Trizol kit

Total RNA was isolated from root tissues using a TRIZOL kit (TransGen, Beijing, China), and cDNA was synthesized using a FastQuant RT kit (Tiangen, Beijing, China). Quantitative RT-PCR was carried out using an Applied Biosystems QuantStudio 7 Flex Real-time PCR system (Applied Biosystems, CA, United States). Gene-specific primer pairs (Supplementary Table 1) were used, and all quantitative RT-PCR assays were carried out using three biological and three technical replicates. The soybean Actin gene was used as an internal control. PCR was performed using the following cycling parameters: 95°C for 3 min, 60°C for 30 s, 40 cycles of 95°C for 15 s, and 60°C for 1 min.

Total RNA was isolated using the TRIzol kit (TransGen Biotech Co., Ltd., Beijing, China). The first-strand cDNA was synthesized with oligo-dT primers using TransScript First-Strand cDNA Synthesis Supermix (TransGen Biotech Co., Ltd., Beijing, China). qRT-PCR was performed in a 20-μl reaction volume using a Roche LightCycler® 480 (Roche Diagnostics GmbH, Mannheim, Germany) for three biological replicates. Wheat β-Actin was used as an internal reference. Relative mRNA levels were calculated using the 2 ^−ΔΔCT method. The qRT-PCR primers for PEG, four phytohormones and FHB are supplied in Additional file 26: Table S14.

Total RNA was extracted from the plant tissues using a TRIzol kit (TransGen Biotech, Beijing, China) and was reverse transcribed into cDNA using a PrimeScript^™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturers’ instructions. The qRT-PCR analysis was performed in a 20 μL reaction volume containing 10 μL of SYBR Premix ExTaq^™ (Takara, Dalian, China), 2 μL of cDNA, and 0.5 μM of each primer. The primer pairs used for qRT-PCR are listed in S1 Table. ACTIN was used as a reference for normalization. The cycle thresholds were determined using a Roche Light Cycler 480 II sequence detection system (Roche, Shanghai, China).

Total RNA was extracted from the plant tissues using a TRIzol kit (TransGen Biotech, Beijing, China) and was reverse into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed in a 20 μL reaction volume containing 10 μL of SYBR Premix ExTaq™ (Takara, Dalian, China), 2 μL of cDNA, and each primer at 0.5 μM. The primer pairs used for qRT-PCR are listed in Table S1. QPto18S was used as the reference for normalization. The cycle thresholds were determined using a Roche Light Cycler 480 II sequence detection system (Roche, Shanghai, China). The levels of mature microRNAs were detected and quantified using a highly sensitive quantitative RT-PCR method^{24 (link)}. The mature microRNAs, including miR156, miR160h, miR858, and miR168, were reverse transcribed and measured using Mir-X miRNA First-Strand Synthesis and a SYBR qRT-PCR kit (Vazyme Biotech, Nanjing, China); miR168 was used as the reference for normalization. The primers used for qRT-PCR are listed in Table S1.

Total RNA was extracted from switchgrass stems using a TRIzol kit (TransGen Biotech, Beijing, China) and was reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed in a 20-μl reaction volume that contained 10 μl of SYBR Premix ExTaq (Takara, Dalian, China), 2 μl of cDNA (first strand cDNA, diluted five times), and 0.5 μM of each primer. The primer pairs used for qRT-PCR are listed in Supplementary Table 1. PvUBQ2 (Pavir.1KG065600) was used as the reference for normalization (Huang et al., 2014 (link)). The cycle thresholds were determined using a Roche Light Cycler 480 II sequence detection system (Roche, Shanghai, China).

The 21-day seedlings of the ZGY1578 hybrid and its parents ZG7A and ZH1578 were treated with 120 mM NaCl solution for 12 h, and then the roots were harvested for RNA extraction. The roots of untreated seedlings were used as the control. The total RNA was extracted using the TRIzol kit, according to the manual instruction (TransGen, www.transgen.com; accessed on 1 June 2022). The construction of the complementary DNA (cDNA) libraries and BGISEQ-500RS sequencing was performed at BGI Shenzhen Co., Ltd., Shenzhen, China. Three biological replications were conducted.