The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

Spruce giga-genomes: structurally similar yet distinctive with differentially expanding gene families and rapidly evolving genes.

Spruces (Picea spp.) are coniferous trees widespread in boreal and mountainous forests of the northern hemisphere, with large economic significance and enormous contributions to global carbon sequestration. Spruces harbor very large genomes with high repetitiveness, hampering their comparative analysis. Here, we present and compare the genomes of four different North American spruces: the genome assemblies for Engelmann spruce (Picea engelmannii) and Sitka spruce (Picea sitchensis) together with improved and more contiguous genome assemblies for white spruce (Picea glauca) and for a naturally occurring introgress of these three species known as interior spruce (P. engelmannii × glauca × sitchensis). The genomes were structurally similar, and a large part of scaffolds could be anchored to a genetic map. The composition of the interior spruce genome indicated asymmetric contributions from the three ancestral genomes. Phylogenetic analysis of the nuclear and organelle genomes revealed a topology indicative of ancient reticulation. Different patterns of expansion of gene families among genomes were observed and related with presumed diversifying ecological adaptations. We identified rapidly evolving genes that harbored high rates of non-synonymous polymorphisms relative to synonymous ones, indicative of positive selection and its hitchhiking effects. These gene sets were mostly distinct between the genomes of ecologically contrasted species, and signatures of convergent balancing selection were detected. Stress and stimulus response was identified as the most frequent function assigned to expanding gene families and rapidly evolving genes. These two aspects of genomic evolution were complementary in their contribution to divergent evolution of presumed adaptive nature. These more contiguous spruce giga-genome sequences should strengthen our understanding of conifer genome structure and evolution, as their comparison offers clues into the genetic basis of adaptation and ecology of conifers at the genomic level. They will also provide tools to better monitor natural genetic diversity and improve the management of conifer forests. The genomes of four closely related North American spruces indicate that their high similarity at the morphological level is paralleled by the high conservation of their physical genome structure. Yet, the evidence of divergent evolution is apparent in their rapidly evolving genomes, supported by differential expansion of key gene families and large sets of genes under positive selection, largely in relation to stimulus and environmental stress response.

Genomic virulence features of Beauveria bassiana as a biocontrol agent for the mountain pine beetle population.

BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae, is an irruptive bark beetle that causes extensive mortality to many pine species within the forests of western North America. Driven by climate change and wildfire suppression, a recent mountain pine beetle (MPB) outbreak has spread across more than 18 million hectares, including areas to the east of the Rocky Mountains that comprise populations and species of pines not previously affected. Despite its impacts, there are few tactics available to control MPB populations. Beauveria bassiana is an entomopathogenic fungus used as a biological agent in agriculture and forestry and has potential as a management tactic for the mountain pine beetle population. This work investigates the phenotypic and genomic variation between B. bassiana strains to identify optimal strains against a specific insect. RESULTS: Using comparative genome and transcriptome analyses of eight B. bassiana isolates, we have identified the genetic basis of virulence, which includes oosporein production. Genes unique to the more virulent strains included functions in biosynthesis of mycotoxins, membrane transporters, and transcription factors. Significant differential expression of genes related to virulence, transmembrane transport, and stress response was identified between the different strains, as well as up to nine-fold upregulation of genes involved in the biosynthesis of oosporein. Differential correlation analysis revealed transcription factors that may be involved in regulating oosporein production. CONCLUSION: This study provides a foundation for the selection and/or engineering of the most effective strain of B. bassiana for the biological control of mountain pine beetle and other insect pests populations.

Transcriptome features of stone cell development in weevil-resistant and susceptible Sitka spruce.

Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.

The Norway spruce genome sequence and conifer genome evolution.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Improved white spruce (Picea glauca) genome assemblies and annotation of large gene families of conifer terpenoid and phenolic defense metabolism.

