Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest

Abstract

Background: The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests.

Results: We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle.

Conclusions: Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

Figure 1

Heterozygosity of an individual male. The individual male mountain pine beetle (MPB) sequence data used for the assembly was mapped back onto the assembly, and the level of heterozygosity (allelic variation) was determined. Inset, A restricted range of single-nucleotide variant (SNV) density. Red markers indicate scaffolds with very low SNV density, which are hypothesized to represent scaffolds on the ancestral × chromosome portion of the neo-X chromosome. These are the six scaffolds shown in Figure 3.

Figure 2

Shared synteny between male and female mountain pine beetle (MPB) assembly scaffolds and Tribolium castaneum linkage groups. Sequences were compared by tBLASTx and regions of significant similarity (e-value <1 × 10^-20) are indicated by lines representing each high-scoring segment pair (appearing as dots at this scale). MPB scaffolds are displayed ordered from shortest to longest. The 20 longest MPB scaffolds are demarcated with faint horizontal lines. (A) Male and (B) female MPB scaffolds. (B) is longer than (A) due to a larger reconstruction size (including Ns) in the female compared with the male. A series of horizontal dots within one T. castaneum linkage group indicates a MPB scaffold sharing similarity with this linkage group. A linkage group for the y_p chromosome in T. castaneum has not been described.

Figure 3

Shared synteny between Tribolium castaneum LG1 = × and scaffolds representing the ancestral × portion of neo-X of the male mountain pine beetle (MPB) assembly. Each trapezoid connects a matching gene model between the two organisms: red trapezoid, parallel orientation; green trapezoid, anti-parallel orientation. Scaffolds Seq_1101913, Seq_1101939, and Seq_1102823 partially overlap, and scaffolds Seq_1102689, Seq_1102308, and Seq_1102713 are contained in one scaffold in the female assembly. The order and orientation of these two groups of scaffolds are otherwise arbitrary.

Figure 4

Horizontal gene transfer. (A) Schematic of the location of the sucrose-6-phosphate hydrolase gene (scrB, red arrow) on the male and female scaffolds. Green arrows indicate adjacent gene models with similarity to insect proteins by BLASTx (post-glycosylphosphatidylinositol (GPI) attachment to proteins factor 2-like (pgap2-like) and hypothetical protein, (hypo)). Blue arrows indicate the location of primers used to amplify intergenic regions between horizontal gene transfer (HGT) and adjacent beetle genes. Grey dashed lines indicate the similar gene models on the different scaffolds. (B) Presence of scrB in different beetle species. Green check marks and red Xs indicate presence and absence of the sucrose-6-phosphate hydrolase gene, respectively. Abbreviations: Dfro, Dendroctonus frontalis; Dmic, Dendroctonus micans; Dpon, Dendroctonus ponderosae; Dpun, Dendroctonus punctatus; Ipin, Ips pini; Ityp, Ips typographus; Pstr, Pissodes strobi; Tcas, Tribolium castaneum. Divergence dates estimated from Sequeira and Farrell [40]. (C) Phylogeny of scrB proteins from Dendroctonus (in red) with Gammaproteobacteria scrB and similar insect proteins. Abbreviations: bFF, beta-fructofuranosidase; Bimp, Bombus impatiens; Blic (in purple), Bacillus licheniformis; Bmor, Bombyx mori; Cfre, Citrobacter freundii; Cint, Commensalibacter intestine; Dfro, D. frontalis; Dmic, D. micans; Dpon, D. ponderosae; Dpun, D. punctatus; Eaer, Enterobacter aerogenes; Ebac, Enterobacteriaceae bacterium 9_2_54FAA; Eclo, Enterobacter cloacae; Ecol, Escherichia coli; Esp, Enterobacter sp.; Etas, Erwinia tasmaniensis; Koxy, Klebsiella oxytoca; Kpne, Klebsiella pneumoniae; Ksp, Klebsiella sp.; Kvar, Klebsiella variicola; Pcar, Pectobacterium carotovorum; Pret, Providencia rettgeri; Prus, P. rustigianii; Raqu, Rahnella aquatilis; Rsp, Rahnella sp.; sacC, glycoside hydrolase; scrB, sucrose-6-phosphate hydrolase; Sfle, Shigella flexneri; Sodo, Serratia odorifera; Sply, Serratia plymuthica; Sson, Shigella sonnei; Ssp, Serratia sp.; Yald, Yersinia aldovae; Yent, Yersinia enterocolitica; Yfre, Yersinia frederiksenii; Yroh, Yersinia rohdei. Branches with dots had greater than 80% bootstrap support. The tree was rooted with Bombyx mori beta-fructofuranosidase (Bmor-bFF, in blue).

Figure 5

Single-nucleotide polymorphism (SNP) density across the scaffolds for eight populations of beetles. Inset shows a restricted range of SNP density.

Figure 6

Orthologs. Comparative analysis of orthologous protein groups between six sequenced insect genomes. Predicted proteins from the genome sequences of mountain pine beetle (MPB), Tribolium castaneum, Apis mellifera (honey bee), Bombyx mori (silk moth), Drosophila melanogaster, and Acyrthosiphon pisum (pea aphid) were clustered into orthologous groups with OrthoMCL [84]. (A) Venn diagram indicates the number of protein groups found in either one or both beetle species among the 12,156 orthologous groups found. Numbers in parentheses indicate the percentage of these groups that were not found in any of the four non-beetle species. (B) Of the 6,663 groups found in both beetle species, 83% had an n to n correspondence between the two beetle species, whereas other groups had more or fewer members in mountain pine beetle (MPB) versus T. castaneum. The histogram indicates the distribution of the ratios of the number of MPB to T. castaneum proteins in each group.

Figure 7

Phylogeny of P450s. Phylogeny of MPB (Dendroctonus ponderosae, Dpon) P450s with those from the honey bee (Apis mellifera, Amel), silk moth (Bombyx mori, Bmor), and red flour beetle (Tribolium castaneum, Tcas). Red arcs indicate areas of expansion of the mountain pine beetle (MPB) CYP4, and both CYP6 and CYP9 P450 families within the CYP4 and CYP3 clades, respectively. Branches with dots had greater than 80% bootstrap support. The tree was rooted with human (Homo sapiens, Hsap) CYP3A4.

Figure 8

Phylogeny of glutathione S-transferases (GSTs). Branches with dots had > 80% bootstrap support. The tree was rooted with human HsapGSTA1.Aaeg, Aedes aegypti; Agam, Aedes gambiae; Amel, Apis mellifera; Apis, Acyrthosiphon pisum; Bmor, Bombyx mori; Cqui, Culex quinquefasciatus; Dmel, Drosophila melanogaster; Dpon, Dendroctonus ponderosae; Hsap; Homo sapiens; Nvit, Nasonia vitripennis; Phum, Pediculus humanus; Tcas, Tribolium castaneum.