Colored horizontal rectangles on panels (A,C) represent the exons, numbered from 1 to 15: dotted rectangles represent extracellular domains of the resulting protein, while blue and striped rectangles represent membrane and cytoplasmic domains, respectively. On panels (A,C) (gDNA), pie charts display, for each SNP along the coding sequence, the genotype frequencies calculated among the 32 cultivars. Panels (C,D) exhibit all non-synonymous nucleic acid substitutions (C) and amino acid replacements (D) identified in CsMLO4 (located on chromosome 2) from the same 32 hemp and drug-type cannabis cultivars as shown on panels (A,B). Panels A and B exhibit all non-synonymous nucleic acid substitutions (A) and amino acid replacements (B) identified in CsMLO1 (located on chromosome 1) from 32 hemp and drug-type cannabis cultivars. Protein structure and polymorphism analysis of the two clade V genes ( CsMLO1 and CsMLO4) in 32 distinct cannabis cultivars aligned against the CBDRx reference genome. MLOs with one asterisk (*) have been experimentally demonstrated to be required for PM susceptibility (Büschges et al., 1997 Feechan et al., 2008 Wan et al., 2020), while MLOs with two asterisks (**) have been identified as main probable candidates for PM susceptibility (Pessina et al., 2016). Number on a node indicates the percentage of bootstrap when higher than 65% (black), or the posterior probabilities of major clades and subclades, according to a Bayesian phylogenetic inference performed on the same alignment (red) (Supplementary Figure 1). The seven defined clades are indicated, as well as potential subclades identified in this study (inner circles). Phylogenetic relationships were estimated using the maximum likelihood method implemented in PhyML + SMS with 1,000 bootstrap independent replicates. Chlamydomonas reinhardtii was used as an outgroup. Phylogenetic tree of manually curated CsMLO proteins (bold) with MLO proteins from selected species ( Arabidopsis thaliana, Prunus persica, Vitis vinifera, Hordeum vulgare, and Zea mays). Phylogenetic relationships of CsMLOs based on maximum likelihood analysis. Those will be useful for breeding purposes in order to develop resistant cultivars.Ĭannabis MLO fungal disease plant–pathogen interactions powdery mildew susceptibility genes. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of seven transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Fifteen Cannabis sativa MLO ( CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. In several studies, the inactivation of those genes resulted in durable resistance to the disease. The Mildew Locus O ( MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes.
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