Piper colubrinum & Piper nigrum Plants challenged with Phytophthora capsici Piper nigrum Piper colubrinum Technology Next Generation Sequencing (Genome

  • View
    229

  • Download
    5

Embed Size (px)

Text of Piper colubrinum & Piper nigrum Plants challenged with Phytophthora capsici Piper nigrum Piper...

  • Piper colubrinum & Piper nigrum Plants challenged with Phytophthora capsici

    Piper nigrum Piper colubrinum TechnologyNext Generation Sequencing(Genome Analyzer IIx Illumina)

  • Sequence Reads(QC and Raw data processing- SeqQC-V2.1)Denovo assembly(Velvet_1.1.04)Assembled contigsAnnotated Transcripts(Oases)Advanced Analysis

  • Summary of NGS DATA

    Summary statisticsSamplePiper colubrinumSamplePiper nigrum Sequence File Size37.70 MB76.06 MBMaximum Sequence Length1576910479Minimum Sequence Length100100Average Sequence Length567.844721.922No. of Sequences62619101284Total Sequences Length3555787573119148Total Number of Non-ATGC Characters13161090Percentage of Non-ATGC Characters0.000040.00001

  • BLAST analyses: Similarities of Piper colubrinum and Piper nigrum transcriptome sequences against different Plant species sequence database.

    Target organismTotal Annotated transcripts Piper colubrinum(mRNA and protein)Total Annotated transcriptsPiper nigrum(mRNA and protein)Total Annotated transcripts (both the species) Arabidopsis thaliana (thale cress)143342799842332Brachypodium distachyon217293510Brassica napus (oilseed rape)65411281782Carica papaya (papaya)4936661159Glycine max (soybean)95111920428715Gossypium hirsutum (Cotton)157628834459Hordeum vulgare (barley)162031424762Lotus japonicus (lotus)326760439310Manihot esculenta (cassava)4036001003Medicago truncatula (barrel medic)4653953114184

  • BLAST analyses continued.

    Target organismTotal Annotated transcripts Piper colubrinum(mRNA and protein)Total Annotated transcriptsPiper nigrum(mRNA and protein)Total Annotated transcripts (both the species)Mimulus guttatus (Mimulus)25-25Oryza sativa (rice)3981738911370Persea americana187304491Physcomitrella patens (Moss)76413381902Phytophthora Sp.73814322170Piper sp.152117269Populus trichocarpa (Poplar)175583377451332Ricinus communis (Ricinus)4033241832821Solanum lycopersicum (tomato)78551536723222Sorghum bicolor (Sorghum)133392508238421Triticum aestivum (Wheat)62941214918443Vitis vinifera (wine grape)195153710556620Zea mays (corn)126622048633148

  • Piper Transcriptome- analysis ESTs IDENTIFIED

    Genes in response to stressbeta 1,3-glucanaseCatalaseChitinases cinnamoyl CoA reductasecinnamyl alcohol dehydrogenaseCu/Zn superoxide dismutasedehydration-induced proteins (dehydrins) delta 1-pyrroline-5-carboxylate synthetasegalactinol synthaseglutathione peroxidaseglutathione reductaseGlutathione s-transferasethaumatin-like proteinpapain-like cysteine proteinase-like proteinperoxisomal betaine-aldehyde dehydrogenasepolygalacturonase inhibiting protein- pgip1

  • Piper Transcriptome- analysis (ESTs IDENTIFIED )

    Genes involved in Metabolic process

    alcohol dehydrogenasealkaline alpha galactosidasebeta-ketoacyl-CoA synthasecitrate synthaseferredoxin-nitrite reductaseisopropyl malate isomerase large subunitglutamine synthetaselipoxygenase (loxA)malate dehydrogenasenicotianamine synthaseomega-3 fatty acid desaturasePhosphoenolpyruvate carboxylasepyruvate 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 dehydrogenasecinnamic acid 4-hydroxylasecaffeoyl CoA O-methyltransferasechalcone synthase (CHS) & Chalcone isomerase Cinnamate 4-hydroxylase (C4H)flavanone 3-hydroxylaseoxidoreductase protein - geranyl geranyl pyrophosphateglucose-6-phosphate 1-dehydrogenaseHMG-CoA reductaseisopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomeraseS-adenosyl-L-methionine synthetaselycopene beta cyclaseO-methyltransferasephenylalanine ammonia lyase

  • Piper Transcriptome analysis ESTs IDENTIFIED

    Other Genes identified

    transcription factorsbHLH transcription factorbZIP transcription factorDRE-binding proteinethylene-responsive transcription factor 2GAGA-binding transcriptional activatorhomeobox transcription factorMYB transcription factormolecular chaperonesChaperone DnaKDNAJ heat shock N-terminal domain-containing proteinheat shock protein 70 & 82peptidyl-prolyl cis-trans isomerase

    Involved insignal transductionlectin protein kinase family proteinPto kinase interactorlectin protein kinase family proteinmitogen-activated protein kinase (MAPK)ras-related protein

  • Gene specific contigs/ transcripts identified from P. colubrinum & P. nigrum transcriptome

