Huang, S.

Warkentin, T.

English

eCommons@USASK

Pea heat stress-responsive transcriptome analysis

University of Saskatchewan Research Archive

www.usask.caPea Heat Stress-Responsive Transcriptome AnalysisShaoming Huang， PhD CandidateSupervisor: Dr. Tom WarkentinWarming CanadaSource: Vincent et al (2012)  Environment Canada                         Saskatchewan Pulse GrowersNegative Yield Response to Higher Temperature at FloweringHuang, 2016. MSc thesis, University of Saskatchewan.y = -67.089x + 2166.3R² = 0.6531010020030040050060070080022 23 24 25 26 27Plot Yield (g/m2)Mean Daily Maximum Temperature (ºC)Pea Yield Response to Seasonal Temperature and PrecipitationBueckert et al. 2015. Can. J. Plant. Sci. 95, 629-639.Heat Stress on Pea • Shortens life cycle• Reduces pollen viability• Induces flower, ovule and pod abortion• Lowers individual seed weight  Jiang et al. 2015. Plant Cell Environ. 38, 2387-2397.Huang et al. 2017. Crop Sci. 57, 1540-1551.Most Heat Sensitive Flowering WindowJiang, 2016. Phd thesis, University of Saskatchewan.Discovery of Heat Responsive Genes via Transcriptome Profiling • Heat responsive transcriptome profiling via microarray has been conducted in A. thaliana; wheat; barley; canola.• The discovered genes are relating to heat shock protein, heat shock factor, reactive oxygen species etc.• The expression of heat responsive genes varies among different species and among different organs within a species.RNA-Seq in Pea – Reports so farRoche 454 based RNA-Seq• First transcriptome atlas (Franssen et al., 2011)• Deep mining of SSR and SNP markers via transcriptome sequencing (Kaur et al., 2012; Duarte et al., 2014)Illumina based RNA-Seq• First de novo assembly of pea transcriptome sequencing data derived from Illumina platform (Sudheesh et al., 2015)• Characterization of nodule transcriptome (Alves-Carvalho et al., 2015)Objectives1. Profile pea leaf and anther responses to high temperature at the transcription level via Illumina based RNA-seq2. Compare the heat responsive gene expression between a heat tolerant and a heat sensitive pea variety3. Build a heat responsive gene expression atlas in peaExperiment ProtocolSequence Data AnalysisDe novo assembly 1. Assembly first method (softwares e.g. Oases, SOAPdenovo)2. Bruijin graph method (softwares e.g. Trinity, Velvet)Functional annotation of assembled contigsBLASTN (threshold E-value <10-10) against published pea transcript sequences as well as NCBI database of M. truncatula, chickpea and soybean  Plant MaterialTwo pairs of heat tolerant and sensitive pea genotypesHeat Tolerant:       CDC Meadow                         PR11-2VS                                        VSHeat Sensitive:         Nitouche PR11-90Preliminary Validation based on RT-PCR of two genes encoding PsHsp 18 and 70▪ Seeds planting ▪ Temperature treatment ▪ Tissue grinding▪ RNA extraction ▪ cDNA synthesis▪ RT-PCR (target genes PsHsp 18 & 70)RCBD (4 genotypes X 4 time points X 2 temperature treatment X 3 reps=96 pots)High temperature (38 /16 C, 16/8 h) vs normal temperature (22/16 C, 16/8 h)Time points:3h, 6h, 12h, 24hTissues: leaf and antherExperimental DesignPhytotron chamber, University of Saskatchewan Some Symptoms of Heat Stress38C              12h                                             24h                                        24h Preliminary Validation based on RT-PCR of two genes encoding PsHsp 18 and 70▪ Seeds planting ▪ Temperature treatment ▪ Tissue grinding▪ RNA extraction ▪ cDNA synthesis▪ RT-PCR (target genes PsHsp 18 & 70)Data AnalysisCt Value: threshold cycleFold Change Formula: 2 (-ΔCt) = 2 -(Ct gene of interest - Ct reference gene)ΔCt = Ct gene of interest – Ct reference geneOutlier Data Check:Q1-1.5*(Q3-Q1) ≤ data ≤ Q3+1.5*(Q3-Q1)Results1. Selection of Reference GeneGH720838 encodes transcription factor IIA 2. Good Data Consistency between Technical RepsAnther Sample Leaf SampleAverage Ct Value 19.44±0.63 20.65±0.66CV (100%) 3.31 3.25Sample Rep1 Rep2 Differential Significant LevelAnther 19.47±0.61 19.41±0.66 0.06 0.56Leaf 20.59±0.73 20.72±0.58 0.13 0.29Results▪ 3. Relative Low Expression of Hsp Genes on Non-heat Stressed PlantLeaf sample Anther sampleGenotype Hsp18 gene Hsp70 gene Hsp18 gene Hsp70 genePR11-2 0.18 0.07 0.41 0.16PR11-90 0.11 0.05 0.26 0.05CDC Meadow 0.19 0.05 0.25 0.07Nitouche 0.30 0.12 0.41 0.11Mean 0.20 0.07 0.33 0.10Values are fold change, which are described as 2 (-ΔCt) = 2 -(Ct gene of interest - Ct reference gene)Results4. Up-regulation of Hsp Genes under High TemperatureLeaf sample Anther sampleTime points Hsp18 gene Hsp70 gene Hsp18 gene Hsp70 gene0h 0.20±0.11c 0.07±0.05c 0.33±0.11d 0.10±0.08c3h 396.07±30.16a 154.68±12.84a 76.88±6.18a 31.77±3.06a6h 130.94±30.51b 33.95±13.92b 58.43±6.18bc 24.20±2.98ab12h 113.85±30.51b 30.60±13.62b 69.36±6.18ab 24.08±3.06ab24h 77.91±31.18b 28.12±13.92b 47.79±7.05c 19.43±3.34bValues are fold change, which are described as 2 (-ΔCt) = 2 -(Ct gene of interest - Ct reference gene)Results050100150200250300350400PR11-2 PR11-90 CDC Meadow NitoucheAnther Hsp18 Gene Expression Fold Change3H 6H 12H 24H0100200300400500600700PR11-2 PR11-90 CDC Meadow NitoucheAnther Hsp70 Gene Expression Fold Change3H 6H 12H 24H05001000150020002500PR11-2 PR11-90 CDC Meadow NitoucheLeaf Hsp18 Gene Expression Fold Change3H 6H 12H 24H05001000150020002500PR11-2 PR11-90 CDC Meadow NitoucheLeaf Hsp70 Gene Expression Fold Change 3H 6H 12H 24HSummary1. For leaf sample, 3-hour is most responsive timing; for anther sample, the most responsive timing seems to be genotype specific. In general, the expression of hsp genes are most up-regulated after 3 hours at 38C.2. For both genes, the relative gene expression differential between PR11-2 & PR11-90 is bigger than the differential between CDC Meadow & Nitouche.RNA-Seq Experiment Underway 3hAcknowledgement▪ Supervisor: Dr. Tom Warkentin▪ Committee: Dr. Rosalind Bueckert, Dr. Bunyamin Tar’an, Dr. Art Davis, and Dr. Yuguang Bai▪ Colleagues: Dr. Kishore Gali, Dr. Amit Deokar, Mandeep Sagi and Mohammad Rezaei▪ Funding

https://harvest.usask.ca/bitstream/10388/8706/1/S.%20Huang%20and%20T.%20Warkentin%2c%2020181.pdf

Pea heat stress-responsive transcriptome analysis

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