Mastercycler ep realplex device

To ensure the libraries’ reliability, we randomly selected 10 DEGs from each library and validated the data by qRT-PCR with three biological replicates. Gene specific primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and the reference gene was the chrysanthemum EF1α (Elongation Factor 1a) gene (Genbank accession number KF305681). The sequences of all primers including those of EF1α were listed in Tables S1 and S2. The amplification with these primers was specific and the efficiency of qPCR was about 100%. qRT-PCR was performed on the Mastercycler ep realplex device (Eppendorf, Hamburg, Germany). Each 20 μL qPCR mixture contained 10 μL SYBR Premix Ex Taq II (Takara), 1 μL forward primer, 1 μL reverse primer, 3 μL ddH₂O and 5 μL cDNA template. The cDNA was synthesized from 1 µg total RNA with PrimeScript^® Reverse Transcriptase (Takara), following the manufacturer’s instructions. The quality of RNA met the criterion that the values of A260/A280 were around 2.0 and the values of A260/A230 were all larger than 2.0. The PCR cycling regime was initially denatured (95 °C/2 min), followed by 40 cycles of 95 °C/10 s, 55 °C/15 s and 72 °C/20 s. A melting curve was followed each assay to confirm the amplicons’ specificity. Relative expression levels of the DEGs were calculated by 2^−ΔΔCt method [41 (link),62 (link)].

To validate the high-throughput sequencing results of chrysanthemum embryo miRNAs, 21 differentially expressed miRNAs were randomly selected and analyzed by qRT-PCR. RNA samples were reverse-transcribed using PrimeScript miRNA qPCR starter kit ver 2.0 (Takara Bio). A final volume of 20 μL was achieved by the addition of 5 pmol of the forward and the reverse primers (S1 Table). The conditions for the PCR amplification were as follows: polymerase activation at 95°C for 3 min, followed by 40 cycles of 95°C for 20 s, 60°C for 20 s and 72°C for 45 s. All reactions were performed in triplicate on a Mastercycler ep realplex device (Eppendorf, Hamburg, Germany) according the kit’s protocol. The EF1α gene was used as the reference gene [39 (link)], and relative expression levels were calculated by the 2^-△△CT method [40 (link)].

To ensure the libraries’ reliability, we randomly selected 29 differentially expressed genes and validated the data by qRT-PCR using three biological replicate samples. The qRT-PCR assays were conducted as described by Song et al [44 (link)] on a Mastercycler ep realplex device (Eppendorf, Hamburg, Germany). In addition, qRT-PCR also performed on the 40 DEGs shown in Tables 5 and 6 using three biological replicates from two crosses. Gene-specific primers (sequences shown in Additional file 7: Table S3) were designed using PRIMER3 RELEASE 2.3.4 [45 (link)]; the reference sequence for the quantitative expression analysis was the Elongation Factor 1a (EF1a) gene, which is stably expressed in chrysanthemum [16 (link), 46 (link)]. Relative transcript abundances were calculated using the 2^−ΔΔCt method [47 (link)].