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Validation of a procedure for BAC end mapping on S. lycopersicum x pennellii ILs

Validation of a procedure for BAC end mapping on S. lycopersicum x pennellii ILs. by: Alessia Fiore, Giulia Falcone ENEA, Casaccia Research Center, Rome. S teps. Choice of 12 sequenced BACs (1/chromosome) with good end sequences on SGN. PCR primer design on BAC ends.

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Validation of a procedure for BAC end mapping on S. lycopersicum x pennellii ILs

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  1. Validation of a procedure for BAC end mapping on S. lycopersicum x pennellii ILs by: Alessia Fiore, Giulia Falcone ENEA, Casaccia Research Center, Rome

  2. Steps Choice of 12 sequenced BACs (1/chromosome) with good end sequences on SGN PCR primer design on BAC ends Amplification of S. lycopersicum and S. pennellii DNA Amplification and sequencing of 72 ILs (single pass, SP6 end) Polymorphism detection and mapping of SP6 end Confirmation of T7 end on selected ILs

  3. Primer design >LE_HBa0051C14_SP6_end CTCATCAACAGCATTGTCAAGGAGCTCTGCCAAGGCTTTAGACATCACAGAACAACAATGGCTATGTTAACACTCCAGTAATAGCATTTGCAATAATCCTATTACCACACAATAATATGGAAACGAAAAATGAATCAGCAATAGCACATACCACCAAATGCCCACTTATGGGAAGTGGCGTTTGAGTGAAGGAACTTCGGGTGAATGTGCAAGAAACTGGAGCCTTCTGTATAATGCCACAAATAAGATGACAACATACATATTAGATGCAAAACACATACTTGAATTACAGATAAAGAATTAAGCTATAAGGCGATATAGAAAGGTAGGCAAAGAAAAGATTGACCAACTACTCAAAACATCAGCACGCATGGTTTATGCATGGTAATGTGTTTGCTAAGAACAATTTTGAAAAAAGGAAAAAATGGGTACTTCAGTGCATAATACTTTTAAGGGTGGACTTGGTAGCATGCCCCTCATCAAATTTCCAGCTTTCCAGAACTGGCGAGATAGGGGTGCTGGACATATTTGTGATGTAGAACATAGGCTTGAATCATCTATCGGAGACTGATCTTGATCCAAGACGGAAGTTCTACTCTGTTCAGTAGCTGTAGAATCTTGCCTCCCACAGCTGCTTGAGGATTTTTTATATGGCAAGGAATCAGCTTCACAGCACTCCATTTGTGGAATTAAACTTTCAACCAATATAGGTTCCAACTTAACAGTCTCTATCTCCCCACCTCCAAGATTGTTACTACACAAGCCAACCATATTTGT Parameters: • End sequences > 500bp available on SGN • Amplification product @400bp • Primer length: 18-20bp Selected BACS

  4. Example of A->G mutation on SP6 end LE_HBa0051C14_SP6_lyc LE_HBa0051C14_SP6_penn

  5. Amplification of IL DNA SP6 end ofLE_HBa0051C14: M: 1Kb ladder, V: empty

  6. Mapping of SP6 end LE_HBa0006E18, SP6 end: maps to Ils 4.2, 4.3, 4.3.2

  7. Confirmation of T7 end

  8. Results

  9. Cost considerations (for 96 BACs)

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