A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis)

Steven G Ralph,Jörg Bohlmann,Natalia Kolosova,Sarah Barber,Dawn Cooper,Marco A Marra,Claire Oddy,Hye Jung E Chun,Robert A Holt,Carol E Ritland,Carl J Douglas,Kermit Ritland,Robert Kirkpatrick,Steven Jm Jones,Richard Moore

doi:10.1186/1471-2164-9-484

Abstract

BackgroundMembers of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation.ResultsAs part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs.ConclusionThis paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.

Highlights

Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests
Subsequent sequence reads from 5'-ends were performed as paired end reads, primarily from clones derived from FLcDNA libraries, to support the identification of a non-redundant FLcDNA set for complete insert sequencing
When we analyzed the 147,146 hq 3'-end expressed sequence tags (ESTs) using the CAP3 program ([44]; assembly criteria: 95% identity, 40 bp window), 120,342 ESTs assembled into 19,941 contigs and the remaining 26,804 ESTs were classified as singletons, suggesting a combined total of 46,745 putative unique transcripts (PUTs) across Sitka spruce, white spruce and interior spruce (Table 2)

Summary

Introduction

Members of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation. Conifers (members of the pine family) have very large genomes (10 to 40 Gb, [1]), and this poses difficulties for both structural and functional genomic studies Their generation times are long and their habitual out-breeding nature prevents the development of inbred strains useful for genetics research. Normalization techniques reduce the frequency of highly expressed genes and increase the rate of rare gene discovery [13,14], providing more comprehensive coverage of the expressed genome

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Conclusion