Bt Research 2024, Vol.15, No.3, 141-153 http://microbescipublisher.com/index.php/bt 144 selection process also considers the ease of amplification and the level of polymorphism provided by the markers, ensuring that the chosen markers can effectively distinguish between closely related strains and reveal evolutionary patterns. 4.2 Commonly used loci in Bt phylogenetics In Bt phylogenetics, several loci are frequently utilized to construct phylogenetic trees and study the genetic relationships among strains. The 16S rRNA gene is a widely used marker due to its presence in all bacteria and its slow evolutionary rate, making it suitable for distinguishing broad taxonomic groups (Rahman et al., 2022). However, for higher resolution, additional loci such as housekeeping genes are employed. MLST schemes often use genes like glpF, gmK, ilvD, pta, pur, pycA, and tpi, which provide detailed insights into the genetic makeup of Bt strains (Wang et al., 2018). The Cry and Cyt toxin genes, responsible for the insecticidal properties of Bt, are also crucial markers. These genes not only help in identifying specific Bt strains but also in understanding their pathogenic potential (Reyaz et al., 2019). Other loci include the vip genes, which encode vegetative insecticidal proteins and are used to study the diversity and phylogenetic relationships of Bt strains with biopesticide potential (Rabha et al., 2018). These commonly used loci provide a comprehensive understanding of the genetic diversity, evolutionary history, and functional capabilities of Bt strains. 4.3 Comparative analysis of marker effectiveness Comparative analysis of different genetic markers is essential to determine their effectiveness in resolving phylogenetic relationships among Bt strains. Housekeeping genes used in MLST, such as those mentioned earlier, offer high-resolution data and are effective in distinguishing closely related strains (Wang et al., 2018). They provide robust phylogenetic trees that can reveal subtle genetic differences and evolutionary patterns. The 16S rRNA gene, while useful for broad classification, often lacks the resolution needed for finer phylogenetic distinctions among closely related strains (Rahman et al., 2022). In contrast, RAPD markers are highly polymorphic and can differentiate strains without prior genomic information, making them useful for initial screening and diversity studies (Qasem et al., 2015). However, RAPD results can sometimes be less reproducible compared to MLST due to their random nature. The Cry and Cyt toxin genes are highly specific and effective in identifying Bt strains with particular insecticidal properties, but they may not provide a complete picture of the overall genetic diversity (Reyaz et al., 2019). Therefore, a combination of different markers is often employed to leverage the strengths of each and provide a comprehensive phylogenetic analysis. This integrative approach ensures accurate and detailed insights into the genetic relationships and evolutionary history of Bt strains. 5 Phylogenetic Tree Construction 5.1 Methods for tree construction Constructing phylogenetic trees is essential for understanding the evolutionary relationships among Bacillus thuringiensis (Bt) strains. Several methodologies are employed to achieve accurate phylogenetic analysis. One of the most commonly used methods is Multi-Locus Sequence Typing (MLST), which involves sequencing multiple housekeeping genes and analyzing their combined data. For instance, Wang et al. (2018) performed MLST using seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA, and tpi) to analyze 233 Bt strains, revealing significant genetic relationships and evolutionary patterns. Another effective method is Whole-Genome Sequencing (WGS), which provides comprehensive genomic data allowing for detailed phylogenetic analysis. Wang and Ash (2015) demonstrated the effectiveness of WGS in constructing phylogenetic trees by comparing 50 complete Bacillus genome sequences using the Feature Frequency Profile (FFP) method, which supported the monophyletic status of Bt. The use of high-throughput sequencing technologies, such as Illumina and PacBio, has facilitated the assembly of complete genomes and the resolution of complex phylogenetic relationships (Lechuga et al., 2020). Tools like MEGA, RAxML, and BEAST
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