Transcription through the eye of a needle: daily and annual cyclic gene expression variation in Douglas-fir needles.

BACKGROUND: Perennial growth in plants is the product of interdependent cycles of daily and annual stimuli that induce cycles of growth and dormancy. In conifers, needles are the key perennial organ that integrates daily and seasonal signals from light, temperature, and water availability. To understand the relationship between seasonal cycles and seasonal gene expression responses in conifers, we examined diurnal and circannual needle mRNA accumulation in Douglas-fir (Pseudotsuga menziesii) needles at diurnal and circannual scales. Using mRNA sequencing, we sampled 6.1 × 109 reads from 19 trees and constructed a de novo pan-transcriptome reference that includes 173,882 tree-derived transcripts. Using this reference, we mapped RNA-Seq reads from 179 samples that capture daily and annual variation. RESULTS: We identified 12,042 diurnally-cyclic transcripts, 9299 of which showed homology to annotated genes from other plant genomes, including angiosperm core clock genes. Annual analysis revealed 21,225 circannual transcripts, 17,335 of which showed homology to annotated genes from other plant genomes. The timing of maximum gene expression is associated with light intensity at diurnal scales and photoperiod at annual scales, with approximately half of transcripts reaching maximum expression +/- 2 h from sunrise and sunset, and +/- 20 days from winter and summer solstices. Comparisons with published studies from other conifers shows congruent behavior in clock genes with Japanese cedar (Cryptomeria), and a significant preservation of gene expression patterns for 2278 putative orthologs from Douglas-fir during the summer growing season, and 760 putative orthologs from spruce (Picea) during the transition from fall to winter. CONCLUSIONS: Our study highlight the extensive diurnal and circannual transcriptome variability demonstrated in conifer needles. At these temporal scales, 29% of expressed transcripts show a significant diurnal cycle, and 58.7% show a significant circannual cycle. Remarkably, thousands of genes reach their annual peak activity during winter dormancy. Our study establishes the fine-scale timing of daily and annual maximum gene expression for diverse needle genes in Douglas-fir, and it highlights the potential for using this information for evaluating hypotheses concerning the daily or seasonal timing of gene activity in temperate-zone conifers, and for identifying cyclic transcriptome components in other conifer species.

Variation in base-substitution mutation in experimental and natural lineages of Caenorhabditis nematodes.

Variation among lineages in the mutation process has the potential to impact diverse biological processes ranging from susceptibilities to genetic disease to the mode and tempo of molecular evolution. The combination of high-throughput DNA sequencing (HTS) with mutation-accumulation (MA) experiments has provided a powerful approach to genome-wide mutation analysis, though insights into mutational variation have been limited by the vast evolutionary distances among the few species analyzed. We performed a HTS analysis of MA lines derived from four Caenorhabditis nematode natural genotypes: C. elegans N2 and PB306 and C. briggsae HK104 and PB800. Total mutation rates did not differ among the four sets of MA lines. A mutational bias toward G:C→A:T transitions and G:C→T:A transversions was observed in all four sets of MA lines. Chromosome-specific rates were mostly stable, though there was some evidence for a slightly elevated X chromosome mutation rate in PB306. Rates were homogeneous among functional coding sequence types and across autosomal cores, arms, and tips. Mutation spectra were similar among the four MA line sets but differed significantly when compared with patterns of natural base-substitution polymorphism for 13/14 comparisons performed. Our findings show that base-substitution mutation processes in these closely related animal lineages are mostly stable but differ from natural polymorphism patterns in these two species.

A genome-wide view of Caenorhabditis elegans base-substitution mutation processes.

Knowledge of mutation processes is central to understanding virtually all evolutionary phenomena and the underlying nature of genetic disorders and cancers. However, the limitations of standard molecular mutation detection methods have historically precluded a genome-wide understanding of mutation rates and spectra in the nuclear genomes of multicellular organisms. We applied two high-throughput DNA sequencing technologies to identify and characterize hundreds of spontaneously arising base-substitution mutations in 10 Caenorhabditis elegans mutation-accumulation (MA)-line nuclear genomes. C. elegans mutation rate estimates were similar to previous calculations based on smaller numbers of mutations. Mutations were distributed uniformly within and among chromosomes and were not associated with recombination rate variation in the MA lines, suggesting that intragenomic variation in genetic hitchhiking and/or background selection are primarily responsible for the chromosomal distribution patterns of polymorphic nucleotides in C. elegans natural populations. A strong mutational bias from G/C to A/T nucleotides was detected in the MA lines, implicating oxidative DNA damage as a major endogenous mutagenic force in C. elegans. The observed mutational bias also suggests that the C. elegans nuclear genome cannot be at equilibrium because of mutation alone. Transversions dominate the spectrum of spontaneous mutations observed here, whereas transitions dominate patterns of allegedly neutral polymorphism in natural populations of C. elegans and many other animal species; this observation challenges the assumption that natural patterns of molecular variation in noncoding regions of the nuclear genome accurately reflect underlying mutation processes.

TileQC: a system for tile-based quality control of Solexa data.

BACKGROUND: Next-generation DNA sequencing technologies such as Illumina's Solexa platform and Roche's 454 approach provide new avenues for investigating genome-scale questions. However, they also present novel analytical challenges that must be met for their effective application to biological questions. RESULTS: Here we report the availability of tileQC, a tile-based quality control system for Solexa data written in the R language. TileQC provides a means of recognizing bias and error in Solexa output by graphically representing data generated by flow cell tiles. The data represented in the images is then made available in the R environment for further analysis and automation of error detection. CONCLUSION: TileQC offers a highly adaptable and powerful tool for the quality control of Solexa-based DNA sequence data.