BAC Library Construction and Microsynteny
Our BAC (bacterial artificial chromosome) library consists of 1,132,480 clones in total; average insert size of ~140kb. This is 3-5X coverage, depending upon what the true genome size of spruce is (20-35 gb). We have finished optimizing the protocol for producing subcloned libraries, for the purpose of shotgun sequencing selected BAC clones.
The first stage of BAC sequencing will involve randomly selected BACs and targeted BACs (a "targeted" BAC contains a specific gene of interest) to a 1x coverage. This will be done by the Vancouver Genome Sciences Centre. Randomly selected BACs will allow insights into the structure of conifer genomes, and may help guide the strategy for a potential collaborative conifer genome sequencing effort in the future. The targeted BACs come from our Functional Genomics research, and include: 3-carene synthase, abietadiene synthase, Cyp720B4, and DXS1 to start. We contract out to BIO S&T the isolation of these targeted genes.
We plan to target 10 genes, many of these in common with the co-funded loblolly pine project (UC Davis) who are planning to sequence at least 10 BACs each containing a gene from this list of genes from the lignin biosynthetic pathway: phenylalanine ammonia-lyase, cinnamate 4-hydroxylase C4H-1, 4-coumarate:CoA ligase, coumarate 3-hydroxylase, caffeoyl CoA O-methyltransferase, cinnamoyl CoA reductase, caffeate O-methyltransferase, cinnamyl alcohol dehydrogenase, s-adenosyl methionine synthetase, glycine hydroxymethyltransferase, LIM transcription factor, cellulose synthase, arabinogalactan protein, and ?-tubulin (some genes contain multiple copies, so in our project we will focus on genes from single-member families, to ensure high probability of homology). Interestingly, many of these lignin pathway genes are also involved with insect defence (Ralph et al., 2006).
Spruce-pine Synteny Mapping
We have tested close to 1000 COS primers on three different conifer species: white spruce, Douglas-fir, and loblolly pine. In spruce, 60% gave products of which 52% were single-banded. Of these, 33% were informative in the spruce pedigrees described above. Of the COS primers tested, 54% amplified in loblolly pine, and 49% gave single-banded products; 19% were informative in the loblolly pine pedigrees that we have. 41% of COS primers amplified single products in both spruce and loblolly pine and we are mapping and genotyping these COS primers on two loblolly pine mapping populations. A paper is currently been written that will provide the first high density map (100+ markers) between spruce and pine (please contact Kermit Ritland for more information).
We have developed a set of shared markers between the Arborea II mapping populations and our own mapping populations. This has allowed us to join the two project's maps resulting in the most extensive genetic map ever constructed for a conifer (manuscript in progress, please contact Kermit Ritland for more information).