Novel PCR markers for discriminating RMia resistance alleles in Prunus rootstock breeding.

Root-knot nematodes are pest of many plant species, particularly in southern Europe, where Meloidogyne incognita, M. arenaria and M. javanica are the most common species. In Prunus rootstock breeding, restrictions on nematicide use has increased importance of incorporating genetic resistance. Three resistance genes have been identified in Prunus: Ma, RMja and RMia. The latter is a TNL gene located on the linkage group 2 that confers resistance to M. arenaria, M. incognita and M. ethiopica. Resistance is expressed in both homozygous and heterozygous dominant genotypes, but not in homozygous recessive individuals. To improve the precision of existing markers and develop markers more closely associated with RMia, an in silico characterization of the genomic region containing this gene was conducted in six Prunus genotypes using 'Texas' almond reference genome v2.0 and a BAC sequence containing RMia. Comparative analysis identified, in the almond reference genome, the resistance-associated insertion previously reported in peach, characterized it as the LTR982 retrotransposon present only in the susceptible RMia haplotype. This previously described structural polymorphism was used to develop novel codominant molecular markers targeting conserved flanking regions. Among the primer pairs tested, three showed high discriminatory capacity. RTP-RMia-F3/R3 amplified a fragment in heterozygous and homozygous recessive individuals, but not in homozygous dominants. The ACI_9660_2 and NEM_6908_4 primers reliably distinguished resistant from susceptible genotypes. Together, these markers provide valuable tools for marker-assisted selection in Prunus rootstock breeding, enabling both resistance screening and allelic discrimination. The molecular results were further validated in hybrid rootstock genotypes through artificial inoculation assays.