Gene Expression in Conifers Revealed on a Comprehensive Scale: Sequencing, Assembly and Classification of the Loblolly Pine Transcriptome

Gymnosperms lack rigorous genomic exploration relative to the widely studied angiosperms, in part because enormous genome sizes have precluded complete sequencing.  In the past year this challenge was overcome with the completion of three conifer genome reference sequences, each over 20 Gb. In these, the number of apparent genic loci is remarkably high, with heterozygosity and pseudogenization further complicating the annotation of transcriptionally active loci. 

Here we present comprehensive transcriptiome sequencing and assembly of loblolly pine, as an essential component of full genome functional annotation.  We utilized all leading RNA reads assembly software applications in parallel, as well as variable inputs, to produce an extensive assembly set whose members were then classified according to protein coding metrics, redundancy, and predicted alternative splicing.  1.1 M transcripts from over 2 billion reads of heterozygous samplings were retained in the broadest representation set, with 146K classified as derived from unique primary loci of expression.  Datasets contributing to this overall transcriptome represented subsets of collections designed for focused experiments, including organ-specificity and stress-responsiveness, each of which revealed deep functional conservation in some genes previously speculated to differ substantially between gymnosperms and angiosperms.  Careful phylogenetic analyses of large gene families demonstrate transcriptome quality and completeness finally available for thorough functional genomics studies in conifers.  We furthermore provide comparative assessments of these RNA assemblies with respect to mapping in the current Pinus taeda genome assembly, and discuss expressed genic variation within this species, whose natural range extends from the mid Atlantic coast to East Texas.