DNA methylation analysis of sterile and fertile CMS-C hybrids and their parents in maize

Original Article


C-type cytoplasmic male sterile (CMS-C) line plays an important role in hybrid seed production in maize. However, mechanisms of pollen abortion and fertility restoration remain unclear. This study aimed to investigate the mechanisms of CMS-C pollen abortion and fertility restoration, particularly based on epigenetics, and to understand the relationship between male fertility performance and DNA methylation status. Methylation-sensitive amplification polymorphism (MSAP) technique was conducted to analyze DNA methylation levels and patterns of tassels at pollen-mother cell, tetrad, mononuclear, and binuclear stages among four half-sib hybrids and their parents with different fertility. Results showed that DNA methylation levels in fertility restored hybrids were higher than those in sterility maintained hybrids. Nine CCGG sites, which displayed methylation polymorphism between male-fertile and male-sterile hybrids, were screened. To validate MSAP results, we performed methylation-sensitive PCR (MS-PCR) and found consistent observations in cloned methylation sites. Interestingly, a specific site named 16–1 was discovered in the region of Rf5 gene, which is one of the restorer genes of CMS-C. Thus, DNA methylation may participate in regulating the expression of fertility restorer genes of maize CMS-C.


Maize CMS-C Fertility restoration DNA methylation 



Cytoplasmic male sterile


C-type cytoplasmic male sterile


Methylation-sensitive PCR


Methylation-sensitive amplification polymorphism


Cetyltrimethyl ammonium bromide



This work was financed by the National Natural Science Foundation of China (Grant No. 30971794).

