Forward and reverse genetics

1. Genetic analysis
It’s all about mutants and their phenotypes

3. From gene to mutant phenotype, to function
From mutant phenotype to gene, from gene to protein function

7. Basic requirements
 Near Isogenic lines differing only for gene of interest.
 Saturated physical map
 Overlapping YAC/BAC library.
 Transformation protocol.

8. Map –based cloning strategies
5 Steps
1. Gene tagging & assigning to particular chromosome
2. High resolution mapping for localization of a gene in
small chromosome region flanked by two tightly linked
makers (<1cM distance)
3. Long distance physical mapping for identification of
Chromosome walking/ jumping library clones carrying
particular gene of interest.
4. Sequencing to identify candidate transcription
units/ORF/Exons in the megabase clone followed by
cDNA library screening.
5. Transformation to check complementation

15.  Involves selecting and hybridizing parental lines that
differ in one or more quantitative traits and
 Analyzing the segregating progeny so as to link the
quantitative trait locus to known DNA markers.

16. Principal Objective of QTL analysis
A principle objective of QTL analysis is confining QTL
to narrow chromosomal regions.
Factors considered
1. Type of experimental design or
2. Segregating population, its size, number,
informativeness and
3. Level of polymorphism of DNA markers and
4. The statistical methodologies both to build up the
linkage map and to perform the QTL analysis

18. Methods to Detect QTL
I. Single Marker Analysis
II. Interval Mapping
 Simple Interval mapping
 Composite Interval Mapping

19. Simple Marker Analysis
1. t-test
2. Annona
3. Regression (R2)Analysis

20. 1.

23. 1. SIMPLE INTERNAL MAPPING
2. COMPLEX INTERVAL MAPPING

31. Applications of QTL analysis
I. MAS in Breeding
 To enhance plant breeding efforts to speed up the creation
of cultivars.
II. Pre-breeding/ germplasm enhancement ,
 It also unveils masked, interesting wild alleles and
 Easier introgression of genes from wild species, without
the usual associated drawbacks.
III. QTL cloning.

35. Basic difference between QTL mapping
and Association Mapping
Mapping Population
Traditional QTL mapping population:
1. Segregating structured bi-parental population
2. Cross over events are limited due to limited no. of
meiosis.
3. Map resolution is determined by the size / population of
the progeny analyzed.

36. Association Mapping
Mapping Population
 Individuals from non-structured populations, with
recombination over many generations in nature.
 It examines variation in the whole population; instead of
limiting to variations present in either of parents.
Assumes that trait of interest is segregating.

37. Association approaches
 SNP- based approaches most productively
employed in partnership with background data
from SSRs to identify ‘functional nucelotide
polymorphism’ in plants

38. Association Mapping Approaches Perennial
Horticultural Crops
↓ Long generation time
↓ Large genomes
↓ Lack of well defined mutants
↓ Availability of large unstructured natural population.
↓ Inefficiencies in transformations
↑ Large unstructured natural populations are ideal.
↑ Linkage disequilibrium is limited in such population,
making candidate gene approach feasible than genome
based approach.

63. Steps
I. Chemical mutagenesis (by EMS) in populations.
II. Screening for point mutations of chemically
mutagenized individuals.
i. The genomic DNA of a queried individual is mixed
with a reference DNA
ii. Mix used to PCR amplify a target region of DNA
with asymmetrically labeled fluorescent primers.
iii. mismatch cleavage protocol
 ssDNA endonucleases used for cleavage assay,
 Cleave non-specifically at bulges in amplified heteroduplexes,
and single-base mismatches derived from induced mutations /
natural variation.