Primer prime plus 5 software version 3

Based on transcriptomes analysis, qPCR was used to confirm the expressed pattern of DRGs in response to DS in two Shanlan upland rice lines. The RNA sample used is same as transcriptome determinations. RNA extraction and reversed cDNA were performed as described previously [25 ]. The qPCR analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems, Forster City, CA, USA, 4309155) and a real-time PCR system (ABI StepOnePlus, Applied Biosystems, USA). Primers for qPCR were designed using Primer Prime Plus 5 Software Version 3.0 (Applied Biosystems, USA). Primers were listed in Table S2. The qPCR running program consists of a reverse transcription step at 48 °C for 30 min and a Taq polymerase activation step at 95 °C for 30 s, followed by PCR: 45 cycles at 95 °C for 15 s, 61 °C for 20 s, and 72 °C for 30 s, ensued by a melting cycle. Assays were performed with three biological samples from each treatment, and measurements were replicated three times. The actin1 gene was used as an expression control (housekeeping gene). Relative expression of a gene against Actin1 was calculated as 2^−ΔΔCT (ΔCT = CT, gene of interest^−CT), as described earlier [26 (link)]. The expression levels of three known drought-resistant genes OsLEA3–2 (LOC4332688) [27 (link)] and RePRP2.2 (LOC4343033) [28 (link)] were also tested as control.

To screen the candidate genes controlling flowering time, we selected 20 accessions with contrasting flowering time values (10 early and 10 late) from experiments performed in 2020 and 2021 at Heilongjiang soybean field base. We further conducted quantitative real-time PCR (qRT-PCR) analysis (ABI StepOnePlus, Applied Biosystems Co., Ltd., USA) for the candidate genes identified by LD-block analysis. For qRT-PCR analysis, samples were collected from the top fully expanded leaves of 60-day old plants. The total RNA was extracted using Ambion PureLink RNA mini kit according to manufacturer’s instruction. Two micrograms of total RNA were reversely transcribed to cDNA with SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Technologies, USA). The qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, USA) with cDNA as a template with the following cycling parameters: 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s. The specific primers for qRT-PCR were designed using Primer Prime Plus 5 Software Version 3.0 (Applied Biosystems, USA). The primer sequences are shown in Table S1. Relative expression of gene against actin, a housekeeping gene, was calculated as follows: 2^−ΔΔCT (ΔCT = CT, gene of interest−CT, Actin1 [27 (link)];). Six biological replicates were performed.