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Molecular Breeding

, Volume 19, Issue 1, pp 15–23 | Cite as

Construction of two Lolium perenne BAC libraries and identification of BACs containing candidate genes for disease resistance and forage quality

  • Kerrie Farrar
  • Torben Asp
  • Thomas Lübberstedt
  • Mingliang Xu
  • Ann M. Thomas
  • Camilla Christiansen
  • Mervyn O. Humphreys
  • Iain S. Donnison
Original Paper

Abstract

Two BAC libraries were constructed for the forage and turf grass species Lolium perenne L. The libraries consisted of 98,304 and 101,376 BAC clones for L. perenne genotypes LTS18 and NV#20F1-30, respectively. The estimated average insert size of both libraries was approximately 100 Kb and L. perenne has a published haploid genome size of 2,034 Mb. Taken together, the two libraries represent almost 10 genome equivalents, so that there is a very high probability of any specific sequence being represented. BAC DNA was isolated and pooled to enable PCR-based screening of both libraries. In addition, BAC clones from the LTS18 genotype were replicated onto filters to enable hybridisation-based screening. To validate the libraries, primers were designed to 20 genes involved in the phenylpropanoid pathway, disease resistance candidate genes and laccases. These primers were used to screen both libraries to verify the genome coverage and to enable the identification of full-length gene and promoter sequences for subsequent single nucleotide polymorphism (SNP) analyses. These sequences will enable studies of gene function and regulation as well as the identification of efficient genetic markers for plant breeders to improve disease resistance and forage quality.

Keywords

BAC library Lolium perenne Promoters QTL SNP 

Abbreviations

BAC

bacterial artificial chromosome

QTL

quantitative trait loci

SNP

single nucleotide polymorphism

Notes

Acknowledgements

The Institute of Grassland and Environmental Research (IGER) is sponsored by the UK Biotechnology and Biological Sciences Research Council of the United Kingdom. The Danish Institute of Agricultural Sciences is funded by Danish Ministry of Food, Agriculture, and Fisheries. The LTS18 genotype library was funded as part of the EU Framework 5 project GRASP: Development of ryegrass allele-specific markers for sustainable grassland improvement. Jakob Hedegaard at the Danish Institute of Agricultural Sciences, Department of Genetics and Biotechnology is acknowledged for assistance in clone picking of the NV#20F1-30 library. Requests to screen the LTS18 library should be addressed to Iain Donnison e-mail: iain.donnison@bbsrc.ac.uk. Requests to screen the NV#20F1-30 library should be addressed to Torben Asp e-mail: torben.asp@agrsci.dk.

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Kerrie Farrar
    • 1
  • Torben Asp
    • 2
  • Thomas Lübberstedt
    • 2
  • Mingliang Xu
    • 2
    • 3
  • Ann M. Thomas
    • 1
  • Camilla Christiansen
    • 2
    • 4
  • Mervyn O. Humphreys
    • 1
  • Iain S. Donnison
    • 1
  1. 1.Department of Plant Genetics & BreedingInstitute of Grassland and Environmental ResearchPlas Gogerddan, AberystwythUK
  2. 2.Department of Genetics and BiotechnologyDanish Institute of Agricultural Sciences, Research Centre FlakkebjergSlagelseDenmark
  3. 3.National Maize Improvement Center of ChinaChina Agricultural UniversityBeijingP.R. China
  4. 4.Plant Biochemistry Laboratory, Department of Plant BiologyThe Royal Veterinary and Agricultural UniversityFrederiksberg CDenmark

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