GBS EcoT22I koa parents and expected progeny
Phyllodes were collected from putative hybrids as well as individual trees and placed on ice in plastic bags. Approximately 130 mg fresh weight of phyllode samples were sprayed with 70% isopropanol, wiped with sterile paper towels, and cut into 2 mm strips for processing. Samples were ground in a sterile mortar and pestle with liquid Nitrogen until they became a fine powder. DNA was extracted using the NucleoSpin Plant II DNA extraction kit (Macherey-Nagel, Bethlehem, PA, USA) following manufacturer s recommendations except as modified by adding an additional centrifugation step and heating of elution buffer. DNA samples were dissolved in TE (10 mM Tris, 0.1 mM EDTA, pH 7.5-8), placed in a 96-well plate, and sent to the Cornell University s Genomic Diversity Facility (Ithaca, NY, USA) for genotyping-by-sequencing. EndonucleaseEcoT22I was used to digest these koa DNA samples.