Theoretical and Applied Genetics

, Volume 110, Issue 1, pp 12–32 | Cite as

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

  • M. J. Faville
  • A. C. Vecchies
  • M. Schreiber
  • M. C. Drayton
  • L. J. Hughes
  • E. S. Jones
  • K. M. Guthridge
  • K. F. Smith
  • T. Sawbridge
  • G. C. Spangenberg
  • G. T. Bryan
  • J. W. Forster
Original Paper


A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST•RFLP loci in the F1(NA6 í AU6) population. A comprehensive set of EST•SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST•RFLP and 71 EST•SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST•RFLP and 58 EST•SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.


Restriction Fragment Length Polymorphism Simple Sequence Repeat Marker Segregation Distortion Perennial Ryegrass Simple Sequence Repeat Locus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We gratefully recognise technical support provided by Benjamin Franzmayr and Deborah Knox. The authors thank Prof. Michael Hayward for his careful critical reading of the manuscript. This work was performed within the Pasture Plant Genomics Program co-funded by the Department of Primary Industries, Victoria, Australia and AgResearch Limited, New Zealand. Funding was provided to AgResearch Ltd by the New Zealand Foundation for Research, Science and Technology under contract number C10X0203. Genetic mapping family development and genomic DNA-derived SSR genotyping was supported by the Molecular Plant Breeding Cooperative Research Centre (CRC). All experiments conducted during this study comply with current Australian laws.


  1. Altschul SF, Madden Tl, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389•3402PubMedGoogle Scholar
  2. Areshchenkova T, Ganal M (2002) Comparative analysis of polymorphism and chromosomal location of tomato microsatellite markers isolated from different sources. Theor Appl Genet 104:229•235CrossRefPubMedGoogle Scholar
  3. Armstead IP, Turner LB, King IP, Cairns AJ, Humphreys MO (2002) Comparison and integration of genetic maps generated from F2 and BC1-type mapping populations in perennial ryegrass. Plant Breed 121:501•507CrossRefGoogle Scholar
  4. Arnold C, Rossetto M, McNally J, Henry RJ (2002) The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae. Am J Bot 89:22•28Google Scholar
  5. Barrett B, Griffiths A, Schreiber M, Ellison N, Mercer C, Bouton J, Ong B, Forster J, Sawbridge T, Spangenberg G, Bryan G, Woodfield D (2004) A microsatellite map of white clover. Theor Appl Genet 109:596•608 PubMedGoogle Scholar
  6. Barth S, Melchinger AE, Devezi-Savula B, Lubberstedt T (2001) Influence of genetic background and heterozygosity on meiotic recombination in Arabidopsis thaliana. Genome 44:971•978CrossRefPubMedGoogle Scholar
  7. Bert PF, Charmet G, Sourdille P, Hayward MD, Balfourier F (1999) A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers. Theor Appl Genet 99:445•452CrossRefGoogle Scholar
  8. Brownstein MJ, Carpten JD, Smith JR (1996) Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20:1004•1010PubMedGoogle Scholar
  9. Buxton DR, Russell JR (1988) Lignin constituents and cell-wall digestibility of grass and legume stems. Crop Sci 28:553•558Google Scholar
  10. Chao S, Baysdorfer C, Heredia-Diaz O, Musket T, Xu G, Coe EH Jr (1994) RFLP mapping of partially sequenced leaf cDNA clones in maize. Theor Appl Genet 88:717•721Google Scholar
  11. Chen M, Presting G, Barbazuk WB, Goicoechea JL, Blackmon B, Fang G, Kim H, Frisch D, Yu Y, Sun S (2002) An integrated physical and genetic map of the rice genome. Plant Cell 14:521•523Google Scholar
  12. Chen X, Salamini F, Gebhardt C (2001) A potato molecular-function map for carbohydrate metabolism and transport. Theor Appl Genet 102:284•295CrossRefGoogle Scholar
