Genomics, proteomics and metabolomics are the three core omics technologies, which respectively deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
2. Introduction:
• Green revolution enabled India to achieve self suffiency in
food production, but still there is a continuous challenge to
futher increase agricultural production to feed the growing
population of India.
• Recent developments in the field of Omics technologies hold
immense potential to reshape India agriculture through green
revolution.
• Genomics, proteomics and metabolomics are the three core
omics technologies, which respectively deal with the analysis
of genome, proteome and metabolome of cells and tissues of
an organism.
3.
4. Genomics
• The comprehensive study of whole sets of genes and their
interactions
• It investigates the variation in genes and how it affects
protein structure and function throughout the life of a cell.
• Genomics is the sub disciplines of genetics devoted to
i. mapping,
ii. sequencing and
iii. functional analysis of genomes
5. From Classical genetics to genomics
Classical genetics
Phenotypic ratio= 3: 1 Genotypic ratio = 1: 2: 1
Drawbacks:
• Linkage between
different genes are not
known.
• Location of genes
unknown
• Simple and qualitative
characters
• Requires more time
because of
morphological selection
• Environmental influences
6.
7. Phenotypic selection vs Marker assisted selection
• Less precise and reliable,
• Time consuming
• Simpler compared to phenotypic
selection,
• Selection at seedling stage
8. Genomic selection for crop improvement
• Two primary
limitations of MAS
1) the biparental
mapping populations
used in most QTL
studies do not readily
translate to breeding
applications
2) statistical methods
used are ill-suited to
the traits polygenic
nature
Genomic selection (GS) has been proposed to address these deficiencies.
GS reduces cycle time & cost by reducing frequency of phenotyping.
10. Overview of a
crop trait
discovery
pipeline
generating a
genome sequence
from fragmented
sequencing reads
to identify the
differences between
the DNA of the
specific individuals
sequenced with the
reference genome
where genomic
sequencing of crop
populations can
allow gene-level
resolution of
agronomic variation
determines the
phenotypic effect of
altered sequences of
specific genes or
regulatory regions
11. Trait mapping using QTL and GWAS
• The QTL analysis bridging the gap between
genomics and the field.
limitations of QTL
mapping
• low resolution caused
by coarse mapping
• only allelic diversity
present in the parents
of the segregating
population can be
assayed
These limitations of QTL
mapping overcomed by
GWAS, employed to
pinpoint genomic regions
linked to traits in diverse,
unrelated populations.
12. Forward and Reverse Genetic Screening
Forward genetic screening
- improve gene cloning and
marker development
- excludes intergenic sequences
Reverse genetic screening
- Targeted Induced Local
Lesions in Genomes (TILLING),
a reverse genetic approach, can
take advantage of conventional
mutation Induction
- providing the capability of
recovering mutations from
any genetic regions and discover
novel phenotypes
13. Advantage of GS over QTL, GWAS or
Reverse genetics
QTL
GWAS
Reverse
genetics
Based on genotyed and phenotyed training
population
Calculating the genomic estimated breeding
values (GEBV) for sets of variants
overcomes inefficient translation of QTL analysis
results from biparental mapping populations
to breeding
combination of GS with automated phenotyping
techniques can further promote prediction
accuracy of GEBV, shortening the breeding cycle
Not essential for
genomics-based
breeding
Difficult to detect
targeting polygenic
agronomic traits
such as yield,
minor effect alleles
Genomic
selection
14.
15. WHAT IS COMPARATIVE GENOMICS?
Analyzing & comparing genetic material from different
species to study
evolution, gene function, and inherited disease
Understand the uniqueness between different species
16. Comparative genomics is a powerful
tool allowing us:
to link genomic changes to
environmental adaptation
to transfer knowledge from
model species to other plants
to trace structural changes
within a genome through time
17. Application of Genomics in Crop
Improvement
• It also reduces the gap between phenotype and genotype.
• It helps in assaying genetic makeup of the individual plants rapidly, so as
to select desirable genotypes in breeding populations, and to design the
superior genotypes for ‘breeding by design’ approach.
