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Forward genetics and map-based cloning approaches

Contents. Forward genetics and reverse genetics. Forward genetics : from phenotype to gene structure Reverse genetics : from gene to phenotype. Mutation. Phenotype. Gene. . . Gene. DisruptionMutation. Phenotype. . . Forward genetics and reverse genetics. Alonso et al., 2006. Reverse genetics.

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Forward genetics and map-based cloning approaches

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    1. Forward genetics and map-based cloning approaches 28 September, 2006 Horticultural Science Molecular Genetics Lab. Won Hee Kang ?????? ?? ??? ???? ?,?? ???? ??? ???. ?? ??? ??? ???? ???? ??? ??? ??? ?? ??? ?? ???, ???? ???? ????? ?? comparative mapping? ?? ??????. ??? ?? ?????, ??? ??? ???? ??? ???? ??? ??? ??? ???????. ??? ? ?? ??? forward genetics and map-based cloning approach???. ?????? ?? ??? ???? ?,?? ???? ??? ???. ?? ??? ??? ???? ???? ??? ??? ??? ?? ??? ?? ???, ???? ???? ????? ?? comparative mapping? ?? ??????. ??? ?? ?????, ??? ??? ???? ??? ???? ??? ??? ??? ???????. ??? ? ?? ??? forward genetics and map-based cloning approach???.

    2. Contents

    3. Forward genetics and reverse genetics Forward genetics : from phenotype to gene structure Reverse genetics : from gene to phenotype ~~~~??? ??? ??? forward genetics? reverse genetics? ??? ???????. ? ?? ??? ?? ????, ??? ????. Forward genetics? ??? ???, ? ??????? ???? ???? ????, Reverse genetics? ?? ??? ?? sequece? ??? ?? ?? ???? ? ???? ???? ???? ?????. ~~~~??? ??? ??? forward genetics? reverse genetics? ??? ???????. ? ?? ??? ?? ????, ??? ????. Forward genetics? ??? ???, ? ??????? ???? ???? ????, Reverse genetics? ?? ??? ?? sequece? ??? ?? ?? ???? ? ???? ???? ???? ?????.

    4. Forward genetics and reverse genetics ??? ??? ????? ?????. Forward genetics? ??? ??? ?????? ??? ?? ??? ?? ?? ????. ? ??? 3?? arabidopsis? seedling?? ???. ??? ???? ????? ?????? ?? ??? ? ????. ??? ???? ?? ??? ???, ???? ???? seedling? ??????. ? ? seedlign?? ??? ??? ?? morphological? trait? ?? ? ? ????. ??? reverse genetics??? sequence? ?? ?? gene?? ?? ?????. ? ??? ???? gene? ??? ?????. ????? reverse forward genetics ?? mutant population? ????. C?? ?? mutagen ??? ? ?? soil?? ??????.(?? family ? 100??? ??) ?? M1? progeny? ???? ?? ? M2? ??? phenotypic screen? ?????.d??? T DNA mutagenesis? ???? (agrobacterium) ????? ?????. ???? ???? ??? ??, T DNA survive? ??? ???? ?? ??? ???? ??. ??? ????? (T1)? hemizygous??, ??? selfing ?? segregating population? ????. ?? T2 ??? screening ?? ? ?????. E??? ???? phenotypic M2 seed? phenitypic forward genetic screen? ?????. ???? ??? ?? ?? ?? ? ??. Reverse genetic screen??? t-dna mutant? ??? 2?? ?? ??? ????. ??? ???, pcr? ?? screen????, t2 individual? ???, insertion site? ??? ??? ???? ??. Insertion ??? sequencing ?? mutant? gene-indexed catalogue? ??.? ??? ??? mutant phenotype? ??? ???? identificaiotn ?? ????. ??g? mapping ??? ?? ? chromosome level?? gene ??? ????. Reverse genetics??? ??? ??? ??? ?? ???. Mutant Gene index? ?? ??? ??? ?? ?? ?? ??? search ???.,? ??? ?? ??? ??. ?, ??? phenotype? ??, ???? ???? ?? frward, ?? sequence? ?? ???? ?? reverse genetics ???.??? ??? ????? ?????. Forward genetics? ??? ??? ?????? ??? ?? ??? ?? ?? ????. ? ??? 3?? arabidopsis? seedling?? ???. ??? ???? ????? ?????? ?? ??? ? ????. ??? ???? ?? ??? ???, ???? ???? seedling? ??????. ? ? seedlign?? ??? ??? ?? morphological? trait? ?? ? ? ????. ??? reverse genetics??? sequence? ?? ?? gene?? ?? ?????. ? ??? ???? gene? ??? ?????. ????? reverse forward genetics ?? mutant population? ????. C?? ?? mutagen ??? ? ?? soil?? ??????.(?? family ? 100??? ??) ?? M1? progeny? ???? ?? ? M2? ??? phenotypic screen? ?????.d??? T DNA mutagenesis? ???? (agrobacterium) ????? ?????. ???? ???? ??? ??, T DNA survive? ??? ???? ?? ??? ???? ??. ??? ????? (T1)? hemizygous??, ??? selfing ?? segregating population? ????. ?? T2 ??? screening ?? ? ?????. E??? ???? phenotypic M2 seed? phenitypic forward genetic screen? ?????. ???? ??? ?? ?? ?? ? ??. Reverse genetic screen??? t-dna mutant? ??? 2?? ?? ??? ????. ??? ???, pcr? ?? screen????, t2 individual? ???, insertion site? ??? ??? ???? ??. Insertion ??? sequencing ?? mutant? gene-indexed catalogue? ??.? ??? ??? mutant phenotype? ??? ???? identificaiotn ?? ????. ??g? mapping ??? ?? ? chromosome level?? gene ??? ????. Reverse genetics??? ??? ??? ??? ?? ???. Mutant Gene index? ?? ??? ??? ?? ?? ?? ??? search ???.,? ??? ?? ??? ??. ?, ??? phenotype? ??, ???? ???? ?? frward, ?? sequence? ?? ???? ?? reverse genetics ???.

