Sybr real master mix

Sampling from 8-week-old plants under the same greenhouse conditions as previously described, qRT-PCR analysis was conducted according to the MIQE guidelines [67 (link), 68 (link)]. Total RNA was isolated using a total RNA isolation system (Tiangen, Beijing, China). First-strand cDNAs were synthesized using a First-strand cDNA Synthesis Kit (Tiangen, Beijing, China). qRT-PCR was performed on a Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (Transgen, Beijing, China) using the following PCR thermal cycle conditions: pre-denaturation at 95 °C for 30 s; followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. 18S RNA (GenBank Accession Number: AJ236016) was used as housekeeping gene. Three biological replicates were performed for each line, and the standard curve method was used for statistical analysis.

Twelve libraries were constructed for transcriptome sequencing that represented four peel samples and three replicates. RNA extraction, transcriptome sequencing, and quantification were performed [21 ]. For library sequencing on the Illumina Hiseq platform, 150 bp paired-end reads were generated for the library. For RNA-Seq., total RNA was extracted and transcribed into cDNA using the PrimeScript™ RT Reagent Kit (TaKaRa) in accordance with the manufacturer’s instructions. qRT-PCR was performed in a Real-time PCR System (ABI QuantStudio 3, ABI) using SYBR Real Master Mix (Transgen, Beijing, China) under the following PCR thermal cycling conditions: predenaturation at 95 °C for 30 s; followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. The sequences of the primers used are listed in Supplementary Table S1. ACT7 and GAPDH (AY123769) bilberry genes were used as the housekeeping gene. Tree biological replicates were performed for each gene, and the standard curve method was applied in statistical analysis.

Samples stored in the freezer at -80 °C were used for total RNA extraction using a total RNA isolation system (Tiangen, Beijing, China). First-strand cDNAs were synthesized using a First-strand cDNA Synthesis Kit (Tiangen, Beijing, China). The full-length coding sequences of genes were isolated from ‘white winter pearmain’ cDNA. Real-time quantitative reverse transcription (qRT-PCR) analysis was performed according to MIQE guidelines [45 (link), 46 (link)]. qRT-PCR was performed on a Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (Transgen, Beijing, China) under the following PCR thermal cycling conditions: predenaturation at 95 °C for 30 s; followed by 39 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. MdActin (GenBank Accession Number: AB638619) was used as the housekeeping gene. The sequences of the primers used are listed in Supplementary Table S1. Three biological replicates were performed for each gene, and the standard curve method was applied in statistical analysis.