  13. Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR, Park WD, Ayres N, Cartinhour S (2000) Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.). Theor Appl Genet 100:713•722CrossRefGoogle Scholar
  14. Cordeiro GM, Casu R, McIntyre CL, Manners JM, Henry RJ (2001) Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to Erianthus and sorghum. Plant Sci 160:1115•1123CrossRefPubMedGoogle Scholar
  15. Decroocq V, Favèc) M, Hagen L, Bordenhave L, Decroocq S (2003) Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor Appl Genet 106:912•922PubMedGoogle Scholar
  16. Dumsday JL, Smith KF, Forster JW, Jones ES (2003) SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f.sp. lolii) in perennial ryegrass (Lolium perenne L.). Plant Pathol 52:628•637CrossRefGoogle Scholar
  17. Endo TR, Gill BS (1996) The deletion stocks of common wheat. J Hered 87:295•307Google Scholar
  18. Eujayl I, Sorrells ME, Baum M, Wolters P, Powell W (2002) Assessment of genotypic variation among cultivated durum wheat based on EST•SSRs. Euphytica 119:39•43CrossRefGoogle Scholar
  19. Feinberg AP, Vogelstein B (1984) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6•13Google Scholar
  20. Forster JW, Jones ES, Kölliker R, Drayton MC, Dupal MP, Guthridge KM, Smith KF (2001) DNA profiling in outbreeding forage species. In: Henry R (ed) Plant genotyping•the DNA fingerprinting of plants. CABI, Wallingford, pp 299•320 Google Scholar
  21. Forster JW, Jones ES, Batley J, Smith KF (2004) Molecular marker-based genetic analysis of pasture and turf grasses. In: Hopkins A, Wang Z-Y, Sledge M, Barker RE (eds) Molecular breeding of forage and turf. Kluwer, Dordrecht, pp 197•239Google Scholar
  22. Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207•209Google Scholar
  23. Giddings G (2000) Modelling the spread of pollen from Lolium perenne. The implications for the release of wind-pollinated transgenics. Theor Appl Genet 100:971•974CrossRefGoogle Scholar
  24. Grattapaglia D, Sederoff R (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics 137:1121•1137PubMedGoogle Scholar
  25. Griffiths AG, Barrett BA, Ellison N, Sawbridge T, Spangenberg G, Bryan GJ, Schreiber M (2002) Getting out there: transportability of white clover EST and genomic DNA-derived microsatellites. In: Proceedings of the 12th Australasian plant breeding conference, Perth, 15•20 September 2002, pp 69•72 Google Scholar
  26. Hackauf B, Wehling P (2002) Identification of microsatellite polymorphisms in an expressed portion of the rye genome. Plant Breed 121:17•25CrossRefGoogle Scholar
  27. Hackett CA, Broadfoot LB (2003) Effects of genotyping errors, missing values and segregation distortion in molecular marker data on the construction of linkage maps. Heredity 90:33•38CrossRefPubMedGoogle Scholar
  28. Hadad RG, Pfeiffer TW, Poneleit CG (1996) Repeatability and heritability of divergent recombination frequencies in the Iowa Stiff Stalk Synthetic (Zea mays L.). Theor Appl Genet 93:990•996CrossRefGoogle Scholar
  29. Haldane JBS (1919) The combination of linkage values and the calculation of distances between the loci of linked factors. J Genet 8:299•309Google Scholar
  30. Hayward MD, Forster JW, Jones JG, Dolstra O, Evans C, McAdam NJ, Hossain KG, Stammers M, Will J, Humphreys MO, Evans GM (1998) Genetic analysis of Lolium. I. Identification of linkage groups and the establishment of a genetic map. Plant Breed 117:451•455Google Scholar
  31. Holton TA, Christopher JT, McClure L, Harker N, Henry RJ (2002) Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat. Mol Breed 9:63•71CrossRefGoogle Scholar
  32. Jenczewski E, Gherardi M, Bonnin I, Prosperi JM, Olivieri I, Huguet T (1997) Insight on segregation distortions in two intraspecific crosses between annual species of Medicago (Leguminosae). Theor Appl Genet 94:682•691CrossRefGoogle Scholar