    5. About 40% genes identified in the Arabidopsis genome are unclassified. Whole genome sequence? ??? arabidopsis? rice? ??? sequence? ?? ???? ??? reverse gentics? ?????, ????? sequence??? ??? ???? ?? ??? ?? ????? forward genetics? ??? ?? ???? ????. ?? whole genome sequece? ??? arabidopsis??? forward genetics ??? ??? ???? ???, sequence? ??? ??? ? ??? ??? ?? ?? ?? 40% ?? ????? ????? forward genetics ??? ?? ???? ????. ???????? Functional genomics? ?? ? ? ?? ??? 40%, ??? ??? ??? ?? ??? reverse? ?? ? ? ??. Forward? ???? ??? ? ??.. ? ??? map-based..Whole genome sequence? ??? arabidopsis? rice? ??? sequence? ?? ???? ??? reverse gentics? ?????, ????? sequence??? ??? ???? ?? ??? ?? ????? forward genetics? ??? ?? ???? ????. ?? whole genome sequece? ??? arabidopsis??? forward genetics ??? ??? ???? ???, sequence? ??? ??? ? ??? ??? ?? ?? ?? 40% ?? ????? ????? forward genetics ??? ?? ???? ????. ???????? Functional genomics? ?? ? ? ?? ??? 40%, ??? ??? ??? ?? ??? reverse? ?? ? ? ??. Forward? ???? ??? ? ??.. ? ??? map-based..

    6. Map-based cloning Positional cloning Markers linked to the mutated gene Delimiting the region containing the gene of interest The amount of effort required for map-based cloning. ?? forward genetics?? ???? ?? ?? ?? ??? map-based cloning ?????. Map-based cloning ??? positional cloning ???? ???, ? ?? ?? ??? ??? ?? ???? ?????. Marker? mutated gene?? ???? ???? ??? ???, ?????? ???? ??? ???? ?? ???? ???? ?????. ??? ? ???? ?? ??? ??? ?????.?? forward genetics?? ???? ?? ?? ?? ??? map-based cloning ?????. Map-based cloning ??? positional cloning ???? ???, ? ?? ?? ??? ??? ?? ???? ?????. Marker? mutated gene?? ???? ???? ??? ???, ?????? ???? ??? ???? ?? ???? ???? ?????. ??? ? ???? ?? ??? ??? ?????.

