Theoretical and Applied Genetics

, Volume 132, Issue 1, pp 227–240 | Cite as

Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris)

  • Takumi Arakawa
  • Sachiyo Ue
  • Chihiro Sano
  • Muneyuki Matsunaga
  • Hiroyo Kagami
  • Yu Yoshida
  • Yosuke Kuroda
  • Kazunori Taguchi
  • Kazuyoshi Kitazaki
  • Tomohiko KuboEmail author
Original Article


Key message

The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele.


Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.



This work was supported in part by JSPS KAKENHI Grant Number 18K05564 (TK) and NARO Bio-oriented Technology Research Advancement Institution (BRAIN) (Research program on development of innovative technology, Grant Number 30001A) (KT, KK and TK). TA is a recipient of a JSPS Research Fellowship for Young Scientists (16J01146).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

122_2018_3211_MOESM1_ESM.pdf (76 kb)
Supplementary Fig. 1. Agarose (2%) gel electrophoresis of PCR products amplified with primers for cytoplasmic DNA markers (PDF 76 kb)
122_2018_3211_MOESM2_ESM.pdf (67 kb)
Supplementary Fig. 2. PCR products amplified with primers for DNA marker o7 (PDF 67 kb)
122_2018_3211_MOESM3_ESM.pdf (58 kb)
Supplementary Fig. 3. DNA gel blot analysis of an NK-305 plant probed with an orf20-like 3′-UTR (PDF 58 kb)
122_2018_3211_MOESM4_ESM.pdf (68 kb)
Supplementary Fig. 4. Alignment of nucleotide sequences of orf20NK-198 and orf20NK-305-1 coding and flanking regions (PDF 68 kb)
122_2018_3211_MOESM5_ESM.pdf (71 kb)
Supplementary Fig. 5. Alignment of nucleotide sequences of orf20LS and orf20NK-305-2 coding and flanking regions (PDF 70 kb)
122_2018_3211_MOESM6_ESM.pdf (33 kb)
Supplementary Fig. 6. Alignment of amino acid sequences of the protein products deduced from orf20NK-198 and orf20NK-305-1 nucleotide sequences (PDF 33 kb)
122_2018_3211_MOESM7_ESM.pdf (33 kb)
Supplementary Fig. 7. Alignment of amino acid sequences of the protein products deduced from orf20LS and orf20NK-305-2 nucleotide sequences (PDF 33 kb)
122_2018_3211_MOESM8_ESM.pdf (47 kb)
Supplementary Fig. 8. Alignment of nucleotide sequences of the upstream regions of orf19 and orf20NK-305-2 (PDF 46 kb)
122_2018_3211_MOESM9_ESM.pdf (61 kb)
Supplementary Fig. 9. Alignment of nucleotide sequences of the upstream regions of orf20LS and orf20NK-305-1 (PDF 60 kb)
122_2018_3211_MOESM10_ESM.pdf (91 kb)
Supplementary Fig. 10. Immunoblot analysis of proteins from transgenic suspension cells separated by SDS-PAGE (PDF 91 kb)
122_2018_3211_MOESM11_ESM.pdf (196 kb)
Supplementary Fig. 11. Immunoblot analysis of proteins from transgenic suspension cells separated by BN-PAGE (PDF 196 kb)
122_2018_3211_MOESM12_ESM.pdf (36 kb)
Supplementary Table 1. Nucleotide sequences of primers used in this study (PDF 35 kb)
122_2018_3211_MOESM13_ESM.pdf (36 kb)
Supplementary Table 2. Segregation of male fertility and o7 marker types in an F2 population (PDF 36 kb)
122_2018_3211_MOESM14_ESM.pdf (38 kb)
Supplementary Table 3. Segregation of male fertility and s17 marker types in an admixture population (PDF 37 kb)
122_2018_3211_MOESM15_ESM.pdf (36 kb)
Supplementary Table 4. Segregation of male fertility and s17 type in a BC2F2 population (PDF 36 kb)


