Download Piper colubrinum

Piper colubrinum & Piper nigrum Plants
challenged with
Phytophthora capsici
Piper nigrum
Technology
Next Generation Sequencing
(Genome Analyzer IIx – Illumina)
Piper colubrinum
Average_Quality_
Score
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
57
59
61
63
65
67
69
71
Transcriptome Assembly and Analysis
Workflow
1. Quality Score at Base Positions - Run28_s_7_1_sequence_trimmed72.txt
40
35
30
Base_Number
Average Quality…
Sequence Reads
(QC and Raw data processing- SeqQC-V2.1)
Denovo assembly
(Velvet_1.1.04)
Assembled contigs
Annotated Transcripts
(Oases)
Advanced Analysis
Summary of NGS DATA
Summary statistics
Sequence File Size
Maximum Sequence Length
Minimum Sequence Length
Average Sequence Length
No. of Sequences
Total Sequences Length
Total Number of Non-ATGC
Characters
Percentage of Non-ATGC
Characters
Sample
Piper colubrinum
Sample
Piper nigrum
37.70 MB
15769
100
567.844
62619
35557875
76.06 MB
10479
100
721.922
101284
73119148
1316
1090
0.00004
0.00001
BLAST analyses: Similarities of Piper colubrinum and Piper nigrum transcriptome sequences against
different Plant species sequence database.
Target organism
Arabidopsis thaliana
(thale cress)
Brachypodium
distachyon
Brassica napus
(oilseed rape)
Carica papaya
(papaya)
Glycine max
(soybean)
Gossypium hirsutum
(Cotton)
Hordeum vulgare
(barley)
Lotus japonicus
(lotus)
Manihot esculenta
(cassava)
Medicago truncatula
(barrel medic)
Total Annotated
transcripts
Piper colubrinum
(mRNA and protein)
14334
Total Annotated
transcripts
Piper nigrum
(mRNA and protein)
27998
Total Annotated
transcripts
(both the species)
217
293
510
654
1128
1782
493
666
1159
9511
19204
28715
1576
2883
4459
1620
3142
4762
3267
6043
9310
403
600
1003
4653
9531
14184
42332
BLAST analyses – continued………….
Target organism
Total Annotated
transcripts
Piper colubrinum
(mRNA and protein)
Total Annotated
transcripts
(both the species)
25
Total Annotated
transcripts
Piper nigrum
(mRNA and
protein)
-
Mimulus guttatus (Mimulus)
Oryza sativa (rice)
3981
7389
11370
Persea americana
187
304
491
Physcomitrella patens (Moss)
764
1338
1902
Phytophthora Sp.
738
1432
2170
Piper sp.
152
117
269
Populus trichocarpa
(Poplar)
17558
33774
51332
Ricinus communis (Ricinus)
403
32418
32821
Solanum lycopersicum (tomato)
7855
15367
23222
Sorghum bicolor (Sorghum)
13339
25082
38421
Triticum aestivum (Wheat)
6294
12149
18443
Vitis vinifera (wine
grape)
19515
37105
56620
Zea mays (corn)
12662
20486
33148
25
Piper Transcriptome- analysis
ESTs IDENTIFIED
Genes
in response to stress
beta 1,3-glucanase
Catalase
Chitinases
cinnamoyl CoA reductase
cinnamyl alcohol dehydrogenase
Cu/Zn superoxide dismutase
dehydration-induced proteins (dehydrins)
delta 1-pyrroline-5-carboxylate synthetase
galactinol synthase
glutathione peroxidase
glutathione reductase
Glutathione s-transferase
thaumatin-like protein
papain-like cysteine proteinase-like protein
peroxisomal betaine-aldehyde dehydrogenase
polygalacturonase inhibiting protein- pgip1
Piper Transcriptome- analysis
(ESTs IDENTIFIED )
Genes involved in Metabolic process
alcohol dehydrogenase
alkaline alpha galactosidase
beta-ketoacyl-CoA synthase
citrate synthase
ferredoxin-nitrite reductase
isopropyl malate isomerase large subunit
glutamine synthetase
lipoxygenase (loxA)
malate dehydrogenase
nicotianamine synthase
omega-3 fatty acid desaturase
Phosphoenolpyruvate carboxylase
pyruvate decarboxylase
pyruvate dehydrogenase
Piper Transcriptome- analysis
(ESTs IDENTIFIED )
Genes involved in Secondary metabolism
3-hydroxy-3-methylglutaryl CoA reductase
4-coumarate-coA ligase