  33. Johnson X, Lidgett A, Chalmers J, Guthridge K, Jones E, Cummings N, Spangenberg G (2003) Isolation and characterisation of an invertase cDNA from perennial ryegrass (Lolium perenne L.). J Plant Physiol 160:903•911Google Scholar
  34. Jones EL, Roberts E (1991) A note on the relationship between palatability and water-soluble carbohydrates in perennial ryegrass. Irish J Agric Res 30:163•167Google Scholar
  35. Jones ES, Dupal MP, Kölliker R, Drayton MC, Forster JW (2001) Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.). Theor Appl Genet 102:405•415CrossRefGoogle Scholar
  36. Jones ES, Mahoney NL, Hayward MD, Armstead IP, Jones JG, Humphreys MO, King IP, Kishida T, Yamada T, Balfourier F, Charmet C, Forster JW (2002a) An enhanced molecular marker-based map of perennial ryegrass (Lolium perenne L.) reveals comparative relationships with other Poaceae species. Genome 45:282•295CrossRefGoogle Scholar
  37. Jones ES, Dupal MD, Dumsday JL, Hughes LJ, Forster JW (2002b) An SSR-based genetic linkage map for perennial ryegrass (Lolium perenne L.). Theor Appl Genet 105:577•584CrossRefGoogle Scholar
  38. Kantety RV, La Rota M, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501•510CrossRefPubMedGoogle Scholar
  39. Knox MR, Ellis THN (2002) Excess heterozygosity contributes to genetic map expansion in pea recombinant inbred populations. Genetics 162:861•873PubMedGoogle Scholar
  40. Kurata N, Nagamura Y, Yamamoto K, Harushima Y, Sue N, Wu J, Antonio BA, Shomura A, Shimizu T, Lin S-Y, Inoue T, Fukuda A, Shimano T, Kuboki Y, Toyama T, Miyamoto Y, Kirihara T, Hayasaka K, Miyao A, Monna L, Zhong HS, Tamura Y, Wang Z-X, Momma T, Umehara Y, Yano M, Sasaki T, Minobe Y (1994) A 300-kilobase-interval genetic map of rice including 883 expressed sequences. Nat Genet 8:365•372CrossRefPubMedGoogle Scholar
  41. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations. Genomics 1:174•181PubMedGoogle Scholar
  42. Li X, Song Y, Century K, Straight S, Ronald P, Dong X, Lassner M, Zhang Y (2001) A fast neutron deletion mutagenesis-based reverse genetics systems for plants. Plant J 27:235•242Google Scholar
  43. Lidgett A, Jennings K, Johnson X, Guthridge K, Jones E, Spangenberg G (2002) Isolation and characterisation of fructosyltransferase gene from perennial ryegrass (Lolium perenne). J Plant Physiol 159:1037•1043Google Scholar
  44. Maliepaard C, van Ooijen JW (1994) QTL mapping in a full-sib family of an outcrossing species. In: van Ooijen JW, Jansen J (eds) Biometrics in plant breeding: applications of molecular markers. In: Proceedings of the 9th meeting of Eucarpia section biometrics, pp 140•146 Google Scholar
  45. McInnes R, Lidgett A, Lynch D, Huxley H, Jones E, Mahoney N, Spangenberg G (2002) Isolation and characterisation of a cinnamoyl-CoA reductase gene from perennial ryegrass (Lolium perenne). J Plant Physiol 159:415•422Google Scholar
  46. Metzgar D, Bytof J, Wills C (2000) Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res 10:72•80PubMedGoogle Scholar
  47. Michell PJ (1973) Relations between fibre and water soluble carbohydrate contents of pasture species and their digestibility and voluntary intake by sheep. Aust J Exp Agric Anim Husb 13:165•170Google Scholar
  48. Mogg R, Batley J, Hanley S, Edwards D, O
tm)Sullivan H, Edwards KJ (2002) Characterisation of the flanking regions of Zea mays microsatellites reveals a large number of useful sequence polymorphisms. Theor Appl Genet 105:532•543CrossRefPubMedGoogle Scholar
  49. Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30:194•200CrossRefPubMedGoogle Scholar
