Molecular Genetics and Genomics

, Volume 286, Issue 5–6, pp 433–447 | Cite as

Comparative sequence analysis of VRN1 alleles of Lolium perenne with the co-linear regions in barley, wheat, and rice

  • Torben Asp
  • Stephen Byrne
  • Heidrun Gundlach
  • Rémy Bruggmann
  • Klaus F. X. Mayer
  • Jeppe R. Andersen
  • Mingliang Xu
  • Morten Greve
  • Ingo Lenk
  • Thomas Lübberstedt
Original Paper


Vernalization, a period of low temperature to induce transition from vegetative to reproductive state, is an important environmental stimulus for many cool season grasses. A key gene in the vernalization pathway in grasses is the VRN1 gene. The objective of this study was to identify causative polymorphism(s) at the VRN1 locus in perennial ryegrass (Lolium perenne) for variation in vernalization requirement. Two allelic Bacterial Artificial Chromosome clones of the VRN1 locus from the two genotypes Veyo and Falster with contrasting vernalization requirements were identified, sequenced, and characterized. Analysis of the allelic sequences identified an 8.6-kb deletion in the first intron of the VRN1 gene in the Veyo genotype which has low vernalization requirement. This deletion was in a divergent recurrent selection experiment confirmed to be associated with genotypes with low vernalization requirement. The region surrounding the VRN1 locus in perennial ryegrass showed microcolinearity to the corresponding region on chromosome 3 in Oryza sativa with conserved gene order and orientation, while the micro-colinearity to the corresponding region in Triticum monococcum was less conserved. Our study indicates that the first intron of the VRN1 gene, and in particular the identified 8.6 kb region, is an important regulatory region for vernalization response in perennial ryegrass.


Lolium perenne Perennial ryegrass VRN1 Comparative genomics Vernalization 



This work was supported by a grant from the framework “Biotechnology and applied plant genetics in plant breeding” from The Directorate for Food, Fisheries and Agricultural Business under the Danish Ministry of Food, Agriculture and Fisheries. Dorthe Strue Nielsen and Kirsten Vangsgaard are acknowledged for their excellent technical assistance.

Supplementary material

438_2011_654_MOESM1_ESM.pdf (24 kb)
Supplementary material 1 (PDF 24 kb)
438_2011_654_MOESM2_ESM.pdf (454 kb)
Supplementary material 2 (PDF 454 kb)


