1. Forward genetics begins with the identification of an organism with an interesting mutant phenotype and aims to discover the function of genes defective in that mutant. It involves mutagenesis, screening for phenotypes of interest, genetic analysis, and chromosome walking to identify the mutated gene. 2. Reverse genetics starts with a known gene and determines the gene's function by modulating its activity through techniques like virus-induced gene silencing, RNA interference, TILLING, and gene deletion and observing resulting phenotypes in the organism. 3. Both forward and reverse genetics are aided by high-throughput approaches like insertional mutagenesis, which allows for rapid screening of large mutant collections to efficiently link phenotypes to genes.

1. Jawaharlal Nehru Krishi
VishwaVidyalaya, Jabalpur
(2016-17)
Credit seminar on
Guided by-
Dr. D.K. MISHRA
Submitted by-
Srishti Singh
Roll no 6527
M.Sc. (Ag) final year

2. FORWARD
GENETICS
REVERSE
GENETICS
A
N
D
TOPICW
E
L
C
O
M
E

3. GENETICS
•Science that deals with the structure,organisation,transmission
and function of genes, and the origin of variation
•Founder -Mendal in 1866
•Terms given by-Bateson in 1905
By:Wikipedia

4. Background
•Genetics is the study of gene function
•Gene function is inferred from the resulting phenotype when the
gene is mutated
•Genomics is changing the way of genetics is performed Globally,i.e.
high-throughput approaches
•Genomics approaches are being applied to both forward and reverse
genetics
GENETICS
FORWARD GENETICS REVERSE GENETICS

5. Forward
Genetics
Forward genetics starts with identification of interesting mutant
phenotype

6. AIM
Then aims to discover the function of genes defective in
mutants by chromosome walking
chromosome contig candidate genes mutation
To find all of the genes involved in a trait

7. •Forward genetics usually starts with mutagenesis of organism Can use
chemicals
e.g., ethyl methyl sulfonate
Or can use radiation
e.g., X rays, gamma rays
•Then screen progeny of mutagenized individuals for phenotypes of
interest
BASICS OF FORWARD GENETICS

8. Visually- abnormal morphology, growth rate, color, flowering, fertility,
etc.
Biochemically— alterations in basic cell
processes (replication, protein synthesis, etc.)
Microscopically (use fluorescent tags
to see overexpression) and image analysis
How do scientists screen phenotypes?
http://www.illuminatedcell.com/autophagosomes.html

9. Genetic analysis
First step in analysis of mutants:
• Precisely describe phenotype
Basis of predicting gene function:
•identify precisely what has malfunctioned (wrong) in the
mutant
•That is what the gene product does in the wild type

10. STEPS IN FORWARD GENETICS
A set of mutants lines are created either by EMS and/or TDNA /
transposon preferably called as knockout for the latter.
These mutant lines are then screened for a particular phenotype usin
g mutant screens or procedures where a large number of
mutants can be screened in a bulk manner
The selected mutants are then carried forward and analyzed by PCR (i
n case of TDNA insertion lines), backcrossed to remove
or dilute any unwanted insertions of TDNA

11. The next logical steps involves cloning of the full length gene an
d cDNA from the wild type plants.
In the final validation step, the gene isolated in the previous step is use
d to transform the mutant plant and phenotyped.
“If
Themutant plant exhibits recovery of wild type phenotype, it is assume
d that the wt type gene is able to rescue the mutant; hence
this validates the gene function.”
The sequence information generated from the PCR is then used to ident
ify the gene where the insertion has taken place and this
becomes the candidate gene for the phenotype tested in previous
steps.