White spruce (Picea glauca), a gymnosperm tree, has been established as one of the models for conifer genomics. We describe the draft genome assemblies of two white spruce genotypes, PG29 and WS77111, innovative tools for the assembly of very large genomes, and the conifer genomics resources developed in this process. The two white spruce genotypes originate from distant geographic regions of western (PG29) and eastern (WS77111) North America, and represent elite trees in two Canadian tree-breeding programs. We present an update (V3 and V4) for a previously reported PG29 V2 draft genome assembly and introduce a second white spruce genome assembly for genotype WS77111. Assemblies of the PG29 and WS77111 genomes confirm the reconstructed white spruce genome size in the 20 Gbp range, and show broad synteny. Using the PG29 V3 assembly and additional white spruce genomics and transcriptomics resources, we performed MAKER-P annotation and meticulous expert annotation of very large gene families of conifer defense metabolism, the terpene synthases and cytochrome P450s. We also comprehensively annotated the white spruce mevalonate, methylerythritol phosphate and phenylpropanoid pathways. These analyses highlighted the large extent of gene and pseudogene duplications in a conifer genome, in particular for genes of secondary (i.e. specialized) metabolism, and the potential for gain and loss of function for defense and adaptation.

Reduced Genetic Diversity and Increased Dispersal in Guigna (Leopardus guigna) in Chilean Fragmented Landscapes.

Landscape fragmentation is often a major cause of species extinction as it can affect a wide variety of ecological processes. The impact of fragmentation varies among species depending on many factors, including their life-history traits and dispersal abilities. Felids are one of the groups most threatened by fragmented landscapes because of their large home ranges, territorial behavior, and low population densities. Here, we model the impacts of habitat fragmentation on patterns of genetic diversity in the guigna (Leopardus guigna), a small felid that is closely associated with the heavily human-impacted temperate rainforests of southern South America. We assessed genetic variation in 1798 base pairs of mitochondrial DNA sequences, 15 microsatellite loci, and 2 sex chromosome genes and estimated genetic diversity, kinship, inbreeding, and dispersal in 38 individuals from landscapes with differing degrees of fragmentation on Chiloé Island in southern Chile. Increased fragmentation was associated with reduced genetic diversity, but not with increased kinship or inbreeding. However, in fragmented landscapes, there was a weaker negative correlation between pairwise kinship and geographic distance, suggesting increased dispersal distances. These results highlight the importance of biological corridors to maximize connectivity in fragmented landscapes and contribute to our understanding of the broader genetic consequences of habitat fragmentation, especially for forest-specialist carnivores.

Evolution of gene structure in the conifer Picea glauca: a comparative analysis of the impact of intron size.

BACKGROUND: A positive relationship between genome size and intron length is observed across eukaryotes including Angiosperms plants, indicating a co-evolution of genome size and gene structure. Conifers have very large genomes and longer introns on average than most plants, but impacts of their large genome and longer introns on gene structure has not be described. RESULTS: Gene structure was analyzed for 35 genes of Picea glauca obtained from BAC sequencing and genome assembly, including comparisons with A. thaliana, P. trichocarpa and Z. mays. We aimed to develop an understanding of impact of long introns on the structure of individual genes. The number and length of exons was well conserved among the species compared but on average, P. glauca introns were longer and genes had four times more intronic sequence than Arabidopsis, and 2 times more than poplar and maize. However, pairwise comparisons of individual genes gave variable results and not all contrasts were statistically significant. Genes generally accumulated one or a few longer introns in species with larger genomes but the position of long introns was variable between plant lineages. In P. glauca, highly expressed genes generally had more intronic sequence than tissue preferential genes. Comparisons with the Pinus taeda BACs and genome scaffolds showed a high conservation for position of long introns and for sequence of short introns. A survey of 1836 P. glauca genes obtained by sequence capture mostly containing introns <1 Kbp showed that repeated sequences were 10× more abundant in introns than in exons. CONCLUSION: Conifers have large amounts of intronic sequence per gene for seed plants due to the presence of few long introns and repetitive element sequences are ubiquitous in their introns. Results indicate a complex landscape of intron sizes and distribution across taxa and between genes with different expression profiles.

Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data.