5-enolpyruvylshikimate 3-phosphate synthase
6-phosphogluconate dehydrogenase
cinnamic acid 4-hydroxylase
caffeoyl CoA O-methyltransferase
chalcone synthase (CHS) & Chalcone isomerase
Cinnamate 4-hydroxylase (C4H)
flavanone 3-hydroxylase
oxidoreductase protein geranyl geranyl pyrophosphate
glucose-6-phosphate 1-dehydrogenase
HMG-CoA reductase
isopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase
S-adenosyl-L-methionine synthetase
lycopene beta cyclase
O-methyltransferase
phenylalanine ammonia lyase
Piper Transcriptome analysis
ESTs IDENTIFIED
Other Genes identified
transcription factors
bHLH transcription factor
bZIP transcription factor
DRE-binding protein
ethylene-responsive transcription factor 2
GAGA-binding transcriptional activator
homeobox transcription factor
MYB transcription factor
molecular chaperones
Chaperone DnaK
DNAJ heat shock N-terminal domain-containing protein
heat shock protein 70 & 82
peptidyl-prolyl cis-trans isomerase
Involved in
signal transduction
lectin protein kinase family protein
Pto kinase interactor
lectin protein kinase family protein
mitogen-activated protein kinase (MAPK)
ras-related protein
Gene specific contigs/ transcripts identified from P. colubrinum &
P. nigrum transcriptome
Reference gene
Aquaporin- Piper
colubrinum
Osmotin - Piper colubrinum
(IIIr)
betaine-aldehyde
dehydrogenase - Glycine
max
Cu/Zn superoxide
dismutase- Gossypium
hirsutum
Mitogen-activated protein
kinase (MAPK)- Gossypium
hirsutum
R gene fragment - Piper
colubrinum
bZIP transcription factorOryza sativa
beta-1,3-glucanase-like
gene- Piper colubrinum
1,3-glucanase-like mRNA,
complete sequence- Piper
colubrinum
Piper colubrinum
Contig Identity Alignme E- Value
length
nt length
2094
99.63
267
2e-149
Piper nigrum
Contig
Identity
length
1229
82.65
Alignment Elength
Value
98
1e-10
297
99.35
155
4e-81
318
94.53
201
4e-87
1698
77.27
726
3e-27
1726
77.61
603
2e-25
865
88.29
401
2e-72
797
82.93
375
5e-64
1798
77.89
995
3e-61
2854
78.35
485
3e-31
3008
98.76
242
1e-129
-
2053
76.62
633
7e-16
1797
78.65
342
3e-21
1074
97.96
490
0
628
93.21
265
3e-108
373
95.76
165
4e-75
628
97.97
345
2e-180
Gene specific contigs/ transcripts – Continued
Reference gene
Piper colubrinum
Contig Ident Alignm Elength ity
ent
Value
length
Piper nigrum
Contig Identity
length
Alignme Ent length Value
CalmodulinA.thaliana
Catalase- A.thaliana
1742
83.79 1497
3e-61
1801
82.93
375
1e-63
1576
78.83 1162
4e-94
1353
79.31
1020
8e-98
Geranylgeranyl
transferaseA.thaliana
Heat shock protein70- A.thaliana
Malate
dehydrogenaseA.thaliana
WRKY
1124
79.9
3e-35
542
81.46
329
1e-39
2401
79.48 1433
7e-146
1857
78.33
1269
1e-88
2304
77.95 1111
1e-70
-
1620
97.92 96
8e-43
977
100
40
9e-17
98.24 227
6e-109
2721
94.95
198
5e-82
98.82 254
4e-135
2482
86.92
237
3e-49
Alpha amylase- Piper 2206
colubrinum
Betaine aldehyde
2595
dehydrogenase- Piper
colubrinum
398
Transcription factor genes identified in the
transcriptomes
Transcriptome
Target species
No. of hits for
transcription factor
genes
Piper colubrinum
Arabidopsis thaliana
165
Populus trichocarpa
12
Vitis vinifera
8
Arabidopsis thaliana
142
Populus trichocarpa
21
Vitis vinifera
9
Piper nigrum
Gene of interest
Expression data for
specific genes
in the
Piper colubrinum
&
P. nigrum
transcriptome
Expression in
Piper colubrinum *
Expression in
Piper nigrum*
ACC oxidase
7.57
0.00
PISTILLATA-like protein PI
13.33
0.00
APETALA3-like protein AP3-2
heat shock protein-70 cognate
protein (ERD2)
41.38
0.00
104.85
3.88
Cinnamoyl CoA reductase
531.15
37020.49
Alpha amylase
2558.75
5379.58
WRKY transcription factor
R gene related sequence (P.