  1. Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44:117–122CrossRefGoogle Scholar
  2. Arakawa T, Uchiyama D, Ohgami T, Ohgami R, Murata T et al (2018) A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus. PLoS ONE 13:e0198409CrossRefGoogle Scholar
  3. Budar F, Pelletier G (2001) Male sterility in plants: occurrence, determinism, significance and use. C R Acad Sci Paris Life Sci 324:543–550CrossRefGoogle Scholar
  4. Chase CD (2007) Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions. Trends Genet 23:81–90CrossRefGoogle Scholar
  5. Chen L, Liu Y-G (2014) Male sterility and fertility restoration in crops. Annu Rev Plant Biol 65:579–606CrossRefGoogle Scholar
  6. Cheng D, Kitazaki K, Xu D, Mikami T, Kubo T (2009) The distribution of normal and male-sterile cytoplasms in Chinese sugar-beet germplasm. Euphytica 165:345–351CrossRefGoogle Scholar
  7. Cheng D, Yoshida Y, Kitazaki K, Negoro S, Takahashi H et al (2011) Mitochondrial genome diversity in Beta vulgaris L. ssp. vulgaris (Leaf and Garden Beet Groups) and its implications concerning the dissemination of the crop. Genet Res Crop Evol 58:553–560CrossRefGoogle Scholar
  8. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  9. Ducos E, Touzet P, Boutry M (2001) The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes. Plant J 26:171–180CrossRefGoogle Scholar
  10. Dufay M, Touzet P, Maurice S, Cuguen J (2007) Modelling the maintenance of male-fertile cytoplasm in a gynodioecious population. Heredity 99:349–356CrossRefGoogle Scholar
  11. Duroc Y, Gaillard C, Hiard S, Defrance MC, Pelletier G et al (2005) Biochemical and functional characterization of ORF138, a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae. Biochimie 87:1089–1100CrossRefGoogle Scholar
  12. Duroc Y, Hiard S, Vrielynck N, Ragu S, Budar F (2009) The Ogura sterility-inducing protein forms a large complex without interfering with the oxidative phosphorylation components in rapeseed mitochondria. Plant Mol Biol 70:123–137CrossRefGoogle Scholar
  13. Duvick DN (1965) Cytoplasmic pollen sterility in corn. Adv Genet 13:1–56CrossRefGoogle Scholar
  14. Fujii S, Bond CS, Small ID (2011) Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution. Proc Natl Acad Sci USA 108:1723–1728CrossRefGoogle Scholar
  15. Geddy R, Brown GG (2007) Genes encoding pentatricopeptide repeat (PPR) proteins are not conserved in location in plant genomes and may be subject to diversifying selection. BMC Genom 8:130CrossRefGoogle Scholar
  16. Hanson MR, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16:S154–S169CrossRefGoogle Scholar
  17. Honma Y, Taguchi K, Hiyama H, Yui-Kurino R, Mikami T et al (2014) Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele. Theor Appl Genet 127:2567–2574CrossRefGoogle Scholar
  18. Hu L, Liang W, Yin C, Cui X, Zong J et al (2011) Rice MADS3 regulates ROS homeostasis during late anther development. Plant Cell 23:515–533CrossRefGoogle Scholar
  19. Kagami H, Kurata M, Matsuhira H, Taguchi K, Mikami T et al (2015) Sugar beet (Beta vulgaris L.). In: Wang K (ed) Agrobacterium protocols. Methods in molecular biology, vol 1223. Springer, New York, pp 335–347Google Scholar
  20. Kagami H, Taguchi K, Arakawa T, Kuroda Y, Tamagake H et al (2016) Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.). Hereditas 153:12CrossRefGoogle Scholar
  21. Kato H, Tezuka K, Feng YY, Kawamoto T, Takahashi H et al (2007) Structural diversity and evolution of the Rf-1 locus in the genus Oryza. Heredity 99:516–524CrossRefGoogle Scholar
