1. Classical mutagenesis and genetic analysis Chromosome walking
Map-Based Cloning
Classical mutagenesis and genetic analysis
Chromosome walking

2. Classical Mutagenesis
The classical mutagenic techniques remain of fundamental importance in the genetic dissection
The phenotypic consequences of mutations provide a rich resource for studies on the function of the WT genes
Chromosomal mutations such as translocation and trisomic and tetrisomic lines are of considerable value for linkage.
Deficiencies (large deletion covering two or more genes) can be used to analyze the functional importance of different chromosomal region, fine scale deletion mapping and integration of the physical and classical genetic maps

3. Types of mutation
Seeds, pollen and a variety of plant tissue have been employ as subjects for mutagenesis
Mutation with a view to gene cloning is most often focused on generating point mutation and small deletion
Seed and tissue culture based mutagenesis are usually employed
Classical mutagenesis techniques have included the use of physical and chemical mutagen

4. Types of mutation
One of the most advantageous is high efficiency of generating mutations
Furthermore, the most widely employed mutagenic methods are at least partly predictable in the outcomes
A particular advantage of chemical mutagenesis lies in the range and subtlety of genetic effect that may be caused by point mutation at different locations in a particular gene, from complete loss of gene function, to altered levels or timing of mRNA expression, or variations in encoded activity
Insertional mutagenesis or radiation-induced deletions are usually more robust in effect

5. Irradiation mutagenesis
It generates chromosomal breaks which following DNA repair, result in a variety of chromosomal aberrations
Gene mutations are less frequent than chromosomal mutations which included translocations, inversions, deletions and deficiencies
It cause single and double strand breaks which following DNA repair, result in chromosomal rearrangements
UV radiation (wavelength 254 nm) has little power to penetrate plant tissues and therefore has most often been employed in mutagenesis or mature pollen or cells in tissue culture

6. Chemical mutagenesis
Most commonly employed chemical for seed mutagenesis is EMS (Ethyl methane sulphonate)
EMS alkylates DNA, causing base pair transitions, most often GC to AT due to the mispairing of O-alkyl-G with T. Alternatively, the formation of O-ethyl-T may result in TA to GC transitions
EMS mutagenesis results primarily in point mutations
EMS may also produce N-alkylation of G and A, leading to depurination of DNA and this in turn result in chromosomal aberrations
NMU (nitrosomethylurea) is an interesting chemical which efficiently produces plastid, as well as, nuclear mutation

7. Mutation Frequency Factors:
the mechanism of mutagen action
target gene size and nucleotide composition
genomic location
chromatin structure
replication timing
efficiency of DNA repair
transcription activation
The recovery of mutants induced by high levels of mutagens is limited by somatic effect, such as reduced viability, growth abnormalities and reduced fertility
Every mutagen has a most effective dose, which produces the maximum level of mutagenesis with minimal somatic effect

8. Frequency of appearance of mutant lines within individual M2 families
The probability of isolating a particular mutation through seed mutagenesis is a product not only of factors affecting the frequency of mutation but also the frequency of mutant detection
Many factors may affect the frequency of appearance of mutant plants within individual M2 families.
the fate of a mutagenised shoot apical cell and the contribution of its cell lineage to the inflorescence of the mature plant,
The frequency of appearance of mutant plants within individual M2 families can range from 6.3 – 25%
Factors determine he efficiency of mutant detection:
the possibility of reduced transmission of reduced transmission of mutant gametes
Variation in the expression of mutant phenotypes and in the viability of mutant plants

9. Frequency of appearance of mutant lines within individual M2 families
The probability (p) of isolating a particular mutation in composed of the probability of inducing the mutation (Pm) and the probability of finding the mutation in an individual family (Pd)
P= Pm x Pd
N N/N1
= [1- (1- m) ][1- (1 – d) ]
m: frequency of M1 plant carrying the particular mutation
d: frequency of appearance of mutant plants in a mutant m2 family
N: the M2 population size
N1: the number of M1 plants

