Precision melt supermix for hrm analysis

For genotyping zebrafish larvae or adults were anesthetized in a solution of tricaine methanesulfonate (MS222, 0.765 mmol/L). Depending on the experiment performed, a fin clip, the trunks or whole larvae were used for the extraction of crude genomic DNA. The samples were lysed in 20 µl 50 mM NaOH by heating them for 10 min at 100°C. Afterwards, the reaction was neutralized by the addition of 2 µl of 100 mM TrisHCl (pH 8). For genotyping, 2 μl of genomic DNA was mixed with 5 μL of the Precision Melt Supermix for HRM analysis (Bio-Rad #172–5112), 0.5 μl of each primer (10 μM) and 2 µl milliQ water. The following primers were used; ATGCGCTGTTTGGTGAGG (depdc5, forward primer), TCC​AGG​AGT​GGG​TGT​TTT​TG (depdc5, reverse primer), GAG​ACC​TGC​CTG​GAC​ATG​AT (tsc2, forward primer) and CTT​GGG​CAG​AGC​AGA​GAA​GT (tsc2, reverse primer). The HRM reaction was performed in the CFX96 touch RT-qPCR detection system (BioRad, RRID:SCR_018064) using CFX Maestro^TM Software (Biorad). The melt peaks were automatically clustered by Precision Melt Analysis^TM Software (Biorad).

The genomic DNA of patients and healthy participants was extracted from EDTA tubes (Sarstedt) using the Qiagen Blood Mini Kit (Qiagen) according to the manufacturer's instructions. The DNA samples were subsequently diluted to a concentration of 10 ng/µL.
The rs6295 (5‐HT1A), rs6311 (5‐HT2A), and rs1049353 (CNR1) were analyzed using high‐resolution melting (HRM) genotyping. HRM analysis uses different denaturation characteristics of the nucleotides to determine the DNA sequence. Before HRM analysis with the Precision Melt Analysis Software (BioRad), the DNA sequence of interest was amplified in a qPCR. The qPCRs were carried out on a CFX384 Touch Real‐Time PCR Detection System (BioRad).
The qPCR reaction for the genotyping contained 20 ng of DNA, 0.2 µM primers (5‐HT1A fw: 5′‐TTG ATG GAA GAA GAC CGA GTG‐3′, rev: 5′‐TTG ATG GAA GAA GAC CGA GTG‐3′; 5‐HT2A fw: 5′‐GTT GGC TTT GGA TGG AAG TGC‐3′, rev: 5′‐GTA TGT CCT CGG AGT GCT GT‐3′; CNR1 fw: 5′‐GTT CAC AGG GCC GCA GAA A‐3′, rev: 5′‐ GTG GAC ACA GAC ATG GTT ACC T‐3′) and 10 μL precision melt supermix for HRM analysis (BioRad) in a total volume of 20 µL. The amplification protocol included an initial denaturation step for 3 min at 95°C, followed by 40 cycles of melting at 95°C for 5 s, annealing and extension at 60°C for 30 s, and a melting curve analysis. All experiments were performed in duplicates and included non‐template controls.

Genomic DNA was extracted from citrus leaves using the Plant DNeasy Prep Kit (Qiagen, Beijing, China). To investigate the genotypes of the CsLOB1 promoter in Wanjincheng orange, the primer pair Hp171‐1f/Hp171‐1R (Table S6) was designed on the basis of the indel of adenine (G) located just downstream of the PthA4 EBE in the CsLOB1 promoter (Hu et al., 2014; Li et al., 2014). The PCR were performed in a final volume of 10 μL, containing 5 μL Precision Melt Supermix for HRM analysis (Bio‐Rad #172–5112), 0.5 μL of each primer (10 μm) and 2 μL genomic DNA. The PCR protocol was 95 °C for 5 min, then 30 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s, followed by 94 °C for 30 s and 25 °C for 60 s. Using Chandler pummelo and Satsuma mandarin as controls, indels in the amplified PCR products were analysed using a LightScanner^® 96 Hi‐Res Melting^® system (Idaho Technology, Salt Lake City, UT). Amplicons were subjected to direct sequencing using the Hp171‐1R primer. The experiment was repeated three times.