Molecular Polymorphism and Evolution of CYP736 Cytochrome P450 Monooxygenase Genes across Wild Vitis Species and Muscadinia rotundifolia

Cytochrome P450 monooxygenases (CYPs) form one of the largest plant enzyme superfamilies and contribute to defense-related secondary metabolism. The grape gene CYP736B is transcriptionally up-regulated in Pierce’s-disease (PD)–resistant grapevines and down-regulated in susceptible grapevines after infection by Xylella fastidiosa , yet its genetic polymorphism across wild Vitis germplasm has remained unexplored. Here we characterized the complete protein-coding sequences of CYP736 homologs from ten wild North American grape accessions representing eight Vitis species, interspecific V. arizonica × V. rupestris hybrids differing in PD phenotype, and Muscadinia rotundifolia . All ten sequences encoded intact open reading frames of 494–496 amino acids that retained the canonical P450 signatures, including the heme-binding decapeptide (FxxGxRxCxG), the ExxR (K-helix) and PERF motifs, and the oxygen-binding I-helix. Pairwise amino-acid identity ranged from 93.3% to 100%. Forty-five variable residues (9.1%) were concentrated in the N-terminal membrane anchor and putative surface/substrate-recognition regions, whereas the catalytic core was invariant. Phylogenetic analysis resolved two well-supported subfamilies—a CYP736B-type clade (eight accessions) and a CYP736A-type clade ( V. rupestris and M. rotundifolia )—nested within a monophyletic CYP736 group (100% bootstrap) distinct from CYP71, CYP76 and CYP716. Pairwise dN/dS ratios (mean ≈ 0.57; 42/44 < 1) indicated predominantly purifying selection with relaxed constraint on surface residues. The PD-resistant and PD-susceptible hybrid haplotypes differed by only two nucleotides, corresponding to a single conservative substitution (Ile218→Val), indicating that CYP736B-associated resistance is unlikely to arise from coding-sequence divergence and instead supports a regulatory basis.