Genomics and Applied Biology 2024, Vol.15, No.4, 200-211 http://bioscipublisher.com/index.php/gab 202 Ultimately, functional validation through genetic and molecular approaches, such as gene overexpression or knockout studies, is essential for confirming the involvement of these genes in anthocyanin biosynthesis and other related pathways (Dai et al., 2012; Kim et al., 2021; Duan et al., 2022). These criteria ensure the accurate identification and characterization of R2R3-MYBgenes in colored rice. 3 Phylogenetic Analysis of R2R3-MYBGenes 3.1 Construction of phylogenetic trees To elucidate the evolutionary history and relationships of R2R3-MYB genes in colored rice, comprehensive phylogenetic trees were constructed initially. The amino acid sequences of R2R3-MYB proteins were retrieved from the rice genome using bioinformatics tools. A multiple sequence alignment was conducted using ClustalW, with appropriate gap penalties and substitution matrices set to ensure high alignment accuracy. The aligned sequences were then employed in the construction of phylogenetic trees utilizing both the Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods. The NJ trees were generated using MEGA X software with 1,000 bootstrap replicates to assess the robustness of the inferred relationships, while ML trees were constructed using RAxML, applying the best-fit model of amino acid substitution as determined by ModelFinder (Jia et al., 2004; Monna et al., 2006; Lachagari et al., 2019). 3.2 Evolutionary relationships among R2R3-MYBgenes The phylogenetic trees revealed the existence of distinct clades, which corresponded to different evolutionary lineages of R2R3-MYB genes. It is noteworthy that the analysis demonstrated a striking prevalence of non-synonymous substitutions in the DNA-binding domains, particularly in the helix 1 and helix 2 regions, indicating adaptive selection (Jia et al., 2004). This indicates that these regions are integral to protein-DNA interactions, which may facilitate the identification of novel DNA-binding sites. Furthermore, a co-evolutionary pattern was identified between the second α-helix of the R2 domain and the second α-helix of the R3 domain, underscoring the functional significance of these interactions (Jia et al., 2004). 3.3 Comparative analysis with other plant species A comparative genomic analysis with other plant species, such as Arabidopsis thaliana, revealed that the R2R3-MYB gene family in rice shares several lineage-specific genes with these species, which may account for functional diversification. A phylogenetic analysis of the Rc locus, a key regulatory gene in the anthocyanin biosynthetic pathway, revealed the existence of a distinct clade that is closely related to the progenitor species O. rufipogon and O. nivara, indicating a conserved evolutionary lineage (Lachagari et al., 2019). This comparative approach highlights the evolutionary conservation and divergence of R2R3-MYB genes across diverse plant species, offering a more comprehensive understanding of their functional roles in rice and other plants. 4 Gene Structure and Chromosomal Location 4.1 Structural features of R2R3-MYBgenes The R2R3-MYB gene family represents one of the most significant transcription factor families in plants, distinguished by the presence of two MYB domains (R2 and R3) that are involved in DNA binding. A comparative analysis of R2R3-MYBgenes in rice has revealed a high frequency of non-synonymous substitutions in the α-helix regions of the DNA-binding domains, indicating adaptive selection and functional diversification (Jia et al., 2004). These structural features are of great consequence for the specific recognition of DNA-binding sites, which in turn affects gene regulation and expression. 4.2 Chromosomal distribution and synteny analysis The chromosomal distribution of R2R3-MYB genes in rice has been extensively mapped. For example, the sequence analysis of rice chromosome 4 has provided insights into the localization of various genes, including transcription factors such as R2R3-MYB. Furthermore, genome-wide analyses have identified the presence of these genes across different chromosomes, with a notable concentration in specific regions that are rich in transposable elements and other repetitive sequences (Temnykh et al., 2001). A comparison of the chromosomal distribution of R2R3-MYBgenes in rice with that of other plant species, such as A. thaliana, has revealed a limited
RkJQdWJsaXNoZXIy MjQ4ODYzMg==