  1. Aamlid TS, Heide OM, Boelt B (2000) Primary and secondary induction requirements for flowering of contrasting European varieties of Lolium perenne. Ann Bot 86:1087–1095CrossRefGoogle Scholar
  2. Andersen JR, Jensen LB, Asp T, Lübberstedt T (2006) Vernalization response in perennial ryegrass (Lolium perenne L.) involves orthologues of diploid wheat (Triticum monococcum) VRN1 and rice (Oryza sativa) Hd1. Plant Mol Biol 60:481–494PubMedCrossRefGoogle Scholar
  3. Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004) Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427:164–167PubMedCrossRefGoogle Scholar
  4. Bennett MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Phil Transac Royal Soc London Series B-Biol Sci 334:309–345CrossRefGoogle Scholar
  5. Bennetzen JL (2000) Transposable element contributions to plant genome evolution. Plant Mol Biol 42:251–269PubMedCrossRefGoogle Scholar
  6. Bruggmann R, Bharti AK, Gundlach H, Lai J, Young S, Pontaroli AC, Wei F, Haberer G, Fuks G, Du C et al (2006) Uneven chromosome contraction and expansion in the maize genome. Genome Res 16:1241–1251PubMedCrossRefGoogle Scholar
  7. Burn JE, Smyth DR, Peacock WJ, Dennis ES (1993) Genes conferring late flowering in Arabidopsis thaliana. Genetica 90:147–155CrossRefGoogle Scholar
  8. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622PubMedCrossRefGoogle Scholar
  9. Choudhuri JV, Schleiermacher C, Kurtz S, Giegerich R (2004) Genalyzer: interactive visualization of sequence similarities between entire genomes. Bioinformatics 20:1964–1965PubMedCrossRefGoogle Scholar
  10. Clarke JH, Dean C (1994) Mapping FRI, a locus controlling flowering time and vernalization response in Arabidopsis thaliana. Mol Gen Genet 242:81–89PubMedGoogle Scholar
  11. Cooper JP (1960) Short-day and low-temperature induction in Lolium. Ann Bot 24:232–246Google Scholar
  12. Corbesier L, Vincent C, Jang SH, Fornara F, Fan QZ, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C (2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316:1030–1033PubMedCrossRefGoogle Scholar
  13. Danyluk J, Kane NA, Breton G, Limin AE, Fowler DB, Sarhan F (2003) TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals. Plant Physiol 132:1849–1860PubMedCrossRefGoogle Scholar
  14. Dubcovsky J, Loukoianov A, Fu D, Valarik M, Sanchez A, Yan L (2006) Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol Biol 60:469–480PubMedCrossRefGoogle Scholar
  15. Evans GM, Rees H, Snell CL, Sun S (1972) The relationship between nuclear DNA amount and the duration of the mitotic cycle. Chromosom Today 3:24–31Google Scholar
  16. Ewing B, Green P (1998) Basecalling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194PubMedGoogle Scholar
  17. Farrar K, Asp T, Lübberstedt T, Xu M, Thomas AM, Christiansen C, Humphreys MO, Donnison IS (2007) Construction of two Lolium perenne BAC libraries and identification of BACs containing candidate genes for disease resistance and forage quality. Plant Breed 19:15–23Google Scholar
  18. Frohman MA, Dush MK, Martin GR (1998) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002CrossRefGoogle Scholar
  19. Fu D, Szücs P, Yan L, Helguera M, Skinner JS, von Zitzewitz J, Hayes PM, Dubcovsky J (2005) Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Mol Gen Genomics 273:54–65CrossRefGoogle Scholar
  20. Gendall AR, Levy YY, Wilson A, Dean C (2001) The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell 107:525–535PubMedCrossRefGoogle Scholar
  21. Gierl A, Saedler H, Peterson PA (1989) Maize transposable elements. Annu Rev Genet 23:71–85PubMedCrossRefGoogle Scholar
  22. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202PubMedGoogle Scholar
  23. Gremme G, Brendel V, Sparks ME, Kurtz S (2005) Engineering a software tool for gene structure prediction in higher organisms. Inf Softw Technol 47:965–978CrossRefGoogle Scholar
  24. Haberer G, Young S, Bharti AK, Gundlach H, Raymond C, Fuks G, Butler E, Wing RA, Rounsley S, Birren B, Nusbaum C, Mayer KF, Messing J (2005) Structure and architecture of the maize genome. Plant Physiol 139:1612–1624PubMedCrossRefGoogle Scholar
