Use of forward and reverse genetics to determine gene function

Forward genetics Wild type \ a amous 0 Generate agamous 9 mutant by T—DNA mutagenesis. 9 Use DNA isolated from agamous T~DNA mutant to construct a genomic 1 library. T:DNA insertion \ I, Genomic DNA li- —_lllllil I: 9 Identify similar sequences in other plant, fungal, and animal species. 10 20 30 40 50 AGOArabidopsis) '1“1 . r r ,H DEF (Antirrhr'num) SRF (Homo sapiens) MCM1 (S. cerevisiae) Conserved amino-acid sequence encodes the MADS box, a DNA-binding domain. 0 Use DNA sequence of AGAMOUS The name MADS rs derived from the first MADS box as a probe on Arabidopsis letters of the names of the four genes genomic DNA aligned here - MCM I , AG, DEE and SRF. AGAMOUS I Related sequences ~ cross-hybridize as w shown on this ' Southern blot. U 6 Clone sequences encoding related MADS box genes in Arabidopsis; construct phylogenetic tree based on MADs box sequences. SEPI SEPZ SEP3 SEP4 AGL6 AGL13 CAL APl FUL Reverse genetics Ancestral AGL79 gene SHPI SZZZAMOUS @ identify mutations in the related genes SEPI, SEPZ. and SEP3 STK using reverse genetic approaches (e.g.. screening knodtout A6112 libraries of T-DNA and transposon mutant lines). V 0 Combine null mutations in each of the three genes by crossing mutants and breeding lines homozygous for mutations in all 59P1 SEPZ seP3 three genes. Analyze the phenotype of the triple null mutant.