    7. Map based cloning Map-based cloning ?? ??? ???? ??? ????. ?? ??? ?? mutation?? ????? ???? ?? ?? ?? ???? ??? ? ??? ?? ????? ?????. Mapping population? ???, marker? ???? ?????? ?????. ??? ??? ?? ?? ??? ???? marker? ??????, ??? ???? ??? ???? ??? ??? ?????? ????? ???? physical map? ?????. ??? ??? ??? ?? ???? ??? ??? ???. ??? ??? ??? ??? ????? ???? ?? ????? ???. ??? ??? ???? ??? ??? ????? ?????.Map-based cloning ?? ??? ???? ??? ????. ?? ??? ?? mutation?? ????? ???? ?? ?? ?? ???? ??? ? ??? ?? ????? ?????. Mapping population? ???, marker? ???? ?????? ?????. ??? ??? ?? ?? ??? ???? marker? ??????, ??? ???? ??? ???? ??? ??? ?????? ????? ???? physical map? ?????. ??? ??? ??? ?? ???? ??? ??? ???. ??? ??? ??? ??? ????? ???? ?? ????? ???. ??? ??? ???? ??? ??? ????? ?????.

    8. Marker identification and genotyping Construction of genetic map ?? ?? ????? mapping ??? ??? ???. ? ?? ?? ?? ? trichome? ?? ????, Trichome? ???? ???? ??? ??? trichome? ?? ?? trichome? ?? ??? ?????. ??? ??? ? parents? homo??? ???. ??? ? ??? ?? F1? ? ?? ??? ??? ??? ?? ??? ??????, ???? hetero? ?????. ??? selfing?? ??? ?? ??? 8?? ?? ??? ?? ???? ??? ?? ??? ?? recombinant inbred line? ???. ??? ??? ??? ??? ???? ???, ?? ??? ???? genetic marker? ?????. ?? ?? ?? AA, ?? ?? ?? aa, ? ? ??? ??? ?? ?? Aa, ?? ?? ?? aa? ?? ??? ?? ??? ???? ?? ??? ???? ?? ??? ? ??, ??? ???? trichome? ??? ??? ??? ????. A genetic map is constructed on the basis of recombination events between two non-sister chromatids of each pair of homologous chromosomes during meiosis. ?? ?? ????? mapping ??? ??? ???. ? ?? ?? ?? ? trichome? ?? ????, Trichome? ???? ???? ??? ??? trichome? ?? ?? trichome? ?? ??? ?????. ??? ??? ? parents? homo??? ???. ??? ? ??? ?? F1? ? ?? ??? ??? ??? ?? ??? ??????, ???? hetero? ?????. ??? selfing?? ??? ?? ??? 8?? ?? ??? ?? ???? ??? ?? ??? ?? recombinant inbred line? ???. ??? ??? ??? ??? ???? ???, ?? ??? ???? genetic marker? ?????. ?? ?? ?? AA, ?? ?? ?? aa, ? ? ??? ??? ?? ?? Aa, ?? ?? ?? aa? ?? ??? ?? ??? ???? ?? ??? ???? ?? ??? ? ??, ??? ???? trichome? ??? ??? ??? ????. A genetic map is constructed on the basis of recombination events between two non-sister chromatids of each pair of homologous chromosomes during meiosis.

    9. Genetic mapping Based on the use of genetic techniques to construct maps showing the position of genes and other sequence features on a genome Genetic marekr? ?????? ??? ???? ???, ??? ?? ????. genetic map??? ??? ???? ??? ???? ??? ??? ?????. ??? ??? ???? ??? ??? ?????. ?? ??? ?? physical map?? physical map? ???????? BAC?? YAC??? ???? library? ?????. BAC(Bacterial artificial chromosome) library? ?? ???? ?? ????(physical mapping) ??? ??? ?? ???? ????? ??? ???? ????? ????. Genetic marekr? ?????? ??? ???? ???, ??? ?? ????. genetic map??? ??? ???? ??? ???? ??? ??? ?????. ??? ??? ???? ??? ??? ?????. ?? ??? ?? physical map?? physical map? ???????? BAC?? YAC??? ???? library? ?????. BAC(Bacterial artificial chromosome) library? ?? ???? ?? ????(physical mapping) ??? ??? ?? ???? ????? ??? ???? ????? ????.

