Pyramiding of major resistance (R) genes through marker-assisted selection (MAS) is a useful way to attain durable and broad-spectrum resistance against Xanthomonas oryzae pv. oryzae pathogen, the causal agent of bacterial blight (BB) disease in rice (Oryza sativa L.). The present study was designed to pyramid four broad spectrum BB-R genes (Xa4, xa5, xa13 and Xa21) in the background of Basmati-385, an indica rice cultivar with much sought-after qualitative and quantitative grain traits. The cultivar, however, is susceptible to BB and was therefore, crossed with IRBB59 which possesses R genes xa5, xa13 and Xa21, to attain broad and durable resistance. A total of 19 F₁ plants were obtained, some of which were backcrossed with Basmati-385 and large number of BC₁F₁ plants were obtained. In BC₁F₂ generation, 31 phenotypically superior genotypes having morphological features of Basmati-385, were selected and advanced up to BC₁F₆ population. Sequence-tagged site (STS)-based MAS was carried out and phenotypic selection was made in each successive generation. In BC₁F₆ population, potentially homozygous recombinant inbred lines (RILs) from each line were selected and evaluated on the bases of STS evaluation and resistance to local Xanthomonas oryzae pv. oryzae (Xoo) isolates. Line 23 was found pyramided with all four BB-R genes i.e., Xa4, xa5, xa13 and Xa21. Five genotypes including line 8, line 16, line 21, line 27 and line 28 were identified as pyramided with three R genes, Xa4, xa5 and xa13. Pathological study showed that rice lines pyramided with quadruplet or triplet R genes showed the highest level of resistance compared to doublet or singlet R genes. Thus, line 23 with quadruplet, and lines 8, 16, 21, 27, and 28 with triplet R genes, are recommended for replicated yield and resistance trials before release as new rice varieties. Further, traditional breeding coupled with MAS, is a solid way to attain highly effective BB-resistant rice lines with no yield cost.

Potato (Solanum tuberosum L.) is one of the emerging staple crops in Pakistan, with Punjab producing over 95% of the country’s potatoes. Wilt is an emerging threat to the potato crop worldwide, including in Pakistan. We identified and characterized Fusarium species associated with potato wilt in Pakistan through morphological and molecular analyses. Samples were collected during the 2020–2022 potato seasons from five major potato-growing regions: Sahiwal, Chichawatni, Pakpattan, Kamalia, and Faisalabad. Morphological characterization, internal transcribed spacer (ITS) sequencing, specific translation elongation factor 1-alpha (TEF) sequencing, and phylogenetic analysis were used to identify six different Fusarium species: F. oxysporum, F. equiseti, F. incarnatum, F. fujikuroi, F. annulatum and F. thapsinum. Pathogenicity tests in a greenhouse revealed that F. oxysporum and F. equiseti were responsible for Fusarium wilt in all sampled regions, with F. oxysporum being more prevalent in wilted samples. This is the first report of F. equiseti on wilted potatoes in Pakistan. In vitro biocontrol tests using Trichoderma harzianum showed 89% inhibition against F. equiseti and 65% inhibition against F. oxysporum. These findings on F. equiseti will aid in developing future control strategies, including biocontrol measures for Fusarium wilt in potatoes.

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.

Monacha cartusiana (O. F. Müller, 1774), native to the Mediterranean region and Europe, is a terrestrial gastropod recognized as a highly destructive agricultural pest that causes significant damage to crop plants, fruit trees, vegetables, ornamentals, and natural ecosystems. Despite its broad geographic distribution, the evolutionary history and phylogeographic relationships of M. cartusiana populations remain globally unexplored. This study reports the first molecularly confirmed record of M. cartusiana in Pakistan and investigates its genetic diversity and phylogeographic structure within a global context using mitochondrial markers. After morphological identification, genomic DNA was extracted from collected specimens using the CTAB method, followed by amplification and sequencing of the mitochondrial COI and 16S rRNA genes. The resulting sequences were subsequently analyzed using DnaSP and PopART software to estimate genetic diversity, perform neutrality tests, and construct haplotype networks. Published sequences of M. cartusiana retrieved from GenBank were incorporated to provide a global comparative framework. The COI dataset (555 bp) revealed 52 haplotypes, whereas the 16S rRNA dataset (269 bp) identified 14 haplotypes across global populations. High haplotype diversity (Hd = 0.946 for COI; Hd = 0.831 for 16S rRNA) and moderate nucleotide diversity (π = 0.010 for COI; π = 0.01253 for 16S rRNA) indicated substantial genetic variability within the species. Neutrality tests produced negative and insignificant values for Tajima’s D for COI and significant values for 16S rRNA (−1.428 for COI; −0.20586 for 16S rRNA) and Fu’s Fs (−29.776 for COI; −1.263 for 16S rRNA), suggesting historical population expansion. Phylogenetic reconstruction and haplotype network analyses identified two major clades (Clade A and Clade B), reflecting genetic relationships among populations from different geographic regions. AMOVA based on COI and 16S rRNA sequences revealed significant population structuring, with 29.98–51.30% of the total genetic variation occurring among populations and high fixation indices (FST = 0.299–0.51398, p = 0.001), indicating pronounced genetic differentiation and restricted gene flow. Pairwise FST analyses indicated that the Pakistani population is most closely related to populations from Italy and Central Europe, suggesting a closer genetic affinity with Southern or Central European populations. However, FST alone does not allow definitive inference of introduction directionality, and additional analyses would be required to robustly identify the source population. Overall, this study provides the first comprehensive molecular and phylogeographic assessment of the M. cartusiana species from Pakistan within a global context. These findings contribute important baseline data for understanding the evolutionary dynamics, dispersal history, and population connectivity of this economically important pest species. The pronounced genetic differentiation among populations and the suggested genetic affinity of the Pakistani population with European lineages have direct implications for biosecurity monitoring, invasion pathway tracing, and targeted pest management strategies. Future research integrating nuclear markers with the mitochondrial data presented here will be essential for a more complete understanding of gene flow and local adaptation in this species.