Bioinformatics and primer design

The genes of interest were obtained by blasting the thale cress protein sequences in NCBI, as well as by querying the Genome Database for Rosaceae (GDR; available at https://www.rosaceae.org/) and the cherry database (available at http://cherry.kazusa.or.jp). Multiple alignments were performed in CLUSTAL-Ω (http://www.ebi.ac.uk/Tools/msa/clustalo/). The primers were designed with Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and checked with OligoAnalyzer 3.1 (http://eu.idtdna.com/calc/analyzer). All the primers and their relative features were previously described⁵, or are reported in Table ​Table6.6. The maximum likelihood phylogenetic tree of XTHs was constructed from full-length protein sequences from sweet cherry and other species, namely poplar, tomato, thale cress, nasturtium⁶⁷. The sequences were aligned with CLUSTAL-Ω and the tree obtained by using IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) with the “Auto” parameter to identify the best-fit substitution model¹⁰¹. The tree was rooted with Bacillus licheniformis lichenase (accession no {"type":"entrez-protein","attrs":{"text":"CAA40547","term_id":"39559","term_text":"CAA40547"}}CAA40547).

List of primers used for gene expression analysis. Details relative to the sequences of the target genes, together with primers’ amplification efficiency %, melting temperature, amplicon sizes and accession numbers are provided