Theoretical and Applied Genetics

, Volume 132, Issue 2, pp 313–322 | Cite as

Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms

  • Bongju Kim
  • Tae-Jin Yang
  • Sunggil KimEmail author
Original Article


Key message

Almost identical mitochondrial genome sequences of two recently diverged male-fertile normal and male-sterile CMS-T-like cytoplasms were obtained in onions. A chimeric gene, orf725 , was found to be a CMS-inducing gene.


In onions (Allium cepa L.), cytoplasmic male-sterility (CMS) has been widely used in hybrid seed production. Two types of CMS (CMS-S and CMS-T) have been reported in onions. A complete mitochondrial genome sequence of the CMS-S cytoplasm has been reported in our previous study. Draft mitochondrial genome sequences of male-fertile normal and CMS-T-like cytoplasms are reported in this study. Raw reads obtained from normal and CMS-T-like cytoplasms were assembled into eight and nine almost identical contigs, respectively. After connection and reorganization of contigs by PCR amplification and genome walking, four scaffold sequences with total length of 339 and 180 bp were produced for the normal cytoplasm. A mitochondrial genome sequence of the CMS-T-like cytoplasm was obtained by mapping trimmed reads of CMS-T onto scaffold sequences of the normal cytoplasm. Compared with the CMS-S mitochondrial genome, the normal mitochondrial genome was highly rearranged with 31 syntenic blocks. A total of 499 single nucleotide polymorphisms (SNPs) or insertions/deletions were identified in these syntenic regions. On the other hand, normal and CMS-T-like mitochondrial genome sequences were almost identical except for orf725, a chimeric gene consisting of cox1 with other sequences. Only three SNPs were identified between normal and CMS-T-like syntenic sequences. These results indicate that orf725 is likely to be the casual gene for CMS induction in onions and that CMS-T-like cytoplasm has recently diverged from the normal cytoplasm by introduction of orf725.



This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program (Vegetable Breeding Research Center), funded by the Ministry of Agriculture, Food and Rural Affairs (710011-03), Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-2-SBB10), and a Grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013400). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The authors declare that all experiments complied with current laws of the Republic of Korea.

