Wheat is a major nutritional cereal crop that has economic and strategic value worldwide. The sustainability of this extraordinary crop is facing critical challenges globally, particularly leaf rust disease, which causes endless problems for wheat farmers and countries and negatively affects humanity’s food security. Developing effective marker-assisted selection programs for leaf rust resistance in wheat mainly depends on the availability of deep mining of resistance genes within the germplasm collections. This is the first study that evaluated the leaf rust resistance of 50 Egyptian wheat varieties at the adult plant stage for two successive seasons and identified the absence/presence of 28 leaf rust resistance (Lr) genes within the studied wheat collection. The field evaluation results indicated that most of these varieties demonstrated high to moderate leaf rust resistance levels except Gemmeiza 1, Gemmeiza 9, Giza162, Giza 163, Giza 164, Giza 165, Sids 1, Sids 2, Sids 3, Sakha 62, Sakha 69, Sohag 3 and Bany Swif 4, which showed fast rusting behavior. On the other hand, out of these 28 Lr genes tested against the wheat collection, 21 Lr genes were successfully identified. Out of 15 Lr genes reported conferring the adult plant resistant or slow rusting behavior in wheat, only five genes (Lr13, Lr22a, Lr34, Lr37, and Lr67) were detected within the Egyptian collection. Remarkedly, the genes Lr13, Lr19, Lr20, Lr22a, Lr28, Lr29, Lr32, Lr34, Lr36, Lr47, and Lr60, were found to be the most predominant Lr genes across the 50 Egyptian wheat varieties. The molecular phylogeny results also inferred the same classification of field evaluation, through grouping genotypes characterized by high to moderate leaf rust resistance in one cluster while being highly susceptible in a separate cluster, with few exceptions.

Cyclotides are cysteine-rich cyclic peptides that have various activities and applications. Mining at the whole-genome scale facilitates the profiling and development of cyclotide-based markers. However, the characterization of cyclotide subfamilies at a genome-scale in plants is an untapped area that needs more studies. Here we developed four degenerate cyclotide subfamily-specific (CSS) markers based on their subfamily’s semi-conserved sequence for identifying each subfamily separately. Initially, an in silico cyclotide mining analysis was performed on 15 plant genomes/transcriptomes. The in silico PCR produced 476 amplicons in the Bracelet, Moebius, Hybrid and Trypsin Inhibitor (TI) subfamilies. In silico profiling results identified 903 cyclotide mature domains (CMDs) in the analyzed genomes/transcriptomes for the four subfamilies. In addition, the prevalence of each subfamily correlated positively with the genome size, particularly in Hybrid, followed by TI and Bracelet subfamilies. The CSS markers were validated in vitro on 19 genotypes/cultivars. The CSS markers generated a total of 175 bands, with a polymorphic percentage of 98%–100%. Cluster analysis showed distinct clusters for cultivars/genotypes of each plant species. The marker efficiency parameters demonstrated the capability of CSS markers to determine the genetic diversity among the studied plant species. This study represents the first report to perform genome/transcriptome-wide mining to characterize the Cyclotide subfamilies in plants. This study proposed potential applications of the novel CSS markers as an effective system for screening cyclotide subfamilies across multiple plant species with no prior sequencing knowledge requirement. Additionally, it proposes an innovative approach for identifying the appropriate cyclotide subfamily used in drug design.