  50. Plummer RM, Hall RL, Watt TA (1990) The influence of crown rust (Puccinia coronata var lolii) on tiller production and survival of perennial ryegrass (Lolium perenne L.) plants in simulated swards. Grass Forage Sci 45:9•16Google Scholar
  51. Potter LR (1987) Effect of crown rust on regrowth, competitive ability and nutritional quality of perennial and Italian ryegrass. Plant Pathol 36:455•461Google Scholar
  52. Potter LR, Cagas B, Paul VH, Birckenstaedt E (1990) Pathogenicity of some European collections (Puccinia coronata Corda) on cultivars of perennial ryegrass. J Phytopathol 130:119•126Google Scholar
  53. Price T (1987) Ryegrass rust in Victoria. Plant Prot Q 2:189Google Scholar
  54. Prioul JL, Pelleschi S, Sèc)ne M, Thèc)venot C, Causse M, de Vienne D, Leonard A (1999) From QTLs for enzyme activity to candidate genes in maize. J Exp Bot 50:1281•1288CrossRefGoogle Scholar
  55. Qi L, Echalier B, Friebe B, Gill BS (2003) Molecular characterisation of a set of wheat deletion stocks for use in chromosome bin mapping of ESTs. Funct Integr Genomics 3:39•55PubMedGoogle Scholar
  56. Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94•1000 CrossRefPubMedGoogle Scholar
  57. Rees H, Thompson JB (1956) Genotypic control of chromosome behaviour in rye. III. Chiasma frequency on homozygotes and heterozygotes. Heredity 10:409•424Google Scholar
  58. Säll T (1990) Genetic control of recombination in barley. II. Variation in linkage between marker genes. Hereditas 112:171•178Google Scholar
  59. Sawbridge T, Ong E-K, Binnion C, Emmerling M, McInnes R, Meath K, Nguyen N, Nunan K, O
tm)Neill M, O
tm)Toole F, Rhodes C, Simmonds J, Tian P, Wearne K, Webster T, Winkworth A, Spangenberg G (2003) Generation and analysis of expressed sequence tags in perennial ryegrass (Lolium perenne L.). Plant Sci 165:1089•1100CrossRefGoogle Scholar
  60. Schneider K, Borchardt DC, Schäfer-Pregl R, Nagl N, Glass C, Jeppsson A, Gebhardt C, Salamini F (1999) PCR-based cloning and segregation analysis of functional gene homologues in Beta vulgaris. Mol Gen Genet 262:515•524CrossRefPubMedGoogle Scholar
  61. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233•234CrossRefPubMedGoogle Scholar
  62. Scott KD, Eggler P, Seaton G, Rossetto M, Ablett EM, Lee LS, Henry RJ (2000) Analysis of SSRs derived from grape ESTs. Theor Appl Genet 100:723•726CrossRefGoogle Scholar
  63. Sebastian RL, Howell EC, King GJ, Marshall DF, Kearsey MJ (2000) An integrated AFLP and RFLP Brassica oleracea linkage map from two morphologically distinct doubled-haploid mapping populations. Theor Appl Genet 100:75•81CrossRefGoogle Scholar
  64. Sharopova N, McMullen MD, Schultz L, Schroeder S, Sanchez-Villeda H, Gardiner J, Bergstrom D, Houchins K, Melia-Hancock S, Musket T, Duru N, Polacco M, Edwards K, Ruff T, Register JC, Brouwer C, Thompson R, Velasco R, Chin E, Lee M, Woodman-Clikeman W, Long MJ, Liscum E, Cone K, Davis G, Coe EH Jr (2002) Development and mapping of SSR markers for maize. Plant Mol Biol 48:463•481CrossRefPubMedGoogle Scholar
  65. Sharp PJ, Kreis M, Shewry PR, Gale MD (1988) Location of β-amylase sequences in wheat and its relatives. Theor Appl Genet 75:286•290Google Scholar
  66. Sorrells ME, Wilson WA (1997) Direct classification and selection of superior alleles for crop improvement. Crop Sci 37:691•697Google Scholar
  67. Sorrells ME, La Rota M, Bermundez-Kandianis CE, Greene RA, Kantety R, Munkvold JD, Miftahudin, Mahmoud A, Ma X, Gustafson PJ, Qi LL, Echalier B, Gill BS, Matthews DE, Lazo GR, Chao S, Anderson OD, Edwards H, Linkiewicz AM, Dubcovsky J, Akhunov ED, Dvorak J, Zhang D, Nguyen HT, Peng J, Lapitan NLV, Gonzalez-Hernandez JL, Anderson JA, Hossain K, Kalavacharla V, Kianian SF, Choi D-W, Close TJ, Bilbirgi M, Gill KS, Steber C, Walker-Simmons MK, McGuire PE, Qualset CO (2003) Comparative DNA sequence analysis of wheat and rice genomes. Genome Res 13:1818•1827 PubMedGoogle Scholar
