Bt Research 2025, Vol.16, No.2, 70-78 http://microbescipublisher.com/index.php/bt 71 amplified polymorphic DNA), AFLP (amplified Fragment length polymorphism), fAFLP (fluorescent AFLP), and rep-PCR (repeat sequence PCR, including primers such as BOX, ERIC, REP, MB1, and GTG5). These methods amplify specific regions of genomic DNA and then use electrophoresis typing to show the genetic differences among Bt strains. For instance, fAFLP and RAPD can effectively distinguish Bt strains from different geographical regions or ecosystems. They can generate polymorphic bands, and then the similarities and differences among strains can be demonstrated through cluster analysis (Pattanayak et al., 2001; Vilas-Bôas and Lemos, 2004; Qasem et al., 2015). rep-PCR has high resolution and good repeatability. It can distinguish different strains within the same species and obtain more complex fingerprint patterns (Da Costa Fernandes et al., 2021). In addition, qPCR is also often used to detect the distribution and typing of specific toxin genes (such as cry, cyt, etc.) (Berçot et al., 2023). 2.2 Phylogenetic approaches for strain classification Phylogenetic analysis is an important tool for studying the classification and genetic relationship of Bt strains. Multilocus sequence typing (MLST) determines the sequences of multiple conformed genes, such as glpF, gmk, ilvD, pta, pur, pycAand tpi, and can reveal the phylogenetic relationships and colony structure of Bt strains with high resolution (Figure 1) (Pheepakpraw et al., 2023). The genetic distance matrix generated by molecular markers (such as rep-PCR, RAPD, AFLP) can also be used to construct phylogenetic trees by methods such as UPGMA and Pearson correlation coefficient to show the kinship and grouping among strains (Pattanayak et al., 2001; Da Costa Fernandes et al., 2021). In addition, the maximum likelihood tree based on genome-wide SNP analysis and core genome alignment can also improve the accuracy of phylogenetic inference (Shikov et al., 2021; Biggel et al., 2022). These methods help us understand the evolutionary history, geographical distribution and the relationship between functional genotypes of Bt strains. Figure 1 Multilocus sequence typing (MLST) analysis of the Bacillus thuringiensis group isolates (Adopted from Pheepakpraw et al., 2023) Image caption: (a) Comparison of UPGMA-based dendrograms of eight isolates and the MLST profiles. The two new MLST profiles were submitted to the PubMLST database and are marked with an asterisk (*). (b) goeBURST analysis performed with Phyloviz2.0, representing the clustering of the B. thuringiensis food poisoning isolates and one reference emetic B. cereus strain into 5 sequence types (STs). The ST identification, according to the MLST database, and the isolate/strain belonging to each ST are indicated inside each circle. The circle size is proportional to the number of isolates belonging to the same ST type. The new STs are shown in red (Adopted from Pheepakpraw et al., 2023)
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