2.4.1. Downy Mildew

Downy mildew, caused by the obligate fungus *Hyaloperonospora parasitica* (Pers. Fr.), is a destructive disease that affects brassica crops, including broccoli [77,78]. Broccoli plants are often stunned or killed when infected with downy mildew at the young seedling stage or infection can result in quality reduction and yield loss at the adult stage [79,80]. The disease is prevalent in cool weather, with initial symptoms of light green-yellow lesions on the upper leaf surface and later on the undersurface; the spot enlarges and turns yellow; white fungi are visible on the undersurface of leaves under high humidity conditions [79,80]. High resistance to downy mildew both at the young and adult stages is present in some broccoli germplasms and is controlled by a single dominant locus [79,81–84]. Resistance loci were mapped and linkage markers were developed for MAS, but the gene has not been cloned [79,82,83]. Giovannelli et al. identified 8 RAPD (random amplification of polymorphic DNA) markers linked to downy mildew resistance in broccoli (cotyledon and true leaf stage), among which two, UBC3596620 and OPM16750, were converted to SCAR (sequence characterized amplified regions) markers linked to the locus with 6.7 and

3.3 cM [82]. Farinhó et al. mapped the locus *Pp523* for downy mildew resistance to adult plants of broccoli and developed flanking RAPD markers OPK17\_980 and AFLP marker AT. CTA\_133/134, with genetic distances of 3.1 cM and 3.6 cM, respectively [83]; in a later study, new AFLP markers were developed and some of them were more user-friendly SCAR and CAPS (cleaved amplified polymorphic sequence) markers; sequencing indicated that *Pp523* is syntenic to the top arm end of *Arabidopsis thaliana* chromosome 1 [79]. We aligned the marker sequences to the broccoli HDEM reference genome [85] and found that the target *Pp523* region is 49.29–50.68 Mb on C8.
