*4.6. Plant Materials, RNA Extraction, and qRT-PCR Analysis*

Take the entire branches of 'Xiariwuniang' and 'Shaoguifei' in the early growth stage, growth stage, and the end of the growth stage to examine the expression patterns of *LiNAC* genes. Three samples of each were collected and the sampling time was 10:00. All samples were immediately frozen in liquid nitrogen and stored at −80 ◦C until needed for RNA isolation.

Total RNA was extracted from plants according to the Instructions of FastPure® Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). Reverse transcription reference HiScript® III All-in-one RT SuperMix perfect for qPCR (Vazyme, Nanjing, China) was used. Then, quantitative real-time PCR (qRT-PCR) analysis was performed on an ABI 7300 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The reaction system is as follows: SYBR® Premix Ex TaqTM (TaKaRa, Dalian, China) 5 μL, cDNA 2 μL, forward and reverse primers 0.4 μL each, ddH2O 2.2 μL. qRT-PCR reaction procedure is: pre-denaturation at 95 ◦C for 30 s, denaturation at 95 ◦C for 5 s, denaturation at 60 ◦C for 30 s, 40 cycles. Three biological replicates were performed for each sample. The relative expression levels of *LiNACs* genes were analyzed by the 2−ΔΔCt method [39] and the experimental data were analyzed by Excel 2010 and SPSS Statistics 20.0 software (IBM Corporation, Armonk, NY, USA). Finally, 21 pairs of gene-specific primers are shown in Table S1 and *LiEF-1α* [40] was used as the internal reference gene.
