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1. Cloning and Characterization
of Full Length Candidate
genes for Specific
Physiological Traits
Presented by
P. L. N. Pravallika
ID. No-BAD/23-08

2. What is a Candidate gene???
• Candidate genes are sequenced genes of known
biological action involved in the development or
physiology of a trait.
• Physiologists consider CG’s as “All genes involved
in the expression of the physiological trait.
• Geneticist consider CG’s as “Polymorphic genes
putatively involved in trait variation”.
• For Plant Geneticist CG’s is “any genes putatively
involved in trait variation based on its biological
function or map localization”.

3. How to develop a candidate
gene?????

6. Application of Candidate gene

8. What is cloning of genes????
• gene cloning (DNA cloning) The production
of exact copies (clones) of a particular gene or
DNA sequence using genetic engineering
techniques.

9. How is the gene cloned??
• The DNA containing the target gene(s) is split into fragments
using restriction enzymes.
• These fragments are then inserted into cloning vectors, such as
bacterial plasmids or bacteriophages, which transfer the recombinant
DNA to suitable host cells, such as the bacterium E. coli.
• Alternatively, complementary DNA is inserted into the vectors, or
‘naked’ DNA fragments can be taken up directly by a host bacterium
from its medium (this is less efficient than vector transfer).
• Inside the host cell the recombinant DNA undergoes replication;
thus, a bacterial host will give rise to a colony of cells containing the
cloned target gene. Various screening methods may be used to
identify such colonies, enabling them to be selected and cultured

10. What is Characterization of genes?
• Characterization is the description of plant
germplasm. It determines the expression of
highly heritable characters ranging from
morphological or agronomical features to seed
proteins or molecular markers.

11. How is Characterization done???
• New methods have made molecular analysis and genotyping
useful techniques for studying diversity.
• Samples of leaf are usually collected from replicated trials.
• A variety of molecular techniques are used, including
cytological markers, biochemical markers and molecular
genetic markers such as SSR, EST-SSR, AFLP, RAPD.
• Their choice depends on the stage of research into molecular
methodologies for the crop, facilities and expertise available in
each gene bank.
• Descriptors are available to describe a genetic marker
technology and collect information about genetic markers in
crops that are standardized and replicable.

13. Case study 1- Cassava

15. Objective
• Cassava is an important root crop to resource-poor farmers in
marginal areas, where its production faces drought stress
constraints.
• This study was carried out to identify candidate drought-
tolerance genes and expression-based markers of drought
stress in cassava.
• One drought-tolerant (improved variety) and one drought-
susceptible (farmer-preferred) cassava landrace were grown in
the glasshouse under well-watered and water-stressed
conditions.

16. Contd………….
• Their morphological, physiological and molecular
responses to drought were characterized.
• Ten genes that have previously been biologically
validated as conferring or being associated with
drought tolerance in other plant species were confirmed
as being drought responsive in cassava.
• Four genes (MeALDH, MeZFP, MeMSD and
MeRD28) were identified as candidate cassava
drought-tolerance genes, as they were exclusively up-
regulated in the drought-tolerant genotype to
comparable levels known to confer drought tolerance in
other species.

19. Conclusion
• Genes underlying MH96/0686 tolerance, which is no doubt
multi-genic, can be divided into two categories:
• those whose baseline expression levels are different in DT
compared with DS (i.e. DT is primed to be less susceptible or
responds more quickly to drought stress) and
• those whose expression levels under drought are significantly
more changed/expressed at higher levels in DT compared with
DS (i.e. they may play a role in the longer-term tolerance of
MH96/0686 observed in the field).
• The more responsive genes (the second category) represent
candidates for the adaptive response of MH96/ 0686 to
drought and therefore important targets for further validation
and for subsequent use in developing cassava varieties with
enhanced drought tolerance.

20. Case study 2- Bajra

22. Objective
• In the present study, they have isolated a dehydrin gene
from P. glaucum, referred to as PgDHN. The
expression of PgDHN is upregulated in response to
drought, salinity, heat stress, and cold stress.
• Its role in rendering tolerance to salinity and heat stress
is further indicated by its expression analysis in
Escherichi coli
• These results indicate that PgDHN plays a protective
role under various abiotic stress conditions and could
be used as a tool to improve the abiotic stress tolerance
of crop plants along with other stress responsive genes.

24. Conclusion
• The sequence analysis of the full length protein
revealed the presence of all the conserved motifs
characteristic of dehydrins.
• Transcript profiles in leaves under different
abiotic stress conditions showed that PgDHN is
upregulated particularly in salinity stress.
• E. coli cells expressing recombinant PgDHN
exhibited higher growth rates as compared to the
control cells (without PgDHN) when exposed to
heat and salinity stress.

25. • These findings suggest that
PgDHN plays a crucial role in
stress adaptation and provides
resistance against different
abiotic stresses in P. glaucum.
• Therefore, PgDHN can be
used to develop transgenic
crop plants to combat multiple
abiotic stress conditions.

26. Case study 3- Turmeric

28. Objective
• Phytocystatin, a type of protease inhibitor (PI), plays major
roles in plant defense mechanisms and has been reported to
show antipathogenic properties and plant stress tolerance.
• Recombinant plant PIs are gaining popularity as potential
candidates in engineering of crop protection and in
synthesizing medicine.
• It is therefore crucial to identify PI from novel sources like
Curcuma longa as it is more effective in combating against
pathogens due to its novelty.
• In this study, a novel cDNA fragment encoding
phytocystatin was isolated using degenerate PCR primers,
designed from consensus regions of phytocystatin from
other plant species.

29. Isolation of Candidate gene responsible
for pest and disease resistance

30. Conclusion
• Cloning and characterization of candidate gene
is one of the powerful tool to combat both
abiotic as well as biotic stress in a specified
manner.
• It also employs identification of novel CG for
having stable food and nutrition security in the
upcoming years if encouraged in proper way.