Genomics Assisted Breeding of

pomegranate for disease

resistance
Leader: Dr. Raghavendra G., Asst. Professor (Molecular Biology)

Associates Dr. Fakruddin B., Prof. & Head, Dept. of BCI, COH-Bangalore
:
Dr. Manjunath G., Asst. Prof. (Plant Path.), DR office, UHS, Bagalkot

Dr. Anand Nanjappanavar, Asst. Prof. (Fruit Science), DR office,

UHS, Bagalkot
 Expt. No.1
(2017-18)

Identification of candidate
resistance genes in
pomegranate against bacterial
blight caused by Xanthomonas
axonopodis pv. punicae
 Bacterial Blight of
Pomegranate
Causal organism:
Xanthomonas axonopodis
pv. punicae
Crop losses
Up to 80%
Eradication of plantation
Management practices
Antibiotics (Streptocyclin)
+ Chemicals
Breeding for resistance
All present day
cultivars: susceptible
Tolerant cultivars:
Nana and Daru
Linkage drag Nana

Polygenic
Genes/markers
not identified
Daru
Project objectives
Candidate genes for bacterial
blight resistance

functional
Gene casettes
markers

Marker
Genetic
assisted back
Engineering/G
cross
enome editing
breeding
Elite pomegranate cultivars
with enhanced resistance to
bacterial blight
 Candidate R genes

PTI TI
E
Classes of R genes

Hammond-Kosack and Kanyuka (2007)

 Open source genomics
data
1. Punica granatum Raw sequence reads
Organism: Punica granatum (Taxonomy ID 22663)
BioProject Accession: PRJNA377402
ID: 377402
2. Transcriptome analysis of moderately bacterial blight resistant and susceptible
genotypes of Punica granatum
Organism: Punica granatum (Taxonomy ID 22663)
BioProject Accession: PRJNA361285
ID: 361285
3. Genome-wide identification of microRNAs in pomegranate (Punica granatum L.)
by high throughput sequencing
Organism: Punica granatum (Taxonomy ID 22663)
BioProject Accession: PRJNA313106
ID: 313106
4. Pomegranate Rhizosphere soil Genome sequencing
Organism: rhizosphere metagenome (Taxonomy ID 939928)
BioProject Accession: PRJNA295001
ID: 295001
5. Punica granatum Transcriptome or Gene expression
Organism: Punica granatum (Taxonomy ID 22663)
BioProject Accession: PRJNA231033
ID: 231033
 Objectives
1.In silico prediction of R gene Analogues
(RGAs) from the pomegranate
transcriptome data
2.Validation of expression of RGAs in
response to Xap challenge inoculation by
qRT-PCR
3.Sequencing of candidate R genes and
identification of sequence polymorphisms
 Methodology
Approx. 55,000 transcripts
assembled (Ophir et al., 2014) reference R-genes,
putative R-Genes
(NCBI)
Putative R genes
(Unigenes)

Transcriptome Catalogue of Putative

In silico prediction pomegranate R genes
of of R genes
Pomegranate Susceptible
Tolerant

Susceptible

Tolerant

R gene expression
analysis
Fukuoka et al. (2013

Sequencing of highly expressed R-genes Challenge inoculation

and functional analysis with Xap and RNA
extraction
 Methodology
In silico prediction of R
genes
Pomegranate transcriptome
Accession: PRJNA231033
Functional annotation
Data Type: Transcriptome or Gene expression (454-
of transcripts using GS-FLX Titanium technology)
Blast2GO
Total No. of Transcripts: 65353
Ref: Ophir R et al., "Single-nucleotide polymorphism
markers from de-novo assembly of the pomegranate
Identification of RGAs transcriptome reveal germplasm genetic diversity.",
through motif PLoS One, 2014 Feb 18;9(2):e88998
search in the
database using
PRgDB PRGdb is web accessible open source
database that holds more than 16000
known and putative R genes belonging
Retrieval of full to 192 plant species challenged by 115
length transcripts different pathogens.
from the
transcriptome data
Validation of functional R
Methodology
genes
Genotypes:
Susceptible: Bhagawa
Tolerant: Daru
Challenge inoculation:
Xap isolated from infected pomegranate
Injection of bacterial suspension to the leaves
Sample collection for RNA collection
0, 12, 24 and 48hpi
Total RNA isolation and cDNA synthesis:
As per RNA isolation kit manufacturers guidelines
qRT-PCR and differential gene expression analysis
Fold change calculation:
 Methodology
Sequencing of R genes and
identification of functional markers
R Genes with higher expression in
Daru will be sequenced in both Daru
and Bhagawa
Multiple sequence alignment
Polymorphic loci at R gene motifs will
be converted into functional markers
 Methodology
Transcriptome data: NCBI
Plant material
Susceptible: Bhagawa
Tolerant: Nana and Daru
Challenge inoculation
Bacterial spores of Xap will be injected to leaves
Sample collection for gene expression
analysis
12, 24, 48 hpi
Nucleotide sequencing
Sequencing highly expressing genes post inoculation
 Budget
Year Particulars Approx. Cost
(Rs.)
I Molecular biology reagents/kits for DNA/RNA 2,00,000.00
extraction, PCR/qPCR, Gel electrophoresis, primer
synthesis
II Gene cloning kits, sequencing and analysis 1,00,000.00
Total (Rs). 3,00,000.00
THANK
YOU