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.2009 Aug 6:9:106.
doi: 10.1186/1471-2229-9-106.

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

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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

Björn Hamberger et al. BMC Plant Biol..

Abstract

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.

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Figures

Figure 1
Figure 1
Structure of white spruce genomic DNA of BAC clones PGB02 and PGB04. The position of the target genes 3CAR and CYP720B4 is indicated. Red and yellow bars represent repeated segments and segments with similarity to DNA transposons, respectively. Transposable elements were identified with the RepeatMasker using theviridiplantaesection of the RepBase Update database. EcIS10,E. coliindividual insertion sequence (IS) of the bacterial transposon Tn10; CSRE, conifer specific repeat element; LB/RB left and right border of pINDIGO; arrows in PGB04 indicate local putative segment duplications. The scale bar represents 10 kbp. (p) pseudogene, based on the accumulation of deleterious mutations and the absence of transcript with >90% identity.
Figure 2
Figure 2
Gene structure of white spruce 3CAR (A) and CYP720B4 (B) and comparison of 3CAR with the grand fir (Abies grandis) limonene synthase (LIM) and pinene synthase (PIN) genes (C). Exons of the 3CAR and CYP720B4 genes matching the cDNA sequences are shown with grey arrows separated by introns. The UTRs are shown with grey lines. ATG, start codon. Putativecis-acting elements were identified using the PlantCare database and positions are highlighted in blue (not to scale): wun-box, wound-responsive element (Brassica oleracea); W-box, fungal elicitor responsive element (Petroselinum crispum); TCRR, TC-rich repeats,cis-acting element involved in defence and stress responsivenes (Nicotiana tabacum); TCA,cis-acting element involved in salicylic acid responsiveness (Brassica oleracea); TGACG,cis-acting regulatory element involved in the MeJA-responsiveness (Hordeum vulgare). LIM, AF326518; PIN, AF326517; roman numbers in part C indicate conserved exons in 3CAR, LIM and PIN; the scale bar represents 1 kbp.
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References

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