• With genomic approaches, the marker-assisted breeding or marker-
assisted selection will gradually evolve into ‘genomics-assisted breeding’
for crop improvement.
• The identification of genes that control economically important traits
provide the basis for new progress in genetic improvement of crop
species, complementing traditional methods based on assisted crosses.
Thus, a genome programme can be envisioned as a highly important tool
for crop improvement.
18. The whole genome contains both the coding and non-
coding genes (that are not related to heritable
phenotypes)
The transcriptomics analysis based only on
coding genes i.e., exons are transcribed and
the translated into proteins.
Limitations of genomics over transcriptomics:
19. Epigenomics
The term epigenetics refers to heritable changes in gene
expression that does not involve changes to the underlying
DNA sequence; a change in phenotype without a change in
genotype.
An epigenome consists of a record of the chemical changes
to the DNA and histone proteins of an organism.
Changes in the epigenome can result in changes to the
structure of chromatin and changes to the function of the
genome.
20. A Lamarckian idea that the act of stretching one's neck
could lead to change in phenotype across generations.
Lamarck's mechanism for evolution is The inheritance of acquired traits.
He believed that traits altered or acquired over an individual's lifetime
could be transferred down to its offspring.
HISTORY
23. Characteristic symptoms of Foc TR4 in
susceptible and resistant banana
WT cavendish
Resistant line RGA2,
from transgenic
cavendish
•Race 1 was involved in the 1960s Panama disease
attacks members of the banana AAB genomic group.
Cavendish cultivars are resistant to Race 1.
•Race 2 infects cooking bananas with ABB genome
and the Bluggoe subgroup.
•Race 3 infecting Heliconia spp. is no longer
considered pathogenic to bananas.
•Race 4 is the causal agent of the current Panama
disease outbreak since it is pathogenic to the
currently used Cavendish cultivars (AAA genome).
Race 4 is further subdivided into Tropical Race 4
(TR4) and Subtropical Race 4 (STR4). The latter
only infects Cavendish
Fusarium oxysporum f. sp. cubense
tropical race 4
24. DNA methylation patterns of banana leaves in response to
Fusarium oxysporum f. sp. cubense tropical race 4
Pathogen infected experiment
on in vitro banana leaves at 0,
4, 12, 24 h, 3 and 6 d post
inoculation. A, mock
inoculated group. B, infected
group.
A
B
In this study, with methylation-sensitive
amplification polymorphism (MSAP) technique,
DNA methylation was compared between the leaves
inoculated with Foc TR4 and the mock-inoculated
leaves at different pathogenic stages
Arrows indicated the polymorphic
fragments of methylation.
25. (contd..)
Results:
• DNA methylation was both
changed and the average
methylated CCGG sequences were
34.81 and 29.26% for the infected
and the mock-inoculated leaves.
• DNA hypermethylation and
hypomethylation were induced by
pathogen infection during all
pathogenic stages.
This results suggest that DNA methylation plays important roles in pathogenic
response to Foc TR4 for banana.
• RT-PCR results of four genes indicated that
their expression patterns were consistent
with their methylation patterns.
26. Transcriptomics
• Transcriptomics deals with the analysis of gene expression
patterns across a wide array of cellular responses,
phenotypes and conditions.
• The identification of candidate genes influencing any
important trait can be approached through an analysis of
gene transcripts or mRNAs.
• It is possible to determine when and where a gene is turned
on or off in various types of cells and tissues by analyzing
the transcriptome.
