Comprehensive Evaluation of Cryptic Juglans Genotypes: Insight from Molecular Markers and Phylogenetic Analysis

<b>Background/Objectives:</b> The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. <b>Methods:</b> Based on morphological traits such as nut size, nut shape, and the number of leaflets, five genotypes were chosen and samples were collected for the current study. For molecular analysis, gDNA was isolated from the fresh leaves, and the five most effective angiosperm-specific markers, ITS2, <i>rbcLa</i>, <i>rbcLc</i>, <i>rpoC1</i>, and <i>UBE3</i>, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and <i>UBE3</i> were recorded as the most efficient markers followed by <i>rbcLa</i>, <i>rbcLc,</i> and <i>rpoC1</i>. <b>Results:</b> During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on <i>UBE3</i> clustered with (KF454101.1-<i>Juglans regia</i>) and (KC870919.1-<i>J. regia</i>) with bootstraps of 56 and 100, respectively. Genotype-3 based on <i>rbcla</i> clustered in a major clade with <i>J. regia</i> L., cultivars (MN397935.1 <i>J. regia</i> 'Vina') and (MN397934.1-<i>J. regia</i> 'Serr'), (MN397933.1 <i>J. regia</i> 'Pedro'), (MN397932.1 <i>J. regia</i> 'Lara'), (MN397931.1 <i>J. regia</i> 'Howard'), and (MN397930.1 <i>J. regia</i> 'Hartley') with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on <i>rbclc</i> and <i>rpoC1</i> clustered with (MN397935.1 <i>J. regia</i> 'Vina') and (MN397934.1 <i>J. regia</i> 'Serr'), across the database sequences. To clarify the taxonomic status of cryptic walnut genotypes, it is necessary to combine diverse DNA barcodes. The results of ITS2 and <i>UBE3</i>, followed by <i>rbcL</i> barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. <b>Conclusions:</b> It has been determined that the genotypes of walnuts in the study area are both <i>J. regia</i> L. and its cultivars and that the accuracy of discrimination regarding the genus <i>Juglans</i> L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of <i>Juglans</i> taxa and its population.