Sequence Analysis and Cloning

To identify GA2ox genes in the peach genome, a BlastP with default parameters of the Phytozome database¹ was performed based on the conserved domain of genes in the 2-oxoglutarate-dependent oxygenase family using the amino acid sequences of seven Arabidopsis GA2oxs as queries. The search results were aligned by ClustalW algorithm with default parameters to reduce duplicate and redundant sequences (Larkin et al., 2007). Gene information from the JGI database was then used to predict the genomic positions of the PpGA2ox genes. The PpGA2ox genes were renamed, plotted on the peach chromosomes, and visualized with MapGene2 Chrom² (Jiangtao et al., 2015).

The amino acid sequences of all GA oxidases used in this study were downloaded from the Phytozome and NCBI databases³. Sequence logos were generated using the online WebLogo platform⁴ (Crooks et al., 2004). A phylogenetic tree was constructed using the neighbor-joining method (NJ) method in MEGA (version 5.1) (Tamura et al., 2011). The main parameters were set as follows: the model was set to “p-distance,” the GAPs was set to “pairwise deletion,” and bootstrapping was performed with 1000 replicates. Default parameters were used for all bioinformatics analyses, except for those specifically mentioned.

The full-length sequences of the PpGA2ox genes were cloned from RNA obtained from peach shoot tips using specific primers and ligated into the pEASY^®-Blunt Vector (TransGen Biotech). PCR products that produced a single band on the gel were purified and sent to Sangon Biotech (Shanghai⁵) for sequencing. All primers used in this study are listed in Supplementary Table S3.