Application of MLVA Analysis of Chloroplast Microsatellite Loci for Differentiating Wild and Cultivated Malus spp. germplasm

Abstract

Background and Aim. The genus Malus comprises wild and cultivated apple forms of major importance for breeding, the conservation of genetic resources, and studies of fruit-crop evolution. Although SSR (Simple Sequence Repeat) and SNP markers are widely used to assess genetic diversity in apples, organellar markers remain important because the chloroplast genome predominantly reflects
a single maternal lineage and has a simpler, more conserved structure than the nuclear genome. The
aim of this study was to investigate the genetic variability of chloroplast loci in representatives of the genus Malus using multiple-locus analysis of tandem repeats and to identify differences between wild and cultivated germplasm.
Materials and Methods. Forty-two chloroplast assemblies deposited in GenBank/ENA and one assembly generated in this study were analysed; the chloroplast genome of Prunus ursina (NC_068527) served as the outgroup. Microsatellite and VNTR-like loci were identified with MISA, orthologous
loci were clustered with custom Python scripts, and primers for the variable loci were designed with Primer3. MLVA profiles were built with MLVA_finder, pairwise distances were calculated under the Stepwise Mutation Model, and a neighbour-joining tree was constructed.
Results. MISA analysis identified 2,918 SSR loci, of which 96.1% were mononucleotide repeats, predominantly poly-A/T. Clustering reduced these to 92 orthologous loci, 48 of which (approximately 52%) varied in repeat number. The eight most informative primer pairs were selected for the cpMLVA panel. The resulting tree showed tight clustering of M. domestica samples, more peripheral positions for
wild species, and clear separation of the Prunus outgroup. BLAST analysis of the complete chloroplast genome confirmed that a record annotated as M. fusca (OR897844.1) is taxonomically questionable.
Conclusion. Chloroplast tandem repeats can serve as a compact marker system for differentiating wild and cultivated representatives of the genus Malus and for detecting erroneous metadata in public databases.