Epigenomics in crop improvement

1. An individual with two eye colours
Coat colour in genetically identical rats
Cloned cat
Rainbow and Copycat
Toad flax flower
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University of Horticultural
Sciences, Bagalkot
Shahenaz begum Khaja hussain
Sr MSc (UHS16PGM740)
Dept. of Biotechnology and Crop Improvement

3. Basic concept in Epigenomics
Epigenetic modifications
Epigenomics of modifications
Comparison of epigenetics with genetics
Applications of epigenetics
Research findings
Conclusion

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DNA
Bases
Genes
Genome
Sequence
*Epigenetics Genetics

5. Term “EPIGENETICS” coined by – Waddington (1942)
He is known as “Father of Epigenetics’’.
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Epigenetics literally means “above”
Or “ on top of” genetics: It refers to
external modification to DNA that
turns genes ‘on’ or ‘off’.
‘’The study of changes in gene
function that are heritable and donot
entail a change in DNA sequence’’
- Corrent wu and morris(2001)
Epigenomics : Study of the complete set of epigenetic modifications on the genetic
material of a cell .

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DNA Histone protein
Chromatin
It is a multitude of chemical compounds that can tell the genome ‘What to do’
Epigenome

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Methylation
DNA methylation abundant in plants and
vertebrates
“Heritable epigenetic enzymatic
modification resulting from the addition of a
methyl group in the cyclic carbon-5 of
cytosine occurs via enzyme called DNA
methyl transferase.”
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DNA methylation-DNMT
Histone methylation-HMT
= Methyl group
DNA methyltransferase
CYTOSINE 5’METHYLCYTIDINE

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DNA methylation
usually inhibits
transcription of
eukaryotic genes
particularly when it
occurs in vicinity of
promoter.
CpG islands occur
near the many
promoters genes .
These CpG islands
are commonly 1000-
2000bp in length and
contain high number of
CpG sites.

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• Ex: DNMT 1Maintenance
methylase
• Ex: DNMT 3a,
DNMT 3b
Denovo
methylase

12. Sequence
specific
enzyme
digestion
Methylated
binding protein
Sodium
bisulphite
conversion
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14. Enzymes
Methylation sensitive enzyme
Ex: HpaII EcoRI, Aat II, Acc II
Methylation insensitive enzyme
Ex : MspI( CCGG) Sphl(CGTAC/g)
and Bbul(CGTAC/G)
and
Agarose gel
banding
pattern
Unmethylated cytosine base
Unmethylated and methylated
cytosine base
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Condensed and
transcriptionally Inactive
Chromatin
Less Condensed (Due to
acetylation of histone or
other remodeling) making
it transcriptionally active
Switching off
Switching on

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Multimeric protein
complexes have being
identified that function
as “cellular memory
keys” that “lock” gene
expression states and
enable their inheritance
over many cell mitotic
or meiotic division
cycles

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Histone
methylation
Histone
acetylation
Histone
phoshorylation
Histone
ubiquitylation

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Modifications Role in transcription Modification site
Acetylation Activation H3(K9,K14,K18,K56)
H4(K5,K8,K12,K16)
H2B(K6,K7,K16,K17)
Strahl and allis(2000)
Methylation Activation H3(K4me2,K4me3,K36me
3,K79me2)
Strahl and allis(2000)
Methylation Repression H3(K9me3,K27me3 and
H4(K20me3)
Balazs(2014)
Phosphorylation Activation H3(S10)
Strahl and allis(2000)

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1) Chromatinimmunoprecipitation(ChIP)
i) Cross-linked ChIP(X ChIP)
ii) Native ChIP(N ChIP)
i)ChIP-Chip
ii)ChIP-seq
iii) ChIP-qPCR
2. Massspectrometry

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A non-coding RNA (ncRNA) is a functional RNA molecule that is transcribed from
DNA but not translated into proteins.
Epigenetic related ncRNAs include miRNA, siRNA. In general, ncRNAs
function to regulate gene expression at the transcriptional and post-transcriptional
level.

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Non coding RNA
Infrastructural
non coding RNA
Regulatory
non coding
RNA
non
coding
siRNA
non
coding
miRNA

24. Single stranded 20- to 25 nucleotide
RNA processed from a stem-loop region of
a longer transcript by Dicer-like enzymes
Bartel. , 2004
MicroRNA (miRNA)
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siRNAs
Double stranded 20-22 nucleotide short
RNA molecules
Produced from long double stranded
RNA by Dicer
Bartel. , 2004

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Properties Epigenetics Genetics
Type of variation Usually does not change in
DNA sequence
Involves change in DNA
sequence
Origin of variation Random changes due to
imperfection of DNA
methylation pattern
Usually random
Unit of variation Activity of gene (Up or
Down)
DNA bases , sequences
Heritability Varies 100%

