Mx3005p real time pcr system

Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Germany). The sequences of anchor markers used for initial mapping were published previously (Song et al., 2015 (link)). For fine mapping the GmPGL2 locus, new primers of InDel markers were synthesized for polymerase chain reaction (PCR; Supplementary Table S1). The candidate genes were amplified by PCR, and the PCR products were sequenced by Sangon Biotech (Shanghai, China). The phylogenetic and syntenic analyses were carried out as described previously (Dai et al., 2018 (link)).
The total RNA was extracted from tissue samples using TRIzol reagent (Qiagen, Germany) according to the manufacturer’s instructions. RNA samples were reverse transcribed using primer Script I (TaKaRa, Japan). An 18-mer oligo (dT) primer for nuclear-encoded genes or random primers for plastid genes were used for first strand cDNA synthesis. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR^® Premix Ex Taq^™ Kit (TaKaRa, Japan) on an MX3005P Real-Time PCR System; the primers used are listed in Supplementary Table S1. The PCR program was as follows: 95°C for 15min, followed by 40cycles at 95°C for 10s, 58°C for 20s, and 72°C for 20s. Actin11 was used as the reference gene (Jian et al., 2008 (link)). Three biological replicates were used for gene expression analysis.

Fifteen genes were selected for validation by Quantitative PCR (qPCR). qPCR was performed in a Mx3005P Real-Time PCR System by using a SYBR Premix kit (Takara, Dalian, China). Fifteen specific primer pairs were designed (Supplementary Table S1). HbACT7 was used as an internal control. qPCR conditions were described as follows: 30 s at 95°C for denaturation, 40 cycles for 10 s at 94°C, 30 s at 60°C, and 15 s at 72°C for amplification. Data obtained from qRT-PCR were clustered in accordance with the instructions provided by Stratagene (Santa Clara, CA, USA). All relative expression data were based on three individual reactions. Analysis of variance (ANOVA) was used to compare the statistical difference based on Fisher’s LSD test. Means were considered significantly different based on the P value (P < 0.05 and P < 0.01), which are indicated by asterisks (*) when P < 0.05, or indicated by (**) when P < 0.01.