Hemerocallis citrina Baroni is a traditional vegetable crop widely cultivated in eastern Asia for its high edible, medicinal, and ornamental value. The phenomenon of codon usage bias (CUB) is prevalent in various genomes and provides excellent clues for gaining insight into organism evolution and phylogeny. Comprehensive analysis of the CUB of mitochondrial (mt) genes can provide rich genetic information for improving the expression efficiency of exogenous genes and optimizing molecular-assisted breeding programmes in H. citrina. Here, the CUB patterns in the mt genome of H. citrina were systematically analyzed, and the possible factors shaping CUB were further evaluated. Composition analysis of codons revealed that the overall GC (GCall) and GC at the third codon position (GC3) contents of mt genes were lower than 50%, presenting a preference for A/T-rich nucleotides and A/T-ending codons in H. citrina. The high values of the effective number of codons (ENC) are indicative of fairly weak CUB. Significant correlations of ENC with the GC3 and codon counts were observed, suggesting that not only compositional constraints but also gene length contributed greatly to CUB. Combined ENC-plot, neutrality plot, and Parity rule 2 (PR2)-plot analyses augmented the inference that the CUB patterns of the H. citrina mitogenome can be attributed to multiple factors. Natural selection, mutation pressure, and other factors might play a major role in shaping the CUB of mt genes, although natural selection is the decisive factor. Moreover, we identified a total of 29 high-frequency codons and 22 optimal codons, which exhibited a consistent preference for ending in A/T. Subsequent relative synonymous codon usage (RSCU)-based cluster and mt protein coding gene (PCG)-based phylogenetic analyses suggested that H. citrina is close to Asparagus officinalis, Chlorophytum comosum, Allium cepa, and Allium fistulosum in evolutionary terms, reflecting a certain correlation between CUB and evolutionary relationships. There is weak CUB in the H. citrina mitogenome that is subject to the combined effects of multiple factors, especially natural selection. H. citrina was found to be closely related to Asparagus officinalis, Chlorophytum comosum, Allium cepa, and Allium fistulosum in terms of their evolutionary relationships as well as the CUB patterns of their mitogenomes. Our findings provide a fundamental reference for further studies on genetic modification and phylogenetic evolution in H. citrina.

中文翻译：

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黄花菜线粒体基因组的密码子使用特征和系统发育分析

黄花菜（Hemerocallis citrina Baroni）是东亚地区广泛种植的传统蔬菜作物，具有较高的食用、药用和观赏价值。密码子使用偏倚（CUB）现象在各种基因组中普遍存在，为深入了解生物体进化和系统发育提供了极好的线索。综合分析柑橘线粒体（mt）基因的CUB，可以为提高柑橘外源基因表达效率、优化分子辅助育种方案提供丰富的遗传信息。在此，系统分析了黄蜂mt基因组中的CUB模式，并进一步评估了影响CUB的可能因素。密码子组成分析显示，mt基因的总体GC（GCall）和第三个密码子位置的GC（GC3）含量低于50%，呈现出对富含A/T的核苷酸和A/T结尾密码子的偏好。 H. 黄水晶。有效密码子数 (ENC) 的高值表明 CUB 相当弱。观察到 ENC 与 GC3 和密码子计数的显着相关性，表明不仅组成限制而且基因长度对 CUB 也有很大影响。组合 ENC 图、中性图和奇偶规则 2 (PR2) 图分析增强了 H. citrina 线粒体基因组的 CUB 模式可归因于多种因素的推论。尽管自然选择是决定性因素，但自然选择、突变压力和其他因素可能在 mt 基因 CUB 的形成过程中发挥重要作用。此外，我们总共鉴定了 29 个高频密码子和 22 个最佳密码子，这些密码子表现出对以 A/T 结尾的一致偏好。随后基于相对同义密码子使用（RSCU）的聚类和基于 mt 蛋白编码基因（PCG）的系统发育分析表明，H. citrina 在进化方面与 Asparagus officinalis、Chlorophytum comosum、Allium cepa 和 Allium fistulosum 接近，反映了一定的进化关系。 CUB 与进化关系之间的相关性。黄蜂线粒体基因组中存在较弱的CUB，且受到多种因素尤其是自然选择的综合影响。研究发现，H. citrina 与 Asparagus officinalis、Chlorophytum comosum、Allium cepa 和 Allium fistulosum 的进化关系及其有丝分裂基因组的 CUB 模式密切相关。我们的研究结果为进一步研究黄蜂的遗传修饰和系统发育进化提供了基础参考。