Identification and diversity assessment of Ziziphus species using DNA barcoding

The genus Ziziphus, belonging to the family Rhamnaceae, is of great economic and medicinal importance worldwide due to the value of its fruits in household income and food security. However, many species within this genus are phenotypically identical, leading to imprecise identification and hindering their conservation, variation in the gene pool, and exploitation. To address this issue, we collected 26 individuals from different regions of Khyber Pakhtunkhwa, Pakistan and Shanxi province, China, to assess the efficiency of DNA barcoding regions in identifying Ziziphus species. We tested the ability of five DNA barcodes regions, namely ITS, matK, rbcL, trnH-psbA, and trnL-UAA, to distinguish between Ziziphus species as an alternative approach for species identification and the phylogenetic reconstruction. The results of the study, as the success rate of PCR amplification of matK, trnL-UAA, and ITS, provided the highest species resolution with 100 % accuracy, except for trnHpsbA (90.78 %). ITS and matK were identified as the most effective single barcode, with a species identification success rate of 87.4 %+/- 0.98 % and 84.89 %+/- 0.66 %. These results suggest that matK, trnL-UAA, and ITS are universal barcode regions for identification of all Ziziphus species identification and their discrimination power to differentiate. In contrast, the combination of trnL-UAA+ITS region displayed the highest species identification rate (92.1 %), the five barcodes region combination, and superior to any barcodes regions combination that included rbcL+trnH-psbA. Additionally, the study found high haplotypes and nucleotide diversity in Pakistani Ziziphus species, mainly in the ITS barcode, with values of 0.84 and 0.0086, respectively. In conclusion, the study establishes that the DNA fragments ITS, trnL-UAA, and matK are effective in identifying and distinguishing Ziziphus species. The approach can be considered as an accurate tool for identifying these species and will be used as a universal barcode's region for Ziziphus species. DNA barcoding is a useful approach and provides a reliable and effective means for Ziziphus discrimination, and in combination with morphology-based taxonomy will be a robust approach for tackling taxonomically complex species. Additionally, this study provides valuable insights into the genomic architecture of Ziziphus species, which has implications for their classification, conservation, and germplasm resource improvement. This research provides a novel approach to the problem of incorrectly identifying economically and medicinally important species, particularly those in the genus Ziziphus.