Supplementary material

122_2018_3218_MOESM1_ESM.tif (145 kb)
Supplementary Fig. 1. PCR amplification patterns of four previously developed molecular markers used for identification of cytoplasm types of onions. The 7th nucleotide ‘G’ in the reverse primer of the marker reported by Havey (1995) was changed into ‘A’ to avoid mismatch with complete chloroplast genome sequences of the normal (GenBank accession: KM088013), CMS-S (KM088014), and CMS-T (KM088015) cytoplasms. (TIFF 145 kb)
122_2018_3218_MOESM2_ESM.tif (111 kb)
Supplementary Fig. 2. Organization of nine contigs assembled from normal and CMS-T-like cytoplasms using trimmed reads produced by next-generation sequencing. Genes transcribed as forward and reverse complements are indicated as boxes on and beneath lines, respectively. Filled and empty boxes indicate exons and introns, respectively. Contig 9 was identified only in the CMS-T-like cytoplasm. (TIFF 110 kb)
122_2018_3218_MOESM3_ESM.tif (250 kb)
Supplementary Fig. 3. Duplicated sequences in scaffold 1 and scaffold 2. A. Positions of duplicated sequences. Duplicated sequences are shown as hatched boxes. Arrow-shaped boxes indicate 5′-to-3′ direction. Exons and introns are shown as gray and empty boxes, respectively. Horizontal arrows indicate primer-binding sites. B. PCR products of primer pairs designed based on duplicated regions and their flanking sequences shown in Supplementary Fig. 2A. A primer pair of nad3-F1 and rps12 was designed for nad3 and rps12 genes, respectively. (TIFF 250 kb)
122_2018_3218_MOESM4_ESM.tif (186 kb)
Supplementary Fig. 4. Duplicated sequences in scaffold 1 and scaffold 3. A. Positions of duplicated sequences. Duplicated sequences are shown as hatched boxes. Filled boxes indicate positions of R6 repeats (Supplementary Table 5). Arrow-shaped boxes indicate 5′-to-3′ direction. Horizontal arrows indicate primer-binding sites. B. PCR products amplified using primer pairs designed based on the duplicated regions and their flanking sequences as shown in Supplementary Fig. 3A. A primer pair of nad3-F1 and rps12 was designed based on nad3 and rps12 genes, respectively. (TIFF 186 kb)
122_2018_3218_MOESM5_ESM.tif (193 kb)
Supplementary Fig. 5. Positions of repetitive sequences larger than 500 base pairs on scaffold sequences. Genes transcribed as forward and reverse complements are indicated as small boxes on and beneath large rectangular boxes, respectively. Exons and introns are shown as dark-gray and empty small boxes, respectively. Repeat sequences are shown as filled boxes in large rectangular boxes. Names of repeats in filled boxes are shown on inverted triangles linked to repeats. Detailed information about repeats is described in Supplementary Table 5. (TIFF 193 kb)
122_2018_3218_MOESM6_ESM.tif (338 kb)
Supplementary Fig. 6. Distribution of repeat sequences in mitochondrial genome sequences of onions and four other plant species. Dot matrix views of sequence alignments performed by BLAST search ( against themselves are presented. GenBank accession numbers of complete mitochondrial genome sequences are KU318712 (Allium cepa, CMS-S cytoplasm), JN375330 (Phoenix dactylifera), KX028885 (Cocos nucifera), JQ804980 (Spirodela polyrhiza), KR559021 (Heuchera parviflora), DQ645536 (Zea mays), and BA000029 (Oryza sativa). (TIFF 337 kb)
122_2018_3218_MOESM7_ESM.tif (138 kb)
Supplementary Fig. 7. Verification of sequence organizations flanking orf725 and cox1 genes using long PCR amplification. A. PCR products of three combinations of primers. Positions of primers are shown in Fig. 2. Sequences of primers are shown in Supplementary Table 1. N, T, S indicate normal, CMS-T-like, and CMS-S mitotypes, respectively. PCR product under the asterisk is a low-copy-number subgenome containing gene organization similar to the corresponding region of CMS-S mitotype. B. Organization of the subgenomic sequence containing orf725 in the CMS-T-like mitotype. Nucleotide sequences of this PCR product were verified by sequencing PCR product which had been further amplified by ten additional cycles. Arrow-shaped boxes indicate 5′-to-3′ direction. Horizontal arrows indicate primer-binding sites. Homologous regions are connected by vertical lines. Filled, hatched, and dotted boxes indicate identical repeat sequences. (TIFF 137 kb)
122_2018_3218_MOESM8_ESM.tif (933 kb)
Supplementary Fig. 8. Location and probability of transmembrane domains in deduced amino acid sequence of orf725. The output of TMHMM Server ( is shown underneath the orf725 diagram. An arrow-shaped box indicates the 5′-to-3′ direction. Gray and black colors indicate cox1-homologous and unknown sequences, respectively. (TIFF 932 kb)
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Supplementary material 9 (DOCX 14 kb)
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Supplementary material 14 (DOCX 14 kb)