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29. Objective:
To know the role of “Epigenetic regulation” for drought tolerance in Tomato by
miRNA analysis in drought tolerant and drought sensitive tomato root and up ground
tissues.
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30. Total RNA isolation (miRNA isolation kit)
Identification of tomato miRNA by deep sequencing
Validation of miRNA expression by qRT PCR
Library construction and sequencing Illumina HiSeq2000 Platform)
Target predictiont (psRNATarget server),
Plant Material – Drought Sensitive- L. esculentum (L.M.I-56),
Drought Tolerant L. esculentum var. cerasiformae(PI187002)
Functional annotation and Gene ontology
Drought treatment-5% PEG-Sensitive and Tolerant Roots and Leaves-4 DNA Samples
Control Condition –Sensitive and Tolerant Leaves and Roots -4 DNA Samples
miRNA Processed
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31. Figure1: Distribution of tomato miRNA across root and upground tissues
in tolerant and sensitive genotypes 31

32. Fig 2:Tomato miRNA distribution in all samples of Drought sensitive and drought tolerant
tissues 32

33. Fig3: Comparisons of conserved miRNA expressing changes after drought exposure33

34. Fig 4 :qRT-PCR Validation of randomly selected drought-responsive eight miRNA in
tolerant root tissues
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35. Figure 5 : Functional Annotation of GO biological process related with
drought stress in tomato by AGRIGO
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36.  Drought responsive miRNA differentially expressed in drought tolerant and drought
sensitive genotypes exhibit tissue specific expression pattern.
 Majority of these miRNAs were involved in fruit, shoot, seed and root devlopment
and few miRNAs were involved in regulation of drought and development
responsive genes like RP, HD-ZIP, MYB, NAC and PSII root and upground tissues.
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37. Objectives:
1.High quality of Denovo assembly of Apple genome with the already available Apple genome
2.DNA methylation data analysis to know their Epigenetic marks which contribute in Apple
fruit development and other agronomically important traits.
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GDDH13 Fruit
Material and Methodology followed
Haploid Plant -GDDH13-Leaves and Fruits
Haploid Plant - GDDH 18-Fruits
DNA Isolation- GDDH 13 (Leaves and Fruits)
GDDH 18 (Fruits) @ 3 days
before pollination and 9 DAP
Sodium Bisulfite treatment (Bisulfite Kit)
Whole genome bisulfite sequencing
(To determine cytosine methylation status)
Identification of Differentially methylated regions (DMR)
Between Leaves and Fruits of GDDH13
Between Fruits of GDDH13 and GDDH18 @ -3DAP and 9DAP
Functional annotation of DMR (Reference Annotated Apple genome and Arabidopsis)

39. Fig.6 DNA Methylation distribution on all 17 chromosomes
DNA Methylation distriubtion
and recombination rates
on Chromosome 11
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Comparison of Methylation level across gene rich regions, Transposable
elements and HODOR (Type of TE)
*TSS-Transcriptional Start Site TES-Transcriptional End Site
Fig.7..Methylation within the gene rich regions is less compared to TE
Transposable element-HODOR showed maximum methylation (90% for CG, 65% for
CHG and 3% for CHH)

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Comparison of global Methylation sites (CG, CHG and CHH) of Arabidopsis,
Soybean and Apple genomes
Fig.8 Higher methylation for CG (86%) sites than CHG (74%) and CHH
(5.4%) sites
Content of Methylation of Soybean and Apple is almost same and less in
Arabidopsis

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Differentially Methylated Regions (DMR) between Leaves
and Fruits of GDDH13
Fig .9 Total of 1,017 significant DMR identified between leaves and fruits
Maximum methylation was for CHH (86%) with 875 DMR followed by CHG sites
with approximately 100 DMR
Increased methylation was seen for developing Fruits than Leaves.
294 DMR were present in the Promoter regions

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Functional analysis of DMR
Table 1..11 DMR were mostly involved In fruit size regulation/fruit devlopment

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Parent Haploid Haploid
Isogenic lines
-3DAP---197 DMR observed between GDDH13 (Large Fruits) and GDDH18 (Small fruits)
47 DMR located in promoter region (fig 11)
9DAP- 148 DMR were identified between GDDH13 (Large Fruits) and GDDH18 (Small fruits)
53 were located in promoter region
Majority of the DMR showing decreased methylation in small fruited genotype
(GDDH18)
and increased methylation in Larger fruits genotype(GDDH18)
22 gene rich- DMR played a role in fruit size regulation- SPL13, ACS8, Cytochrome P456 etc

45. Methylation plays an important role in Fruit size regulation
Higher methylation occurs in CG sites than others
Limited methylation occurs in the promoter regions
Increased methylation occurs in larger fruits
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46.  Some Tomatoes do not ripen and remain green
 These tomatoes have a heritable cytosine hyper methylation on the CNR, NOR and
RIN gene promoter, which inhibits the expression of these genes leading to no
ripening.
 When the unripe tomato is injected with the enzymes that inhibit the methylase
enzymes-Lead to ripening of the tomato indicating the strong regulation of
epigenetics in Tomato ripening process
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Objective:
Role of DNA methylation in tomato ripening related genes such as RIN, NOR and
CNR & their association with ripening/non-ripening.

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Figure. 12

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50. Role of Epigenetic regulations in biological process in
plants
Phenylpropanoid synthesis
N & S assimilation
Light perception
Photosynthesis
Flowering time Sugar metabolism
Cell elongation
Cold responses
Starch mobilization
Hormone signaling
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