This document discusses how post-genomic tools like transcriptomics, proteomics, and metabolomics can be used for genetic enhancement of germplasm. It provides an introduction to each omics technique, examples of technologies used, and applications in understanding biological processes and identifying genes/proteins involved in traits. The conclusion states that omics expression analysis of germplasm will help characterize genome function and restore traits from wild varieties, aiding development of more sustainable crop varieties.

1. POST GENOMIC TOOLS FOR
GENETIC ENHANCEMENT OF
GERMPLASM
VISHAL REDDY
PA1 TBZ123
DEPT OF GENETICS AND
PLANT BREEDING

2. CONTENTS
• INTRODUCTION
• NEED FOR GENETIC ENHANCEMENT
• PLANT OMICS TECHNOLOGIES
• TRANSCRIPTOMICS
• PROTEOMICS
• METABOLOMICS
• CONCLUSION
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3. INTRODUCTION
• The term "enhancement" was first used by Jones (1983) which according to him
can be defined as transferring useful genes from exotic or wild types into
agronomically acceptable background.
• Rick (1984) used the term pre-breeding or developmental breeding to
describe the same activity.
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4. Need for Genetic Enhancement
• In the past, crop improvement has led to narrowing down of genetic base
resulting in slower progress in plant breeding and increased risk of genetic
vulnerability.
• To meet the market requirement, plant breeders have to develop improved
cultivars, In improving the level of resistance to biotic and abiotic stress.
• It will also help in value addition of different genotypes through genetic
enhancement.
• Genetic enhancement will result in the accumulation of unused potential
germplasm and to break these bottlenecks and to create superior gene pools.
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6. • Post genomics technologies imply the holistic approach for quantification and
characterization of transcripts, proteins and metabolites of a biological organism
known as transcriptomics, proteomics and metabolomics, respectively.
• The analytical procedures involved in omics studies have been made easier, faster,
precise and cost effective by revolutionary technological advancement in last few
decades.
• Multi-dimensional omics studies lead to better understanding of factors affecting the
phenotypic traits, metabolic process and role of biomolecules in various
biosynthetic pathways of crop plants.
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Post genomics technologies

7. Transcriptomics
• Transcription is the process where the single stranded RNA is formed
from DNA.
• In this process DNA must be read and transcribed. These gene readouts
are called transcripts, and the collection of all gene readouts present in a
cell is called transcriptome.
• The study of the complete set of RNAs (transcriptome) encoded by the
genome of a specific cell or organism at a specific time or under a
specific set of conditions is called Transcriptomics.
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8. List of transcriptomics techniques with their applications
Techniques Application Reference
DNA microarray Study of circadian clock, plant defense,
environmental stress response, fruit ripening
Aharoni and Vorst, 2001
EST Sequence information for pre microarray
design
Marra et al., 1998
SAGE Expression analysis of plants with less
characterized genome
Velculescu et al., 2000
MPSS Identify and quantify the RNA transcript Brenner et al., 2000
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9. Transcriptomics in genetic enhancement of germplasm
• The study of transcriptome helps in understanding the function of genes,
transcription levels and molecular mechanism of cellular metabolism.
• Useful in identification of candidate genes for a particular biological trait and
can help in identifying key enzymes of a metabolic pathway.
• The analysis of transcriptome assemblies gives the information about various
functional markers such as SSR and SNP related with stress resistant response.
• After obtaining the quantitative counts of each transcript, differential gene
expression could be analyzed by normalizing the data with help of statistical
modeling.
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10. Crop/Plant
Cell
studied
Technique
utilized
Biomolecule
studied
Key findings
Solanum
lycopersicum
Fruit
tissues
Laser capture
microdissection,
RNA-Seq
RNA Tomato ripening involves
gene expression gradients
that start in internal
tissues radiating outward,
and basipetally along a
latitudinal axis
Arabidopsis
thaliana
Root tip
cells
RNA-Seq RNA Regeneration of root
follows the embryonic
developmental stages
Solanum
lycopersicum
Fruit cells
and cell
types
transcriptome
profiling
RNA Identification of
differentially expressed
genes among cell or
tissue types
Cotton
(Gossypium
barbadense L.)
Cotton
fiber cells
Total RNA-Seq
and QRT-PCR
DNA, RNA Dynamic DNA
methylation regulates
lipid biosynthesis
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Transcriptomics studies in different crops

