Supplementary file

Description

Supplementary tables show the WOX family members and characteristics of Rosa chinensis ‘Old Blush’, Rosa wichuraiana ‘Basye's Thornless’ and Rosa rugosa , as well as the analysis of the collinear pair Ka/Ks existing in them. The supplementary figures include the phylogenetic tree of WOX family members of the three Rosa species and Arabidopsis thaliana, as well as the remaining supplementary maps to verify the gene function.

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1.Identification and structural domain of WOX proteins We used two methods to identify WOX genes of R. chinensis, R. rugosa and R. wichuraiana ‘Basye's Thornless’ plants. First, the 15 WOX sequences of A. thaliana (http://www.arabidopsis.org) were downloaded as reference sequences, and a local BLAST (version: 2.6.0+; parameters; -evalue: 1e-6) search was performed using the genome database (https://lipm-browsers.toulouse.inra.fr/pub/RchiOBHm-V2/; https://www.Rosaceae.org/). Second, the Hidden Markov Model (HMM) of the WOX family (PF00046) was downloaded and searched with HMM (version: 3.0; parameters: -domE: 1e-6). The protein sequences were subjected to an NCBI CD search (https://www.ncbi.nlm.nih.gov/Structure/cdd/) for structural domain screening, which resulted in the identification of WOXs in the three Rosa species. Conserved structural domains of the WOXs were analyzed with SMART and MEGA7.0, after which WebLogo (https://weblogo.threeplusone.com/create.cgi) was used to construct logo plots. 2. Phylogenetic tree construction and gene nomenclature To explore evolutionary relationships among R. chinensis, R. rugosa and R. wichuraiana ‘Basye's Thornless’, we performed phylogenetic analysis using MEGA7.0 and the neighbor-joining (NJ) method with a bootstrap set to 1000 . They were subsequently used to construct evolutionary trees with Arabidopsis for naming WOXs. Eventually, the WOX genes of the three Rosa species were combined to construct phylogenetic trees to determine the evolutionary relationships between the WOX genes of these plants. 3. Gene amplification and collinearity analysis TBtools (version: 2.016) was applied to obtain the loci of WOXs from GFF3. The genome and GFF3 cell line were constructed to extract collinearity information and highlight regions related to the collinearity of the target genes to construct a collinearity map of the WOXs. The simple Ka/Ks calculator program was used to estimate the synonymous substitution rate (Ks) and nonsynonymous substitution rate (Ka). 4. Virus-induced gene silencing (VIGS) The VIGS experiment was based on a previous report . The conserved RcWUS1 sequence was cloned and inserted into the tobacco rattle virus (pTRV2) vector To verify the efficiency of silencing, TRV was used as a negative control, and RcWUS1 was used as a positive control. A. tumefaciens strain EHA105 carrying the silencing vector was injected into single-bud stem segments with viable buds; cells were collected and resuspended in buffer after overnight culture (OD600 = 1.0). During the infection process, a vacuum pump was used for infection of the stem segments, with an air pressure of 0.08 MPa, suction for 10 min, pressure holding for 10 min, and gas release for 10 min. This process was repeated three times. Cuttings were collected after three days at 8 °C in the dark, and the phenotype was observed at room temperature.

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