Abstract: The genes codon usage of species has a bias,which can avoid the wrong incorporation of amino acids. The bias of Medicago codon has not been reported. In order to reveal the codon bias characteristics of Medicago genome,we used software to analyze the codon composition of the coding region,the relative synonymous codon use frequency (RSCU),and other parameters of four Medicago species to explore the codon bias characteristics. At the same time,we used various methods to analyze the influencing factors causing the codon bias of Medicago,and calculated the optimal codon of Medicago. Finally,the evolutionary relationship of codon usage bias among species was studied by cluster analysis. The results showed that the codon bias of four Medicago species was relatively weak,of which ‘Zhongmu No.1’ was the weakest,and the third position of Medicago codon prefered A/T;The codon bias of four Medicago species was greatly affected by natural selection pressure,and most of the optimal codons ended in A/T. The bias of four Medicago species was similar and close to Nicatiana tabacum L.and Solanum lycopersicum L.. This study provides a theoretical basis for the application of Medicago codon optimization,especially through the optimization to improve the expression of Cas9 protein to promote gene editing.

Key words: Medicago, Codon usage bias, Optimal codon, Gene editing, Natural selection

摘要： 物种基因的密码子使用具有偏好性，能避免氨基酸的错误掺入。苜蓿(Medicago)密码子的偏好性尚未见报道。为揭示苜蓿属基因组密码子偏好性特点，我们利用软件分析四种苜蓿基因编码区密码子组成、相对同义密码子使用频率(RSCU)等参数探讨密码子偏好性特点，同时用多种方法分析造成苜蓿密码子偏好性的影响因素，计算确定苜蓿的最优密码子，最后通过聚类分析研究物种间密码子使用偏好性进化关系。结果表明：四种苜蓿的密码子偏好性相对较弱，其中‘中苜1号’最弱，苜蓿密码子第三位偏爱使用A/T；四种苜蓿密码子偏好性受自然选择压力的影响大，最优密码子多以A/T结尾；四种苜蓿的偏好性相似，且与烟草(Nicatiana tabacum L.)、番茄(Solanum lycopersicum L.)偏好性接近。本研究为苜蓿密码子优化应用提供了理论基础，特别是通过优化可以提高Cas9蛋白表达量促进基因编辑。

关键词: 苜蓿, 密码子偏好性, 最优密码子, 基因编辑, 自然选择