Background/Objectives: The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. Methods: 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, rbcLa, rbcLc, rpoC1, and UBE3, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and UBE3 were recorded as the most efficient markers followed by rbcLa, rbcLc, and rpoC1. Results: During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on UBE3 clustered with (KF454101.1-Juglans regia) and (KC870919.1-J. regia) with bootstraps of 56 and 100, respectively. Genotype-3 based on rbcla clustered in a major clade with J. regia L., cultivars (MN397935.1 J. regia ‘Vina’) and (MN397934.1-J. regia ‘Serr’), (MN397933.1 J. regia ‘Pedro’), (MN397932.1 J. regia ‘Lara’), (MN397931.1 J. regia ‘Howard’), and (MN397930.1 J. regia ‘Hartley’) with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on rbclc and rpoC1 clustered with (MN397935.1 J. regia ‘Vina’) and (MN397934.1 J. regia ‘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 UBE3, followed by rbcL barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. Conclusions: It has been determined that the genotypes of walnuts in the study area are both J. regia L. and its cultivars and that the accuracy of discrimination regarding the genus Juglans L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of Juglans taxa and its population.

The Himalayan region is recognized as one of the world’s major biodiversity hotspots due to its remarkable altitudinal variation, high species richness, and exceptional endemism. Despite its ecological significance, the endemic flora of the western Himalayas remains insufficiently explored at the molecular level. This study aimed to evaluate the performance of multiple nuclear and chloroplast DNA barcode loci (ITS2, rbcL, trnH-psbA, trnA, trnV, rpoB, ycf3, and rbcLa) for the identification and phylogenetic assessment of 32 endemic and threatened plant species representing 31 genera and 24 families from the western Himalayan region of Pakistan, such as Thalictrum secundum Edgew. Aquilegia pubiflora Wall. ex Royle, Anemone obtusiloba D.Don and Delphinium cashmerianum Royle of family (Ranunculaceae), Pimpinella stewartii (C.B. Clarke) Nasir (Umbeliferaceae), Bistorta amplexicaulis (D.Don) Greene, and Bistorta affinis (D.Don) Greene (Polygonaceae) Abelia triflora R.Br. ex Wall. And Lonicera japonica Thunb. (Caprifoliaceae), Aesculus indica (Wall. ex Cambess.) Hook. (Sapindaceae), Arisaema utile Nakai (Araceae), Coriaria nepalensis Wall. (Coriaraceae), Desmodium elegans DC (Papilionacea), Daphne papyracea Wall. ex Steud. (Thymelaceae), Epimedium elatum C.Morren. (Berberidaceae), Galium tetraphyllum. Nazim. & Ehrend. (Rubiaceae), Bupleurum lanceolatum Wall. ex August. and Heracleum polyadenum Franchet (Apiaceae), Impatiens edgeworthii Hook.f. (Balsaminaceae), Incarvillea emodi Chatterjee (Bignoniaceae), Potentilla erecta (L.) Raeusch., Cotoneaster frigidus var. affinis (Lindl.) Wenz., and Spiraea hazarica R.Parker (Rosaceae), Phytolacca latbenia (Moq.) H.Walter. (Phytolaccaceae), Rhamnus parvifolius Bunge. (Rhamnaceae), Thymus linearis Benth. (Lamiaceae), Jasminum leptophyllum Wall. ex G.Don (Oleaceae), Rhododendron lepidotum Wall. ex Hook.f. (Ericaceae), Swertia ciliata Roxb. ex Fleming. (Gentianaceae), Zanthoxylum armatum DC. (Rutaceae), Dioscorea balcanica Kosanin (Dioscoreaceae), and Deutzia staminea R.Br. ex Wall. (Hydrangeaceae). Among the tested markers, rbcL showed the highest PCR amplification (100%) and sequencing success (96%), followed by trnH-psbA (100% amplification, 80% sequencing success), whereas ITS2 exhibited comparatively lower sequencing efficiency (52%). BLAST analysis demonstrated ≥ 97% sequence identity for most taxa, and best Match/Best Close Match analysis indicated higher discrimination efficiency for rbcL and trnH-psbA compared with other loci. Multilocus phylogenetic analysis further confirmed taxonomic placement at the family, genus, and species levels. Overall, this study demonstrates that a multilocus DNA barcoding approach provides reliable species authentication and phylogenetic resolution for endemic Himalayan flora, contributing valuable molecular reference data to global databases and supporting conservation and taxonomic efforts in this biodiversity-rich yet understudied region.

Chaenomeles japonica (Thunb.) Lindl., an ornamental species belonging to the family Rosaceae, was molecularly identified from a cultivated population in the Hazara region, Khyber Pakhtunkhwa (KP), Pakistan. Fresh leaf samples were collected from a cultivated garden, and genomic DNA was extracted for molecular analysis. Four chloroplast DNA markers ( matK, rbcLa, trn A , and ycf3 ) were amplified and sequenced. PCR amplification showed a 100% success rate, generating sequence lengths ranging from 400–1600 bp depending on the marker. BLAST analysis revealed 99–100% sequence identity, 100% query coverage, and E-values of 0.0 when compared with reference sequences available in GenBank, primarily from China, Japan, and the United States. Phylogenetic analysis using Neighbour Joining method demonstrated that the obtained sequences clustered with authenticated Chaenomeles japonica accessions, supported by bootstrap values. The results confirm the molecular identity of cultivated C. japonica in Pakistan and highlight the utility of chloroplast DNA barcoding markers for accurate species identification of ornamental plants.

Falconeria insignis Royle leaf extract was utilized for the first time as a natural reducing and stabilizing agent in a green precipitation method to synthesize silver nanoparticles devoid of hazardous chemicals. A change in hue from light yellow to pale brown showed that AgNPs were formed. This was confirmed by UV-visible spectroscopy, which showed a unique surface plasmon resonance peak at 300–350 nm. The FTIR investigation found hydroxyl, carbonyl, amide, C–N, and C–O functional groups. This showed that proteins, phenolics, and flavonoids can bio-reduce and cap. XRD confirmed that the silver was nanocrystalline and had a mean crystallite size of 19 nm. The SEM showed predominantly non-spherical and heterogeneous in overall shape. Biogenic AgNPs are different from crude extract and silver nitrate, had much higher antibacterial activity against Staphylococcus aureus . ANOVA verified statistically significant differences between treatments. According to these findings, F. insignis Royle is a viable, sustainable bioresource for the manufacture of green AgNP with possible biomedical antibacterial uses.