Genetic engineering of Glxuronoxylan Methyltransferase genes in Populus alba x tremula using clustered regularly interspaced short palindromic repeat (CRISPR) - Cas9 (CRISPR associated protein 9)

Figure S2.1 GXM gene models based on Populus trichocarpa V4.1 sequence.

The gene models are generated using the latest genomic sequences and the annotation available in PLAZA 5.0 (Van Bel et al. 2022).

Figure S2.2. Golden Gate Assembly confirmation of PtaGXM targeting CRISPR vector & Figure S2.3. Gateway Assembly confirmation of PtaGXM targeting CRISPR vector.

CRISPR-Cas9 construct targeting the GXM genes in Populus alba x tremula were assembled as described in Lowder et al. 2015. Successful cloning was confirmed using restriction enzyme digestion and gel electrophoresis (1%).

Figure S2.4. Propagation of ptagxm mutant lines for greenhouse trial.

Successfully transformed and edited Populus alba x tremula plantlets were propagated via root cutting and grown on hygromycin selection media for six weeks prior to greenhouse transplant.

Figure S2.5. Plant height of ptagxm mutants during the greenhouse growth trial and post-harvest growth.

Heights of the plants undergone greenhouse growth trial were measured every 4 weeks. Growth variation of the ptagxm transgenic plants were subjected to Roche's t-test to evaluate the statistical significance against the wild type plants.

Figure S2.6. Microscopy analysis of ptagxm mutant stems.

The ptagxm plants apical meristem at the third and fourth node were harvested after four-month of greenhouse growth trial. The stem samples were subjected to light microscopy using a standard wax-embedding and toluidine blue staining.

Table S2.12. Sequence similarity among the four GXM genes in Populus trichocarpa.

Protein sequences of the GXM genes were aligned using Clustal Omega, and their sequence similarity was calculated.