Trait variation, molecular identification and genetic diversity analysis of F1 generation of interspecific hybridization of Rubus

Interspecific hybridization is an effective approach for creating novel Rubus germplasms, but early hybrid authentication is difficult when parental genetic backgrounds are complex and ploidy levels differ. In this study, we evaluated the F1 progeny derived from a cross between the hexaploid primocane-fruiting blackberry cultivar ‘Freedom’ (female parent) and the diploid Rubus hirsutus male parents via seven SSR primers. To account for parentage effects, hybrid identification was summarized by cross combination: Freedom × R-1/2 (n = 5), Freedom × R-3 (n = 35), and Freedom × R-4 (n = 41). Across all crosses, 62 seedlings (3 + 27 + 32) were authenticated as true hybrids (76.5%). Flow cytometry profiles of two vigorous F1 individuals revealed that the nuclear DNA fluorescence peak positions were intermediate between those of the diploid male parent and the hexaploid ‘Freedom’, which was consistent with a tetraploid level. From the authenticated hybrids, 26 vigorous seedlings were selected for phenotypic and physiological evaluation. Considerable variation was observed among the selected hybrids in terms of plant height, SPAD, total nitrogen content, and antioxidant-related traits (CAT, SOD, GSH, VC, TP, and TF). SSR-based diversity analysis revealed moderate polymorphism (18 polymorphic bands; expected heterozygosity 0.27–0.64, mean 0.44; Nei's genetic diversity 0.26–0.64). These results provide SSR marker references for early hybrid authentication in wide Rubus crosses and support subsequent selection of promising interspecific hybrid germplasms.