Bt Research 2024, Vol.15, No.3, 141-153 http://microbescipublisher.com/index.php/bt 143 For example, in a study conducted in Kuwait, researchers isolated 109 Bt strains from soil samples using culture and serological methods, ultimately identifying 15 subspecies of Bt thuringiensis (Qasem et al., 2015). The isolation process often includes selective media and biochemical tests to differentiate Bt from other Bacillus species. Once isolated, the strains are identified using a combination of morphological characteristics and molecular techniques. Techniques such as 16S rRNA sequencing are commonly employed to confirm the identity of Bt strains. In addition to morphological and biochemical identification, advanced methods like serotyping and analysis of flagellin proteins are used to classify the strains further (Shikov et al., 2021). This comprehensive approach ensures accurate identification and categorization of Bt strains, which is essential for subsequent phylogenetic analysis. 3.2 DNA extraction and sequencing DNA extraction and sequencing are fundamental to phylogenetic studies. The extraction process involves breaking down the cell walls of Bt strains to release DNA, which is then purified. Various kits and methods, such as phenol-chloroform extraction and commercial DNA extraction kits, are used to obtain high-quality DNA. Once extracted, the DNA undergoes sequencing to analyze the genetic material. Whole-genome sequencing and targeted sequencing of specific genes, such as those encoding for Cry and Cyt proteins, are commonly used. For instance, Wang et al. (2018) utilized multi-locus sequence typing (MLST) to analyze seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA, andtpi) in 233 Bt strains. This method allowed the researchers to establish genetic relationships and identify new sequence types (STs). Another study employed the random amplified polymorphic DNA (RAPD) technique to assess genetic diversity among Bt strains, demonstrating the variability in DNA patterns (Qasem et al., 2015). High-throughput sequencing technologies, such as Illumina and PacBio, provide detailed genomic data, facilitating in-depth phylogenetic analysis and evolutionary studies. 3.3 Computational tools and software Computational tools and software are essential for analyzing sequencing data and constructing phylogenetic trees. These tools help in aligning sequences, detecting genetic variations, and visualizing evolutionary relationships. One commonly used software is MEGA (Molecular Evolutionary Genetics Analysis), which facilitates sequence alignment, model testing, and phylogenetic tree construction. In their study, Rabha et al. (2018) used MEGA for phylogenetic analysis of Bt isolates from Assam, identifying unique sequence types and analyzing vegetative insecticidal protein (vip) genes. Another tool, the Composition Vector Tree (CVTree) method, was employed by Wang et al. (2023) for high-resolution typing of Bt strains, proving effective for genomic variability analysis. Software like BioEdit and ClustalW are used for multiple sequence alignments, while RAxML and BEAST provide robust platforms for phylogenetic inference and evolutionary analysis. Phylogenetic trees can be visualized using tools like FigTree and Dendroscope. These computational tools enable researchers to interpret complex genomic data and understand the genetic relationships and evolutionary history of Bt strains comprehensively. 4 Genetic Markers and Loci 4.1 Selection of genetic markers The selection of appropriate genetic markers is critical for the effective phylogenetic analysis of Bacillus thuringiensis (Bt) strains. Genetic markers are specific sequences of DNA that can be used to identify and differentiate between various strains. These markers are chosen based on their ability to provide clear, reproducible, and polymorphic data that reflect the genetic diversity and evolutionary relationships among Bt strains. Commonly selected markers include housekeeping genes, which are essential for basic cellular functions and are highly conserved across different strains. For instance, multi-locus sequence typing (MLST) often employs housekeeping genes such as glpF, gmK, ilvD, pta, pur, pycA, and tpi to assess genetic variability and establish phylogenetic relationships (Wang et al., 2018). Random amplified polymorphic DNA (RAPD) markers are used to analyze genetic diversity without prior knowledge of the genome, offering a quick and effective means of strain differentiation (Qasem et al., 2015). The
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