Sybr premix

qPCR analysis was performed at a series of cDNA dilutions, including 10, 10², 10³, 10⁴, 10⁵ and 10⁶. The primers (10 µM) were used at gradient concentrations of 0.25, 0.5, 0.75, 1, 1.25 and 1.5 µl. The amplification efficiency of each primer pair ranged from 0.9–1.1. The primer concentration with the highest amplification efficiency was selected for subsequent quantitative analysis. The qPCR reaction system included 2X of 10 µl SYBR Premix (Tiangen Biotech Co., Ltd), 10 µM forward and reverse primers (0.6 µl of each) and 1 µl cDNA, with total volume adjusted to 20 µl using ddH₂0. The prepared reaction solution was analyzed using Bio-Rad fluorescence PCR (Bio-Rad Laboratories, Inc.). Reaction conditions were as follows: Pre-denaturation at 95°C for 15 min and 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec, and extension at 72°C for 20 sec. The fluorescent signal was recorded during the extension phase of each cycle using the CFX96 real-time PCR detection system (Bio-Rad PCR; Bio-Rad Laboratories, Inc.). Melting curve analysis (95–65°C) was performed following the reaction.

Predicted target genes were obtained from the B. napus Sequence database (http://www.genoscope.cns.fr/brassicanapus/). The software Primer Premier 5.0 (PREMIER Biosoft Int., Palo Alto, CA, USA) was used to design specific primers for quantitative RT-PCR (Table S2), and qPCR was performed using a CFX96 Real-time System (BIO-RAD, USA) with SYBR® Premix (TIANGEN, Beijing, China). Briefly, each 20 μL PCR reaction contained ~100 ng cDNA, 8 μL 2.5 × RealMasterMix/20 × SYBR solution, and 200 nM each primer. The PCR conditions were as follows: 94°C for 2 min, followed by 40 cycles of 94°C for 15 s and 60 °C for 30 s. A final ramping stage of 65–95°C was performed to confirm the absence of multiple products and primer dimers. Actin was used for each sample as an endogenous control. All samples were subjected to at least three technical replicates. Data were analyzed using the Ct (2^−ΔΔCt) method described above. Moreover, the software SPSS19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis in this study.

Total RNA was extracted and subjected to reverse transcription using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan). Quantitative real-time PCR (qPCR) was performed using the SYBR Premix (Tiangen, Beijing, China). Relative mRNA levels were normalized to that of GAPDH. The primers used in qPCR analyses were as follows: UBAP1 (F: 5’-GTTGGGTGCAGATTTTCATGG-3’, R: 5’- CTGTACTTCTCTGACAACCTG-3’); and GAPDH (F: 5’-AGCCACATCGCTCAGACACCA-3’, R: 5’-ATGTAGTTGAGGTCAATGAA-3’).

The putative candidate genes were searched within the interval of the associated SNPs (± 200 kb) based on the Darmor-bzh B. napus reference genome v4.1. Functional annotation was implemented to predict the function of candidate genes using a Blastp program against to the Arabidopsis thaliana TAIR10 protein database.
The expression levels of silique length-related genes were validated by real-time PCR (qRT–PCR) using a CFX96 Real-time System (BIO-RAD, USA). In brief, 1 µg of RNA from each sample was used for cDNA synthesis; the expression of the silique length-related genes in different rapeseed samples was evaluated using SYBR® Premix (TIANGEN, Beijing, China) and a CFX96 Real-time System (BIO-RAD, USA). Gene-specific primers are listed in Additional file 19: Table S9. Average Cq values were calculated from three replicates, and expression levels were normalized to the reference gene Bna.Actin7 using the 2^−ΔΔCt method.

Total RNA was isolated from various tissues and buds of TE5A and homozygous TE5 plant lines using a plant RNA extraction kit (TIANGEN¹) as recommended by the manufacturer. RNA (5 μg) was synthesized to first-strand cDNA using the ReverTra Ace-a-First-Strand cDNA Synthesis Kit (TIANGEN¹). The reverse-transcription products from various tissues were then used as templates for RT-PCR and qRT-PCR. qRT-PCR was performed in 96-well optical plates containing SYBR Premix (TIANGEN) on an OPTICON 2 PCR instrument (MJ Research). Actin was used as a control for normalization. The primers RT1-F/R were used for the detection of MS5^d transcripts (Supplementary Table S1). All reactions were performed in three independent experiments. To determine the full-length transcript of MS5^d, 5’-RACE and 3’-RACE were performed using total RNA from young buds with the SMART RACE cDNA amplification kit (Takara; Supplementary Table S1).