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

Abstract

BackgroundConifers 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.ResultsWe 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.ConclusionWe 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.

The first insight into the Taxus genome via fosmid library construction and end sequencing

“…DNA-based transposons have been implicated in the capture and transduplication of host genes in rice, Lotus japonicus, and Arabidopsis (Hoen et al 2006). The proximity of DNA transposons to the protein coding 3CAR and CYP720B4 genes is consistent with the possibility that a DNA transposon-mediated translocation mechanism may contribute to the diversification of the conifer gene families (Hamberger et al 2009). …”

“…Only 88 sequences showed identity to sequences from Taxus, 57 of them hit to different microsatellite-containing sequences of Taxus species and 8 of them hit to different T. baccata SSR markers. Among the remaining 23 hits, 14 sequences hit to P. glauca cultivar PG29 clone BAC PGB02 3-carene synthase gene (3CAR; Hamberger et al 2009), three to P. glauca clone BAC PGB04 CYP720B4 gene (Hamberger et al 2009), three to T. cuspidata taxadien-5-alpha-ol-O-acetyltransferase (T5aH) gene, two to P. glauca clone BAC PGB09 (-)-ent-kaurene synthase gene (Keeling et al 2010), and one to T. cuspidata taxadiene 5-alpha hydroxylase gene.…”

“…In order to compare the composition of repetitive sequences, 14 BAC clone sequences of Pinus taeda (accession nos. GU477256-GU477262, and GU477264-GU477266; Kovach et al 2010) and Picea glauca (FJ609174, FJ609175, GU059904, and GU059905; Keeling et al 2010;Hamberger et al 2009) were retrieved from NCBI GSS database and analysed with the above programs. SPSS v17.0 was used in the statistical tests and the significance level was set as 0.05.…”

The heterogeneous levels of linkage disequilibrium in white spruce genes and comparative analysis with other conifers

“…It is unlikely that all necessary variants will be represented on an array in the near future. Simply to cover the gene space estimated at 32 700 genes for spruce (Rigault et al, 2011), and at a rate of one SNP per 85 bp and an average gene size of 3-3.5 kb (Hamberger et al, 2009), a total of 1.1-1.3 million SNPs would be necessary. Hence, a priori information about the genes involved in specific physiological processes underlying ecological or economical characters seems essential to reduce the number of genes and SNPs that could be considered in association scans involving unrelated individuals.…”

Extended Linkage Disequilibrium in Noncoding Regions in a Conifer, Cryptomeria japonica

“…Thus, it is important to know the extent of LD in regions other than the coding genes and their surrounding regions. However, the extent of LD in such regions has not been investigated in conifers to our knowledge possibly because of the lack of sequence information on those regions [but see Hamberger et al (2009) ;Keeling et al (2010); Kovach et al (2010)]. …”