Slide 1

Gene Expression in Loblolly Pine Early DevelopmentKeithanne MockaitisCarol LoopstraIndiana UniversityCenter for Genomics and BioinformaticsTexas A & M University

http://pinegenome.org/pinerefseq/Progressive Transcript ProfilingBuild a useful transcriptome reference early in project: generate long reads for ease of assembly, scaffolding of existing shorter data integrate community data into assemblies

Vegetative Organsvegetative budscandlesstemsneedlesroots

Early Stress Signaling Responsescoldheatelevated UVcompressionReproductive DevelopmentmegastrobilimicrostrobiliEarly Developmentseedsyoung seedlings

http://pinegenome.org/pinerefseq/Sequencing of Early Development Collections, Stage 1

embryos dissected from germinating seedsseeds immediately after stratificationmegagametophytes dissected from germinating seedsLib 1Lib 2cDNA libraries optimized for 454 sequencing, partially normalized GS XLR Plus http://pinegenome.org/pinerefseq/Sequence reads length distribution of libraries

seed/embryo pool megagametophyte pool http://pinegenome.org/pinerefseq/Data Assembled

http://pinegenome.org/pinerefseq/Coverage of Assembled Transcripts > 1 kb average coveragelengthhttp://pinegenome.org/pinerefseq/Transcripts with no blastx hit to NCBI dbEST: 2,173Transcripts with blastx hit to NCBI dbEST: 49,386Hits not to Pinus genus: 6,322Hits not to gymnosperm: 653

Hits to Pinus transcripts in dbEST: 43,064

Most transcripts from new assembly contribute substantial length to older data~2000 selected Pinus transcriptslengthEstimated Gene Discovery

http://pinegenome.org/pinerefseq/Estimated Maternal ExpressionFull Assembly Isogroups: 24,688Megagametophyte Isogroups Mapped (>80% length, 98% id): 12,478 (51%)

Homology EstimationFully Assembly Transcripts (Isotigs): 51,513Transcripts with significant blastx hit to TAIR10: 41,187 (80%) Unique: 12,233Transcripts with significant blastx hit to Populus trichocarpa v2: 41,291 Unique: 12,768

Unique OrthoMCL groups represented: 7,075Paralog Groups: 5,362

http://pinegenome.org/pinerefseq/Most Highly Represented Gene FamiliesOrtholgous Groupprotein family/superfamilymembers 126569, 126570, 126572, 126573, 126767, 127916histone80135110, 164369PPR or TPR containing72 126935heat shock37127136LRR transmembrane protein kinase33 126574ABC transporter28 136634transducin family, WD40 repeat containing 21 126615plasma membrane intrinsic18OrthoMCL: Li et al., 2003 Genome Res. 13, 2178http://pinegenome.org/pinerefseq/Many expected transcripts are well covered

Vuosku et al., 2009 J Exp Bot 60, 1375RAD5198.5%KU8099.4%DNA ligase IV67.3%TatD DNAse63.9%MCA100%http://pinegenome.org/pinerefseq/Progressive Transcript ProfilingEarly Development, Stage 2seeds embryos from 20-1010seedlings young tissues, stages from 20-1010

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 http://pinegenome.org/pinerefseq/Progressive Transcript ProfilingReproductive Developmentmegastrobili: 4 stagesmicrostrobili: 4 stages

http://pinegenome.org/pinerefseq/

ThanksIU CGB

James FordZach SmithAaron BuechleinTexas A & M

Jeff Puryearhttp://pinegenome.org/pinerefseq/