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

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

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

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

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

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

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

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

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 N1 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

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

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

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

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

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

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

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

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

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