Molecular Genetics and Genomics

, Volume 288, Issue 9, pp 445–457 | Cite as

RNA editing events in mitochondrial genes by ultra-deep sequencing methods: a comparison of cytoplasmic male sterile, fertile and restored genotypes in cotton

  • Hideaki Suzuki
  • Jiwen Yu
  • Scott A. Ness
  • Mary A. O’Connell
  • Jinfa ZhangEmail author
Original Paper


Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in failure to produce functional pollen and is widely used in the production of hybrid seed. Improper RNA editing is implicated as the molecular basis for some CMS systems. However, the mechanism of CMS in cotton is unknown. This study compared RNA editing events in eight mitochondrial genes (atp1, 4, 6, 8, 9, and cox1, 2, 3) among three lines (maintainer B, CMS A, and restorer R). These events were quantified by ultra-deep sequencing of mitochondrial transcripts and sequencing of cloned versions of these genes as cDNAs. A comparison of genomic PCR and RT-PCR products detected 72 editing sites in coding sequences in the eight genes and four partial editing sites in the 3′-untranslated region of atp6. The most frequent alteration (61.4 %) resulted in changes of hydrophilic amino acids to hydrophobic amino acids and the most common alteration was proline (P) to leucine (L) (26.7 %). In atp6, RNA editing created a stop codon from a glutamine in the genomic sequence. Statistical analysis of the frequencies of RNA editing events detected differences between mtDNA genes, but no differences between cotton cytoplasms that could account for the CMS phenotype or restoration. This study represents the first work to use next-generation sequencing to identify RNA editing positions and efficiency, and possible association with CMS and restoration in plants.


Cotton  Cytoplasmic male sterility  Restoration  RNA editing  Ultra-deep sequencing 



The research was supported in part by New Mexico Agricultural Experiment Station. The authors thank Anthony Aragon and Jeremy Edwards for help with the Ion Torrent sequencing. Some of the experiments used the Keck-UNM Genomics Resource in the University of New Mexico Cancer Center. Parts of this work were supported by USPHS/NIH grant 1R01CA170250-01 (to SAN).

