Background: Synonymous codon usage bias (CUB) is a pervasive feature of plant nuclear genomes, shaped by lineage-specific base composition and additional evolutionary forces. Despite its well-documented role in genome evolution, the codon-preference landscape of Fabaceae relative to non-Fabaceae lineages remains unclear. This study establishes a comparative framework across 30 angiosperm species, centered on Fabaceae, to delineate lineage-scale differentiation patterns and identify key determinants of synonymous codon usage. 
 
Methods: A total of 5,434 bHLH genes were identified from 30 species and codon-bias profiling was performed under standardized quality-control protocols. Key indices of codon bias, including GC3s, the effective number of codons (ENc/Nc), codon adaptation index (CAI), codon bias index (CBI) and frequency of optimal codons (Fop), were calculated. ENc-GC3s analyses were integrated with the wright expected curve and strand-asymmetric base usage at the third codon position was examined using PR2-bias metrics. Multivariate techniques, such as hierarchical clustering, principal component analysis (PCA) and correspondence analysis (CA), were employed to validate global codon-preference structures and assess lineage-specific variability. 
 
Result: Species-level analyses revealed consistent phylogenetic stratification in codon-preference strength and third-position base composition. Poaceae exhibited higher GC3s (up to 0.764) and lower Nc (minimum 42.32), whereas Fabaceae species clustered within a lower GC3s range (~0.32-0.44) with differentiated Nc values (47-52), establishing GC3s as a dominant factor influencing codon-preference divergence. Despite adherence to the Wright expectation, deltaENC medians were predominantly negative, indicating a &ldquo;stronger-than-expected&rdquo; codon bias. PR2 analysis identified substantial interspecific differences in third-position base asymmetry, with distinct directional biases in A/T and G/C usage in Fabaceae and Poaceae. Multivariate analysis confirmed these findings, with PCA explaining 86.5% of the variance along the primary axis of codon-preference, and CA independently validating species clustering.

Comparative Analysis of Codon Usage Bias in bHLH Genes Across 30 Angiosperm Species Centered on Fabaceae

Transgenic &nbsp;food &nbsp;animal &nbsp;production &nbsp;is one &nbsp;of &nbsp;the &nbsp;potential and &nbsp;need &nbsp;oriented &nbsp;research &nbsp;to &nbsp;mitigate &nbsp;the &nbsp;food &nbsp;crises &nbsp;of &nbsp;the &nbsp;world. &nbsp;In &nbsp;vitro &nbsp;gene &nbsp;silenced &nbsp;animal &nbsp;cells &nbsp;and &nbsp;making &nbsp;use of &nbsp;these &nbsp;cells &nbsp; for &nbsp;transgenesis &nbsp;one of the suitable way to produce &nbsp;productive animals. &nbsp;Myostatin is a negative regulator of muscle growth, has the potential to increase the muscle mass upon its silencing. Four Hush 29-mer &nbsp;anti- myostatin (MSTN) &nbsp;shRNA &nbsp;constructs &nbsp;were checked for myostatin &nbsp;gene silencing in caprine foetal fibroblast cells &nbsp;and &nbsp;its &nbsp;subsequent effect &nbsp;on &nbsp;basic &nbsp;helix&ndash; loop&ndash;helix (bHLH) transcription &nbsp;factors. These factors are necessary for the terminal differentiation, proliferation, and homeostasis of muscle development. Different &nbsp;shRNA constructs displayed 55.1 &nbsp;to &nbsp;91.5% (p&lt; 0.01) of &nbsp;myostatin &nbsp;silencing &nbsp;in &nbsp;caprine &nbsp;foetal &nbsp;fibroblast &nbsp;cells &nbsp;and &nbsp;upregulation of &nbsp;myogenic gene. Upregulation of 7.97 &nbsp;to &nbsp;111.67 % &nbsp;for MyoD, 77.0 % &nbsp;to &nbsp;319.47 % for myogenin, &nbsp;16.67 % &nbsp;to &nbsp;138.0 % &nbsp;for Myf5 were observed . The &nbsp;Pearson &nbsp;correlation &nbsp;established &nbsp;a &nbsp;negative &nbsp;correlation &nbsp;between &nbsp;myostatin &nbsp;and &nbsp;genes under study. &nbsp; Result &nbsp;suggests &nbsp;that &nbsp;knockdown &nbsp;of &nbsp;MSTN a &nbsp;potential approach &nbsp;to &nbsp;improve &nbsp;caprine musculatures.

Myostatin silencing effect on basic helix&ndash;loop&ndash;helix transcription factors in caprine foetal fibroblast cells