Abstract
BackgroundMassively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies.ResultsWe obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening.ConclusionsThis sequence collection represents a major genomic resource for P. contorta, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.
Highlights
Parallel sequencing of cDNA is an efficient route for generating enormous sequence collections that represent expressed genes
454 sequencing and assembly We created a normalized cDNA pool based on RNA extracted from needles and developing conelets that were sampled from four individual P. contorta trees in the Medicine Bow National Forest in Wyoming
A large number of sequences (10.4%) were most similar to fungal proteins (Table 3), likely indicating the presence of endophytic fungi in our sampled tissues. This seemingly low percentage of Expressed Sequence Tag (EST) with BLAST hits is partially due to a high frequency of short sequences in our ESTs, annotation of only 30-40% of sequences is common in analyses of large EST collections [5,16,38]
Summary
Parallel sequencing of cDNA is an efficient route for generating enormous sequence collections that represent expressed genes This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. Transcriptome, or Expressed Sequence Tag (EST), sequencing is an efficient means to generate functional genomic level data for non-model organisms or those with genome characteristics prohibitive to whole genome sequencing. Large collections of EST sequences have proven invaluable for gene annotation and discovery [2,4], comparative genomics [5], development of molecular markers [6,7], and for population genomic studies of genetic variation associated with adaptive traits [8]. Until recently, traditional laboratory methods for the development of EST resources have required costly and time consuming approaches involving cloning, cDNA library construction, and many labor intensive Sanger sequencing runs [2]
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