الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Cyclotides are short-length plant family peptides (28-37 amino acid residues) with a cyclic cysteine knot (CCK) motif as their basis. Cyclotide precursors are multi-domain precursors (five domains) with a genetic structure that is highly conserved. With the aid of the strong bioinformatics tools currently available, the finished and published genomes give scientists the opportunity to mine multiple cyclotide motifs that could be utilized to generate productive cyclotide-based markers. Cyclotide subfamily mining at the genome level or scale in plants is considered an untouched area with relatively few reports. Therefore, this study focused on the validation of cyclotide-based markers in addition to the genome-wide cyclotide profiling and characterization of their subfamily’s frequencies in several plant genomes. The available cyclotide amino acid and nucleotide sequences belonging to each subfamily (Bracelet, Moebius, Hybrid, and Trypsin Inhibitor), in addition to fifteen plant genomes and transcriptomes were collected from the UniProt and NCBI databases to be used in detecting the conserved or semi-conserved sequences in each cyclotide subfamily, evaluation of the developed cyclotide primers at the genome and 62 transcriptome level, and cyclotide genome-wide mining analysis, respectively. The collected nucleotide and amino acid sequences of each cyclotide subfamily were aligned independently in MegAlign software using the Clustal-W method. Thereafter, degenerate cyclotide subfamily-specific (CSS) markers were designed based on the alignment of each cyclotide subfamily due to the high sequence variability of the cyclotide mature domain (CMD). All primers had an average size of 20-mer, an average degeneracy of 48%, and an average GC percentage of 52%. Notably, particular nucleotide sequences (TGY codons, which stand for cysteine amino acid residues) were fixed in the sequences of all primer pairs. In-silico PCR analysis was achieved using the EMBOSS software to estimate the number of the expected CMD products, and the amplifiable sequences located between two adjacent CMDs in a plant genome or transcriptome. To simulate the PCR reality, the 15 plant genomes and transcriptomes were used separately against the designed CSS markers with many mismatch percentages (5%, 10%, and 15%), and the products were filtered based on their size (4Kb). The 10% mismatch percentage generated the most logical or reasonable product number in the obtained in silico PCR results compared to other mismatch percentages. The in-silico 63 PCR results produced a total of 476 amplifiable amplicons, including 117, 43, 218, and 98 amplicons for the Bracelet, Moebius, Hybrid, and Trypsin Inhibitor (TI) subfamilies, respectively. The correlation analysis that was performed between the abundance of each cyclotide subfamily in genomes and the genome size of each plant showed that the Hybrid subfamily exhibited the highest positive correlation with the genome size, followed by the Trypsin Inhibitors subfamily, and then the Bracelet subfamily. Meanwhile, the Moebius subfamily exhibited a negative correlation to genome size. The developed primers (as singles rather than pairs) that target each subfamily’s CMD were used as query sequences against the 15 plant genomes and transcriptomes to perform cyclotide profiling analysis to determine the prevalence of each cyclotide subfamily within the obtained plant genomes and transcriptomes. The presence of cyclotides was confirmed by Python scripts as well as the in- silico CyPerl tool. The findings of the in-silico profiling successfully identified 903 CMDs in all genomes and transcriptomes, comprising 191, 206, 49, and 457 CMDs for the Bracelet, Moebius, Hybrid, and TI subfamilies, respectively, with the Trypsin Inhibitors as the most prevalent and common subfamily in the studied plant genomes and transcriptomes. 64 The in-vitro validation of developed CSS markers was performed on 19 genotypes or cultivars belonging to seven plant species. The genomic DNA was isolated from all cultivars using the DNeasy Plant DNA Extraction Kit (Qiagen, Germany). The CSS primer pairs were used in the PCR amplification to screen cyclotide subfamilies (Bracelet, Hybrid, and Trypsin Inhibitor) inside the selected cultivars of each plant species. All cyclotide amplification fragments were separated on agarose and polyacrylamide non-denaturing gels for visualization. The developed CSS markers generated a total of 175 bands, with a percentage of polymorphism ranging from 98 to 100%. Noteworthy, among all the studied cyclotide subfamilies, the faba bean species had the fewest bands. While the tomato species exhibited the largest number of bands in the Hybrid subfamily, the sesame species showed the largest number of bands in the Trypsin Inhibitors and Bracelet subfamilies. The CSS technique relies on two primers (forward and reverse primers) that amplify the CMD regions of each subfamily. The CSS primers can either amplify a single CMD or the interspaced regions between two or more adjacent CMDs (<4-5 kb) for each cyclotide subfamily. Each CSS primer pair is developed to anneal with almost 66% of the cysteine residues existing in each CMD. However, the PCR reaction 65 amplifies the CSS primers located inside the genic regions on the two DNA strands. Based on the PCR amplification products, a construction of a dendrogram was relying on the unweighted pair group method of the arithmetic averages (UPGMA) method using Past Software. The dendrogram showed discrete clusters for cultivars/genotypes belonging to each plant species. Furthermore, the similarity matrix for the combined data of all CSS markers was calculated relying on the Jaccard similarity coefficient. The similarity matrix showed results that were similar to the dendrogram results. Based on the analysis of the acquired marker results, seven informative indices were calculated in order to estimate the efficiency of the CSS markers. Hence, the results of marker efficiency parameters revealed the CSS markers’ capability to determine the genetic diversity of the studied plant species. Eventually, the CSS marker technique could be utilized effectively in cyclotide subfamily screening and cyclotide-based pharmaceutical drug design.