Powergen high throughput homogenizer

Five leaf samples were collected from Arabidopsis 3 days after recovery from treatment with 0 μg/mL (MS only), 0.15 μg/mL, or 0.3 μg/mL Tm. The leaves were frozen in liquid nitrogen and homogenized in a high-throughput homogenizer (Powergen High Throughput Homogenizer, Fisher), then washed with 1 mL methanol, homogenized for 2 min, and centrifuged for 2 min at 13,000 g. Chlorophyll was measured by adding 200 μL of supernatant to a 96-well polystyrene plate (Fisher) and read using a microplate reader (Epoch, Biotek) at 652 and 665 nm wavelengths. Absorbance corrections and chlorophyll calculations were completed as described in Ritchie (2006) (link). Data were subjected to statistical analyses (Tukey’s HSD test) as described above.

For the discovery populations and validation population in both years, 30–60 mg of unexpanded leaf tissue from each individual was collected into 96-well plates and frozen at − 80 °C until extraction. Immediate parents and other pedigree-connected individuals with discovery populations were also included. DNA extraction was performed using a modified Integrated DNA Technologies (IDT) Plant DNA Extraction Protocol (Keb-Llanes et al. 2002 (link)). Prior to DNA extraction, frozen samples were ground with a Fisher Scientific PowerGen high-throughput homogenizer (Pittsburgh, PA) twice for 1 min with a 3-min refreezing at −  80 °C between grindings. Genotyping  was performed using the 38K Axiom^® IStraw35 384HT array (Verma et al. 2017a (link)). Genotyping errors were detected by comparing, within full-sib families, SNP calls of each individual with that of the parental genotypes, and replaced with ‘no call’ if not matching possible genotypes. If the correct parent was not obvious, the incorrect parent was replaced with an ‘undetermined’ parent. In addition, markers with inheritance errors, as determined in the ‘mconsistency’ file of FlexQTL™ outputs, were removed from the data and the data re-analyzed in FlexQTL™ until errors were minimized.