Molecular Pathogens 2024, Vol.15, No.3, 155-169 http://microbescipublisher.com/index.php/mp 160 genomes of different PM pathogens has revealed unusual bimodal GC distributions and exceptionally long cytochrome b genes, which are indicative of complex evolutionary processes (Zaccaron and Stergiopoulos, 2021). 5 Localization of Genes Resistant to PM 5.1 Techniques used for gene mapping in Cucurbitaceae Recent advancements in gene mapping techniques have significantly contributed to the identification of PM resistance genes in Cucurbitaceae plants. CRISPR/Cas9-mediated mutagenesis has been effectively used to generate PM resistance in cucumber by targeting the CsaMLO8 gene, which encodes susceptibility factors for the pathogen P. xanthii (Figure 3) (Shnaider et al., 2022). Additionally, QTL-seq combined with bulked segregant analysis has been employed to identify resistance loci in Korean cucumber inbred lines, leading to the discovery of two QTLs on chromosomes 5 and 6 (Zhang et al., 2020). High-resolution melting markers (HRM) have also been used to map resistance regions in melon, narrowing down candidate intervals to specific chromosomal regions (López-Martín et al., 2022). Figure 3 Generation of CsaMLO8mutant cucumber plants using CRISPR/Cas9 (Adopted from Shnaider et al., 2022) Image caption: Panel A illustrates the CsaMLO8 gene structure with exons represented by blue boxes and sgRNA target sites indicated by black arrows, where the sequence of each sgRNA and the position of the PAM site are shown above. Underlined text denotes restriction sites used for screening mutant plants. Panel B shows the screening of F1 progeny for mutations at the target site through PCR amplification followed by restriction enzyme digestion, with nondigested bands indicating mutations. Panel C displays the detection of a significant deletion (1 280 bp) and a small insertion (10 bp) in the CsaMLO8-cr-2 line via PCR of the region between exon 1 and exon 5. Panel D presents sequence alignments comparing the 'Ilan' control, a single T0 plant with a 2-bp substitution in exon 5, and various F1 CsaMLO8mutant lines (Adapted from Shnaider et al., 2022)
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