Comparative Codon Bias Analysis of WRKY Gene Family in Oryza sativa subsp. indica and japonica

Abstract

Codon usage bias (CUB) is an important molecular evolutionary feature shaped by various forces such as mutational pressure and natural selection. In this study, we performed a comprehensive codon usage bias analysis of WRKY transcription factor (TF) genes in Oryza sativa subsp. indica and japonica to explore their underlying evolutionary dynamics. A total of 109 and 128 valid WRKY CDSs were retrieved and analyzed respectively. Several indices including nucleotide composition, GC content, RSCU (Relative Synonymous Codon Usage), ENC (Effective Number of Codons), and neutrality plot analysis were used to assess the CUB patterns. Our results revealed that both subpopulations exhibited moderate codon bias, with the majority of genes displaying ENC values above 40, indicating weak bias. The ENC vs. GC3s plot showed most data points clustered around the expected curve, suggesting mutational pressure as the primary force shaping codon usage. Neutrality plot regression slopes (~0.28) and strong correlations between GC3 and GC12 further supported the dominance of mutational bias. PR2 plots demonstrated slight deviations in A/T and G/C proportions at the third codon position, indicating minor influences of natural selection. Additionally, correspondence analysis (COA) showed that the first axis, largely influenced by GC3s, explained over 90% of codon usage variation, reinforcing the role of base composition. Comparative analysis with existing literature highlighted that our findings align with previous reports on codon usage in stressresponsive gene families in rice. These results collectively suggest that although natural selection may play a minor role, mutational pressure predominantly governs codon usage patterns in WRKY TF genes of O. sativa, offering insights for evolutionary genomics and crop improvement.