Transcriptome sequencing - a case study in Piper JOHNSON GEORGE K

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

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

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 567.844 721.922 No. of Sequences 62619 101284 Total Sequences Length 35557875 73119148 Total Number of Non-ATGC Characters 1316 1090 Percentage of Non-ATGC Characters 0.00004 0.00001

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

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

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

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

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

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

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

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

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

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 37020.49 Alpha amylase 2558.75 5379.58 WRKY transcription factor 5197.90 14193.71 R gene related sequence (P. colubrinum) 6161.33 Hydroxyproline-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 Expression data for specific genes in the Piper colubrinum & P. nigrum transcriptome *based on average read depth

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

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

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

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