Combining Traditional Mutagenesis with New High-Throughput Sequencing and Genome Editing to Reveal Hidden Variation in Polyploid Wheat

1. 1

2. Combining Traditional Mutagenesis with New High-Throughput
Sequencing and Genome Editing to Reveal Hidden Variation in
Polyploid Wheat
UNIVERSITY OF AGRICULTURAL SCIENCES, BENGALURU
COLLEGE OFAGRICULTURE, V C FARM, MANDYA
SEMINAR - II
SURESH YADAV
I.D. No. PALM-6005
Dept. of Genetics and Plant Breeding
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3. Introduction
SELECTION OF DIFFERENT TYPES OF
VARIATION IN DIPLOID AND POLYPLOID SPECIES
Targeting induced local lesions in genomes
(TILLING)
Genome Editing
Case Studies
Conclusion
CONTENTS
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4. TERMINOLOGY
• Polyploidy :-
• Polyploidization :-
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5. Diploidization :-
Homeologous chromosomes :-
Example hexaploid
wheat 2n = 6x = 42
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6. 6

7. Application of polyploidy in Crop Improvement
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8. 8

9. SELECTION OF DIFFERENT TYPES OF VARIATION IN
DIPLOID AND POLYPLOID SPECIES
Uauy et al., (2017) 9

10. GENERATING INDUCED MUTATIONS IN
POLYPLOID WHEAT
• The rate of visible mutants recovered after mutagenesis in diploids
was proportional to the radiation intensity.
• Polyploid oat and wheat species yielded fewer no visible mutant
phenotypes when subjected to otherwise lethal doses in diploid
species ?
L. J. Stadler (1929) 10

11. Till et al., (2012) 11

12. Targeted induced local lesions in genomes
(TILLING)
• TILLING is a reverse genetics approach for mutation generation and
discovery.
• The TILLING method relies on the formation of DNA
heteroduplexes that are formed when multiple alleles are amplified by
PCR and are then heated and slowly cooled.
• Slade et al., (2005) created a TILLING library in both bread and
durum wheat to determine its utility in a complex genome like wheat.
• Using locus-specific PCR primers, they were able to identify 246
alleles of the waxy genes by TILLING.
• This made available novel genetic diversity at waxy loci and provided
a way for allele mining in important germplasm of wheat.
Banik et al., (2007) 12

13. Maleki et al., (2007)
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14. Rashid et al., (2011) 14

15. COMPLEXITY REDUCTION TO IDENTIFY INDUCED,
RANDOM MUTATIONS IN TARGETED GENOMIC
REGIONS
• Whole genome shotgun (WGS) sequencing :-
Martin et al., (2017) 15

16. Exome capture :-
Krasileva et al., (2017) 16

17. STRATEGIES TO LINK INDUCED VARIATION WITH
PHENOTYPES
• Sequenced Mutant Populations :-
• Reverse genetic approaches, such as TILLING, are well suited to
polyploid species ?
Watson et al., (2011) 17

18. • Disadvantages of TILLING :-
• PCR amplification and sequencing requires the development
of efficient genome-specific primers.
• Laborious
• Time consuming
Solution ?
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19. Genome Editing
Epstein et al., (2009) 19

20. Zinc Finger Nucleases (ZFNs)
Bae et al., (2017) 20

21. Bae et al., (2017)
Transcription Activator-Like Effector Nuclease
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22. Clustered regularly interspaced short palindromic
repeats CRISPR/Cas9
Bae et al., (2017) 22

23. Gene-edited CRISPR mushroom escapes US regulation
Sugano et al., (2017)
“I am confident we’ll see more gene-edited
crops falling outside of regulatory authority.”
by. Caixia Gao 23

24. Comparison between Sequenced mutant populations and
gene editing
Features Sequenced mutant population Gene editing
Ease of getting started Mutations are searchable online ,
immediate access to mutant seed
Requires additional time for
construct design and optimization;
delivery into wheat is dependent
on access to technology; relatively
high cost
Achieving specificity Specific for C to T and G to A
transitions; local sequence
dependent bias affects the
probability that C/G positions
will be mutated
Dependent on presence of PAM (5-
NGG-3); new Cas9 specificities
have been published new nucleases
[Cpf1] have a different range of
PAMs
Off-target effects Thousands of mutations outside
gene of interest with many
potential deleterious mutations
Very specific with more limited off-
target effects
Epstein et al., (2009) 24

25. Features Sequenced mutant population Gene editing
Developing triple mutants Mutants in individual
homoeologs can be Combined
through traditional crossing and
marker-assisted selection
Triple mutants in first generation
not likely (approximately 0.5%);
requires crossing of single
homoeologs
Range of varieties Original mutants restricted to
sequenced populations; can be
transferred to locally relevant
germplasm by crossing
Dependent on transformation
efficiency of variety; requires
crossing to locally relevant
germplasm to deploy in
agriculture
Use in breeding Currently deployed and not
subject to regulation
Nontransgenic classification is
still uncertain in many countries;
may be problematic for globally
traded crops
Epstein et al., (2009)
Cont..
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26. STRATEGIES FOR THE USE OF MUTANTS IN GENE
ANALYSIS AND BREEDING
• Multiple Independent Lines :- To avoid the potentially confounding
effect of other mutations present in the same plant.
• Sibling Lines :- In tetraploid wheat, homozygous null mutants and
wild-type sibling plants can be selected using molecular markers from
segregating F2 individuals from the cross between A and B genome
mutants.
• Backcrossing :- Background mutations can not only confound gene–
trait associations, but can also have detrimental effects on overall plant
performance
Ziems et al., (2017) 26

27. Uauy et al., (2017) 27

28. Case Studies…
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29. Genomics as the key to unlocking the polyploid potential
of wheat
Borrill et al., (2015)Norwich
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30. Borrill et al., (2015) 30

31. Borrill et al., (2015) 31

32. Accessing variation in polyploid wheat
1.Variation at the genome-wide scale :-
2. Targeted approaches for variation discovery :-
Epstein et al., (2009) 32

33. Bulked segregant analysis
• Strong linkage – possibility to analyze in bulk
phenotype r phenotype R
Rr
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34. Borrill et al., (2015) 34

35. Borrill et al., (2015) 35

36. Phenotypic consequences of polyploidy
Borrill et al., (2015) 36

37. Borrill et al., (2015) 37

38. Simultaneous editing of three homoeoalleles
in hexaploid bread wheat confers heritable
resistance to powdery mildew
Beijing, China Wang et al., (2014)
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39. Wang et al., (2014) 39

40. Wang et al., (2014) 40

41. Wang et al., (2014) 41

42. Wang et al., (2014) 42

43. 43

44. THANK YOU
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