Gene Expression in Loblolly Pine Early Development - PowerPoint PPT Presentation

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Gene Expression in Loblolly Pine Early Development

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  1. Gene Expression in Loblolly Pine Early Development KeithanneMockaitis Carol Loopstra Indiana University Center for Genomics and Bioinformatics Texas A & M University

  2. Progressive Transcript Profiling Build a useful transcriptome reference early in project:  generate long reads for ease of assembly, scaffolding of existing shorter data  integrate community data into assemblies Early Development seeds young seedlings Reproductive Development megastrobili microstrobili Early Stress Signaling Responses cold heat elevated UV compression Vegetative Organs vegetative buds candles stems needles roots

  3. Sequencing of Early Development Collections, Stage 1 embryos dissected from germinating seeds seeds immediately after stratification megagametophytes dissected from germinating seeds Lib 1 Lib 2 cDNA libraries optimized for 454 sequencing, partially normalized GS – XLR Plus

  4. Sequence reads length distribution of libraries megagametophyte pool seed/embryo pool

  5. Data Assembled

  6. Coverage of Assembled Transcripts > 1 kb average coverage length

  7. Estimated Gene Discovery Transcripts with no blastx hit to NCBI dbEST: 2,173 Transcripts with blastx hit to NCBI dbEST: 49,386 Hits not to Pinus genus: 6,322 Hits not to gymnosperm: 653 ~2000 selected Pinus transcripts Hits to Pinus transcripts in dbEST: 43,064 Most transcripts from new assembly contribute substantial length to older data length

  8. Estimated Maternal Expression Full Assembly Isogroups: 24,688 MegagametophyteIsogroups Mapped (>80% length, 98% id): 12,478 (51%) Homology Estimation Fully Assembly Transcripts (Isotigs): 51,513 Transcripts with significant blastx hit to TAIR10: 41,187 (80%) Unique: 12,233 Transcripts with significant blastx hit to Populustrichocarpav2: 41,291 Unique: 12,768 Unique OrthoMCL groups represented: 7,075 Paralog Groups: 5,362

  9. Most Highly Represented Gene Families OrthoMCL: Li et al., 2003 Genome Res. 13, 2178

  10. Many expected transcripts are well covered RAD51 98.5% KU80 99.4% DNA ligase IV 67.3% TatDDNAse 63.9% MCA 100% Vuoskuet al., 2009 J Exp Bot 60, 1375

  11. Progressive Transcript Profiling Build a useful transcriptome reference early in project:  generate long reads for ease of assembly, scaffolding of existing shorter data  integrate community data into assemblies  generate deeper stage-specific sequencing of samples within original pools, additional collections  attribute source specificities through comparative mapping  refine assemblies of alternatively spliced transcripts Early Development, Stage 2 seeds embryos from 20-1010 seedlings young tissues, stages from 20-1010

  12. Progressive Transcript Profiling Reproductive Development megastrobili: 4 stages microstrobili: 4 stages

  13. Thanks Texas A & M Jeff Puryear IU CGB James Ford Zach Smith Aaron Buechlein