Integrated Phenotypic, Cytotypic, and Microsatellite Diversity Analysis of Wild-Growing/Naturalized Ber (Ziziphus mauritiana Lam.) Across Pakistan: Implications for Germplasm Conservation and Breeding

Ziziphus mauritiana Lam. (ber or Indian jujube) is a stress-tolerant dryland fruit tree valued for its nutritious fruit and ability to grow on marginal land. However, the phenotypic, cytotypic and genetic structure of its wild-growing/naturalized germplasm in Pakistan remains poorly characterized. This study provides an integrated assessment of phenotypic, cytotypic and simple-sequence-repeat (SSR) diversity in 100 wild-growing/naturalized accessions collected from Khyber Pakhtunkhwa, Punjab and Sindh to establish a baseline for conservation and germplasm management. We recorded 37 morphological and biochemical traits, estimated ploidy levels by flow cytometry (using diploid Z. jujuba ‘Dongzao’ as a reference), and genotyped a representative subset of 60 accessions with 14 SSR markers scored as a binary presence/absence matrix. Substantial phenotypic variation was observed, especially in canopy architecture, leaf traits and stone-related characteristics; fruit quality traits (total soluble solids, vitamin C, and acidity) varied within a narrower range. Province explained only a modest proportion of phenotypic variation (PERMANOVA R2 = 0.059–0.109; p < 0.01), with extensive overlap among regions. Flow cytometry revealed polyploid diversity: hexaploid (2n = 6x = 72) accessions dominated (46.7%), followed by octoploid (2n = 8x = 96; 31.7%) and tetraploid (2n = 4x = 48; 21.7%) cytotypes. SSR analysis showed moderate within-province diversity (Nei’s H ≈ 0.51) but negligible genetic differentiation among provinces (R2 = 0.030; p = 0.60; Φ ≈ −0.011), indicating weak geographic structuring. Wild-growing/naturalized Z. mauritiana in Pakistan forms a diverse, weakly structured gene pool in which most variation occurs within rather than among provinces. Sampling for conservation and germplasm management should, therefore, prioritize phenotypic distinctiveness, cytotype representation and ecological context rather than geographic origin alone. Experimental validation of any adaptive or agronomic advantages of particular cytotypes is needed before breeding recommendations can be made.