Exploring genetic diversity and genomic insights of Bacillus subtilis isolates from cassava rhizosphere using molecular barcoding and whole genome sequencing.

Bacillus subtilis plays a significant role in both agriculture and industry. It is commonly isolated from agricultural environments, particularly various soil types. This study aimed to investigate DNA barcoding and whole-genome sequencing of B. subtilis strains, focusing on those specific to the cassava rhizosphere. Genetic identification and diversity of B. subtilis strains isolated from the rhizosphere of the Piroon 2 cassava cultivar were initially characterized using 16 S rDNA, a molecular marker for species-level identification. To explore strain-specific biodiversity within B. subtilis, repetitive DNA elements-specifically extragenic palindromic and BOX sequences-were analyzed across the genomes of the isolated strains. These repetitive sequences revealed two main structural patterns, providing clear and distinct genomic fingerprints for biodiversity analysis. The results showed that both REP and BOX sequences were highly conserved within specific regions of the B. subtilis genome, resulting in reliable and reproducible DNA patterns suitable for whole-genome phylogenetic analysis. While the 16 S rDNA approach showed a high sequence similarity among the B. subtilis strains (99.98%), whole-genome analysis using repetitive sequences allowed for clearer differentiation, with phylogenetic distances exceeding 97%. Whole-genome sequencing of the elite strain BsPr8 was performed using the Illumina MiSeq platform. The sequencing results yielded 56 contigs, with an average GC content of 43.67% and a total genome size of approximately 4,050 Kbp. Genome annotation identified 3,575 proteins with functional assignments, including 1,055 enzymes classified by Enzyme Commission numbers. The PATRIC database further annotated 3,937 genus-specific protein families. Additionally, 45 genes homologous to known antibiotic resistance genes were identified within the BsPr8 genome. These findings have important implications for sustainable agricultural practices and cassava cultivation. By elucidating the genetic diversity and genomic characteristics of B. subtilis strains, this study facilitates the identification of beneficial traits-such as plant growth promotion, pathogen suppression, and improved nutrient uptake. These strains hold potential for development as biofertilizers or biopesticides, offering an environmentally friendly alternative to conventional chemical inputs.