  1. Abdelnoor RV, Christensen AC, Mohammed S, Munoz-Castillo B, Moriyama H, Mackenzie SA (2006) Mitochondrial genome dynamics in plants and animals: convergent gene fusions of a MutS homologue. J Mol Evol 63:165–173CrossRefGoogle Scholar
  2. Albert B, Godelle B, Gouyon PH (1998) Evolution of the plant mitochondrial genome: dynamics of duplication and deletion of sequences. J Mol Evol 46:155–158CrossRefGoogle Scholar
  3. Allen JO, Fauron CM, Mink P, Roark L, Oddiraju S, Lin GN, Meyer L, Sun H, Kim K, Wang C, Du F, Xu D, Gibson M, Cifrese J, Clifton SW, Newton KJ (2007) Comparisons among two fertile and three male-sterile mitochondrial genomes of maize. Genetics 177:1173–1192CrossRefGoogle Scholar
  4. Alverson AJ, Rice DW, Dickinson S, Barry K, Palmer JD (2011) Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber. Plant Cell 23:2499–2513CrossRefGoogle Scholar
  5. Archibald JM (2015) Endosymbiosis and eukaryotic cell evolution. Curr Biol 25:R911–R921CrossRefGoogle Scholar
  6. Arrieta-Montiel M, Lyznik A, Woloszynska M, Janska H, Tohme J, Mackenzie SA (2001) Tracing evolutionary and developmental implications of mitochondrial stoichiometric shifting in the common bean. Genetics 158:851–864Google Scholar
  7. Backert S, Neilsen BL, Börner T (1997) The mystery of the rings: structure and replication of mitochondrial genomes from higher plants. Trends Plant Sci 2:477–483CrossRefGoogle Scholar
  8. Bellaoui M, Martin-Canadell A, Pelletier G, Budar F (1998) Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids. Mol Gen Genet 257:177–185CrossRefGoogle Scholar
  9. Bohra A, Jha UC, Adhimoolam P, Bisht D, Singh NP (2016) Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep 35:967–993CrossRefGoogle Scholar
  10. Bonhomme S, Budar F, Ferault M, Pelletier G (1991) A 2.5 kb Nco I fragment of Ogura radish mitochondrial DNA is correlated with cytoplasmic male-sterility in Brassica cybrids. Curr Genet 19:121–127CrossRefGoogle Scholar
  11. Bonhomme S, Budar F, Lancelin D, Small I, Defrance M, Pelletier G (1992) Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids. Mol Gen Genet 235:340–348CrossRefGoogle Scholar
  12. Budar F, Touzet P, De Paepe R (2003) The nucleo-mitochondrial conflict in cytoplasmic male sterilities revised. Genetica 117:3–16CrossRefGoogle Scholar
  13. Courcel AGL, de Vedel F, Boussac JM (1989) DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions. Theor Appl Genet 77:793–798CrossRefGoogle Scholar
  14. Cui X, Wise RP, Schnable PS (1996) The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science 272:1334–1336CrossRefGoogle Scholar
  15. Engelke T, Terefe D, Tatlioglu T (2003) A PCR-based marker system monitoring CMS-(S), CMS-(T) and (N)-cytoplasm in the onion (Allium cepa L.). Theor Appl Genet 107:162–167CrossRefGoogle Scholar
  16. Fauron CMR, Moore B, Casper M (1995) Maize as a model of higher plant mitochondrial genome plasticity. Plant Sci 112:11–32CrossRefGoogle Scholar
  17. Forde BG, Oliver RJC, Leaver CJ (1978) Variation in mitochondrial translation products associated with male-sterile cytoplasm s in maize. Proc Natl Acad Sci USA 75:3841–3845CrossRefGoogle Scholar
  18. Fujii S, Toriyama K (2009) Suppressed expression of RETROGRADE-REGULATED MALE STERILITY restores pollen fertility in cytoplasmic male sterile rice plants. Proc Natl Acad Sci USA 106:9513–9518CrossRefGoogle Scholar
  19. Gaborieau L, Brown GG, Mireau H (2016) The propensity of pentatricopeptide repeat genes to evolve into restorers of cytoplasmic male sterility. Front Plant Sci 7:1816CrossRefGoogle Scholar
  20. Hanson MR, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16:S154–S169CrossRefGoogle Scholar