  22. Kim Y-J, Zhang D (2018) Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci 23:53–65CrossRefGoogle Scholar
  23. Kitazaki K, Kubo T, Kagami H, Matsumoto T, Fujita A et al (2011) A horizontally transferred tRNACys gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is amino acylated. Plant J 68:262–272CrossRefGoogle Scholar
  24. Kitazaki K, Arakawa T, Matsunaga M, Yui-Kurino R, Matsuhira H et al (2015) Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris). Plant J 83:290–299CrossRefGoogle Scholar
  25. Lane N (2011) Mitonuclear match: optimizing fitness and fertility over generations drives ageing within generations. BioEssays 33:860–869CrossRefGoogle Scholar
  26. Laporte V, Merdinoglu D, Saumitou-Laprade P, Butterlin G, Vernet P et al (1998) Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. maritima. Theor Appl Genet 96:989–996CrossRefGoogle Scholar
  27. Lee J, Yoon JB, Park HG (2008) Linkage analysis between the partial restoration (pr) and the restorer-of-fertility (Rf) loci in pepper cytoplasmic male sterility. Theor Appl Genet 117:383–389CrossRefGoogle Scholar
  28. Li X-Q, Jean M, Landry BS, Brown GG (1998) Restorer genes for different forms of Brassica cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several mitochondrial genes. Proc Natl Acad Sci USA 95:10032–10037CrossRefGoogle Scholar
  29. Luo D, Xu H, Liu Z, Guo J, Li H et al (2013) A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice. Nat Genet 45:573–577CrossRefGoogle Scholar
  30. Mackenzie SA (2005) The influence of mitochondrial genetics on crop breeding strategies. In: Janick J (ed) Plant breeding reviews. Wiley, New York, pp 115–138Google Scholar
  31. Majewska-Sawka A, Rodriguez-Garcia MI, Nakashima H, Jassen B (1993) Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.). Sex Plant Reprod 6:22–32CrossRefGoogle Scholar
  32. Matsuhira H, Kagami H, Kurata M, Kitazaki K, Matsunaga M et al (2012) Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.). Genetics 192:1347–1358CrossRefGoogle Scholar
  33. Melonek J, Stone JD, Small I (2016) Evolutionary plasticity of restorer-of-fertility-like proteins in rice. Sci Rep 6:35152CrossRefGoogle Scholar
  34. Mora JRH, Rivals E, Mireau H, Budar F (2010) Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo). BMC Plant Biol 10:35CrossRefGoogle Scholar
  35. Moritani M, Taguchi K, Kitazaki K, Matsuhira H, Katsuyama T et al (2013) Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype. Mol Breed 32:91–100CrossRefGoogle Scholar
  36. Nishizawa S, Kubo T, Mikami T (2000) Variable number of tandem repeat loci in the mitochondrial genomes of beets. Curr Genet 37:34–38CrossRefGoogle Scholar
  37. Ohgami T, Uchiyama D, Ue S, Yui-Kurino R, Yoshida Y, Kamei Y, Kuroda Y et al (2016) Identification of molecular variants of the nonrestoring restorer-of-fertility 1 allele in sugar beet (Beta vulgaris L.). Theor Appl Genet 129:675–688CrossRefGoogle Scholar
  38. Owen FV (1945) Cytoplasmically inherited male-sterility in sugar beets. J Agric Res 71:423–440Google Scholar
  39. Papini A, Mosti S, Brighigna L (1999) Programmed-cell-death events during tapetum development of angiosperms. Protoplasma 207:213–221CrossRefGoogle Scholar
  40. Parish RW, Li SF (2010) Death of a tapetum: a programme of developmental altruism. Plant Sci 178:73–89CrossRefGoogle Scholar
  41. Pillen K, Steinrücken G, Herrmann RG, Jung C (1993) An extended linkage map of sugar beet (Beta vulgaris L.) including nine putative lethal genes and the restorer gene X. Plant Breed 111:265–272CrossRefGoogle Scholar