27. Post sequencing analysis
TOPHAT2
Cufflink program
Cuffdiff program
BLAST2GO program
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Transcripts
C S vs C R UR vs C R US vs C S US vs UR
C ombination
Neutral
UP
Down
LB
hpt
expression
cassette
pCaMV35S
Musa DIR11
CDS
RB
tnos
PmlINcoI
TRANSCRIPTOMIC APPROACH FOR SIGATOKA RESISTANCE
Resistant responsive pathways
Susceptible responsive pathways
Differential Gene Expression Gene Ontology
Resistant mechanism
Sigatoka Component
R genes
1. LRR
2. LRR/serine/threonine-
protein kinase
28. Creation of simulated drought stress &
stress imposition on selected banana
cultivars
Comparative Transcriptomics
Transcriptome sequencing –Illumina
Hi-seq
162.36 million reads (CT,DT) = nr unigenes-23,096
126.58 million reads (CS,DS)= nr unigenes -23,079
Selection of putative drought
tolerance genes
Diff. gene expn.
CT vs. DT, CS vs. DS
NCBI- SRP087441
Validation of some of the genes
by 2 D PAGE for Proteins (43)
q RT-PCR Validation for sel.
Candidate genes (6 DEGs)
Long non-coding RNAs
(lncRNAs)- 905
Full length gene isolation
Protein coding genes/mRNAs
2268 (T) and 2963 (S)
A.TIP3-1
B. GST
C. Wax syn
D. CUT-1
E. HSP
F. ABC I-17
Eval. of genetically transformed
plants for drought tolerance
Development of transgene
construct
pBM GF-DC-34
11730 bp
NPTII (Becks)
APsy2a
Left border 1
Left Border 2
Right Border
pVS1 sta
pBR322 bom
Nos promoter
Maize Ubiquitin Promoter
pBR322 ori
pVS1 rep
Nos Terminator
nos terminator
SmaI (6976)
XmaI (6974)
XmnI (5539)
EcoRI (3455)
HindIII (6943)
StuI (4200)
StuI (6989)
BamHI (4934)BamHI (6955)
KpnI (6941)
KpnI (6953)
Tolerant cv. Saba (ABB) ;
Sensitive cv. Grand Naine
(AAA)
Identification of Molecular targets for Drought Tolerance
Comparative
Proteomics
Drought Component
30. Limitations of transcriptomics over proteomics:
• All the transcribed mRNA are not translated into proteins
because of transcriptional gene silencing, RNA interference,
transposon gene silencing.
• This caused the RNA unable to make protein during
translation.
31. Proteomics
• The study of proteome, the structure and function of
complete set of protein in a cell at a given time.
• It is used to know plant-insect interactions that help identify
candidate genes involved in the defensive response of plants
to herbivore.
32.
33. Studies on molecular basis of somatic embryogenesis
and its manipulation in recalcitrant banana cultivars
34. Methodology
Different Stages of SE
SDS- PAGE
2DE
MALDI-TOF MS/MS
Annotation
RNA
qRT-PCR
Gene expression
Phytohormone Analysis Proteomic Analysis
TranscriptAnalysis
Antioxidantenzyme
Assay
POX
CAT
SOD
Media
Manipulation
1
23
4
5
ResultAnalysis
36. Results
Spot
No
Protein Cultivar Triggering
component
20
9
Indole-3-pyruvate
monooxygenase
Glutathione S-
transferase
Grand
Naine
Rasthali
*Tryptophan
*Increased conc.
of IAA
16 adenylate
isopentenyltransfe
rase
Grand
Naine
*Applying
cytokinins like
BAP and Kinetin.
