The number of sequenced genes having unknown function continues to climb with the continuing decrease in the cost of genome sequencing. In Reverse Genetics (RG), functions of known genes are investigated with targeted modulation of gene activity, and hypothesis regarding gene function directly tested in vivo. Several RG approaches like insertional mutagenesis, fast neutron mutagenesis, TILLING and RNA interference have led to the identification of mutations in candidate genes and subsequent phenotypic analysis of these mutants. Okabe et al. (2011) employed TILLING technique to screen six ethylene receptor genes in tomato (SlETR1–SlETR6) and two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) with reduced ethylene response were identified. Using fast neutron mutagenesis, Li et al. (2001) obtained arabidopsis deletion mutants for bZIP transcription factor viz. AHBP 1b and OBF 5, a key regulator for systemic acquired resistance but their role were compensated by other regulatory factors in mutants. Terada et al. (2007) successfully blocked the expression of the Adh 2 gene through homologous recombination followed by transgenesis in rice however phenotype could not be determined since no differences were observed between wild and transgenic plants. RNA interference (RNAi) works as sequence-specific gene regulation and has been used in determination of function of many genes. Saurabh et al. (2014) reviewed the impact of RNAi in crop improvement and found its application in improvement of nutritional aspects, biotic and abiotic stresses, morphol¬ogy, crafting male sterility, enhanced secondary metabolite synthesis. In addition, new advances in technology and reduction in sequencing cost may soon make it practical to use whole genome sequencing or gene targeting like ZFN technology and TAL effectors technology on a routine basis to identify or generate mutations in specific genes. Scholze and Boch (2011) mentioned that TAL effectors technology is more specific and predictable than ZFN. RG techniques have their own advantages and disadvantages depending on the species being targeted and the questions being addressed. Finally, with the continuous development of new technologies, the most efficient RG technique in the future may involve high throughput direct sequencing of part or complete genomes of individual plants followed by efficient novel tools to determine the function for utilization in crop improvement.

1. Manjeet Kumar
ID. No. 44071
Doctoral Seminar - II

2. Genetic Analysis
It’s all about making relationship between genetic makeup
and their phenotypes!

3. Approaches…

5. Reverse Genetics: Basics…..
.

6. Ways to modulate gene activity
1. Identify gene by mutagenesis
 Chemical mutagenesis i.e. TILLING
 Insertional mutagenesis
 Fast neutron mutagenesis
2. RNA interference
3. Gene targeting
 Homologous recombination
 Zinc Finger Nuclease
 TAL effector molecules

7. TILLING: Targeting Induced Local Lesions IN Genomes
TILLING is a general reverse genetic method that combines
chemical mutagenesis with PCR based screening to identify
point mutations in regions of interest (McCallum et al., 2000).
 Make EMS-mutagenized population
 Target gene of interest with PCR primers
 Search for rare mutants among many individuals

9. Tilling vs. Ecotilling
Tilling
 Screen mutagenized population
at one locus
 Search for rare mutants among
many individuals
 Not good for heavy
mutagenized small population
Ecotilling
 Screen natural variable
population at one locus
 Discover rare haplotypes
 Not good for few individuals
for many loci

10. Case Study….
Cont…

11. Mutation frequency in the Micro-Tom EMS mutant population
Sletr 1 mutant alleles exhibited
dominant inheritance
Cont…

12. F
r
u
i
t
s
h
e
l
f
l
i
f
e
Delayed petal abscission and fruit ripening
Ethylene insensitivity at seedling level
Ethylene insensitive SLETR 1 mutants

13. Applications:
 Detection of SNPs
 Detection of both induced and natural DNA polymorphisms
 The general applicability of TILLING makes it appropriate for
genetic modification of crops without introducing foreign DNAs.

14. Worldwide Tilling Projects

15. Crop Method Genes Function References
Wheat Tilling GBSS 1
Eleven genes
Sgp-1 and
Wx involved in
starch biosynthesis
Near waxy
phenotype
WKS 1 & WKS 2-
Yellow rust
resistance
Novel alleles of
genes
Slade et al. (2005)
Uayu et al. (2009)
Sestili et al. (2010)
Rice Tilling Ten genes 57 mutations in two
population
Till et al. (2007)
Peanut Ecotilling Ara h2 decreased
allergenicity
Ramos et al. (2009)
B. napus Ecotilling FAE 1 association with
Erusic acid content
Wang et al. (2010)
Maize Tilling DMT 102
chromomethylase
allelic series of 3
missense mutation,
deletarious
Till et al. (2004)

16. 2. FAST NEUTRON BOMBARDMENT: DELETE-A-GENE
DELETION DELETION LIBRARY CONSTRUCTION

17. Arabidopsis ‘bZIP’ transcription factor Mutant Screen- Pooling of lines
Individual lines (51840 lines)
Sub pools -2880 (18 lines/ pool)
Pools -1440 (2 sub pools/ Pool)
Super pools -180 (8 pools/ Superpool)
Mega pools - 20 (9 superpools/ Megapool)
Li X. et al. (2001)
Cont..

