Sampling and DNA sequencing
In the 1970s, the Forest Service’s Pacific Southwest Regional Genetic Resources Program planted clones of 302 wild ponderosa pines from diverse climate conditions in the central portion of the Sierra Nevada mountains in an orchard located in Chico, California. We chose 223 individual P. ponderosa genotypes from the orchard for the GEA analysis whose collection locations span the full climatic range included in the collection. The source locations for these genotypes (Fig. 1) fall within just one of the several genetic subdivisions previously identified in ponderosa pine (Conkle & Critchfield 1988; Williams 2009; Potter et al. 2015). Fresh needles were collected from these individuals and placed in labeled tea bags over silica gel to dry them and quickly preserve the DNA for extraction.
DNA was extracted from the dried needles using a modified Qiagen plant kits protocol by adding proteinase K and quantified using an Eppendorf BioSpectrometer (Eppendorf, AG, Germany). Samples were frozen and sent to the UC Davis Genome Center for library construction. Four 48-plex GBS libraries consisting of 47 DNA samples and negative control (no DNA) and one 36-plex GBS library composed of 35 DNA samples and negative control were prepared. The pool was quantified via qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) for Illumina sequencing platforms, with 0.9X bead cleanup to remove small fragments (<250 bp). Additional DNA purification using the Zymo DNA Clean & Concentrator kit (Zymo Research, Irvine, CA) was performed to increase the purity of the extracted DNA. The libraries were then sequenced (single-end read 90 bp or 100 bp) using an Illumina HiSeq 4000 (Illumina, San Diego, CA), one library per lane.