Fx96 touch real time pcr detection system

Segregation analysis was performed to confirm that the identified mutations were causal mutations of the aco1a phenotype. Approximately 300 BC₂S₁ plants segregating for the mutation were genotyped using Kompetitive allele-specific PCR (KASP) technology. Primers were synthesized by LGC Genomics ®³, and the KASP assay was performed in the FX96 Touch Real−Time PCR Detection System (Bio-Rad ^®) using the LGC protocol⁴. The multiplex PCRs were run with 10 μL final reaction volume containing 5 μL KASP V4.0 2× Master mix standard ROX (LCG Genomics ^®), 0.14 μL KASP-by-Design primer mix (LCG Genomics ^®), 2 μL of 10–20 ng/μL genomic DNA, and 2.86 μL of water. The PCR thermocycling conditions were 15 min at 94°C (hot-start activation) followed by 10 cycles of 94°C for 20 s and 61°C for 1 min (dropping −0.6°C per cycle to achieve a 55°C annealing temperature) followed by 26 cycles of 94°C for 20 s and 55°C for 1 min. Data were then analyzed using CFX Maestro™ Software (Bio-Rad ^®) to identify SNP genotypes.

Kompetitive allele-specific PCR (KASP) technology was used to genotype 322 plants from a BC₁S₁ segregating population and to validate the causal mutation of the dwfcp phenotype. The LGC protocol and the FX96 Touch real-time PCR detection system (Bio-Rad®) were used to perform the KASP assay. LGC Genomics® synthesized the primers. A final reaction volume of 10 μl was used for multiplex PCRs, containing 5 μl KASP V4.0 2x Master Mix Standard ROX (LCG Genomics®), 0.138 μl KASP-by-Design Primer Mix (LCG Genomics®), and 4.862 μl of 20–50 ng/μl genomic DNA. SNP genotypes were finally identified using CFX Maestro™ software (Bio-Rad®).

qRT-PCR experiments were performed using an FX96 Touch™ Real-Time PCR Detection System (Bio-Rad) with a SYBR Green I Master Kit (Tiangen). JrACT2 (NCBI Reference: XM_018972062.1) was used as the reference gene to calculate relative fold differences based on the comparative cycle threshold (2^-∆∆ct). The PCR mixture (20 μL) contained 10 μL SYBR-Green PCR Master Mix (Tiangen), 0.2 μM each primer, 100 ng cDNA template, and nuclease-free water. The PCR procedure was as follows: 95 °C for 5 min; 30 cycles of 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s; followed by a final extension at 72 °C for 3 min.