1. Chiriya
7
Sonalika
Seedling resistance test against B. sorokiniana
under glasshouse conditions
Multilocation evaluation of wheat germplasm
against Bipolaris sorokiniana to select highly
resistant and susceptible genotypes
The plates showing clones of subtracted secondary PCR cDNAs product followed by
electroporation mediated transformation. (a) Blue white selection on Luria Agar medium, (b)
Colonies streaked on Luria Agar medium
S.
N
Genotypes
Disease
severity
Delhi
Ludhiana
Wellington
1
Sonalika
47 57
73
2
Milan
00 12
00
3
Chiriya
3
00 0
0
00
4
Chiriya
7
00 00
01
5
Chiriya
1
00 00
01
6
SW
89-‐5427
10 24
02
7
Shuzoe
8
-‐
00
-‐
Clone
ID
Blast
n
Matching
accession
no.
Gene
discrip>on
Size
E
value
50-‐39
AY704445.1
Calcium
dependent
protein
kinase
3-‐
like
(TriHcum
aesHvum)
853
0
50-‐50
XM_003577327.1
ABC
transporter
F
family
member-‐5
like
(Brachypodium
distachyon)
750
0
60-‐77
XM_003570240.1
Nudix
hydrolase
3-‐like
(Brachypodium
distachyon)
620
0
SSHJP17
U32428.1
Lipoxygenase
(TriHcum
aesHvum)
751
0
SSHJP42
XR_137902.1
Predicted
dual
specificity
protein
kinase
pyk1-‐like
(Brachypodium
distachyon)
374
0
SSHJP45
AY841792.1
Cysteine
protease
(CP)
(TriHcum
aesHvum)
686
0
50-‐57
FJ394355.1
PutaHve
phospholipase
D
alpha
1
precursor
(Aegilops
speltoides)
679
0
60-‐11
XM_003564941.1
Serine
palmitoyltransferase
2-‐like
(Brachypodium
distachyon)
933
0
60RA33
XM_003561955.1
Predicted
omega-‐amidase
NIT2-‐like(Brachypodium
distachyon)
472
1e-‐105
50RA3
HM037186.1
PutaHve
calreHculin
(TriHcum
aesHvum)
592
0
60-‐43
XM_003558279.1
Predicted
topless-‐related
protein
1-‐like
(Brachypodium
distachyon)
750
0
50-‐35
AF508970.1
TranslaHonally
controlled
tumor
protein
(TriHcum
aesHvum)
701
0
List of identified SSH clones of Defense related genes of Chiriya 7
Functional cataloguing of differentially expressed
genes in resistant wheat genotypes Chiriya 7 post
inoculation with B. sorokiniana
Cataloguing of differentially expressed
genes in resistant wheat genotypes Chiriya
7 post inoculation with B. sorokiniana
based on host
Identification of putative defense related genes and their expression profiling in wheat
(Triticum aestivum) against Bipolaris sorokiniana
Introduc>on
Materials
&
Methods
Results
Bipolaris sorokiniana, the casual agent of spot blotch of wheat,
significantly reduces grain yield worldwide. Wheat germplasm
comprising of Milan, Chiriya3, Chiriya7, Chiriya 1, SW89-5427,
Suzhoe and Sonalika evaluated against virulent isolate BS-75
showed Sonalika as most susceptible and Chiriya 7 as resistant
genotype.
Differential induction of defense genes response in resistant/ susceptible
genotype to understand compatible/incompatible interactions against
virulent pathotype of B. sorokiniana was studied by preparing cDNA
library through suppression subtractive hybridization technique. Total
RNA was isolated from the leaves of resistant and susceptible
genotypes at different time intervals post inoculation using Trizol
reagent. Positive clones were sequenced and Time course expression
analysis of MAP kinases was done by using semi quantitative and Real-
Time PCR.
Subtraction product after primary and secondary PCR. Agarose gel (1.2%) showing the
PCR Amplified subtracted cDNA
Time course expression analysis of TaDSPK amplified through semi-quantitative RT-PCR in
genotype Sonalika and Chiriya 7 at specified period of post inoculation with B. sorokiniana BS-75
isolate. Lane U- uninoculated; Lane1- 2 hours post inoculation (hpi); Lane2- 19 hpi; Lane3- 24 hpi;
Lane4- 48 hpi; Lane5- 4 days post inoculation(dpi); Lane6- 6 dpi; Lane7- 8 dpi; and Actin is taken
as internal control.
Expression pattern of TaDSPK in wheat genotypes Sonalika and Chiriya7 on inoculation with
B. sorokiniana BS-75 isolate using RT-PCR at different periods indicating the changing fold of
the gene transcript over the control.
Multiple alignment of TaDSPK protein showing conserved region of amino acids sequence. Red box
represents TxY motif, where phosphorylation of threonine and tyrosine residue is required, in order
to lock the kinase domain in a catalytically competent confirmation. Blue box represents ‘DFG’
motif that is conserved amongst most protein kinases.
Conclusion
More than 1500 clones were obtained from SSH cDNA library, out of
which more than 150 clones have been sequenced. These differentially
expressed genes were categorized based on their functional annotations.
12 were categorized under defense responsive genes, and rest of the
genes were categorized under signal transduction genes, cellular
transport, translation and miscellaneous activities. Maximum genes
(83.75%) were characterized from wheat and allied species such as
Brachypodium, Aegilops and Hordeum. Further, time-course expression
analysis indicated the role of TaDSPK in defense responses against B.
sorokiniana.
(a)
(b)
Chiriya 7 has been identified as resistant genotype. cDNA sequences
obtained by SSH technique has shown 12 genes related to defense
responses. Real-Time PCR analysis confirmed the role of TaDSPK gene
in defense response in wheat against B. sorokiniana.
Selected
References
Diatchenko,
L.,
Chris
Lau,
Y.,
Campbell,
P.A.,
Chenchik,
A.,
Moqadam,
F.,
Huang,
B.,
Lukyanov,
S.,
Lukyanov,
K.,
Gurskaya,
N.,
Sverdlov,
E.D
and
Siebert,
P.S.
(1996).
Suppression
SubtracHon
HybridizaHon:
A
method
for
generaHng
differenHally
regulated
or
Hssue-‐specific
cDNA
probes
and
libraries.
Proc.
Natl.
Acad.
Sci.
USA.
93:
6025-‐6030
Sagar Banerjee*, Rashmi Aggarwal,, Prasanna Kumari, Sangeeta Gupta, Sapna Sharma, B.M. Bashyal, Harita Babbar
Division of Plant Pathology, Indian Agriculture Research Institute, New Delhi-110012
*sagar.iari@yahoo.com