18. Arabidopsis ‘bZIP’ transcription factor Mutant Screen
Li X. et al. (2001)
Homozygous deletion plants inoculated with Pseudomonas syringae pv. tomato
DC3000 to test for enhanced susceptibility. BUT no difference was observed
between the mutant and the wild-type plant.

19. INSERTIONAL MUTAGENESIS
IM: A reverse genetics strategy
It is not possible to target specific genes for disruption
But possible to saturate the genome with mutations
Maintain a resource of seeds representing interruptions in every single gene
Researcher can search for insertional mutants affecting the gene of interest

21. T-DNA Mutagenesis
Integration of T-DNA carrried on plasmid into nuclear genome
• Directly generates stable insertions
• Doesn’t require additional steps for
making it stable
Features …
• Completely random
• No T-DNA integration hotspots or
integration preferences
Insertion of a piece of T-DNA (5 to 25 kb in length) produces disruption of gene
function.

22. Saturating the Genome with Mutations
Generate a population large enough to ensure that every
single gene has been mutated : Not practical
So, Perform some calculations to estimate how many T-
DNA–transformed lines are realistically necessary and
sufficient.

23. P: probability of finding one T-DNA insert
within a given gene
x: length of the gene in kilobases,
n: number of T-DNA inserts present in the
population.
Assumptions:
1. Haploid Arabidopsis genome is 120 Mb
2. T-DNA insertion is random.
P=1- [1-(x/120,000)]n
Formula
 5-kb gene requires 110,000 T-DNA inserts to achieve a 99% probability
of being mutated
 1-kb gene correspondingly necessitates 550,000 T-DNA inserts.

24. Screening
 maintenance of a genomic DNA
bank that can be screened by
PCR.
 Screening: one PCR primer
representing the gene of
interest and one primer
representing the insertion
element
Identification of
insertion in specific
gene

25. Pooling strategy
 Primary pool (9 lines/PP)
 Super pool (25 PP/SP)
 Mega pool (9 SP/MP)
• T-DNA border primers
• Gene specific primer
Krysan P.J. et al. (1999)

26. Characterization of Phenotypes

28. 2 sets of functions
movement of the transposon from one
piece of “host” DNA to the next
(transposition functions).
provide an advantage for the host of the
transposon — antibiotic resistance.
Transposon mutagenesis
 Gene ‘Knock out’: disrupts and completely inactivates the gene
 Polar mutations: genes downstream of the transposon on the same transcript are not
expressed efficiently.

29. Transposable elements generally used
 Activator/Dissociation (Ac/Ds) from corn : Arabidopsis, tobacco,
rice, potato, petunia, tomato
 Enhancer/Suppressor-mutator (En/Spm) from corn: Arabidopsis
 Mutator (Mu) from corn (Zea mays)
 Tam3 from Antirrhinum majus
 Tos17 from rice (Oryza sativa)
 Sleeping Beauty (SB): used in vertebrates.

30. Genes identified using Tos17 tagged mutants in Rice

31. Comparison
reversion of the mutation: not possible in T-DNA

32. RNA interference
the ability of exogenous/
endogenous dsRNA
suppresses the expression
of its corresponding gene.
RNA interference
mi RNA si RNA

33. Carolyn Napoli,' Christine Lemieux, and Richard Jorgensen (1990)
Historical background

34. 1995, Guo and Kemphues: injection of either antisense or sense RNAs in the
germline of C. elegans was equally effective at silencing homologous target
genes.
1998, Mello and Fire: extension of above experiments, combination of sense
and antisense RNA (= dsRNA) was 10 times more effective than single strand
RNA.

35. mi RNA…

36. si RNA...

