Exploring secondary structures within the nuclear ribosomal DNA internal transcribed spacers (nr DNA ITS) for enhanced phylogenetic insights in Medicago sativa (alfalfa)

ITS sections, which are situated inside the ribosomal DNA operon, are recognized for their high levels of diversity and slow rate of evolution, which makes them important for plant phylogenetic studies. The objective of this work is to use the internal transcribed spacer (ITS) regions of 18 alfalfa (Medicago sativa) genotypes/varieties to determine their genetic relationships and to verify possible genetic markers for variety identification and classification. This has been done by validating the secondary structures of the ITS regions and observing the effects of the relationships among the lowest energy state, the total number of possible secondary structure hairpins, and (GC) content. The results indicated that the length of the ITS sequences varied between 426 and 629 nucleotides and that the G + C content of the ITS region varied between 46.6 and 50%. In this study, the analysis of molecular variance (AMOVA) was used to validate the effectiveness of configuration analysis and revealed that fluctuations in the data accounted for a substantial amount of the observed total variance. Moreover, 94 percent of the genotypes analyzed exhibited a significant degree of variety. This shows that a large fraction of the observed variation is likely attributable to genetic variables. Population four, comprising Bilensoy80, Emily, Quin, Vendor, and Felicia, had the highest degree of heterozygosity at 68.8%, whereas population two, including Gozlu1, Prosementi, Nimet and Local (Van), had the lowest level at 37.5%. The original Shannon’s approach as an unbiased estimator employed in population genetics research validated the differences among alfalfa genotypes with the AMOVA analysis results of this study. Individual differences were found to be 59%, whereas population differences were found to be 41%. There have been a few ITS studies on Medicago sativa that have utilized ITS as a phylogenetic marker to estimate connections and define new taxonomic categories (e.g., tribes). However, our research also includes an analysis of the secondary structure of these sequences and the results of this study imply that ITS sequence and secondary structure data can be utilized to understand the intraspecific genetic makeup of different alfalfa varieties.