Supplementary material

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Supplementary material 1 (DOCX 50 kb)
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  1. Araya A, Domec C, Bégu D, Litvak S (1992) An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts. Proc Natl Acad Sci USA 89:1040–1044PubMedCrossRefGoogle Scholar
  2. Bégu D, Graves PV, Domec C, Arselin G, Litvak S, Araya A (1990) RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing. Plant Cell 2:1283–1290PubMedGoogle Scholar
  3. Bentolila S, Elliott LE, Hanson MR (2008) Genetic architecture of mitochondrial editing in Arabidopsis thaliana. Genet 178:1693–1708CrossRefGoogle Scholar
  4. Binder S, Marchfelder A, Brennicke A, Wissinger B (1992) RNA editing in trans-splicing intron sequences of nad2 mRNAs in Oenothera. Mitochondria 267:7615–7623Google Scholar
  5. Bonen L (2008) Cis- and trans-splicing of group II introns in plant mitochondria. Mitochondrion 8:26–34PubMedCrossRefGoogle Scholar
  6. Castandet B, Choury D, Begu D, Jordana X, Araya A (2010) Intron RNA editing is essential for splicing in plant mitochondria. Nucleic Acids Res 38:7112–7121PubMedCrossRefGoogle Scholar
  7. Chapdelaine Y, Bonen L (1991) The wheat mitochondrial gene for subunit I of the NADH dehydrogenase complex: a f/ww-splicing model for this gene-in-pieces. Cell 65:465–472PubMedCrossRefGoogle Scholar
  8. Chase CD (2007) Cytoplasmic male sterility: a window to the world of plant mitochondrial–nuclear interactions. Trends Genet 23:81–90PubMedCrossRefGoogle Scholar
  9. Corneille S, Lutz K, Maliga P (2000) Conservation of RNA editing between rice and maize plastids are most editing events dispensable? Mol Gen Genet 264:419–424PubMedCrossRefGoogle Scholar
  10. Covello PS, Gray MW (1989) RNA editing in plant mitochondria. Nature 341:662–666PubMedCrossRefGoogle Scholar
  11. Covello PS, Gray MW (1990) Differences in editing at homologous sites in messenger RNAs from angiosperm mitochondria. Nucleic Acids Res 18:5189–5196PubMedCrossRefGoogle Scholar
  12. Das S, Sen S, Chakraborty A, Chakraborti P, Maiti MK, Basu A, Basu D, Sen SK (2010) An unedited 1.1 kb mitochondrial orfB gene transcript in the wild abortive cytoplasmic male sterility (WA-CMS) system of Oryza sativa L. subsp. Indica. BMC Plant Biol 10:39PubMedCrossRefGoogle Scholar
  13. Fujii S, Small I (2011) The evolution of RNA editing and pentatricopeptide repeat genes. New Phytol 191:37–47PubMedCrossRefGoogle Scholar
  14. Gallagher LJ, Betz SK, Chase CD (2002) Mitochondrial RNA editing truncates a chimeric open reading frame associated with S male-sterility in maize. Curr Genet 42:179–184PubMedCrossRefGoogle Scholar
  15. Giegé P, Brennicke A (1999) RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs. Proc Natl Acad Sci USA 96:15324–15329PubMedCrossRefGoogle Scholar
  16. Grewe F, Herres S, Viehöver P, Polsakiewicz M, Weisshaar B, Knoop V (2011) A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii. Nucleic Acids Res 39:2890–2902PubMedCrossRefGoogle Scholar
  17. Gualberto JM, Lamattina L, Bonnard G, Weil JH, Grienenberger JM (1989) RNA editing in wheat mitochondria results in the conservation of protein sequences. Nature 341:660–662PubMedCrossRefGoogle Scholar
  18. Gualberto JM, Weil JH, Grienenberger JM (1990) Editing of the wheat coxIII transcript: evidence for twelve C to U and one U to C conversions and for sequence similarities around editing sites. Nucleic Acids Res 18:3771–3776PubMedCrossRefGoogle Scholar
  19. Handa H (2003) The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana. Nucleic Acids Res 31:5907–5916PubMedCrossRefGoogle Scholar
  20. Hanson MR, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16:S154–S169PubMedCrossRefGoogle Scholar
  21. Hanson MR, Sutton CA, Lu BW (1996) Plant organelle gene expression: altered by RNA editing. Trends Plant Sci 1:57–64CrossRefGoogle Scholar
  22. Hiesel R, Wissinger B, Schuster W, Brennicke A (1989) RNA editing in plant mitochondria. Science 246:1632–1634PubMedCrossRefGoogle Scholar
  23. Howad W, Kempken F (1997) Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor. Proc Natl Acad Sci USA 94:11090–11095PubMedCrossRefGoogle Scholar
  24. Hu J, Wang K, Huang W, Liu G, Gao Y, Wang J, Huang Q, Ji Y, Qin X, Wan L, Zhu R, Li S, Yang D, Zhu Y (2012) The rice pentatricopeptide repeat protein RF5 restores fertility in Hong-Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162. Plant Cell 24:109–122PubMedCrossRefGoogle Scholar
  25. Jiang W, Yang S, Yu D, Gai J (2011) A comparative study of ATPase subunit 9 (Atp9) gene between cytoplasmic male sterile line and its maintainer line in soybeans. Afr J Biotech 10:10387–10392Google Scholar
  26. Knoop V, Schuster W, Wissinger B, Brennicke A (1991) Trans splicing integrates an exon of 22 nucleotides into the nad5 mRNA in higher plant mitochondria. EMBO J 10:3483–3493PubMedGoogle Scholar
  27. Kubo T, Nishizawa S, Sugawara A, Itchoda N, Estiati A, Mikami T (2000) The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNA(Cys)(GCA). Nucleic Acids Res 28:2571–2576PubMedCrossRefGoogle Scholar
  28. Kudla J, Bock R (1999) RNA editing in an untranslated region of the Ginkgo chloroplast genome. Gene 234:81–86PubMedCrossRefGoogle Scholar
