| Summary: | This study was carried out to evaluate genetic diversity, phenolic compound composition, and biological activity of <i>Setaria italica</i> L. collected from different parts of South Korea. Antioxidant potential of seeds was estimated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and antimicrobial activity was determined by evaluating the minimum inhibitory concentration (MIC). Eight phenolic acids and 3 flavonoids were identified and quantified, among which myricetin and salicylic acid were the most dominant phytochemical compounds detected in the majority of accessions. The antioxidant potential of the leaf extracts of all the accessions was significantly higher (ranging from 32.33 ± 1.53 µg mL<sup>−1</sup> in SI-03 to 87.87 ± 1.63 µg mL<sup>−1</sup>) in SI-10 than that of the root, stem, or seeds. Among the 15 accessions, methanolic extracts of the SI-15 accession strongly suppressed the growth of <i>Escherichia coli</i> (250 µg mL<sup>−1</sup>). Accessions SI-14 and SI-15 showed positive antimicrobial activity against all gram-positive bacteria. Interestingly, extracts of all accessions were more sensitive towards <i>E. coli</i> and <i>Staphylococcus aureus</i>, with MICs ranging from 250 to 1000 µg mL<sup>−1</sup>. Three phenolic acids, namely chlorogenic acid, catechin, caffeic acid, naringin, hesperetin, and myricetin, were found to be moderately positively correlated with antioxidant activities. A wide range of diversity was observed in morphological traits, namely plant height (99.33 to 201.33 cm), culm length (67.10 to 160.00 cm), spike length (12.80 to 24.00 cm), 1000 seeds weight 1.44 to 2.91 g), bloom beginning (93.67 to 128.00 days), and full bloom (99.67 to 135 days). A dendogram generated from unweighted pair group method with arithmetic mean clustering (UPGMA) cluster analysis based on the morphological traits and inter simple sequence repeat (ISSR) marker data revealed three major groups. However, no clear correlation between these two different approaches was found. The average Shannon’s information index value (I) was 0.492, and it ranged from 0 to 0.693. The average expected heterozygosity (He) was 0.335, and it ranged from 0 to 0.499. The substantial variation in the morphological traits, bioactive properties, and genetic diversity among the accessions may provide useful information for breeding programs attempting to obtain <i>S. italica</i> with improved bioactive properties.
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