UNLABELLED: White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commercially valuable tree will help improve forest management and conservation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. Here, we describe a whole-genome shotgun sequencing strategy using two Illumina sequencing platforms and an assembly approach using the ABySS software. We report a 20.8 giga base pairs draft genome in 4.9 million scaffolds, with a scaffold N50 of 20,356 bp. We demonstrate how recent improvements in the sequencing technology, especially increasing read lengths and paired end reads from longer fragments have a major impact on the assembly contiguity. We also note that scalable bioinformatics tools are instrumental in providing rapid draft assemblies. AVAILABILITY: The Picea glauca genome sequencing and assembly data are available through NCBI (Accession#: ALWZ0100000000 PID: PRJNA83435). http://www.ncbi.nlm.nih.gov/bioproject/83435.

Genetic architecture of terpene chemistry and growth traits and the impact of inbreeding on these traits in western redcedar (Thuja plicata).

Western redcedar (WRC; Thuja plicata) is a conifer of the Pacific Northwest of North America prized for its durable and rot-resistant wood. WRC has naturally low outcrossing rates and readily self-fertilizes in nature. Challenges faced in WRC breeding and propagation involve selecting trees for accelerated growth while also ensuring enhanced heartwood rot resistance and resistance to ungulate browsing, as well as mitigating potential effects of inbreeding depression. Terpenes, a large and diverse class of specialized metabolites, confer both rot and browse resistance in the wood and foliage of WRC, respectively. Using a Bayesian modelling approach, we isolated single nucleotide polymorphism (SNP) markers estimated to be associated with three different foliar terpene traits and four different heartwood terpene traits, as well as two growth traits. We found that all traits were complex, being associated with between 1700 and 3600 SNPs linked with putatively causal loci, with significant polygenic components. Growth traits tended to have a larger polygenic component while terpene traits had larger major gene components; SNPs with small or polygenic effect were spread across the genome, while larger-effect SNPs tended to be localized to specific linkage groups. To determine whether there was inbreeding depression for terpene chemistry or growth traits, we used mixed linear models for a genomic selection training population to estimate the effect of the inbreeding coefficient F on foliar terpenes, heartwood terpenes and several growth and dendrochronological traits. We did not find significant inbreeding depression for any assessed trait. We further assessed inbreeding depression across four generations of complete selfing and found that not only was inbreeding depression not significant but that selection for height growth was the only significant predictor for growth during selfing, suggesting that inbreeding depression due to selfing during operational breeding can be mitigated by increased selection intensity.

Assembly and annotation of the black spruce genome provide insights on spruce phylogeny and evolution of stress response.

Black spruce (Picea mariana [Mill.] B.S.P.) is a dominant conifer species in the North American boreal forest that plays important ecological and economic roles. Here, we present the first genome assembly of P. mariana with a reconstructed genome size of 18.3 Gbp and NG50 scaffold length of 36.0 kbp. A total of 66,332 protein-coding sequences were predicted in silico and annotated based on sequence homology. We analyzed the evolutionary relationships between P. mariana and 5 other spruces for which complete nuclear and organelle genome sequences were available. The phylogenetic tree estimated from mitochondrial genome sequences agrees with biogeography; specifically, P. mariana was strongly supported as a sister lineage to P. glauca and 3 other taxa found in western North America, followed by the European Picea abies. We obtained mixed topologies with weaker statistical support in phylogenetic trees estimated from nuclear and chloroplast genome sequences, indicative of ancient reticulate evolution affecting these 2 genomes. Clustering of protein-coding sequences from the 6 Picea taxa and 2 Pinus species resulted in 34,776 orthogroups, 560 of which appeared to be specific to P. mariana. Analysis of these specific orthogroups and dN/dS analysis of positive selection signatures for 497 single-copy orthogroups identified gene functions mostly related to plant development and stress response. The P. mariana genome assembly and annotation provides a valuable resource for forest genetics research and applications in this broadly distributed species, especially in relation to climate adaptation.

Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms.