10. Maximising the efficiency of mutant isolation
Optimal M2/M1 ratio (figure 1)
Minimum M2 population size required for the isolation of a new mutant (figure 2)
Comparison of different approach towards maximising mutant isolation efficiency (figure 3)
Probability of obtaining a mutant by following optimal M2/M1 approach will be reduced somewhat when the practicabiliies of harvesting and sampling of M1 populations are considered
The pooling of M2 progenies from individual M1 plant doesnot have a serious impact on the probability of mutant isolation, if the contribution of each M1 plant in the pool is almost the same
Pooling can considerably simplify seed sampling and this can be one of the most important practical factors in a large-scale mutagenesis experiment
Care must be taken to select the most suitable plant varieties in order to maximize the use of growth space and to ease the harvest of the seed

14. The problem of multiple induced mutation and obtaining plant carrying a single gene mutation
Mutagenized M1 plants may be chimeric for a large number of heterozygous mutations, many of which will be transmitted to the M2
Multiple mutations can influence the phenotypic expression of a wonted mutation and complicate further analysis
To ensure that the new mutant phenotype is clean and due to single mutation, newly isolated mutant must be repeatedly backcrossed to the original WT lines

15. Chromosome walking
Technique for the isolation of genes defined only by mutation or naturally occurring polymorphism
It involved the construction of a series of overlapping clones starting from a DNA segment known to map close to the desire gene and proceeding in steps until the gene is reached
Any gene that can be mapped can be cloned
No prior knowledge of the gene product is required
It is much easier in organism with a short generation time, a high density genetic map, a small simple genome, a high map unit/kb ratio and good transformation technology

16. The basic procedure Identification flanking DNA markers
Obtaining linked recombinational breakpoints
Isolation a series of contiguous DNA clones between the flanking markers using flanking markers as start points and using recombinational breakpoints to both orient and monitor the progress of the walk
Gene identification from within the series of clones

17. Identifying flanking markers
Preliminary mapping and the choice of markers
Initially, an approximate chromosomal location for the gene of interest must be established
Visible or molecular marker can be sued for mapping
Generating mapping populations
The basic mapping procedure involve crossing a line mutant in the gene of interest with a WT line of a different genetic background
In F2 populations, marker will be segregating, as will the phenotype conferred by the mutation
Detail mapping
Identification of the most closely linked DNA markers available and their relative order with respect to the gene of interest
Plant from the original F2 sample that carry chromosome with recombinational breakpoints between the gene of interest and linked molecular or visible markers are scored with respect to other DNA markers known to map to the region

18. Obtaining linked recombinational breakpoints
The importance:
The direction and extent of the chromosome walk can be established by mapping DNA clones in the walk relative to the recombinational breakpoints
The desired gene can be tightly delimited by mapping the position of the two recombinational breakpoint that most closely flank the gene
Assuming the probability of a breakpoint lying close to the desired gene increases as the size of the recombinant collection increases.
It is therefore useful to generate a substantial number of recombinants at the outset particularly since at least three plant generation are required for he isolation

19. Using flanking visible markers to reduce the workload
There are number of useful strategies for identifying individuals carrying recombinational breakpoints near gene of interest. One of the most efficient methods is to pre-select plant known to have breakpoint flanking the gene of interest using flanking visible markers
Briefly, recombinant individuals are identified, based on phenotype, from the F2 of crosses between a line mutant in the gene of interest and lines carrying proximal and/or distal visible mutations
There are some general points concerning the visible markers selected. Two genetic backgrounds must be included in the cross to allow DNA polymorphisms to be scored

20. Using flanking visible markers to reduce the workload
Although the cis configuration gives a far higher proportion of recombinants than the trans configuration, a disadvantage is that it can time consuming to construct the line needed.
In most cases, the mutant alleles of the gene of interest and the flanking marker gene are both recessive. In order to place them in cis, three generations are required. In all, six generations will be needed to isolate the recombinants
It is very helpful if the phenotype of the flanking mutations chosen can be scored early, so that non-recombinants can be weeded out

21. Alternative methods for recombinant isolation
DNA markers can be used to score F2 individuals for recom