Comprehensive Transcriptome Analysis of Loquat Fruit

Total RNA from the fruit samples described above was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) and treated with TURBO DNA-free^™ kit (Ambion, Austin, TX, USA) to remove DNA contamination, following the manufacturer’s protocol. Purified RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was evaluated using 1% agarose gel electrophoresis. First-strand complementary DNA synthesis was conducted using a reverse transcription kit containing a PrimeScript^TM RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China).
Genes related to OA metabolism were selected according to the loquat whole genome sequence [22 (link)]. The internal reference genes for normalization of relative gene expression were obtained from [19 (link)]. PEPC2, NAD-MDH, NADP-ME2, VHA-A, and VHP1 were obtained from [23 (link)]. ALTMs were obtained from [24 (link)]. The detailed sequences of primers used are listed in Supplementary Table S1.
qRT-PCR was performed on a CFX96 Touch Real-Time PCR C1000 Thermal Cycler system (Bio-Rad, Hercules, CA, USA) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies Inc., Santa Clara, CA, USA), following the manufacturer’s recommendations. For each biological replicate, three technical replicates of each PCR were performed.