  68. Squirrel J, Hollingsworth PM, Woodhead M, Russell J, Lowe AJ, Gibby M, Powell W (2003) How much effort is required to isolate nuclear microsatellites from plants? Mol Ecol 12:1339•1348CrossRefPubMedGoogle Scholar
  69. Tanksley SD, Ganal MW, Prince JP, de Vicente MC, Bonierbale MW, Broun P, Fulton TM, Giovannoni JJ, Grandillo S, Martin GB, Messeguer R, Miller JC, Miller L, Paterson AH, Pineda O, Röder MS, Wing RA, Wu W, Young ND (1992) High-density molecular genetic linkage maps of the tomato and potato genomes. Genetics 132:1141•1160PubMedGoogle Scholar
  70. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res 11:1441•1452CrossRefPubMedGoogle Scholar
  71. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterisation of gene-derived SSR markers in barley (Hordeum vulgare L.) Theor Appl Genet 106:411•422PubMedGoogle Scholar
  72. Thornsberry JM, Goodman MM, Doebley J, Kresovich S, Nielsen D, Buckler ES IV (2001) Dwarf8 polymorphisms associate with variation in flowering time. Nat Genet 28:286•289CrossRefPubMedGoogle Scholar
  73. Thorogood D, Kaiser WJ, Jones JG, Armstead I (2002) Self-incompatibility in ryegrass 12. Genotyping and mapping the S and Z loci of Lolium perenne L. Heredity 88:385•390CrossRefPubMedGoogle Scholar
  74. Tyler BF, Jones EL (1982) Evaluation of forage genetic resources in relation to breeding objectives and varietal assessment. In: Hayward MD (ed) The utilisation of genetic resources in fodder crop breeding. Proceedings of the Eucarpia fodder crops section meeting, Aberystwyth Institute for Grassland and Environmental Research (IGER), pp 148•162Google Scholar
  75. Vance V, Vaucheret H (2001) RNA silencing in plants•defence and counterdefence. Science 292:2277•2280CrossRefPubMedGoogle Scholar
  76. Wilson ID, Barker GL, Edwards KJ (2003) Genotype to phenotype: a technological challenge. Ann Appl Bot 142:33•39Google Scholar
  77. Yamada T, Jones ES, Nomura T, Hisano H, Shimamoto Y, Smith KF, Hayward MD, Forster JW (2004) QTL analysis of morphological, developmental and winter hardiness-associated traits in perennial ryegrass (Lolium perenne L.). Crop Sci 44:925•935Google Scholar
  78. Zamir D, Tadmor Y (1986) Unequal segregation of nuclear genes in plants. Bot Gaz 147:355•358CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • M. J. Faville
    • 1
  • A. C. Vecchies
    • 2
    • 3
  • M. Schreiber
    • 4
  • M. C. Drayton
    • 2
    • 3
  • L. J. Hughes
    • 2
    • 3
    • 6
  • E. S. Jones
    • 2
    • 3
    • 7
  • K. M. Guthridge
    • 2
    • 3
    • 8
  • K. F. Smith
    • 3
    • 5
  • T. Sawbridge
    • 2
    • 3
    • 9
  • G. C. Spangenberg
    • 2
    • 3
  • G. T. Bryan
    • 1
  • J. W. Forster
    • 2
    • 3
  1. 1.Grasslands Research CentreAgResearch Ltd.Palmerston NorthNew Zealand
  2. 2.Primary Industries Research Victoria, Plant Biotechnology CentreLa Trobe UniversityBundooraAustralia
  3. 3.Molecular Plant Breeding Cooperative Research CentreAustralia
  4. 4.AgResearch Limited, School of Biological SciencesUniversity of AucklandAucklandNew Zealand
  5. 5.Primary Industries Research Victoria, Hamilton CentrePrivate Bag 105, HamiltonAustralia
  6. 6.School of Biological Sciences and Biotechnology, Division of Science and EngineeringMurdoch UniversityMurdochAustralia
  7. 7.Crop Genetics Research and DevelopmentPioneer Hi-Bred InternationalJohnstonUSA
  8. 8.Cell Cycle and Development LaboratoryPeter MacCallum Cancer CentreEast MelbourneAustralia
  9. 9.Molecular Bioscience Technologies, Department of Primary Industries and Fisheries, Queensland Biosciences PrecinctThe University of QueenslandSt. LuciaAustralia

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