Genetic diversity and nucleotide sequence analysis of powdery mildew marker and Vf2RAD resistant gene in apple (Malus domestica) land races

Introduction: DNA sequencing-based methods and nucleotide sequence analysis have become the most common molecular approaches currently used for molecular typing purposes and phylogenetic diversity analysis. Methods: In this study, the nucleotide sequence variations of Powdery mildew resistance gene marker (CH03c02) and the apple scab resistance gene (Vf2RAD) beside phylogenetic diversity of seven apple landraces have been investigated. The two-locus have been successfully cloned and their nucleotide sequences were determined across all studied landraces. Results: Results of sequence alignment of the Powdery mildew resistant locus (CH03c02), compared with that of the published sequence of the same locus of Discovery genotype (HiDRAS), revealed that the nucleotide variations of this locus ranged from 1 to 28 nucleotide substitutions across all seven apple landraces. Whilst, the nucleotide variations of VF2RAD ranged from 2-8 nucleotide substitutions across all the investigated landraces. The highest genetic distance (0.062) was between Amara and Barwari. Whereas, the lowest genetic distance (0.0015) was found between each of the Lubnani, Rechard, Ispartal, and the Ahmadagha. The nucleotide sequences of the two loci were concatenated and implemented to build a Neighbor-Joining tree. The seven apple landraces were successfully grouped into two main genetic clusters (C1 and C2) in the phylogenetic tree. Conclusions: It can be concluded that the cloning approach used in the current study was found to be very successful and helpful for obtaining the full nucleotide sequences of these two loci. The investigated loci were displayed nucleotide variations among the studied landraces. And, finding of these variations was allowed the distinguishing and discrimination of these landraces.