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Map Based Cloning- way to find candidate genes

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Published on February 18, 2014

Author: ckcr

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Map-Based Cloning – way to find candidate genes: Map-Based Cloning – way to find candidate genes Presented by: chethana , c. k I Ph.D, Dept. of GPB UAS, Dharwad Contents: Contents Map-based cloning 1 Examples 2 Forward genetics and reverse genetics: Forward genetics and reverse genetics Forward genetics : from phenotype to gene structure Reverse genetics : from gene to phenotype Mutation Phenotype Gene Gene Disruption Mutation Phenotype Forward genetics and reverse genetics: Forward genetics and reverse genetics Alonso et al ., 2006 Select a biological process Generate a random and highly redundant mutant population Screen a large number of mutagenized M2 plants Map and clone the mutation Reverse genetics Forward genetics Select a gene or genes Generate a collection of mutants or tools Catalogue the mutants in the collection Select mutants in the gene or genes of interest About 40% genes identified in the Arabidopsis genome are unclassified.: About 40% genes identified in the Arabidopsis genome are unclassified. By Prof. Lee Map-based cloning: 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. Map based cloning : Map based cloning Experimental design Mapping population Marker identification genotyping Phenotyping Fine mapping Gene confirmation Candidate gene analysis Marker identification and genotyping: Marker identification and genotyping Construction of genetic map Mauricio, 2001 Genetic mapping: Genetic mapping Based on the use of genetic techniques to construct maps showing the position of genes and other sequence features on a genome Bendahmane et al ., 1997 Physical mapping and the identification of candidate sequence: 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 map and physical map: Genetic map and physical map Map based cloning of the VTC2 gene Jander et al ., 2002 Candidate genes confirmation: 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 PowerPoint Presentation: Map-based Cloning 1. Use genetic techniques to find marker near gene Gene Marker 2. Find cosegregating marker Gene/Marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker 4. Find ORFs on contig Gene/Marker 5. Prove one ORF is the gene by transformation or mutant analysis Mutant + ORF = Wild type? Yes? ORF = Gene Contents: Contents Map-based cloning 1 Examples 2 Examples: Examples Tomato – Pto Pepper – Bs2 Pto: Pto Provides resistance against bacterial speck disease of tomato caused by Pseudomonas syringae pv. tomato Cloned by Martin et al . Pto: Pto Martin et al ., 1991, Martin et al.,1993 (A) RFLP map of chromosome 5 of tomato showing the location of Pto gene and linked markers (B)) Genetic map of the Pto region on chromosome5 A B Pto: 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 Martin et al ., 1993 Pto: 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 Martin et al ., 1993 Pto: Pto Physical map of CD186 cDNA insert and predicted amino acid sequence of Pto The position and orientation of ORF1 ( Pto ) and the 35S CaMV promoter on pPTC8 are indicated Deduced amino acid sequence of ORF1 ( Pto ) shown in one-letter code Martin et al ., 1993 Examples: Examples Tomato – Pto Pepper – Bs2 Bs2: 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. Bs2: Bs2 Map-based cloning of the bacterial spot resistance gene Bs2 from pepper Contig of the three ECW-123R pepper library YAC clones that span the Bs2 locus Contig of cosmid clones that span the Bs2 region from YAC contig Tai et al ., 1999 Bs2: Bs2 Pepper (ECW), tobacco ( N. benthamiana ), and toamto (VF36) are shown 48hr postinoculation with Agrobacterium containing binary plasmid 35S: avrBs2 (A), mixture of 35S: avrBs2 (B), and 35: Bs2 (C) Tai et al ., 1999 Bs2: Bs2 Kinetics of bacterial growth in tomato pants transformed with 35S: Bs2 Disease symptoms on tomato (VF36) transformed with or without 35S: Bs2 Tai et al ., 1999 Comparison of effort involved in map-based cloning: Comparison of effort involved in map-based cloning Jander et al ., 2002 First-pass mapping (20cM resolution) Generate physical map Fine-scale mapping (40kbp resolution) Consider candidate genes Final identification of mutation Key steps in map-based Cloning process 2002 Total effort: <1 person-year Standard molecular marker set is available Physical map exists Choose marker from Cereon database Identify candidate genes from Col-0 sequence Design PCR primers from Col-0 then sequence 1995 Total effort: 3 to 5 person-years Mixture of DNA and visible markers Build physical map (YACs or cosmids) Develop markers from YACs or cosmids No DNA sequence for candidate genes available Clone and complement, the de novo sequencing References : 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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