colubrinum)
Hydroxyproline-rich
glycoprotein
5197.90
14193.71
6161.33
0.00
11175.07
26616.99
Peroxidase
30732.80
183601.93
beta-1,3-glucanase
66399.50
2825.04
Aquaporin
75066.67
6042.32
Osmotin
2016108.51
394.81
*based on average read depth
Identification of Resistance Gene Analogues (RGAs) in the P. colubrinum transcritome
S. No
1
Transcript No.
(PN)
Locus_22875
Length
(bp)
143
2
Locus_51895
127
3
Locus_14276
178
4
Locus_19163
134
5
Locus_34918
148
6
Locus_52055
183
7
Locus_11301
1086
8
Locus_48409
127
9
Locus_53603
122
Most similar R gene related sequence
coverage
E-value
Vitis vinifera probable disease resistance
protein At5g63020-like mRNA
Brassica rapa subsp. pekinensis isolate
BrCNL5 disease resistance protein gene,
complete cds
Vitis vinifera putative disease resistance
RPP13-like protein 1 mRNA
Vitis vinifera putative disease resistance
protein mRNA
Vitis vinifera putative disease resistance
protein mRNA
Ipomoea batata isolate S1_C09
resistance gene analog genomic
sequence
Populus trichocarpa NBS resistance
protein, mRNA
Solanum demissum isolate 286O21c
disease resistance protein R3a-like
protein pseudogene, complete sequence
Medicago truncatula TIR-NBS-LRR type
disease resistance protein mRNA,
complete cds
94%
3e-10
99%
4e-10
79%
2e-07
99%
2e-08
99%
2e-08
83%
6e-09
38%
3e-51
97%
1e-10
91%
6e-12
Identification of SNPs
Piper colubrinum - 50972 nos. & Piper nigrum -231680 nos of
SNPs Identified
.
Identification of a putative Phytophthora Resistance gene
Steps followed
An R gene fragment isolated from Piper
colubrinum using primers based on
Conserved sequence motifs
One Contig (3008bp, 913 aa) was found to
be matching with R gene fragment
Advanced analysis of the Contig showed
high similarity to R genes identified in
different crops
Blast results : PSI-Blast
Conserved Domain Search
FingerPRINTScan
EXPRESSION OF OSMOTIN GENE IN
Piper colubrinum challenge inoculated with Phytophthora capsici.
EXPRESSION OF BETA 1,3 GLUCANASE GENE IN
Piper colubrinum challenge inoculated with Phytophthora capsici.
Expression analysis (real-time quantitative RT-PCR ) of R- gene from
Piper colubrinum challenge inoculated with P. capsici
Relative expression
Expression analysis of wrky gene from Piper colubrinum
challenge inoculated with P. capsici
Relative expression
OTHERS
• Resistance related gene sequences amplified from Piper colubrinum utilizing
Motif - Directed Profiling and Rolling Circle Amplifications (RCA). Allelic variations
and INDELs found in R gene analogues.
• Full length wrky gene ( of Piper colubrinum) deduced from transcriptome data
and found to have a coding sequence of 303 amino acids
corresponding to 909 nucleotides. Introns and other variants recovered
through amplification from DNA.
Studies on allelic variations
QRT-PCR (Quantitative RT-PCR) & cDNA Microarrays
Functional validation of the genes: RNA silencing
Proteomics approaches