1. Transcriptome sequencing - a case study in Piper
JOHNSON GEORGE K

2. Piper colubrinum & Piper nigrum Plants challenged with
Phytophthora capsici
Piper nigrum
Technology
Next Generation Sequencing
(Genome Analyzer IIx – Illumina)
Piper colubrinum

3. Transcriptome Assembly and Analysis Workflow
Sequence Reads
(QC and Raw data processing- SeqQC-V2.1)
Transcriptome Assembly and Analysis Workflow
Denovo assembly
(Velvet_1.1.04)
Assembled contigs
Annotated Transcripts
(Oases)
Advanced Analysis

4. Summary of NGS DATA Summary statistics Sequence File Size 37.70 MB
Sample
Piper colubrinum
Piper nigrum
Sequence File Size
37.70 MB
76.06 MB
Maximum Sequence Length
15769
10479
Minimum Sequence Length
100
Average Sequence Length
No. of Sequences
62619
101284
Total Sequences Length
Total Number of Non-ATGC Characters
1316
1090
Percentage of Non-ATGC Characters

5. Gene function annotation
BLAST analyses: Similarities of Piper colubrinum and Piper nigrum transcriptome sequences against
different Plant species sequence database.
Target organism
Total Annotated transcripts
Piper colubrinum
(mRNA and protein)
Piper nigrum
(both the species)
Arabidopsis thaliana (thale cress)
14334
27998
42332
Brachypodium distachyon
217
293
510
Brassica napus (oilseed rape)
654
1128
1782
Carica papaya (papaya)
493
666
1159
Glycine max (soybean)
9511
19204
28715
Gossypium hirsutum (Cotton)
1576
2883
4459
Hordeum vulgare (barley)
1620
3142
4762
Lotus japonicus (lotus)
3267
6043
9310
Manihot esculenta (cassava)
403
600
1003
Medicago truncatula (barrel medic)
4653
9531
14184

6. BLAST analyses – continued………….
Target organism
Total Annotated transcripts
Piper colubrinum
(mRNA and protein)
Piper nigrum
(both the species)
Mimulus guttatus (Mimulus) 25 -
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

7. Piper Transcriptome- analysis in response to stress beta 1,3-glucanase
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

8. Piper Transcriptome- analysis alcohol dehydrogenase
(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

9. Genes involved in Secondary metabolism
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

10. transcription factors
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
mitogen-activated protein kinase (MAPK)
ras-related protein

11. Gene specific contigs/ transcripts identified from P. colubrinum &
P. nigrum transcriptome
Reference gene
Piper colubrinum
Piper nigrum
Contig length
Identity
Alignment length
E- Value
Aquaporin- Piper colubrinum
2094
99.63
267
2e-149
1229
82.65 98
1e-10
Osmotin - Piper colubrinum (IIIr)
297
99.35
155
4e-81
318
94.53
201
4e-87
betaine-aldehyde dehydrogenase - Glycine max
1698
77.27
726
3e-27
1726
77.61
603
2e-25
Cu/Zn superoxide dismutase- Gossypium hirsutum
865
88.29
401
2e-72
797
82.93
375
5e-64
Mitogen-activated protein kinase (MAPK)- Gossypium hirsutum
1798
77.89
995
3e-61
2854
78.35
485
3e-31
R gene fragment - Piper colubrinum
3008
98.76
242
1e-129 -
bZIP transcription factor- Oryza sativa
2053
76.62
633
7e-16
1797
78.65
342
3e-21
beta-1,3-glucanase-like gene- Piper colubrinum
1074
97.96
490
628
93.21
265
3e-108
1,3-glucanase-like mRNA, complete sequence- Piper colubrinum
373
95.76
165
4e-75
97.97
345
2e-180