BACKGROUND: Comparative genomics can inform us about the processes of mutation and selection across diverse taxa. Among seed plants, gymnosperms have been lacking in genomic comparisons. Recent EST and full-length cDNA collections for two conifers, Sitka spruce (Picea sitchensis) and loblolly pine (Pinus taeda), together with full genome sequences for two angiosperms, Arabidopsis thaliana and poplar (Populus trichocarpa), offer an opportunity to infer the evolutionary processes underlying thousands of orthologous protein-coding genes in gymnosperms compared with an angiosperm orthologue set. RESULTS: Based upon pairwise comparisons of 3,723 spruce and pine orthologues, we found an average synonymous genetic distance (dS) of 0.191, and an average dN/dS ratio of 0.314. Using a fossil-established divergence time of 140 million years between spruce and pine, we extrapolated a nucleotide substitution rate of 0.68 × 10(-9) synonymous substitutions per site per year. When compared to angiosperms, this indicates a dramatically slower rate of nucleotide substitution rates in conifers: on average 15-fold. Coincidentally, we found a three-fold higher dN/dS for the spruce-pine lineage compared to the poplar-Arabidopsis lineage. This joint occurrence of a slower evolutionary rate in conifers with higher dN/dS, and possibly positive selection, showcases the uniqueness of conifer genome evolution. CONCLUSIONS: Our results are in line with documented reduced nucleotide diversity, conservative genome evolution and low rates of diversification in conifers on the one hand and numerous examples of local adaptation in conifers on the other hand. We propose that reduced levels of nucleotide mutation in large and long-lived conifer trees, coupled with large effective population size, were the main factors leading to slow substitution rates but retention of beneficial mutations.

Technical brief: isolation of total DNA from postmortem human eye tissues and quality comparison between iris and retina.

BACKGROUND: Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies. METHODS: DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene. RESULTS: Regarding concentration, the retina yielded 936 ng/µl of DNA, while the iris yielded 78 ng/µl of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/µl/mg vs. 7.42 ng/µl/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies. CONCLUSIONS: The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye tissues. Retinal tissue provides DNA of excellent quantity and quality for genotyping and downstream genomic studies. However, DNA isolated from iris tissues, and treated with bovine serum albumin, may also be a valuable source of DNA for genotyping and genomic studies.

Genomic selection reveals hidden relatedness and increased breeding efficiency in western redcedar polycross breeding.

Western redcedar (WRC) is an ecologically and economically important forest tree species characterized by low genetic diversity with high self-compatibility and high heartwood durability. Using sequence capture genotyping of target genic and non-genic regions, we genotyped 44 parent trees and 1520 offspring trees representing 26 polycross (PX) families collected from three progeny test sites using 45,378 SNPs. Trees were phenotyped for eight traits related to growth, heartwood and foliar chemistry associated with wood durability and deer browse resistance. We used the genomic realized relationship matrix for paternity assignment, maternal pedigree correction, and to estimate genetic parameters. We compared genomics-based (GBLUP) and two pedigree-based (ABLUP: polycross and reconstructed full-sib [FS] pedigrees) models. Models were extended to estimate dominance genetic effects. Pedigree reconstruction revealed significant unequal male contribution and separated the 26 PX families into 438 FS families. Traditional maternal PX pedigree analysis resulted in up to 51% overestimation in genetic gain and 44% in diversity. Genomic analysis resulted in up to 22% improvement in offspring breeding value (BV) theoretical accuracy, 35% increase in expected genetic gain for forward selection, and doubled selection intensity for backward selection. Overall, all traits showed low to moderate heritability (0.09-0.28), moderate genotype by environment interaction (type-B genetic correlation: 0.51-0.80), low to high expected genetic gain (6.01%-55%), and no significant negative genetic correlation reflecting no large trade-offs for multi-trait selection. Only three traits showed a significant dominance effect. GBLUP resulted in smaller but more accurate heritability estimates for five traits, but larger estimates for the wood traits. Comparison between all, genic-coding, genic-non-coding and intergenic SNPs showed little difference in genetic estimates. In summary, we show that GBLUP overcomes the PX limitations, successfully captures expected historical and hidden relatedness as well as linkage disequilibrium (LD), and results in increased breeding efficiency in WRC.

Physical disturbance by recovering sea otter populations increases eelgrass genetic diversity.

Most knowledge regarding the role of predators is ecological in nature. Here, we report how disturbance generated by sea otters (Enhydra lutris) digging for infaunal prey in eelgrass (Zostera marina) meadows increases genetic diversity by promoting conditions for sexual reproduction of plants. Eelgrass allelic richness and genotypic diversity were, respectively, 30 and 6% higher in areas where recovering sea otter populations had been established for 20 to 30 years than in areas where they had been present <10 years or absent >100 years. The influence of sea otter occupancy on the aforementioned measures of genetic diversity was stronger than those of depth, temperature, latitude, or meadow size. Our findings reveal an underappreciated evolutionary process by which megafauna may promote genetic diversity and ecological resilience.