12. Genomics and forward genetics
High-throughput genetic screens
•Candidate-gene approach
To go from phenotype to gene
•Insertional mutagenesis
Loss-of-function mutation Activation tagging
•Enhancer trapping

13. High-throughput genetic screen
Paradigm Genetics, Inc.
performs “phenotypic
profiling”
Take measurements of
mutants’ physical and
chemical parameters
e.g., plant height, leaf size,
root density, and nutrient
utilization
Different developmental
times: compare to wild type

14. From phenotype to gene
Once an interesting
mutant is found and
characterized, we
want to find the gene
in which the mutant
occurred
Positional cloning
•First use genetic
mapping
•Then use
chromosome
walking
chromosome contig
candidate genes mutation

15. Candidate-gene approach
If the mutated gene is localized to a sequenced region
of the chromosome, then look for genes that could be
involved in the process under study
Last step:
•confirm gene identification
•Rescue of phenotype
•Mutations in same gene in different alleles

16. Insertional mutagenesis
•Alternative to chromosome walking
•To reduce time and effort required to identify mutant gene
•Insert piece of DNA that disrupts genes
•Inserts randomly in chromosomes
•Make collection of individuals
•Each with insertion in different place
•Screen collection for phenotypes
•Use inserted DNA to identify mutated gene :Researchgate

17. Insertional mutagenesis in Arabidopsis
• T-DNA inserts into plant
chromosome
• Screen for mutations
that affect flower
formation
• Make genomic library
from mutant DNA
• Probe with T-DNA
• Identify mutant gene
T-DNA
inserts
into
gene
probe library
made from
T-DNA tagged
mutant with
T-DNA
sequence
DNA flanking
T-DNA to
identify gene

18. Enhancer trapping
• Type of insertional mutagenesis
– Used to find genes with interesting expression
patterns
• Insert carries a reporter gene
– Expresses foreign protein
– No effect on organism
• Enhancer trap
– Has minimal promoter in front of reporter gene
– Enhancer near point of insertion acts on it

19. Enhancer trapping in Drosophila
• Use transposon P
element
• Carries reporter gene
– b-galactosidase
• Hops into genome
• When lands near
enhancer, activates gene
expression
• Expression similar to that
of neighboring gene
P element recognition sites
enhancer gene Y
enhancer b-galactosidase
b-galactosidase
gene Y

20. Advantages and disadvantages of Forward genetics
 It is a tested method and many genes have been isolated in plants.
 It works very well in phenotypes that can be distinguished
quickly, for eg. color, shape etc.
 It works in a unbiased manner because it is random in natureThe
mutant lines created are quite stable and T-
DNA is known to preferentially integrate in and around the genic
region
 On the other hand, phenotypes that are difficult to evaluate mak
e it very tedious.
 Only one gene/trait can be analyzed
 Some genes may be missed and the procedure works only in plan
ts that are amenable to Agrobacterium transformation

21. REVERSE GENETICS
Reverse genetics starts with a known gene and alters
its function by transgenic technology

22. AIM
Then aims to determine the role of the gene from the
effects on the organism
AGCTCAATAGATAATC
TCGAGTTAGTCTATTAG
Sequence/mutation DNA Phenotype

23. Basics of reverse genetics
To find out the function of a known gene
• Reverse genetics starts with known genes
– e.g., from genomic sequencing
• to determine function through targeted
modulation of gene activity
Decrease
Increase

24. Reverse Genetics Techniques
Virus indused gene silencing
Chemical mutagenesis or TILLING
RNA mediated interfernce (RNAi)
DELETEAGENES

25. Virus indused gene silencing(VIGS) eg: zebra fish
Injection of zebra fish embryo with retrovirus preparation
Raising of founder embryo generation
F1 generation by inbreeding/outbreeding of founder
Identification of F1 fish with multiple insertions

26. Raising F2 families by inbreeding selected F1 fishes
Sperms were collected and archived from 2nd and 3rd
generation
DNA extracted from fin clips
Insertion sites were identified by LM-PCR and
sequencing
Eg. Arabidopsis

28. RNA mediated interfernce (RNAi)
•Suppresses gene expression
•Post-transcriptional biological process
•mRNA degradation/ inhibition of
translation
•Target - dsRNA

29. The Mechanism of RNA Interference

30. DELETEAGENES useful for small genes and tandem repeats
Deletion library construction
Treat wild type seeds with fast neutron
Plant M1 seeds and grow up population
Collect M2 seeds from individual plants
Plant some seeds from each line
Collect tissue and extract DNA
Fast Neutron Deletion Mutagenesis-based Reverse Genetics
Approach for Plants

31. TILLING is a general reverse genetic technique that
combines chemical mutagenesis with PCR based screening to
identify point mutations in regions of interest. (McCallum
et.al, 2000)
(Targeting Induced Local Lesions IN Genomes)
TILLING
TILLING first began in the late 1990’s by McCallum
who worked on characterizing the function of two
chromomethylase gene in Arabidopsis.