  29. Kurek I, Ezra D, Begu D, Erel N, Litvak S, Breiman A (1997) Studies on the effects of nuclear background and tissue specificity on RNA editing of the mitochondrial ATP synthase subunits α, 6 and 9 in fertile and cytoplasmic malesterile (CMS) wheat. Theor Appl Genet 95:1305–1311CrossRefGoogle Scholar
  30. Lamattina L, Weil JH, Grienenberger JM (1989) RNA editing at a splicing site of NADH dehydrogenase subunit IV gene transcript in wheat mitochondria. FEBS Lett 258:79–83PubMedCrossRefGoogle Scholar
  31. Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTech 43:649–656CrossRefGoogle Scholar
  32. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681PubMedCrossRefGoogle Scholar
  33. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463PubMedCrossRefGoogle Scholar
  34. Mazers GR, Moyret C, Jeanteur P, Theillet C-G (1991) Direct sequencing by thermal asymmetric PCR. Nucleic Acids Res 19:4783CrossRefGoogle Scholar
  35. Mower JP, Palmer JD (2006) Patterns of partial RNA editing in mitochondrial genes of Beta vulgaris. Mol Gen Genomics 276:285–293CrossRefGoogle Scholar
  36. Okuda K, Hammani K, Tanz SK, Peng L, Fukao Y, Myouga F, Motohashi R, Shinozaki K, Small I, Shikanai T (2010) The pentatricopeptide repeat protein OTP82 is required for RNA editing of plastid ndhB and ndhG transcripts. Plant J 61:339–349PubMedCrossRefGoogle Scholar
  37. Pang M, Stewart JMcD, Zhang J (2011) A mini-scale hot borate method for the isolation of total RNA from a large number of cotton tissue samples. Afr J Biotech 10:15430–15437Google Scholar
  38. Picardi E, Horner DS, Chiara M, Schiavon R, Valle G, Pesole G (2010) Large-scale detection and analysis of RNA editing in grape mtDNA by RNA deep-sequencing. Nucl Acids Res 38:4755–4767PubMedCrossRefGoogle Scholar
  39. Salazar RA, Pring DR, Kempken F (1991) Editing of mitochondrial atp9 transcripts from two sorghum lines. Curr Genet 20:483–486PubMedCrossRefGoogle Scholar
  40. Schnable PS, Wise RP (1998) The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci 3:175–180CrossRefGoogle Scholar
  41. Schuster W, Unseld M, Wissinger B, Brennicke A (1990) Ribosomal protein S14 transcripts are edited in Oenothera mitochondria. Nucleic Acids Res 18:229–233PubMedCrossRefGoogle Scholar
  42. Schuster W, Ternes R, Knoop V, Hiesel R, Wissinger B, Brennicke A (1991) Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs. Curr Genet 20:397–404PubMedCrossRefGoogle Scholar
  43. Sosso D, Mbelo S, Vernoud V, Gendrot G, Dedieu A, Chambrier P, Dauzat M, Heurtevin L, Guyon V, Takenaka M, Rogowsky PM (2012) PPR2263, a DYW-subgroup pentatricopeptide repeat protein, is required for mitochondrial nad5 and cob transcript editing, mitochondrion biogenesis, and maize growth. Plant Cell 24:676–691PubMedCrossRefGoogle Scholar
  44. Suzuki H, Yu J, Wang F, Zhang J (2013) Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton. Theor Appl Genet. doi: 10.1007/s00122-013-2070-4 PubMedGoogle Scholar
  45. Tan HQ, Singh J (2011) High-efficiency thermal asymmetric interlaced (HE-TAIL) PCR for amplification of Ds transposon insertion sites in Barley. J Plant Mol Biol Biotechnol 2:9–14Google Scholar
  46. Terauchi R, Kahl G (2000) Rapid isolation of promoter sequences by TAIL-PCR: the 5′-flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet 263:554–560PubMedCrossRefGoogle Scholar
  47. Unseld M, Marienfeld JR, Brandt P, Brennicke A (1997) The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924. Nat Genet 15:57–61PubMedCrossRefGoogle Scholar
  48. Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12PubMedCrossRefGoogle Scholar
  49. Wang F, Feng CD, O’Connell MA, Stewart JMcD, Stewart JF (2010) RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton. Euphytica 172:93–99CrossRefGoogle Scholar
  50. Wei L, Yan ZX, Ding Y (2008) Mitochondrial RNA editing of F0-ATPase subunit 9 gene (atp9) transcripts of Yunnan purple rice cytoplasmic male sterile line and its maintainer line. Acta Physiol Plant 30:657–662CrossRefGoogle Scholar
  51. Wilkins TA, Smart LB (1996) Isolation of RNA from plant tissue. In: Krieg PA (ed) A laboratory guide to RNA: isolation, analysis, and synthesis. Wiley-Liss Inc., New York, pp 21–42Google Scholar
  52. Yura K, Go M (2008) Correlation between amino acid residues converted by RNA editing and functional residues in protein three-dimensional structures in plant organelles. BMC Plant Biol 8:79PubMedCrossRefGoogle Scholar
  53. Zhang J, Stewart JMcD (2000) Economical and rapid method for extracting cotton genomic DNA. J Cott Sci 4: 193-201Google Scholar
  54. Zhang J, Stewart J McD (2001) Inheritance and genetic relationships of the D8 and D2-2 restorer genes for cotton cytoplasmic male sterility. Crop Sci 41:289–294Google Scholar
  55. Zhang JF, Turley RB, Stewart JMcD (2008) Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display. Plant Cell Rep 27:553–561PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Hideaki Suzuki
    • 1
    • 3
  • Jiwen Yu
    • 1
    • 2
  • Scott A. Ness
    • 3
  • Mary A. O’Connell
    • 1
  • Jinfa Zhang
    • 1
    Email author
  1. 1.Department of Plant and Environmental SciencesNew Mexico State UniversityLas CrucesUSA
  2. 2.State Key Laboratory in Cotton Biology, Cotton Research InstituteChinese Academy of Agricultural ScienceAnyangChina
  3. 3.Molecular Genetics and MicrobiologyUniversity of New Mexico Health Sciences CenterAlbuquerqueUSA

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