Molecular Assessment of Clonal Fidelity in Micropropagated Grape (Vitis spp.) Rootstock Genotypes Using RAPD and ISSR Markers

Abstract

Micropropagated plantlets derived from three different grape rootstock genotypes namely, Dogridge (Vitis champini), SO4 (V. berlandieri×V. rupestris) and ARI-H-144 (V. vinifera×V. labrusca) were subjected to randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analyses in order to evaluate their genetic stability and/or detect likely existing variations among in vitro derived plantlets. A dozen RAPD (10-mer) and ten ISSR (dinucleotide contained repeats) primers were used for PCR and reproducible band profiles were obtained. The 84 and 81 distinct and scorable band classes (a total of 1,914 and 1,980 scorable bands) with an average of 7.0 and 8.1 bands per primer were obtained by RAPD and ISSR, respectively. Although higher numbers of bands were obtained by ISSR rather than RAPD, but none of the primers showed polymorphism among micropropagated plantlets and their respective mother plants. The profiles generated based on the two marker systems were found to be highly uniform and monomorphic. Cluster analysis further confirmed genetic stability of micropropagated plantlets. Jaccard's similarity coefficients obtained for both markers in mother plants and their in vitro regenerants were estimated to be 1.00 but three sets of genotypes were grouped into two major clusters with similarity coefficients of 0.53 (RAPD) and 0.63 (ISSR). The molecular analyses precisely proved the production of genetically stable grape plantlets and certified the application of micropropagation protocol to be developed on a commercial scale.