  25. Heide OM (1994) Control of flowering and reproduction in temperate grasses. New Phytol 128:347–362CrossRefGoogle Scholar
  26. Higgins JA, Bailey PC, Laurie DA (2010) Comparative genomics of flowering time pathways using Brachypodium distachyon as a model for the temperate grasses. PLoS ONE 5(4):e10065PubMedCrossRefGoogle Scholar
  27. Jensen LB, Andersen JR, Frei U, Xing Y, Taylor C, Holm PB, Lübberstedt T (2005) QTL mapping of vernalization response in perennial ryegrass (Lolium perenne L.) reveals co-location with an orthologue of wheat VRN1. Theor Appl Genet 110:527–536PubMedCrossRefGoogle Scholar
  28. Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C (2000) Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290:344–347PubMedCrossRefGoogle Scholar
  29. Keller B, Feuillet C (2000) Colinearity and gene density in grass genomes. Trends Plant Sci 5:246–251PubMedCrossRefGoogle Scholar
  30. Koornneef M, Blankestijn-de Vries H, Hanhart C, Soppe W, Peeters T (1994) The phenotype of some late-flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Lansberg erecta wild-type. Plant J 6:911–919CrossRefGoogle Scholar
  31. Lee I, Bleecker A, Amasino R (1993) Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol Gen Genet 237:171–176PubMedCrossRefGoogle Scholar
  32. Lee I, Michaels SD, Masshardt AS, Amasino RM (1994) The late-flowering phenotype of FRIGIDA and LUMINIDEPENDENS is suppressed in the Lansberg erecta strain of Arabidopsis. Plant J 6:903–909CrossRefGoogle Scholar
  33. Levy YY, Mesnage S, Mylne JS, Gendall AR, Dean C (2002) Multiple roles of Arabidopsis VRN1 in vernalization and flowering time control. Science 297:243–246PubMedCrossRefGoogle Scholar
  34. Lewin B (1997) Transposons. In: Lewin B (ed) Genes VI. Oxford University Press, New York, pp 563–595Google Scholar
  35. Li C, Dubcovsky J (2008) Wheat FT protein regulates VRN1 transcription through interactions with FDL2. Plant J 55:543–554PubMedCrossRefGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene Expression data using real-time quantitative PCR and the 2 Δ Δ CT method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  37. Lübberstedt T, Andreasen BS, Holm PB et al (2003) Development of ryegrass allele-specific (GRASP) markers for sustainable grassland improvement—a new framework V project. Czech J Genet Plant Breed 39:125–128Google Scholar
  38. Lukashin AV, Borodovsky M (1998) GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26:1107–1115PubMedCrossRefGoogle Scholar
  39. McCarthy EM, McDonald JF (2003) LTR_STRUC: a novel search and identification program for LTR retrotransposons. Bioinformatics 19:362–367PubMedCrossRefGoogle Scholar
  40. Michaels SD, Amasino RM (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11:949–956PubMedCrossRefGoogle Scholar
  41. Napp-Zinn K (1987) Vernalization: environmental and genetic regulation. In: Atherton JG (ed) Manipulation of flowering. Butterworths, London, pp 123–132Google Scholar
  42. Osborn TC, Kole C, Parkin IAP, Sharpe AG, Kuiper M, Lydiate DJ, Trick M (1997) Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana. Genet Soc Am 146:1123–1129Google Scholar
  43. Pereira A, Cuypers H, Gierl A, Sommer ZS, Saedler H (1986) Molecular analysis of the En/Spm transposable element system in Zea mays. EMBO J 5:835–841PubMedGoogle Scholar
  44. Salamov A, Solovyev V (2000) Ab initio gene finding in Drosophila genomic DNA. Genome Res 10:516–522PubMedCrossRefGoogle Scholar
  45. SanMiguel PJ, RamaKrishna W, Bennetzen JL, Busso C, Dubovsky J (2002) Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5Am. Funct Integr Genomics 2:70–80PubMedCrossRefGoogle Scholar
  46. Schranz ME, Quijada P, Sung SB, Lukens L, Amasino R, Osborn TC (2002) Characterization and effects of the replicated flowering time gene FLC in Brassica rapa. Genetics 162:1457–1468PubMedGoogle Scholar
