Molecular characterization of apple (Malus × domestica borkh.) genotypes originating from three complementary conservation strategies

Abstract

Apple (Malus × domestica Borkh.) genotypes originating from different plant collections (field collection, in vitro plant collections undergoing or not undergoing cryopreservation) were screened and characterized by SSR markers. Shoot tips excised from plants grown in vitro were successfully cryopreserved by encapsulation-dehydration. The highest regrowth frequency (69%, cultivar Goldrush) of cryopreserved apices was achieved after 24 h of osmoprotection in 0.5 M sucrose, 3 h of desiccation, and 24% water content of alginate beads. No differences in morphological characteristics including shoot length and number and length of roots were observed between controls and plants recovered after cryopreservation. SSR markers were used for calculation of genetic similarities between plants from the field collection, in vitro-micropropagated plants, or plants regenerated after liquid nitrogen storage. The set of microsatellite markers showed a low level of polymorphism among the studied genotypes, which could be distinguished by a specific combination of alleles generated by CH03g07, CH05c02, CH05d11, and CH05e03 primers. The CH03g07, CH05c02, CH05d11, CH05e03, GD96, GD147, and GD162 SSR markers exhibited low levels of polymorphism, while CH04AE07, CH04g10, GD100, and GD142 were nonpolymorphic. The Dice coefficient confirmed the effectiveness of SSRs for distinguishing between plants from ex situ collections and preserved plants. No major differences between ex situ plants, micropropagated plants, and plants recovered after cryopreservation were observed.