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.