  47. Schwartz S, Zhang Z, Frazer KA, Smit A, Riemer C, Bouck J, Gibbs R, Hardison R, Miller W (2000) PipMaker—a web server for aligning two genomic DNA sequences. Genome Res 10:577–586PubMedCrossRefGoogle Scholar
  48. Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, Peacock WJ, Dennis ES (1999) The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11:445–458PubMedCrossRefGoogle Scholar
  49. Shirasu K, Schulman AH, Lahaye T, Schulze-Lefert P (2000) A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res 10:908–915PubMedCrossRefGoogle Scholar
  50. Skøt L, Humphreys MO, Armstead I, Heywood S, Skøt KP, Sanderson R, Thomas ID, Chorlton KH, Sackville Hamilton NR (2005) An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.). Mol Breed 15:233–245CrossRefGoogle Scholar
  51. Sung S, Amasino RM (2004a) Vernalization and epigenetics: how plants remember winter. Cur Opin Plant Biol 7:4–10CrossRefGoogle Scholar
  52. Sung S, Amasino RM (2004b) Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427:159–164PubMedCrossRefGoogle Scholar
  53. Sung S, Amasino RM (2005) Remembering winter: toward a molecular understanding of vernalization. Annu Rev Plant Biol 56:491–508PubMedCrossRefGoogle Scholar
  54. Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a protein is a mobile flowering signal in rice. Science 316:1033–1036PubMedCrossRefGoogle Scholar
  55. Trevaskis B, Bagnall DJ, Ellis MH, Peacock WJ, Dennis ES (2003) MADS box genes control vernalization-induced flowering in cereals. Proc Natl Acad Sci USA 100:13099–13104PubMedCrossRefGoogle Scholar
  56. Trevaskis B, Hemming MN, Peacock WJ, Dennis ES (2006) HvVRN2 responds to daylength, whereas HvVRN1 is regulated by vernalization and developmental status. Plant Physiol 140:1397–1405PubMedCrossRefGoogle Scholar
  57. Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59:573–594PubMedCrossRefGoogle Scholar
  58. von Zitzewitz J, Szucs P, Dubcovsky J, Yan L, Francia E, Pecchioni N, Casas A, Chen TH, Hayes PM, Skinner JS (2005) Molecular and structural characterization of barley vernalization genes. Plant Mol Biol 59:449–467CrossRefGoogle Scholar
  59. Xing Y, Frei U, Schejbel B, Asp T, Lübberstedt T (2007) Nucleotide diversity and linkage disequilibrium in 11 expressed resistance candidate genes in Lolium perenne. BMC Plant Biol 7:43PubMedCrossRefGoogle Scholar
  60. Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J (2003) Positional cloning of the wheat vernalization gene VRN1. Proc Natl Acad Sci USA 100:6263–6268PubMedCrossRefGoogle Scholar
  61. Yan L, Helguera M, Kato K, Fukuyama S, Sherman J, Dubcovsky J (2004a) Allelic variation at the VRN-1 promoter region in polyploid wheat. Theor Appl Genet 109:1677–1686PubMedCrossRefGoogle Scholar
  62. Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, SanMiguel P, Bennetzen JL, Echenique V, Dubcovsky J (2004b) The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science 303:1640–1644PubMedCrossRefGoogle Scholar
  63. Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik M, Dubcovsky J (2006) The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc Natl Acad Sci USA 103:19581–19586PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Torben Asp
    • 1
  • Stephen Byrne
    • 1
  • Heidrun Gundlach
    • 2
  • Rémy Bruggmann
    • 3
  • Klaus F. X. Mayer
    • 2
  • Jeppe R. Andersen
    • 1
  • Mingliang Xu
    • 4
  • Morten Greve
    • 5
  • Ingo Lenk
    • 5
  • Thomas Lübberstedt
    • 6
  1. 1.Department of Molecular Biology and Genetics, Research Centre FlakkebjergAarhus UniversitySlagelseDenmark
  2. 2.Institute for Bioinformatics and Systems BiologyHelmholtz Zentrum MünchenNeuherbergGermany
  3. 3.Department of BiologyUniversity of BernBernSwitzerland
  4. 4.National Maize Improvement Center of ChinaChina Agricultural UniversityBeijingPeople’s Republic of China
  5. 5.Research DivisionDLF-Trifolium LtdStore HeddingeDenmark
  6. 6.Department of AgronomyIowa State UniversityAmesUSA

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