  21. Havey MJ (1993) A putative donor of S-cytoplasm and its distribution among open-pollinated populations of onion. Theor Appl Genet 86:128–134CrossRefGoogle Scholar
  22. Havey MJ (1995) Identification of cytoplasms using the polymerase chain reaction to aid in the extraction of maintainer lines from open-pollinated populations of onion. Theor Appl Genet 90:263–268CrossRefGoogle Scholar
  23. Havey MJ (2000) Diversity among male-sterility-inducing and male-fertile cytoplasms of onion. Theor Appl Genet 101:778–782CrossRefGoogle Scholar
  24. Holford P, Croft JH, Newbury HJ (1991) Differences between, and possible origins of, the cytoplasms found in fertile and male-sterile onions (Allium cepa L.). Theor Appl Genet 82:737–744CrossRefGoogle Scholar
  25. 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 24:109–122CrossRefGoogle Scholar
  26. Janska H, Sarria R, Woloszynska M, Arrieta-Montiel M, Mackenzie SA (1998) Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility. Plant Cell 10:1163–1180CrossRefGoogle Scholar
  27. Jones HA, Emsweller SL (1936) A male-sterile onion. Proc Am Soc Hort Sci 34:582–585Google Scholar
  28. Kim S (2013) Identification of hypervariable chloroplast intergenic sequences in onion (Allium cepa L.) and their use to analyse the origins of male-sterile onion cytotypes. J Hortic Sci Biotech 88:187–194CrossRefGoogle Scholar
  29. Kim S (2014) A codominant molecular marker in linkage disequilibrium with a restorer-of-fertility gene (Ms) and its application in reevaluation of inheritance of fertility restoration in onions. Mol Breed 34:769–778CrossRefGoogle Scholar
  30. Kim S, Lim H, Park S, Cho K, Sung S, Oh D, Kim K (2007) Identification of a novel mitochondrial genome type and development of molecular makers for cytoplasm classification in radish (Raphanus sativus L.). Theor Appl Genet 115:1137–1145CrossRefGoogle Scholar
  31. Kim S, Lee E, Cho DY, Han T, Bang H, Patil BS, Ahn YK, Yoon M (2009) Identification of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing three cytoplasm types in onion (Allium cepa L.). Theor Appl Genet 118:433–441CrossRefGoogle Scholar
  32. Kim S, Bang H, Patil BS (2013) Origin of three characteristic onion (Allium cepa L.) mitochondrial genome rearrangements in Allium species. Sci Hortic 157:24–31CrossRefGoogle Scholar
  33. Kim S, Kim C, Park M, Choi D (2015a) Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq. Theor Apple Genet 128:2289–2299CrossRefGoogle Scholar
  34. Kim S, Park J, Yang T (2015b) Comparative analysis of the complete chloroplast genome sequences of a normal male-fertile cytoplasm and two different cytoplasms conferring cytoplasmic male sterility in onion (Allium cepa L.). J Hortic Sci Biotech 90:459–468CrossRefGoogle Scholar
  35. Kim B, Kim K, Yang T, Kim S (2016) Completion of the mitochondrial genome sequence of onion (Allium cepa L.) containing the CMS-S male-sterile cytoplasm and identification of an independent event of the ccmF N gene split. Curr Genet 62:873–885CrossRefGoogle Scholar
  36. Kitazaki K, Arakawa T, Matsunaga M, Yui-Kurino R, Matsuhira H, Mikami T, Kubo T (2015) Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris). Plant J 83:290–299CrossRefGoogle Scholar
  37. Kmiec B, Woloszynska M, Janska H (2006) Heteroplasmy as a common state of mitochondrial genetic information in plants and animals. Curr Genet 50:149–159CrossRefGoogle Scholar
  38. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL (2004) Versatile and open software for comparing large genomes. Genome Biol 5:R12CrossRefGoogle Scholar
  39. L’Homme Y, Brown GG (1993) Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus. Nucleic Acids Res 8:1903–1909CrossRefGoogle Scholar
  40. Laser KD, Lersten NR (1972) Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms. Bot Rev 38:425–454CrossRefGoogle Scholar