37 calcium-binding
mitochondrial
carrier protein
Rasthali *Increased
concentration of
Calcium chloride
7 calcium-dependent
protein kinase
Rasthali *Increased
concentration of
Calcium chloride
Spot No Protein Cultivar Triggering
component
1 and 13 SAUR-like auxin-
responsive family
protein
Grand
Naine
NAA
13 and 55 Glutaredoxin Rasthali NAA
9 Glutathione S
transferase
Rasthali NAA , ethryl, cold
treatment
19, 61 and
67
Ethylene responsive
transcription factor
Rasthali Methionine, Ethryl
56 Calcium dependent
protein kinase
Rasthali CaCl2, cold
treatment
63 methylthioalkyl
malate synthase
Rasthali Methionine
27 and 28 cysteine proteinase
inhibitor
Grand
Naine
cold treatment
3 3-ketoacyl-CoA
synthase 11
Grand
Naine
cold treatment
8 Oleosin 18.5 kDa Grand
Naine
cold treatment
Stimulation of highly expressed proteins via exogenous application Induction of highly expressed proteins via exogenous application
37. Establishment of ECS in Recalcitrant banana cultivarsGrandNaineRedBananaMonthanNeyPoovanKarpuravalliSabri
Cultivar Media
Augmentation
Improved EC
induction
(%) over
control
Grand
Naine
IAA (11.41
µM)
> 200
Red
Banana
Tryptophan
(2.32 µM )
> 250
Monthan Tryptophan
(489.64 µM)
> 200
Ney
Poovan
Tryptophan
(489.64 µM)
> 300
Karpurav
alli
CaCl2 (10
mM)
> 400
Sabri IAA (11.41
µM)
>700
Flower Buds Embryogenic callus Cells with starch Cells with Nucleus ECS
38. Effect of Cold treatment @ 4 ˚C for 24 hr on Germination
Modified germination
medium
Shooting medium
Well Rooted plants
Primary hardening
Secondary hardening
Genetic fidelity
cv. Ney Poovan cv. Red Banana cv. Sabri
39. Application of Proteomics
• In Arabidopsis, while studying the role of GAs during
initial stages of seed germination, and the impact of
scarification on seed germination, application of
proteome analysis resulted in better understanding of the
complex cellular events.
• Similarly, in barley, the proteome analysis revealed new
insights into cellular mechanisms underlying seed
development during grain filling and seed maturation
phases.
40. Contd..
• In rice, proteome studies have helped in detecting novel
traits useful for breeding.
• Both abiotic and biotic stresses can bring about
dramatic changes to the plant proteome, andthese are
manifested as the up- or down- regulation of proteins,
or their post translation modification.
41. Metabolomics
• Study of metabolome, collection of all metabolites in a cell,
tissue, organ or organism.
• The metabolites determine the flavour, aroma, colour and
texture of crops, their storage properties and performance in
field (Memelink, 2005).
42. Global metabolomics analysis reveals distinct tolerance
mechanism of different plant organs of lentils
(Skliros et al., 2018)
Varieties used:
• F-56, a Greek variety
from Samos island,
• LC-960254, a variety
originating from the
U.S.A.)
Studied differences in their global
metabolite profile
• using gradual or initial application of
salt stress,
• between leaves and roots and
• between the varieties.
Experimental designs:
• For the purpose of gradually acclimation
(GA), 50 mM NaCl (GA 50) and 75 mM
NaCl (GA 75).
• For the initial application (IA) treatment, 25
mM NaCl (IA 25), 50 mM NaCl (IA 50) and
75 mM NaCl (IA 75)
Results:
• Demonstrated the driver of
deleterious Cl− accumulation in
leaves, a defensive mechanism for
withstanding salinity stress in plants.
• Finally, a model is suggested of how
legumes upregulate a metabolic
pathway, which involves purines
catabolism in order to assimilate
carbon and nitrogen, which are
limited during salinity stress.
43. . (A) Plant figure
shows the
compartmentalizati
on of deleterious
ions in vacuole of
leaf cells.
(B) Heat maps
represent
comparative
metabolic response
of L-asparagine, D-
trehalose and
Lactobionic acid in
roots and putative
osmoprotectants in
leaves for each
variety and
treatment (GA
stands for gradual
acclimation; IA for
initial application)
separately.
(C) Increase of salinity stress, oxidative stress and purine catabolism and
decrease of respiration and photosynthetic carbon accumulation over time
is shown as well. (D) Part of the purine catabolism pathway in the root cells
is shown
44. Applications of metabolomics
• Characterization of metabolism
• Identification of regulated key sites in network
• Biofortification and genetic modification
• Investigation of gene function under stress
conditions
47. Future concerns
• Reduction in cost of technology usage.
• Development of bioinformatic tools for data
analysis and storage of databases.
• Human resource development for an overall
purview of technology to apply in crop breeding.