37. Applications in crop improvement
 Seedless fruit development
 Enhanced shelf life
 Male sterility and fertility
 Nutritional improvement
 Defence improvement
 Abiotic stress tolerance

38. Species Gene Action Function References
tobacco NtDHD/SHD Down regulation Growth retardation Ding et al. (2007)
Potato StSPP1 and StSPP2 Down regulation decreased
hexose-to-sucrose
ratio under cold
treatment
Chen et al. (2008)
Cotton Gh PEL Down regulation decreased fibre
elongation
Wang et al.
(2010)
Soyabean Gm MYB 176 Down regulation reduced isoflavinoid
biosynthesis
Yi et al. (2010)
Sugarcane PFP Down regulation enhanced sucrose
accumulation in
culm
Van der Merve et
al. (2010)
Wheat CSFL 6 Down regulation decreased β-glucan
in endosperm
Nemeth et al.
(2010)

39. Gene Targeting
 Gene targeting involves the integration or removal of a
piece of DNA from a specific target sequence in the host
plant.
1. Homologous recombination
2. Zinc-Finger Nucleases
3. TAL effectors technology

40. HOMOLOGOUS RECOMBINATION
Using an “O” or “Insertion” Type
Construct
Using an “W” or “Replacement” Type
Construct
Non-homologous DNA Flanked by Homologous DNA

41. Gene Targeting using Positive/Negative Selection:
1 2 3 4
1 3 4
1 3 4
NeomycinR HSV-TK
NeomycinR

42. Gene Targeting by Homologous Recombination in Rice
Strategy for the modification of the Adh locus
A, Genomic structure of the Adh locus containing Adh3, Adh2, Copia and Gypsy-like retroelements, and
Adh1 on chromosome 11. B, Structure of the vector pJHYAd2. C, Structure of the modified adh2 gene
having the hpt sequence inserted in front of its initiation codon. The pink regions represent the
homologous sequences that correspond to the flanking Adh2 segments carried by pJHYAd2.
Terada R. et al. (2007)

43. Expression of the Adh genes in fertile transformed plants
No Adh2 expression was observed in these adh2- disrupted mutants BUT also no
apparent phenotypic alterations could be detected in the adh2 mutants.

44. Zinc-Finger Nucleases
 ZFN is engineered by combining two zinc finger proteins that recognise a
specific DNA sequence, with an endonuclease FOK 1, that causes non-
specific double-stranded breaks in DNA.
 Zinc-finger nucleases (ZFNs) breaks the specific targeted DNA.

45. Repair outcomes of a genomic double strand
Break
 targeted mutagenesis
 repaired by nonhomologous end
joining,
 targeted gene replacement
 In presence of homologous donor
DNA

47. TAL effector technology….
 NLS and AD function as transcriptional activators.
 Central tandem repeat domain (red) confers DNA binding
specificity.
 Repeat types have specificity for one or several DNA bp.

48. Function:
 As transcription activator
 As genome editor
TAL nucleases (TALNs) promote genome editing
(a) TALNs are fusions between TAL effectors and
the FokI endonuclease domain. A tailored TAL
repeat domain controls DNA-binding
specificity.
(b) Two TALNs bind neighbouring DNA boxes and
FokI dimerization induces DNA cleavage in
the spacer region between the boxes.

49.  The binding specificity of a ZF array is not completely predictable, because
specificities of neighbouring ZFs inter-depend which results in highly
laborious screening of libraries to identify suitable candidates.
 In contrast, TALs have an obvious advantage, because the TAL–DNA-
binding specificity is unambiguously predictable and TAL repeat specificity
is obviously neighbour independent.
ZFN vs. TAL effectors

50. Method Advantages Disadvantages
Homologous
recombinatio
n
•Allows for exact replacement or
modification of the targeted gene
• Highly specific to the target gene
• Results in stable mutations
• Very low efficiency
• Low throughput
Gene
silencing
• Possibility of restricting the alterations to
specific tissues or developmental stages
• Study of gene families with high degree of
functional redundancy
• The degree of gene silencing is
unpredictable
• Risk of off-target effects
• Instability of phenotypes
TILLING • Allows the identification of loss-of-
function alleles, hypomorphs and gain-of-
function alleles
• Can be used in non-transformable species
• Results in stable mutations
• Based on random mutagenesis,
so the desired mutation might
never be found
• Low to medium throughput

51. Method Advantages Disadvantages
Deleteagene • Allows the identification of two or
more genes in close proximity
• Can be used in non-transformable
species
• Results in stable mutations
• Based on random mutagenesis, so the
desired mutation might never be found
• Limited to loss-of-function mutations
• Low to medium throughput
Insertional
mutagenesis
• High throughput
• Can be adapted for both loss-of-
function and gain-of-function studies
• Results in stable mutations
• Few unwanted mutations
• Based on random (T-DNA) or non-
targeted (transposon) mutagenesis, so
the desired mutation might never be
found
• Cannot be used to study tandemly
repeated genes (T-DNA mutagenesis)
• Only limited information can be
obtained for essential genes
Zinc-finger
Nucleases &
TAL effectors
• Highly specific
• Results in stable mutations
• Low throughput
• Its use is limited to transformable
species

52. Conclusion

53. Thank You