Ciliates represent higher unicellular animals, and several species are also important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome in ciliates can not only promote a better understanding of the genetic mode and evolution history of these organisms but also help optimize codons to improve the gene editing efficiency of model ciliates. In this study, macronuclear genome sequences of nine free-living ciliates were analyzed with CodonW software to calculate the following indices: the guanine-cytosine content (GC); the frequency of the nucleotides U, C, A, and G at the third position of codons (U3s, C3s, A3s, G3s); the effective number of codons (ENC); the correlation between GC at the first and second positions (GC12); the frequency of the nucleotides G + C at the third position of synonymous codons (GC3s); the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, neutrality plot analysis, and correlation analysis were performed to explore the factors that influence codon preference. The results showed that the GC contents in nine ciliates’ MAC genomes were lower than 50% and appeared AT-rich. The base compositions of GC12 and GC3s are markedly distinct and the codon usage pattern and evolution of ciliates are affected by genetic mutation and natural selection. According to the synonymous codon analysis, the codons of most ciliates ended with A or U and eight codons were the general optimal codons of nine ciliates. A clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) expression vector of Stylonychia lemnae was constructed by optimizing the macronuclear genome codon and was successfully used to knock out the Adss gene. This is the first such extensive investigation of the MAC genome CUB of ciliates and the initial successful application of the CRISPR/Cas9 technique in free-living ciliates.

Background: Codon usage pattern is an important evolutionary feature in genomes widely observed in many organisms. Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: a germline micronucleus and a vegetative macronucleus. Analysis of codon usage pattern of S. lemnae macronucleus genome helps in understanding evolution at molecular level and acquires significance in mRNA translation, design of transgenic and new gene discovery. Results: The codons of the macronucleus genome sequence of S. lemnae were analyzed and 20,750 coding sequences (CDS) were screened. The overall codon usage of S. lemnae is similar and slightly biased. The value of effective number of codons (ENC) showed that the overall extent of codon usage bias in S. lemnae is relatively high. Nucleotide analysis showed that the overall codon usage is biased toward A- and U-ending codons. The phylogenetic analysis indicated that ciliate is independent evolutionary origins from a common ancestor. The RSCU analysis showed that the codon usage pattern of S. lemnae is more similar to that of Thtrahymana thermophila and Paramecium caudatum . Correlation analysis, ENC-GC 3S plot, and PR2 plot indicated that the codon usage patterns of S. lemnae are influenced by both mutational pressure and natural selection, neutrality plot analysis showed that those two factors play major roles. C onclusions : Codon usage patterns in eukaryotes are not determined by translational efficiency, but also are determined by the genome. Our study is the first attempt to evaluate the codon usage pattern of S.lemnae macronucleus genome to better understand the evolutionary changes. These results built the base for further research on the molecular evolution of S. lemnae .

Background: Codon usage pattern is an important evolutionary feature in genomes widely observed in many organisms. Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: a germline micronucleus and a vegetative macronucleus. Analysis of codon usage pattern of S. lemnae macronucleus genome helps in understanding evolution at molecular level and acquires significance in mRNA translation, design of transgenic and new gene discovery. Results: The codons of the macronucleus genome sequence of S.lemnae were analyzed and 20,750 coding sequences (CDS) were screened. The overall codon usage of S. lemnae is similar and slightly biased. The value of effective number of codons (ENC) showed that the overall extent of codon usage bias in S. lemnae is relatively high. Nucleotide analysis showed that the overall codon usage is biased toward A-and U-ending codons. The phylogenetic analysis indicated that ciliate is independent evolutionary origins from a common ancestor. The RSCU analysis showed that the codon usage pattern of S. lemnae is more similar to that of Thtrahymana thermophila and Paramecium caudatum . Correlation analysis, ENC-GC 3S plot, and PR2 plot indicated that the codon usage patterns of S. lemnae are not only influenced by mutational pressure but also by natural selection, but neutrality plot analysis showed that the latter plays a major role. C onclusions : Codon usage patterns in eukaryotes are not determined by translational efficiency, but also are determined by the genome. Our study is the first attempt to evaluate the codon usage pattern of S.lemnae macronucleus genome to better understand the evolutionary changes. These results built the base for further research on the molecular evolution of S. lemnae . BackgroundThe ciliate S. lemnae is a classical model single-celled eukaryote, which widespread in ponds, rivers and marshes. S. lemnae possesses both a macronucleus (MAC), specialized for gene expression, and a micronucleus (MIC), containing the germline genome that permits recombination and transmission of genetic information across sexual generations [1]. The macronucleus genome of S. lemnae is 50.2-Mb, contains 80 ribosomal relate genes [2].As we all know, the codons encoding the same amino acid are called the synonymous codons, and all