Spatial and temporal changes in genetic structure of greenhouse and field populations of cabbage looper, Trichoplusia ni.

Trichoplusia ni is a subtropical moth that migrates annually from southern California to southern British Columbia, Canada where it invades vegetable greenhouses and field crops. The heated greenhouse environment has altered the natural extinction-recolonization dynamics of T. ni populations, and allows year-round persistence in some locations. In addition, the extensive use of the biopesticide, Bacillus thuringiensis subspecies kurstaki (Bt) in some greenhouses has selected for resistance. Here we investigated the genetic structure of T. ni populations in British Columbia greenhouses and in field populations in California and British Columbia using amplified fragment length polymorphisms (AFLP) as related to patterns of Bt resistance. The majority of British Columbia field populations were similar to the California field populations, the potential source of migrants. However populations in two geographic areas with high concentrations of greenhouses showed local genetic differentiation. Some of these populations experienced severe bottlenecks over-winter and following Bt sprays. Greenhouse populations showed a pattern of isolation by distance and a strong positive relationship between genetic differentiation and levels of Bt resistance. These patterns indicate that greenhouses that sometimes support year-round populations of T. ni and the ensuing strong bottlenecking effects following winter cleanups and Bt application cause genetic differentiation of T. ni populations. Long distance migrants to field populations contribute to genetic homogeneity of these.

Genomic selection for resistance to spruce budworm in white spruce and relationships with growth and wood quality traits.

With climate change, the pressure on tree breeding to provide varieties with improved resilience to biotic and abiotic stress is increasing. As such, pest resistance is of high priority but has been neglected in most tree breeding programs, given the complexity of phenotyping for these traits and delays to assess mature trees. In addition, the existing genetic variation of resistance and its relationship with productivity should be better understood for their consideration in multitrait breeding. In this study, we evaluated the prospects for genetic improvement of the levels of acetophenone aglycones (AAs) in white spruce needles, which have been shown to be tightly linked to resistance to spruce budworm. Furthermore, we estimated the accuracy of genomic selection (GS) for these traits, allowing selection at a very early stage to accelerate breeding. A total of 1,516 progeny trees established on five sites and belonging to 136 full-sib families from a mature breeding population in New Brunswick were measured for height growth and genotyped for 4,148 high-quality SNPs belonging to as many genes along the white spruce genome. In addition, 598 trees were assessed for levels of AAs piceol and pungenol in needles, and 578 for wood stiffness. GS models were developed with the phenotyped trees and then applied to predict the trait values of unphenotyped trees. AAs were under moderate-to-high genetic control (h 2: 0.43-0.57) with null or marginally negative genetic correlations with other traits. The prediction accuracy of GS models (GBLUP) for AAs was high (PAAC: 0.63-0.67) and comparable or slightly higher than pedigree-based (ABLUP) or BayesCπ models. We show that AA traits can be improved and that GS speeds up the selection of improved trees for insect resistance and for growth and wood quality traits. Various selection strategies were tested to optimize multitrait gains.

Complete Chloroplast Genome Sequence of a Black Spruce (Picea mariana) from Eastern Canada.

Here, we present the chloroplast genome sequence of black spruce (Picea mariana), a conifer widely distributed throughout North American boreal forests. This complete and annotated chloroplast sequence is 123,961 bp long and will contribute to future studies on the genetic basis of evolutionary change in spruce and adaptation in conifers.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

BACKGROUND: Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. RESULTS: We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. CONCLUSION: We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Complete Chloroplast Genome Sequence of a White Spruce (Picea glauca, Genotype WS77111) from Eastern Canada.

Here, we present the complete chloroplast genome sequence of white spruce (Picea glauca, genotype WS77111), a coniferous tree widespread in the boreal forests of North America. This sequence contributes to genomic and phylogenetic analyses of the Picea genus that are part of ongoing research to understand their adaptation to environmental stress.