11. Future Perspectives of Transcriptomic
Technologies
• Expression profile coupled with phenotypic characters would lead to
functional annotations of uncharacterized genes.
• Analysis of multiple data sets simultaneously would enable for comparing
the gene expression patterns among different species.
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12. Proteomics
Proteomics is a newly emerging field of life science research that uses High
Throughput (HT) technologies to display, identify and/or characterize all the
proteins in a given cell, tissue or organism (i.e. the proteome)
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13. List of proteomics techniques with their applications
Techniques Application Reference
2D-PAGE for separation of protein
components of cellular extract
Klose, 1975
MS for analysis of peptide sequence
of proteins expressed at a
particular time
Fenn et al., 1989
MALDI TOF is a procedure, which analyses
the mass of peptide ions in mass
spectrometry and peptide
mapping
Kersten et al., 2002
LC-MS and HPLC applied for multidimensional
analysis of proteins since the
high ionic charge of peptides
enhances the separation of
peptides
Washburn et al., 2001
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14. • Proteomics deals with the analysis of amino acid sequence for
determination of their relative concentration and various post-
translational modifications.
• The study of proteomics helps in understanding the complex biological
procedures and cellular response against environmental stress.
• It explains the function of proteins maintaining homeostasis inside cells,
involved in cell signaling pathways and required for structural
maintenance.
Proteomics in genetic enhancement of germplasm
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Crop/Plant
Cell
studied
Technique
utilized
Biomolecule
studied
Key findings
Solanum
lycopersicum
Root tip
tissues
Laser capture
microdissection
followed by nano
LC-MS/MS
Proteins of
root
epidermal
cells
Varying effects of Al
toxicity on various layers
of root cells can be
studied through
proteomics
Arabidopsis
thaliana
Vascular
and
epidermal
cells
western blotting,
affinity
enrichment
Proteins Overall number and types
of proteins vary in specific
leaf tissues
Oryza sativa L.
cv. Nipponbare
Rice egg,
sperm,
callus cells
SDS-PAGE
followed by LC-
MS/MS
Proteins Preferential occurrence of
specific proteins
expressed in gamete cells
Proteomics studies in different crops

16. Future Perspectives of Proteomic Technologies
• The organ and time-specific proteomic analysis can identify the proteins
accumulated during stress response, which would lead to development of stress
resistant variety.
• The comparative proteome analysis of different organs can identify the proteins
involved in organ-specific metabolic pathways and thus can help in identifying
unique targets for inhibiting a particular pathway.
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17. Metabolomics
• Metabolomics is an analytical profiling technique for
measuring and comparing large numbers of metabolites
present in biological samples. Combining high-throughput
analytical chemistry and multivariate data analysis
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18. Applications of Metabolomics
• Metabolic profiles have been successfully applied for determining the
impact of seasonal changes, geographical region and natural variation.
• Metabolic flux analysis has helped in the identification of orthologous
enzymes with similar catalytic properties.
• Metabolic studies have also been performed for characterizing the
growth profile, developmental stages and chemo taxis analysis.
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Crop/Plant Cell studied Technique utilized
Biomolecule
studied
Key findings
Brassica
napus var.
Globa
Leaf guard cells GC-MS, UPLC-MS
and MRM HPLC-
MS
Phytohormones Low CO2 promotes
increased stomatal
opening controlled
through multiple
hormone
Allium cepa Epidermal single-
cell of bulbs
Probe ESI-MS Metabolites Continuous monitoring
of a live single-cell can
be achieved by using
specialized miniaturized
probes
Vicia faba Water and
wound-stressed
single cells from
leaf tissue
Nano spray
Ionization Tandem
MS using stable
isotope labelling
followed by LC-
MS/MS
Isoleucine and
Abscisic acid
hormones
Stress-induced
accumulation of ABA
and JA-Ile in single cells
Metabolomics studies in different crops

20. Future Perspectives of Metabolomic Technologies
• Metabolome analysis has helped in identification of metabolites, which provides
resistance to abiotic and biotic stresses. More such metabolites can be identified in
future that can be used as biomarkers for the study of stress tolerance response.
• Metabolic contents are more closely associated with particular phenotypic trait of
agricultural crops and could be used for QTL mapping in breeding programs.
• Metabolomics studies have been found useful for development of association map.
And could be utilized for genetically engineering a metabolic pathway.
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• To conquer the problems of growing population, changing climatic patterns and
environmental stress there is a requirement of developing the novel crop
varieties with higher yield, thermo-tolerance and less pesticide consumption.
• The expression analysis of germplasm by omics technologies will play very
crucial role in genome function characterization, for example, gene ontology,
pathway analysis and system modelling.
• However, the use of post genomic tools will increase the restoration of
environmentally favourable traits of wild varieties of crop plants and hence,
enhance their sustainability.

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