  41. Mackenzie SA, Chase CD (1990) Fertility restoration is associated with loss of a portion of the mitochondrial genome in cytoplasmic male-sterile common bean. Plant Cell 2:905–912CrossRefGoogle Scholar
  42. Oldenburg DJ, Bendich AJ (2001) Mitochondrial DNA from the Liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5′ protein. J Mol Biol 310:549–562CrossRefGoogle Scholar
  43. Park JY, Lee Y, Lee J, Choi B, Kim S, Yang T (2013) Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm. Theor Appl Genet 126:1763–1774CrossRefGoogle Scholar
  44. Rice DW, Alverson AJ, Richardson AO, Young GJ, Sanchez-Puerta MV, Munzinger J, Barry K, Boore JL, Zhang Y, dePamphilis CW, Knox EB, Palmer JD (2013) Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella. Science 342:1468–1473CrossRefGoogle Scholar
  45. Sakai T, Imamura J (1993) Evidence for a mitochondrial sub-genome containing radish AtpA in a Brassica napus cybrid. Plant Sci 90:95–103CrossRefGoogle Scholar
  46. Sandhu AP, Abdelnoor RV, Mackenzie SA (2007) Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc Natl Acad Sci USA 104:1766–1770CrossRefGoogle Scholar
  47. Sato Y (1998) PCR amplification of CMS-specific mitochondrial nucleotide sequences to identify cytoplasmic genotypes of onion (Allium cepa L.). Theor Appl Genet 96:367–370CrossRefGoogle Scholar
  48. Satoh M, Kubo T, Nishizawa S, Estiati A, Itchoda N, Mikami T (2004) The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs. Mol Genet Genomics 272:247–256CrossRefGoogle Scholar
  49. Schnable PS, Wise RP (1998) The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci 3:175–180CrossRefGoogle Scholar
  50. Schweisguth B (1973) Étude d’un nouveau type de stérilité male chez l’oignon, Allium cepa L. Ann Amélior Plant 23:221–233Google Scholar
  51. Skippington E, Barkman TJ, Rice DW, Palmer JD (2015) Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes. Proc Natl Acad Sci USA 112:E3515–E3524CrossRefGoogle Scholar
  52. Sloan DB (2013) One ring to rule them all? Genome sequencing provides new insights into the ‘master circle’ model of plant mitochondrial DNA structure. New Phytol 200:978–985CrossRefGoogle Scholar
  53. Sloan DB, Alverson AJ, Chuckalovcak JP, Wu M, McCauley DE, Palmer JD, Taylor DR (2012) Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates. PLoS Biol 10:e1001241CrossRefGoogle Scholar
  54. Small I, Suffolk R, Leaver CJ (1989) Evolution of plant mitochondrial genomes via substoichiometric intermediates. Cell 58:69–76CrossRefGoogle Scholar
  55. Treangen TJ, Salzberg SL (2011) Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet 13:36–46CrossRefGoogle Scholar
  56. Wang D, Wu YW, Shih AC, Wu CS, Wang YN, Chaw SM (2007) Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA. Mol Biol Evol 24:2040–2048CrossRefGoogle Scholar
  57. Wang W, Wu Y, Messing J (2012) The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PLoS ONE 7:e46747CrossRefGoogle Scholar
  58. Woloszynska M, Trojanowski D (2009) Counting mtDNA molecules in Phaseolus vulgaaris: sublimons are constantly produced by recombination via short repeats and undergo rigorous selection during substoichiometric shifting. Plant Mol Biol 70:511–521CrossRefGoogle Scholar
  59. Wu Z, Cuthberta JM, Taylorb DR, Sloana DB (2015) The massive mitochondrial genome of the angiosperm Silene noctiflora is evolving by gain or loss of entire chromosomes. Proc Natl Acad Sci USA 112:10185–10191CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Plant Biotechnology, Biotechnology Research InstituteChonnam National UniversityGwangjuRepublic of Korea
  2. 2.Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life SciencesSeoul National UniversitySeoulRepublic of Korea

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