Forward genetics and map-based cloning approac.PPTVIP

Forward genetics and map-based cloning approaches 28 September, 2006 Horticultural Science Molecular Genetics Lab. Won Hee Kang Contents Forward genetics and reverse genetics Forward genetics : from phenotype to gene structure Reverse genetics : from gene to phenotype Forward genetics and reverse genetics About 40% genes identified in the Arabidopsis genome are unclassified. 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 Marker identification and genotyping Construction of genetic map Genetic mapping Based on the use of genetic techniques to construct maps showing the position of genes and other sequence features on a genome 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 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 Contents Examples Pto Provides resistance against bacterial speck disease of tomato caused by Pseudomonas syringae pv. tomato Cloned by Martin et al. 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 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 Pto Examples Bs2 Provides resistance against bacterial spot disease of tomato and pepper, caused by Xanthomonas campestris pv. vesi