Primer mix

Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized using random hexamers (Qiagen, Hilden, Germany), and Superscript III Reverse Transcriptase (Invitrogen, Life Technologies, Paislay, UK) according to the manufacturer’s instructions. qPCR mixture consisted of 3.5 µl Light Cycler 480 Probe master (Roche Diagnostics, Mannheim, Germany) 1 µl 10 µM Primer mix (Sigma-Aldrich, Taufkirchen, Germany) and 0.05 µl UPL probe (Roche Diagnostics, Mannheim, Germany). 2.5 µl of a 1:10 dilution of cDNA served as template. Expression analysis was performed on the LightCycler 480-2 (Roche) system with the following progression: 10 min 95 °C and 45 cycles of 10 s 95 °C, 20 s 55 °C, 1 s 72 °C. Alternative to the UPL system, Sybr green qPCR was performed in a total volume of 10 µl including 5 µl Prima Quant Mix (Steinbrenner, Wiesenbach, Germany), 0.6 µl Primermix (10 µM each, Sigma-Aldrich) and 2 µl of 1:10 dilution of the template cDNA. Samples were incubated at 95 °C for 15 min and 15 s at 95 °C, 30 s at 55 °C, and 10 s at 72 °C for 45 cycles. Target gene expression was normalized to the housekeeping gene GAPDH using the ΔCT method (relative expression is equal to 2^-ΔCT). All used primers are listed in Supplementary Data 5.

A subset of the positively identified SNP markers from the BSA were selected for genotyping the F₂ populations from the AC × PI 207262 and AC × G 2333 crosses. Genotyping was accomplished using kompetitive allele specific PCR (KASP) assays. The KASP primer sequences were designed using Primer3 [52 (link),53 (link)], and the KASP reactions were conducted following the manufacturer's instructions in 10 μL reactions with 5 μL of 2X premade KASP master mix (LGC, Middlesex, UK), 0.14 μL of primer mix (Sigma-Aldrich, St. Louis, USA), and 20–40 ng of genomic DNA. PCR parameters were performed as described by LGC on standard thermocycling machines using white semiskirted polypropylene 0.2 mL 96-well PCR plates (USA Scientific) and sealed with Microseal B (Bio-Rad, Hercules, CA, USA). After PCR amplification was completed, the PCR plates were scanned using the Mx3000P qPCR machine (Agilent, Santa Clara, CA), and allele calls for each genotype were obtained using KlusterCaller software (LGC, Middlesex, UK).