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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. - PowerPoint PPT Presentation
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Piper colubrinum & Piper nigrum Plants challenged with
Phytophthora capsici
Piper nigrum
Piper colubrinum
Technology
Next Generation Sequencing(Genome Analyzer IIx – Illumina)
Tran
scrip
tom
e As
sem
bly
and
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
.
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
Re
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exp
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Re
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exp
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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
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