2. Forward genetics and reverse genetics
There are basically two ways to link the sequence and function of a specific gene: forward and reverse genetics
Forward genetics: from phenotype to gene sequence
Reverse genetics : from gene sequence to phenotype
Mutation
Phenotype
Gene
Gene
Mutation
Phenotype

3. Forward genetics and reverse genetics
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
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
Jose M. Alonso et al., 2006

4. Generation of genetic diversity
Jose M. Alonso et al., 2006

5. Tandem gene multiplications pose a challenge for functional analysis
In sequenced plant genomes, 15% or more of the identified genes are members of tandem-arrayed gene Families.
Assigning function to unknown plant genes is to study phenotypic changes associated with knockout mutations.
Mutating only one gene in a duplicated pair often produces no measurable phenotype, this poses a particular challenge for functional analysis.
It is often necessary to knock out more than one member of a plant gene family to obtain a mutant phenotype.
Here describe alternate strategies that can be used to identify knockouts of two or more tandem-arrayed plant genes for functional analysis.

6. Table 1. Advantages and disadvantages of approaches for generating mutations in tandem-arrayed genes

7. Selection for deletions spanning multiple genes
Lox P : locus of X-over P1
Cre : Cyclization Recombination
Figure 1. Deletion of tandem-arrayed genes.

8. Silencing gene families by RNA interference
RNA interference allows down-regulation of gene expression in transgenic plants, including the genes of interest are located in tandem arrays.
RNA interference was used to silence all members of the gene family.
Silencing efficiency depends on the type of construct that is used, the site of integration, and the particular target genes.
Gene silencing is generally not 100% effective, mutant phenotypes that require the complete knockout of a gene family might not be detected using the described silencing approaches.

9. Selection for meiotic recombination between adjacent genes
Figure 2. Selection for recombination between T-DNA or transposon insertions

10. Sequential mutagenesis to create a double mutant
Figure 3. Localized transposon mutagenesis.
Ds elements tend to transpose to nearby genetic locations, allowing screens for insertions in adjacent genes. Imperfect DNA repair can leave the gene at the original insertion site inactivated .

11. Prospects for site-directed mutagenesis in higher plants
zinc finger DNA binding domains
FokⅠ DNA nuclease
Figure 4. Site-directed mutagenesis with a zinc finger nuclease (ZFN). Double-strand cleavage by a ZFN leads to target gene knockout by imperfect repair or homologous recombination

12. High-throughput TILLING
Trenton Colbert et al., 2001

13. Total 50,000 lines
Xin Li et al., 2001