A Comparative Expression Analysis of Selected Genes Involved in Secondary Metabolites Biosynthesis Among Three Alfalfa Cultivars Grown In Iraq

Because of its abundance in proteins, vital amino acids, vitamins, and minerals that promote balanced nutrition, Medicago sativa L., also known as alfalfa, is regarded as a nutritionally excellent forage crop. In addition to its nutritional value, alfalfa produces a range of bioactive secondary metabolites, including flavonoids, alkaloids, and phenolic acids, further supporting its use in medicinal and nutritional applications. Three alfalfa cultivars grown in Iraq were examined for gene expression levels of the primary genes involved in secondary metabolite biosynthesis (PAL, COMT, CHS, and OMT). Among these cultivars are Victoria (VC), Green Gold (GC), and a local cultivar (LC). The reference cultivar, LC, has its expression level standardized to 1. Gene identity and sequence conservation among the cultivars under study were confirmed by DNA sequencing, which revealed high similarity between the amplified fragments and their corresponding reference genes in GenBank. The findings showed that the three cultivars' levels of gene expression varied significantly. While their expression was significantly downregulated in GC and VC, PAL and CHS showed higher expression in LC. The OMT gene, on the other hand, showed significant overexpression in GC, with a fold change of 68.59 compared to LC, suggesting a marked increase in transcriptional activity in GC. VC consistently showed low expression levels for all genes under investigation. Therefore, potential epigenetic mechanisms, such as DNA methylation and histone modifications, which alter transcriptional activity without altering the underlying gene sequences, may contribute to the notable variations in gene expression among the three alfalfa cultivars. Rather than being definitively shown in the current investigation, these pathways are suggested as potential regulatory elements.