Validation of Chrysanthemum Gene Expression

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)].