    10. Physical mapping and the identification of candidate sequence Uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features including genes Bacterial artificial chromosome (BAC) library Yeast artificial chromosome (YAC) library Genetic marekr? ?????? ??? ???? ???, ??? ?? ????. genetic map??? ??? ???? ??? ???? ??? ??? ?????. ??? ??? ???? ??? ??? ?????. ?? ??? ?? physical map?? physical map? ???????? BAC?? YAC??? ???? library? ?????. BAC(Bacterial artificial chromosome) library? ?? ???? ?? ????(physical mapping) ??? ??? ?? ???? ????? ??? ???? ????? ????. Genetic marekr? ?????? ??? ???? ???, ??? ?? ????. genetic map??? ??? ???? ??? ???? ??? ??? ?????. ??? ??? ???? ??? ??? ?????. ?? ??? ?? physical map?? physical map? ???????? BAC?? YAC??? ???? library? ?????. BAC(Bacterial artificial chromosome) library? ?? ???? ?? ????(physical mapping) ??? ??? ?? ???? ????? ??? ???? ????? ????.

    11. Genetic map and physical map ??? ? genetic map? physical map? ??? ?? ??? ????. ??? ? genetic map? physical map? ??? ?? ??? ????.

    12. Candidate genes confirmation The functional experiment of a candidate gene - genetic engineering - overexpressing the target gene - down-regulating the target gene - genetic complementation of a known mutant

    13. Contents

    14. Examples

    15. Pto Provides resistance against bacterial speck disease of tomato caused by Pseudomonas syringae pv. tomato Cloned by Martin et al.

    16. Pto

    17. Pto (A) DNA blot analysis of the Pto gene family in tomato (B) DNA blot analyisis of YAC PTY538-1 (C) DNA blot analysis of Pto gene homologs in other species

    18. Pto (A) Representative plants of (left) the susceptible backcross progeny and (right) the resistant backcross progeny (B) Growth of P. syringae pv. Tomato in leaves of tomato

    19. Pto

    20. Examples

    21. Bs2 Provides resistance against bacterial spot disease of tomato and pepper, caused by Xanthomonas campestris pv. vesicatoria (Xcv) In pepper, several single loci - Bs1, Bs2, Bs3 Cloned by Tai et al.

    22. Bs2

    23. Bs2

    24. Bs2

    25. Comparison of effort involved in map-based cloning

    26. References Alonso et al. (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat. Rev. Genet. 1-12 Borevitz et al. (2004) Genomics tools for QTL analysis and gene discovery. Curr. Opin. Plant Biol. 7, 132-136 Bortiri et al. (2006) Advanced in maize genomics: the emergence of positional cloning. Curr. Opin. Plant Biol. 9. 164-171 Huang et al. (2005) Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant J. 42, 251-261 Jander et al. (2002) Arabidopsis map-based cloning in the post-genome era. Plant physiol. 129, 440-450 Keller et al. (2005) Map-based isolation of disease resistance genes from bread wheat: cloning in a surprise genome. Genet. Res. 85, 93-100 Lukowitz et al. (2000) Positional cloning in Arabidopsis. Why it feels good to have a genome initiate working for you. Plant Physiol. 123, 795-805 Martin et al. (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262, 1432-1436 Mauricio (2001) Mapping quantitative trait loci plants: uses and caveats for evolutionary biology. Nat. Rev. Genet. 2, 370-381 Peters et al. (2003) Forward genetics and map-based cloning approaches. Trends Plant Sci. 8, 484-491 Salvi et al. (2005) To clone or not to clone plant QTLs: present and future challenges. Trends Plant Sci. 10. 297-304 Tai et al. (1999) Expression of Bs2 pepper gene confers resistance to bacterial spot disease in tomato. PNAS 96, 14153-14158

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