Supplementary material

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  1. Carlsson J, Lagercrantz U, Sundstrom J, Teixeira R, Wellmer F, Meyerowitz EM, Glimelius K (2007) Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers. Plant J 49(3):452–462PubMedCrossRefGoogle Scholar
  2. Fieldes MA, Schaeffer SM, Krech MJ, Brown JC (2005) DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax. Theor Appl Genet 111(1):136–149PubMedCrossRefGoogle Scholar
  3. Guo XC (2001) How to understand nuclear DNA in cytoplasmic male sterility. Chin J Appl Environ Biol 7(3):297–301Google Scholar
  4. Hanson MR, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16(Suppl):S154–S169PubMedPubMedCentralCrossRefGoogle Scholar
  5. Jaroslav Fulneček, Aleš Kovařík (2014) How to interpret methylation sensitive amplified polymorphism (MSAP) profiles? BMC Genetics 15:2. http://www.biomedcentral.com/1471-2156/15/
  6. Kakutani T, Munakata K, Richards EJ, Hirochika H (1999) Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana. Genet 151(2):831–838Google Scholar
  7. Kohls S, Stamp P, Knaak C, Messmer R (2011) QTL involved in the partial restoration of male fertility of C-type cytoplasmic male sterility in maize. Theor Appl Genet 123(2):327–338PubMedCrossRefGoogle Scholar
  8. Lu YL, Rong TZ, Cao MJ (2008) Analysis of DNA methylation in different maize tissues. J Genet Genom 35(1):41–48CrossRefGoogle Scholar
  9. Lu YL, Xi Y, Wang J, Cao MJ, Rong TZ (2010) Variation and patterns of DNA methylation in maize C-type CMS lines and their maintainers. J Plant Biochem Biotechnol 19(1):43–50CrossRefGoogle Scholar
  10. McClelland M, Nelson M, Raschke E (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 22:3640–3659PubMedPubMedCentralCrossRefGoogle Scholar
  11. Peredo EL, Revilla MA, Arroyo Garc R (2006) Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organism genic calli. J Plant Physiol 163:1071–1079PubMedCrossRefGoogle Scholar
  12. Reyna-Lopez GE, Simpson J, Ruiz-Herrera J (1997) Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms. Mol Gen Genet 253(6):703–710PubMedCrossRefGoogle Scholar
  13. Richards EJ (1997) DNA methylation and plant development. Trends Genet 13:319–323PubMedCrossRefGoogle Scholar
  14. Santi DV, Garrett CE, Barr PJ (1983) On the mechanism of inhibition of DNA cytosine methyltransferases by cytosine analogs. Cell 33(1):9–10PubMedCrossRefGoogle Scholar
  15. Sha AH, Lin XH, Huang JB, Zhang DP (2005) Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP. Mol Genet Genomics 273:484e490CrossRefGoogle Scholar
  16. Shaked H, Kashkush K, Ozkan H, Feldman M, Levy AA (2001) Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat. Plant Cell 13(8):1749–1759PubMedPubMedCentralCrossRefGoogle Scholar
  17. Stamp P, Chowchond S, Menzi M, Weingartner U, Kaeser O (2000) Increase in the yield of cytoplasmic male sterile maize revisited. Crop Sci 40:1586–1587CrossRefGoogle Scholar
  18. Tonelli C, Consonni G, Faccio Dolfini S, Dellaporta SL, Viotti A, Gavazzi G (1991) Genetic and molecular analysis of Sn, a light inducible, tissue specific regulatory gene in maize. Mol Gen Genet 225:401–410PubMedCrossRefGoogle Scholar
  19. Vidakovic M (1988) Genetics of fertility restoration in cytoplasmic male sterility of the C-type (CMS-C) in maize. Maydica 33:51–64Google Scholar
  20. Vldakovic M, Vancetovie J (1997) The existence of a duplicated or paral1el genetic system for fertility restoration in CMS-C of maize (Zea mays L.). Maydica 42(3):313–316Google Scholar
  21. Walder RY, Langtimm CJ, Chatterjee R, Walder JA (1983) Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII. J Biol Chem 258(2):1235–1241PubMedGoogle Scholar
  22. Xiong LZ, Xu CG, Maroof MAS, Zhang Q (1999) Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation sensitive amplification polymorphism technique.Mol. Genet Genom 261(3):439–446Google Scholar
  23. Yi ZB, Sun Y, Niu TT, Liang XH, Liu LL, Zhao WJ, Li BL (2005a) Patterns of DNA cytosine methylation between hybrids and their parents in sorghum genome. Acta Agron Sin 31(9):1138–1143Google Scholar
  24. Yi ZB, Sun Y, Niu TT, Liang XH, Liu LL, Yan M, Zhao WJ (2005b) Genomic DNA cytosine methylations of corn hybrids and their parents. Acta Bot Boreali-occidentalia Sin 25(12):2420–2425Google Scholar
  25. Zhang XY (2008) The epigenetic landscape of plants. Science 320(5875):489–492PubMedCrossRefGoogle Scholar
  26. Zhang YH, Yi HY, Fang M, Rong TZ, Cao MJ (2014) Cytological observation and DNA methylation analysis of two new cytoplasmic male sterile lines of maize during microspore genesis. Hereditas 36(10):1021–1026PubMedGoogle Scholar
  27. Zhao XX, Chai Y, Liu B (2007) Epigenetic inheritance and variation of DNA methylation level and pattern in maize intra-specific hybrids. Plant Sci 172(5):930–938CrossRefGoogle Scholar
  28. Zheng YL, Liu JL (1992) Relativity between mitochondria DNA and fertility instability of CMS-S in maize. J Hua Zhong Agric Univ 11(2):120–126Google Scholar
  29. Zhu XX, Wang BH, Guo WZ, Zhang TZ (2009) Inheritance of DNA methylation in two cotton hybrid derived from CRI-12. Acta Agron Sin 35(12):2150–2158CrossRefGoogle Scholar
  30. Zilberman D, Gehring M, Tran RK, Ballinger T, Henikoff S (2007) Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat Genet 39(1):61–69PubMedCrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2015

Authors and Affiliations

  1. 1.Key Laboratory of Maize Biology and Genetic Breeding on Southwest of China, Ministry of AgricultureMaize Research Institute of Sichuan Agricultural UniversityChengduChina
  2. 2.Agronomy Department of Xichang CollegeXichangChina

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