32.  Development of mutagenized population
EMS mutagenesis
Development of M2 population
 DNA preparation and pooling of individuals
 Mutation Discovery
PCR amplification of a region of interest
Mismatched cleavage
Detection of Heteroduplexes as extra peak or band (HPLC
or Acrylamide gel)
Identification of the mutant individual
Sequencing of Mutant PCR product
The TILLING Methodology

33. The TILLINGpopulation-MUTAGENESIS
Mutagenizing seed with EMS done by soaking seeds in an EMS
solution (14-18 h in a 30-100 mM (0.3%-1%) EMS solution.)
Planting the seeds in field
Mutagenized population (M1generation) is grown to maturity
allowed to self-fertilize to produce M2 seeds.
M2 seeds can be maintained as lines or bulked
If the M2 seed is bulked then lines need to be established using M3 seed.
In either case, when sampling M2 plants to establish population

34. Arraying the population for TILLING
Genomic DNA is isolated from individuals M2/M3 plants
Standardized the DNA concentration of each sample
Arrayed in a 96-well microtiter plate
Allowing up to 8-fold pooling of diploid plants to increase
TILLING throughput

35. Tilling in soybean

36. Advantages
Its applicability to virtually any organism.
Its facility for high-throughput and its independence of genome
size, reproductive system or generation time.
Since it uses Chemical mutagenesis virtually all genes can be
targeted by screening few individuals.
High degree of mutational saturation can be achieve without
excessive collateral DNA damage.
Eco- TILLING is useful for association mapping study and linkage
disequilibrium analysis.
Ecotilling can be used not only to determine the extent of variation
but also to assay the level of heterozygosity within a gene.

37. EcoTILLING
The first publication of the EcoTILLING method in which
TILLING was modified to mine for natural polymorphisms
was in 2004 from work in Arabidopsis thaliana.
EcoTILLING is similar to TILLING, except that its objective
is to identify natural genetic variation as opposed to induced
mutations.
Many species are not amenable to chemical mutagenesis;
therefore, EcoTILLING can aid in the discovery of
natural variants and their putative gene function
This approach allows one to rapidly screen through many
samples with a gene of interest to identify naturally occurring
SNPs and / or small INs/DELS.

38. TILLING and EcoTILLING

39. CASE STUDY- TILLING

40. CASE STUDY
Forward genetics in a reverse way

41. REFERENCES
Azpiroz-Leehan, R., and Feldmann, K.A. (1997).T-DNA insertion
mutagenesis in Arabidopsis: Going back and forth. Trends Genet. 13,
152–159.
Bradelly J Till et al.,(.2004) Discovery of induced mutation in maize by
TILLING:BMC Plant biology.4,471-2229
Krysan, P.J., Young, J.C., and Sussman, M.R. (1999). T-DNA as an
insertional mutagen in Arabidopsis. Plant Cell 11, 2283–2290
Parinov, S., and Sundaresan, V. (2000). Functional genomics in
Arabidopsis: Large-scale insertional mutagenesis complements the
genome sequencing project. Curr. Opin. Biotechnol. 11, 157–161.
Sussman, M.R., Amasino, R.M., Young, J.C., Krysan, P.J., and Austin-
Phillips, S. (2000). The Arabidopsis knockout facility at the University of
Wisconsin-Madison. Plant Physiol. 124, 1465–1467.

42. Also seen
• www.apsnet.org/edcenter/advan
ced/topics/Pages/ReverseGeneticT
ools.aspx
•http://www.illuminatedcell.com/a
utophagosomes.html
•https://www.ncbi.nlm.nih.gov ›
NCBI › Literature › PubMed Central
(PMC)