Piper colubrinum & Piper nigrum Plants challenged with

Phytophthora capsici

Piper nigrum

Piper colubrinum

Technology

Next Generation Sequencing(Genome Analyzer IIx – Illumina)

Tran

scrip

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e As

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Anal

ysis

W

orkfl

owSequence 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 SamplePiper colubrinum

SamplePiper nigrum

Sequence File Size 37.70 MB 76.06 MBMaximum Sequence Length 15769 10479Minimum Sequence Length 100 100Average Sequence Length 567.844 721.922No. of Sequences 62619 101284Total Sequences Length 35557875 73119148Total Number of Non-ATGC Characters 1316 1090Percentage of Non-ATGC Characters 0.00004 0.00001

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)

Total Annotated transcriptsPiper nigrum(mRNA and protein)

Total Annotated transcripts (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

Target organism Total Annotated transcripts Piper colubrinum(mRNA and protein)

Total Annotated transcriptsPiper nigrum(mRNA and protein)

Total Annotated transcripts (both the species)

Mimulus guttatus (Mimulus) 25 - 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

BLAST analyses – continued………….

Piper Transcriptome- analysis ESTs IDENTIFIED

Genes

in response to stressbeta 1,3-glucanase

CatalaseChitinases

cinnamoyl CoA reductasecinnamyl alcohol dehydrogenase

Cu/Zn superoxide dismutasedehydration-induced proteins (dehydrins) delta 1-pyrroline-5-carboxylate synthetase

galactinol synthaseglutathione peroxidaseglutathione reductase

Glutathione s-transferasethaumatin-like protein

papain-like cysteine proteinase-like proteinperoxisomal betaine-aldehyde dehydrogenase

polygalacturonase inhibiting protein- pgip1

Piper Transcriptome- analysis (ESTs IDENTIFIED )

Genes involved in Metabolic process

alcohol dehydrogenasealkaline alpha galactosidasebeta-ketoacyl-CoA synthase

citrate synthaseferredoxin-nitrite reductase

isopropyl malate isomerase large subunitglutamine synthetaselipoxygenase (loxA)

malate dehydrogenasenicotianamine synthase

omega-3 fatty acid desaturasePhosphoenolpyruvate carboxylase

pyruvate decarboxylasepyruvate 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 synthase6-phosphogluconate dehydrogenase

cinnamic acid 4-hydroxylasecaffeoyl CoA O-methyltransferase

chalcone synthase (CHS) & Chalcone isomerase Cinnamate 4-hydroxylase (C4H)

flavanone 3-hydroxylaseoxidoreductase protein -

geranyl geranyl pyrophosphateglucose-6-phosphate 1-dehydrogenase

HMG-CoA reductaseisopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase

S-adenosyl-L-methionine synthetaselycopene beta cyclaseO-methyltransferase

phenylalanine ammonia lyase

Piper Transcriptome analysis ESTs IDENTIFIED

Other Genes identified

transcription factorsbHLH transcription factorbZIP transcription factor

DRE-binding proteinethylene-responsive transcription factor 2

GAGA-binding transcriptional activatorhomeobox transcription factor

MYB transcription factor

molecular chaperonesChaperone DnaK

DNAJ heat shock N-terminal domain-containing proteinheat shock protein 70 & 82

peptidyl-prolyl cis-trans isomerase

Involved in

signal transductionlectin protein kinase family protein

Pto kinase interactorlectin 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 Piper colubrinum Piper nigrumContig length

Identity Alignment length

E- Value 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 0 628 93.21 265 3e-108

1,3-glucanase-like mRNA, complete sequence- Piper colubrinum

373 95.76 165 4e-75 628 97.97 345 2e-180

Reference gene Piper colubrinum Piper nigrumContig length

Identity

Alignment length

E- Value

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

Gene specific contigs/ transcripts – Continued

Transcriptome Target species No. of hits for transcription factor genes

Piper colubrinum Arabidopsis thaliana 165

Populus trichocarpa 12

Vitis vinifera 8

Piper nigrum Arabidopsis thaliana 142

Populus trichocarpa 21

Vitis vinifera 9

Transcription factor genes identified in the transcriptomes

Gene of interest 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 41.38 0.00heat shock protein-70 cognate protein (ERD2) 104.85 3.88

Cinnamoyl CoA reductase 531.15 37020.49

Alpha amylase 2558.75 5379.58

WRKY transcription factor 5197.90 14193.71R gene related sequence (P. colubrinum) 6161.33 0.00Hydroxyproline-rich glycoprotein 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

Expression data for specific genes in the Piper colubrinum &P. nigrum transcriptome

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

99% 2e-08

5 Locus_34918 148 Vitis vinifera putative disease resistance protein mRNA

99% 2e-08

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 127 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

Identification of SNPs

Piper colubrinum - 50972 nos. & Piper nigrum -231680 nos of SNPs Identified

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Steps followed

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

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

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.

Studies on allelic variationsQRT-PCR (Quantitative RT-PCR) &  cDNA Microarrays

Functional validation of the genes: RNA silencingProteomics approaches