  42. Ran Z, Michaelis G (1995) Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris). Theor Appl Genet 91:836–840CrossRefGoogle Scholar
  43. Rhoads DM, Brunner-Neuenschwander B, Levings CS III, Siedow JN (1998) Cross-linking and disulfide bond formation of introduced cysteine residues suggest a modified model for the tertiary structure of URF13 in the pore-forming oligomers. Arch Biochem Biophys 354:158–164CrossRefGoogle Scholar
  44. Rogers HJ (2006) Programmed cell death in floral organs: how and why do flowers die? Ann Bot 97:309–315CrossRefGoogle Scholar
  45. Ryan MT, Hoogenraad NJ (2007) Mitochondrial-nuclear communications. Annu Rev Biochem 76:701–722CrossRefGoogle Scholar
  46. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  47. Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu Y-C et al (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11:297–322CrossRefGoogle Scholar
  48. Schnable PS, Wise RP (1998) The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci 3:175–180CrossRefGoogle Scholar
  49. Stone JD, Koloušková P, Sloan D, Štorchová H (2017) Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris. J Exp Bot 68:1599–1612CrossRefGoogle Scholar
  50. Taguchi K, Hiyama H, Yui-Kurino R, Muramatsu A, Mikami T et al (2014) Hybrid breeding skewed the allelic frequencies of molecular variants derived from restorer-of-fertility 1 locus for cytoplasmic male sterility in sugar beet (Beta vulgaris L.). Crop Sci 54:1407–1412CrossRefGoogle Scholar
  51. Tang H, Xie Y, Liu Y-G, Chen L (2017) Advances in understanding the molecular mechanisms of cytoplasmic male sterility and restoration in rice. Plant Reprod 30:179–184CrossRefGoogle Scholar
  52. Touzet P (2012) Mitochondrial genome evolution and gynodioecy. In: Marechal-Drouard L (ed) Mitochondrial genome evolution. Academic Press, Oxford, pp 71–98CrossRefGoogle Scholar
  53. Touzet P, Hueber N, Bürkholz A, Barnes S, Cuguen J (2004) Genetic analysis of male fertility restoration in wild cytoplasmic male sterility G of beet. Theor Appl Genet 109:240–247CrossRefGoogle Scholar
  54. van der Blienk AM, Sedensky MM, Morgan PG (2017) Cell biology of the mitochondrion. Genetics 207:843–871CrossRefGoogle Scholar
  55. Wilson ZA, Zhang D-B (2009) From Arabidopsis to rice: pathways in pollen development. J Exp Bot 60:1479–1492CrossRefGoogle Scholar
  56. Wise RP, Dill CL, Schnable PS (1996) Mutator-induced mutations of the rf1 nuclear fertility restorer of T-cytoplasm maize alter the accumulation of T-urf13 mitochondrial transcripts. Genetics 143:1383–1394PubMedPubMedCentralGoogle Scholar
  57. Yamamoto MP, Kubo T, Mikami T (2005) The 5′-leader sequence of sugar beet mitochondrial atp6 encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility. Mol Genet Genom 273:342–349CrossRefGoogle Scholar
  58. Zhang H, Cheng X, Zhang L, Si H, Ge Y et al (2018) Rf4 has minor effects on the fertility restoration of wild abortive-type cytoplasmic male sterile japonica (Oryza sativa) lines. Euphytica 214:49CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Takumi Arakawa
    • 1
  • Sachiyo Ue
    • 1
  • Chihiro Sano
    • 1
  • Muneyuki Matsunaga
    • 1
  • Hiroyo Kagami
    • 1
  • Yu Yoshida
    • 1
  • Yosuke Kuroda
    • 2
  • Kazunori Taguchi
    • 2
  • Kazuyoshi Kitazaki
    • 1
  • Tomohiko Kubo
    • 1
    Email author return OK on get
  1. 1.Research Faculty of AgricultureHokkaido UniversitySapporoJapan
  2. 2.Hokkaido Agricultural Research CenterNational Agriculture and Food Research OrganizationMemuroJapan

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