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Transcriptome sequencing - a case study in Piper

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.

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Transcriptome sequencing - a case study in Piper

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  1. Transcriptome sequencing - a case study in Piper JOHNSON GEORGE K

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

  3. Transcriptome Assembly and Analysis Workflow Sequence Reads (QC and Raw data processing- SeqQC-V2.1) Denovo assembly (Velvet_1.1.04) Assembled contigs Annotated Transcripts (Oases) Advanced Analysis

  4. Summary of NGS DATA

  5. Gene function annotation BLAST analyses: Similarities of Piper colubrinum and Piper nigrum transcriptome sequences against different Plant species sequence database.

  6. BLAST analyses – continued………….

  7. Piper Transcriptome- analysis ESTs IDENTIFIED

  8. Piper Transcriptome- analysis (ESTs IDENTIFIED )

  9. Piper Transcriptome- analysis (ESTs IDENTIFIED )

  10. Piper Transcriptome analysis ESTs IDENTIFIED

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

  12. Gene specific contigs/ transcripts – Continued

  13. Transcription factor genes identified in the transcriptomes

  14. Expression data for specific genes in the Piper colubrinum & P. nigrum transcriptome *based on average read depth

  15. Identification of Resistance Gene Analogues (RGAs) in the P. colubrinumtranscritome

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

  17. Steps followed

  18. Blast results : PSI-Blast

  19. Conserved Domain Search

  20. FingerPRINTScan

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

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

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

  24. Expression analysisof wrky gene from Piper colubrinum challenge inoculated with P. capsici Relative expression

  25. 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.

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

  27. T H A N K S

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