    Reference genePiper colubrinumPiper nigrumContig lengthIdentityAlignment lengthE- ValueContig lengthIdentityAlignment lengthE- ValueAquaporin- Piper colubrinum209499.632672e-149122982.65981e-10Osmotin - Piper colubrinum (IIIr)29799.351554e-8131894.532014e-87betaine-aldehyde dehydrogenase - Glycine max169877.277263e-27172677.616032e-25Cu/Zn superoxide dismutase- Gossypium hirsutum86588.294012e-7279782.933755e-64Mitogen-activated protein kinase (MAPK)- Gossypium hirsutum179877.899953e-61285478.354853e-31R gene fragment - Piper colubrinum300898.762421e-129-bZIP transcription factor- Oryza sativa205376.626337e-16179778.653423e-21beta-1,3-glucanase-like gene- Piper colubrinum107497.96490062893.212653e-1081,3-glucanase-like mRNA, complete sequence- Piper colubrinum37395.761654e-7562897.973452e-180

  • Gene specific contigs/ transcripts Continued

    Reference genePiper colubrinumPiper nigrumContig lengthIdentityAlignment lengthE- ValueContig lengthIdentityAlignment lengthE- ValueCalmodulin- A.thaliana174283.7914973e-61180182.933751e-63Catalase- A.thaliana157678.8311624e-94135379.3110208e-98Geranylgeranyl transferase- A.thaliana112479.93983e-3554281.463291e-39Heat shock protein-70- A.thaliana240179.4814337e-146185778.3312691e-88Malate dehydrogenase- A.thaliana230477.9511111e-70-WRKY162097.92968e-43977100409e-17Alpha amylase- Piper colubrinum220698.242276e-109272194.951985e-82Betaine aldehyde dehydrogenase- Piper colubrinum259598.822544e-135248286.922373e-49

  • Transcription factor genes identified in the transcriptomes

    TranscriptomeTarget species No. of hits for transcription factor genesPiper colubrinumArabidopsis thaliana 165Populus trichocarpa12Vitis vinifera8Piper nigrumArabidopsis thaliana 142Populus trichocarpa21Vitis vinifera9

  • *based on average read depthExpression data for specific genes in the Piper colubrinum &P. nigrum transcriptome

    Gene of interest Expression in Piper colubrinum *Expression in Piper nigrum* ACC oxidase 7.570.00PISTILLATA-like protein PI 13.330.00APETALA3-like protein AP3-2 41.380.00heat shock protein-70 cognate protein (ERD2) 104.853.88Cinnamoyl CoA reductase 531.1537020.49Alpha amylase 2558.755379.58WRKY transcription factor5197.9014193.71R gene related sequence (P. colubrinum)6161.330.00Hydroxyproline-rich glycoprotein 11175.0726616.99Peroxidase 30732.80183601.93beta-1,3-glucanase 66399.502825.04Aquaporin 75066.676042.32Osmotin 2016108.51394.81

  • Identification of Resistance Gene Analogues (RGAs) in the P. colubrinum transcritome

    S. NoTranscript No. (PN)Length (bp)Most similar R gene related sequencecoverageE-value1Locus_22875143Vitis vinifera probable disease resistance protein At5g63020-like mRNA94%3e-102Locus_51895127Brassica rapa subsp. pekinensis isolate BrCNL5 disease resistance protein gene, complete cds99% 4e-103Locus_14276178Vitis vinifera putative disease resistance RPP13-like protein 1 mRNA79% 2e-074Locus_19163134Vitis vinifera putative disease resistance protein mRNA99%2e-085Locus_34918148Vitis vinifera putative disease resistance protein mRNA99%2e-086Locus_52055183Ipomoea batata isolate S1_C09 resistance gene analog genomic sequence83% 6e-097Locus_113011086Populus trichocarpa NBS resistance protein, mRNA38% 3e-518Locus_48409127Solanum demissum isolate 286O21c disease resistance protein R3a-like protein pseudogene, complete sequence97% 1e-109Locus_53603122Medicago truncatula TIR-NBS-LRR type disease resistance protein mRNA, complete cds91% 6e-12

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

  • Steps followed

  • Blast results : PSI-Blast

  • Conserved Domain Search

  • FingerPRINTScan

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

    Chart1

    11control

    1794hours

    11248 hours

    192516 hours

    1150024 hours

    1110048 hours

    15672 hours

    copy numbers

    control

    treatments

    Series 3

    Sheet1

    controltreatmentsSeries 3treatments

    control112

    4hours179279

    8 hours11243124

    16 hours19255

    24 hours11500

    48 hours11100

    72 hours156

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

    Chart1

    11control

    174hours

    1408hours

    17016hours

    129624hours

    196848hours

    12772hours

    copy numbers

    control

    treatments

    Sheet1

    controltreatmentsSeries 3

    control112

    4hours172

    8hours1403

    16hours1705

    24hours1296

    48hours1968

    72hours127

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

    Chart1

    11controlcontrol

    3412hour2hour

    1514hour4hour

    1218hour8hour

    5116 hour16 hour

    10124 hour24 hour

    10148 hour48 hour

    3172 hour72 hour

    Treatments

    control

    Series 3

    Series 4

    Sheet1

    TreatmentscontrolSeries 3Series 4

    control112

    2hour3412

    4hour1513

    8hour1215

    16 hour51

    24 hour101

    48 hour101

    72 hour31