A total of 25 SSR primers were screened on 37 putative F₁s derived from the five different crosses. Identified cross specific highly informative SSRs primers, i.e., 14 for the first cross, 10 for the second, 12 for the third and 6 each for fourth and fifth crosses. For the first cross Bhagwa × Daru 17, four primers (HvSSRT_375, NRCP_SSR9, NRCP_SSR12 and NRCP_SSR92) were found to be highly informative with higher 100% hybrid purity index (HPI), PIC (~0.52), and observed heterozygosity (Ho, range 0.87–0.93) values, and two F₁s namely H₁ and H₂ were found to be highly heterotic with a heterozygosity index (HI) of 92.85%. Similarly, for Bhagwa × Nana, three primers (HvSSRT_375, HvSSRT_605 and NRCP_SSR19) had higher HPI (70%–100%), PIC (0.52–0.69), and Ho (0.75–0.33) values, and three F₁s H₁, H₂, and H₄ had 70% (HI). For Bhagwa × IC318712, four SSRs (HvSSRT_254, HvSSRT_348, HvSSRT_826 and NRCP_SSR95) had higher Ho (~0.83), HPI (100%) and PIC (~0.52) values, and four F₁s H₂, H₇, H₉, and H₁₀ showed 91.66% (HI). For Bhagwa × Nayana, HvSSRT_605, HvSSRT_826, and HvSSRT_432, and for Ganesh × Nayana, HVSSRT_375, HVSSRT_605, and HvSSRT_826 were found informative. These markers will be highly useful in developing maps of populations.

Low temperature is one of the most significant environmental factors that threaten the survival of subtropical and tropical plant species. By conducting a study, which was arranged in a completely randomized design with three replicates, the relative freezing tolerance (FT) of four Iranian pomegranate cultivars, including ‘Alak Torsh’, ‘Tabestaneh Torsh’, ‘Poost Sefid’, and ‘Poost Syah’, as well as its correlation with some biochemical indices, were investigated. From each cultivar, pieces of one-year-old shoot samples were treated with controlled freezing temperatures (−11, −14, and −17 ℃) to determine lethal temperatures (LT₅₀) based on survival percentage, electrolyte leakage, phenolic leakage, and tetrazolium staining test (TST) methods. Results showed that FT was higher in the second year with a lower minimum temperature and a higher concentration of cryoprotectants. The stronger correlation of electrolyte leakage with survival percentage (r = 0.93***) compared to the other three indices explained that this index could be the most reliable injury index in determining the pomegranate FT to investigate freezing effects. Of all four cultivars, ‘Poost Syah’ was the hardest by presenting a higher FT than ~ −14 ℃ in mid-winter. Accordingly, this pomegranate cultivar seems to be promising to grow in regions with a higher risk of freezing and to be involved in breeding programs to develop novel commercial cultivars.