Ultrasybr green 1 mixture

The specific primers of the PtFAD genes were designed to identify the expression patterns using a RT-qPCR (Supplemental Table S8). The total RNA was extracted using the hexadecyl trimethyl ammonium bromide (CTAB) method, as previously described, [71 (link)] for all poplar samples. The concentration and quality of the RNA were investigated using a NanoDrop One/OneC spectrophotometer (Thermo Scientific, Waltham, MA, USA). For the reverse-transcription, the PrimeScript™ RT Master Mix (TaKaRa, Kusatsu, Japan) was applied to reverse-transcribe 1 μg of RNA. The gene expression levels were quantified using the ABI 7500 Fast Real-Time PCR System (Applied Biosystem, Waltham, MA, USA) and the UltraSYBR Green I Mixture (CWBIO, Beijing, China). The RT-qPCR amplification reactions were performed in 20 mL volume with the following procedure: pre-denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The melting curve conditions were 60 °C for 60 s and 95 °C for 15 s. Three experimental repetitions were conducted per selected sample. The 2^−ΔΔCt values were estimated to indicate the relative expression levels of the PtFAD genes. The TBtools was used to generate heatmaps according to the 2^−ΔΔCt values.

The samples were manually collected from poplar leaf, stem, and root and immediately flash frozen and stored in a freezer for RNA extraction. The CTAB method, as previously described [51 (link)], was applied to carry out RNA extraction. The 2% agarose gel electrophoresis and a NanoDrop One/OneC spectrophotometer (Thermo Scientific, Waltham, MA, USA) were used to determine the integrity and concentration of RNA, respectively. Total RNA (1 μg) was reverse-transcribed to the first-strand cDNA using the PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). The gene specific primers (Table S2) were designed and chosen to analyze the expression levels of PtLACS genes. The qRT-PCR assays were performed using the UltraSYBR Green I Mixture (CWBIO, Beijing, China) and the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The PCR was performed with the following procedure: thermal cycling, including 95 °C for 5 min; 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s; and dissociation-curve analysis, including 60 °C for 60 s and 95 °C for 15 s.

The leaves, stems, and roots harvested from the P. trichocarpa, ‘Shanxinyang’ (P. davidiana × P. bolleana Loucne), and ‘Nanlin 895’ (P. deltoides × P. euramericana) were used for tissue-specific gene expression analysis. Additionally, the leaves of ‘Nanlin 895’ grown on MS medium were treated with 2 mM of H₂O₂, 10% PEG₆₀₀₀, 200 mM of NaCl, and 200 μM of ABA, and leaves were collected at 0 (untreated leaves served as a control), 1, 6, 12, 24, and 48 h after each treatment. The abiotic stress treatments were performed with three replicates, with three poplars per replicate. Leaves harvested from treated and untreated poplars were stored at −80 °C.
Total RNA was extracted from various tissues and treated and untreated leaves using an RNA extraction kit (Takara, Japan). The reverse transcriptase (Takara, Japan) was used to synthesize first-strand cDNA of poplar. The UltraSYBR Green I Mixture (CWBIO, China) with 10 μL of Green I Mixture in a 20-μL reaction volume was applied to identify the PtCCD gene expression patterns. The Primer3.0 online tool was used to design the primers used in qRT–PCR. The qRT–PCR procedure was as follows: 95 °C for 10 min; 40 cycles of 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The relative PtCCD expression levels were identified according to the 2^−ΔΔCT method, with the Ptactin (XM-006370951), considered the internal control.