Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information


DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Alm V, Fang C, Busso CS, Devos KM, Vollan K, Grieg Z, Rognli OA (2003) A linkage map of meadow fescue (Festuca pratensis Huds.) and comparative mapping with other Poaceae species. Theor Appl Genet 108:25–40

  2. Armstead I, Donnison I, Aubry S, Harper J, Hortensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2006) From crop to model to crop: identifying the genetic basis of the staygreen mutation in the Lolium/Festuca forage and amenity grasses. New Phytol 172:592–597

  3. Armstead I, Donnison I, Aubry S, Harper J, Hortensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2007) Cross-species identification of Mendel’s I locus. Science 315:73

  4. Bertin I, Zhu JH, Gale MD (2005) SSCP-SNP in pearl millet—a new marker system for comparative genetics. Theor Appl Genet 110:1467–1472

  5. Chen C, Sleper DA, Johal GS (1998) Comparative RFLP mapping of meadow and tall fescue. Theor Appl Genet 97:255–260

  6. Choi HK, Kim D, Uhm T, Limpens E, Lim H, Mun JH, Kalo P, Penmetsa RV, Seres A, Kulikova O, Roe BA, Bisseling T, Kiss GB, Cook DR (2004) A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics 166:1463–1502

  7. Feuillet C, Keller B (2002) Comparative genomics in the grass family: molecular characterization of grass genome structure and evolution. Ann Bot 89:3–10

  8. Fredslund J, Madsen LH, Hougaard BK, Nielsen AM, Bertioli D, Sandal N, Stougaard J, Schauser L (2006) A general pipeline for the development of anchor markers for comparative genomics in plants. BMC Genomics 7:207

  9. Gale MD, Devos KM (1998) Comparative genetics in the grasses. Proc Natl Acad Sci USA 95:1971–1974

  10. Humphreys MW, Thomas HM, Morgan WG, Meredith MR, Harper JA, Thomas H, Zwierzykowski Z, Ghesquiére M (1995) Discriminating the ancestral progenitors of hexaploid Festuca arundinacea using genomic in situ hybridization. Heredity 75:171–174

  11. Humphreys MW, Pašakinskiene I, James AR, Thomas H (1998) Physically mapping quantitative traits for stress-resistance in the forage grasses. J Exp Bot 49:1611–1618

  12. Ikeda S, Takahashi W, Onishi H (2004) Generation of expressed sequence tags from cDNA libraries of Italian Ryegrass (Lolium multiflorum Lam.). Grassl Sci 49:593–598

  13. International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436:793–800

  14. Ishikawa G, Yonemaru J, Saito M, Nakamura T (2007) PCR-based landmark unique gene (PLUG) markers effectively assign homoeologous wheat genes to A, B and D genomes. BMC Genomics 8:135

  15. Jones ES, Mahoney NL, Hayward MD, Armstead IP, Jones JG, Humphreys MO, King IP, Kishida T, Yamada T, Balfourier F, Charmet G, Forster JW (2002) An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes. Genome 45:282–295

  16. King IP, Morgan WG, Armstead IP, Harper JA, Hayward MD, Bollard A, Nash JV, Forster JW, Thomas HM (1998) Introgression mapping in the grasses. I. Introgression of Festuca pratensis chromosomes and chromosome segments into Lolium perenne. Heredity 81:462–467

  17. King J, Armstead I, Donnison S, Roberts L, Harper J, Skøt K, Elborough K, King I (2007) Comparative analyses between Lolium/Festuca introgression lines and rice reveal the major fraction of functionally annotated gene models is located in recombination-poor/very recombination-poor regions of the genome. Genetics 177:597–606

  18. Kopecký D, Lukaszewski AJ, Doležel J (2008) Cytogenetics of Festulolium (Festuca × Lolium hybrids). Cytogenet Genome Res 120:370–383

  19. Lauvergeat V, Barre P, Bonnet M, Ghesquiere M (2005) Sixty simple sequence repeat markers for use in the Festuca-Lolium complex of grasses. Mol Ecol Notes 5:401–405

  20. Lem P, Lallemand J (2003) Grass consensus STS markers: an efficient approach for detecting polymorphism in Lolium. Theor Appl Genet 107:1113–1122

  21. Lyons LA, Laughlin TF, Copeland NG, Jenkins NA, Womack JE, Obrien SJ (1997) Comparative anchor tagged sequences (CATS) for integrative mapping of mammalian genomes. Nat Genet 15:47–56

  22. Momotaz A, Forster JW, Yamada T (2004) Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism. Plant Breed 123:370–376

  23. Morgante M, Olivieri AM (1993) PCR-amplified microsatellites as markers in plant genetics. Plant J 3:175–182

  24. Murray MG, Thompson WF (1980) Rapid isolation of high molecular-weight plant DNA. Nucleic Acids Res 8:4321–4325

  25. Ohyanagi H, Tanaka T, Sakai H, Shigemoto Y, Yamaguchi K, Habara T, Fujii Y, Antonio BA, Nagamura Y, Imanishi T, Ikeo K, Itoh T, Gojobori T, Sasaki T (2006) The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Nucleic Acids Res 34:D741–D744

  26. Pašakinskiene I, Griffiths CM, Bettany AJE, Paplauskiene V, Humphreys MW (2000) Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses. Theor Appl Genet 100:384–390

  27. Shinozuka H, Hisano H, Ponting RC, Cogan NOI, Jones ES, Forster JW, Yamada T (2005) Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 α-subunit genes. Theor Appl Genet 112:167–177

  28. Sim S, Chang T, Curley J, Warnke SE, Barker RE, Jung G (2005) Chromosomal rearrangements differentiating the ryegrass genome from the Triticeae, oat, and rice genomes using common heterologous RFLP probes. Theor Appl Genet 110:1011–1019

  29. Stammers M, Harris J, Evans G, Hayward M, Forster J (1995) Use of random PCR (RAPD) technology to analyse phylogenetic relationships in the Lolium/Festuca complex. Heredity 74:19–27

  30. Wei H, Fu Y, Arora R (2005) Intron-flanking EST-PCR markers: from genetic marker development to gene structure analysis in Rhododendron. Theor Appl Genet 111:1347–1356

  31. Yamada T, Forster JW, Humphreys MW, Takamizo T (2005) Genetics and molecular breeding in Lolium/Festuca grass species complex. Grassl Sci 51:89–106

  32. Yuan QP, Shu OY, Wang AH, Zhu W, Maiti R, Lin HN, Hamilton J, Haas B, Sultana R, Cheung F, Wortman J, Buell CR (2005) The institute for genomic research Osa1 rice genome annotation database. Plant Physiol 138:17–26

Download references


We gratefully acknowledge Dr. M.W. Humphreys for having provided of F. pratensis ‘S215’ genomic DNA, and Ms. Mayumi Hata and Ms. Azusa Kameyama for their technical assistance. This work was supported, in part, by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project DM-1406) and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

Author information

Correspondence to Toshihiko Yamada.

Additional information

Communicated by E. Guiderdoni.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tamura, K., Yonemaru, J., Hisano, H. et al. Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information. Theor Appl Genet 118, 1549–1560 (2009).

Download citation


  • Simple Sequence Repeat Marker
  • Cleave Amplify Polymorphic Sequence
  • Polymerase Chain Reaction Fragment
  • Indel Marker
  • Intergeneric Hybrid