12. Gene specific contigs/ transcripts – Continued
Reference gene
Piper colubrinum
Piper nigrum
Contig length
Identity
Alignment length
E- Value
Calmodulin- A.thaliana
1742
83.79
1497
3e-61
1801
82.93
375
1e-63
Catalase- A.thaliana
1576
78.83
1162
4e-94
1353
79.31
1020
8e-98
Geranylgeranyl transferase- A.thaliana
1124
79.9
398
3e-35
542
81.46
329
1e-39
Heat shock protein-70- A.thaliana
2401
79.48
1433
7e-146
1857
78.33
1269
1e-88
Malate dehydrogenase- A.thaliana
2304
77.95
1111
1e-70 -
WRKY
1620
97.92 96
8e-43
977
100 40
9e-17
Alpha amylase- Piper colubrinum
2206
98.24
227
6e-109
2721
94.95
198
5e-82
Betaine aldehyde dehydrogenase- Piper colubrinum
2595
98.82
254
4e-135
2482
86.92
237
3e-49

13. Transcription factor genes identified in the transcriptomes
Target species
No. of hits for transcription factor genes
Piper colubrinum
Arabidopsis thaliana
165
Populus trichocarpa 12
Vitis vinifera 8
Piper nigrum
142 21 9

14. Expression data for specific genes in the Piper colubrinum & P. nigrum
Gene of interest
Expression in
Piper colubrinum *
Piper nigrum*
ACC oxidase
7.57
0.00
PISTILLATA-like protein PI
13.33
APETALA3-like protein AP3-2
41.38
heat shock protein-70 cognate protein (ERD2)
104.85
3.88
Cinnamoyl CoA reductase
531.15
Alpha amylase
WRKY transcription factor
R gene related sequence (P. colubrinum)
Hydroxyproline-rich glycoprotein
Peroxidase
beta-1,3-glucanase
Aquaporin
Osmotin
394.81
Expression data for
specific genes
in the
Piper colubrinum
&
P. nigrum
transcriptome
*based on average read depth

15. Identification of Resistance Gene Analogues (RGAs) in the P
Identification of Resistance Gene Analogues (RGAs) in the P. colubrinum transcritome
S. No
Transcript No. (PN)
Length (bp)
Most similar R gene related sequence
coverage
E-value 1
Locus_22875
143
Vitis vinifera probable disease resistance protein At5g63020-like mRNA
94%
3e-10 2
Locus_51895
127
Brassica rapa subsp. pekinensis isolate BrCNL5 disease resistance protein gene, complete cds
99%
4e-10 3
Locus_14276
178
Vitis vinifera putative disease resistance RPP13-like protein 1 mRNA
79%
2e-07 4
Locus_19163
134
Vitis vinifera putative disease resistance protein mRNA
2e-08 5
Locus_34918
148 6
Locus_52055
183
Ipomoea batata isolate S1_C09 resistance gene analog genomic sequence
83%
6e-09 7
Locus_11301
1086
Populus trichocarpa NBS resistance protein, mRNA
38%
3e-51 8
Locus_48409
Solanum demissum isolate 286O21c disease resistance protein R3a-like protein pseudogene, complete sequence
97%
1e-10 9
Locus_53603
122
Medicago truncatula TIR-NBS-LRR type disease resistance protein mRNA, complete cds
91%
6e-12

16. Identification of SNPs
Piper colubrinum nos. & Piper nigrum nos of
SNPs Identified .

17. 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

18. Blast results : PSI-Blast

19. Conserved Domain Search

21. FingerPRINTScan

22. EXPRESSION OF OSMOTIN GENE IN
Piper colubrinum challenge inoculated with Phytophthora capsici.

23. EXPRESSION OF BETA 1,3 GLUCANASE GENE IN
Piper colubrinum challenge inoculated with Phytophthora capsici.

24. Expression analysis (real-time quantitative RT-PCR ) of R- gene from Piper colubrinum challenge inoculated with P. capsici
Relative expression

25. Expression analysis of wrky gene from Piper colubrinum challenge inoculated with P. capsici
Relative expression

26. O T H E R S
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.

27. Studies on allelic variations
FUTURE PROGRAMME
Studies on allelic variations
QRT-PCR (Quantitative RT-PCR) &  cDNA Microarrays
Functional validation of the genes: RNA silencing
Proteomics approaches

28. T H A N K S