To sequence the complete chloroplast genome, Katie first carries out a biochemical isolation of the intact chloroplast organelles from fresh tissue samples, using a high-salt-plus-saline Percoll gradient method. Once the samples are enriched for the chloroplast genomic material—a crucial step due to P. rubens’ enormous nuclear genome size—a DNA extraction can be completed. DNA is sequenced using the Illumina Miseq high-throughput sequencing method.
Katie plans to perform chloroplast extraction and sequencing on only 30 of our 340 individuals, but it’s still possible to construct 340 complete genomes using bioinformatics. Partial coverage of the chloroplast genome in all 340 trees was produced during the exome sequencing, allowing us to impute missing bases using the whole-genome sequence data as a reference. This wealth of sequence data will make it possible to analyze the genetic diversity, population size, and gene flow of red spruce populations in the context of climatic changes since the last glacial maxima using a coalescent theory-based analysis. The insight gained into population dynamics throughout history will inform ongoing spruce conservation efforts.