学报, 39(5):712-729.
[30]	Pedro O M, Luis S C, Clelia D L P. 2018. 5-Azacytidine:A promoter of epigenetic changes in the quest to improve plant somatic embryogenesis. International Journal of Molecular Sciences, 19(10):3182.
[31]	Pilar A O, Teresa M, Rafael R A, Teresa R A. 2008. Arabidopsis DEMETER-LIKE proteins DML2 and DML 3 are required for appropriate distribution of DNA methylation marks. Plant Molecular Biology, 67(6):671-681.
[32]	Qi Chunjie, Gu Yumeng, Zeng Yan. 2021. Progress of citrus industry economy in China. Journal of Huazhong Agricultural University, 40(1):58-69. (in Chinese)
	祁春节, 顾雨檬, 曾彦. 2021. 我国柑橘产业经济研究进展. 华中农业大学学报, 40(1):58-69.
[33]	Sano H, Kamada I, Youssefian S, Katsumi M, Wabiko H. 1990. A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA. Molecular and General Genetics, 220:441-447.
[34]	Teyssier E, Bernacchia G, Maury S, Kit A, Stammitti-ber L, Rolin D, Gallusci P. 2008. Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening. Planta, 228(3):391-399.