WU Yan-qing, Ll Zhi-yuan, ZHAO Da-qiu, TAO Jun

1 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China

2 Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, P.R.China

1. lntroduction

A codon is the nucleotide sequence that corresponds to a specific amino acid or signal during protein synthesis.Codons which define the same amino acid are referred to as synonymous codons; the usage of synonymous codons is not uniform in different species or different genes of the same species. A usage bias exists in some specific synonymous codons known as codon usage bias (CUB),which can reflect the origin of species or genes, evolutionary law and mutation pattern. Meanwhile, CUB is important for the study of protein expression and protein structure (Bulmer 1991; Mcinerney 1998; Songet al. 2015). There are two CUB therioes: neutral theory and selection-mutation-drift theory (Bulmer 1991). Neutral theory suggests that base mutation of synonymous codons is the result of neutral genetic shift, and is not under the influence of natural selection pressure. However, selection-mutation-drift theory involves the preferential use of the optimal synonymous codon with stronger selection for effective translation(Romeroet al. 2000). The study of CUB has increased in recent years with the addition of new whole genome sequences to of NCBI (https://www.ncbi.nlm.nih.gov/). Prior to this, studies mainly focused on the relationship between codon bias and gene expression in prokaryotes and lower eukaryotes (Gustafssonet al. 2004), and more recently,higher organisms, such as mammals and plants, which will aid effective analysis of gene expression in animals and plants and molecular evolution mechanisms (Angellottiet al. 2007). Phylogenetic relationships among species can be analyzed through the comparison of differential degrees of codon bias. In recent years, studies on CUB have been carried out on plant gene families or single function genes like south mustard, tea tree, grape and rice(Muhammadet al. 2012; Xuet al. 2012; Panet al. 2013; Liet al. 2014; Liuet al. 2015). CUB is a unique characteristic of the genome, which varies between the genes of a genome and specific genes between species (Duret and Mouchiroud 1999; Supek and Vlahovicek 2005). Gene codon usage patterns depend on amino acid sequences as well as mutational pressure and natural selection (Mandliket al. 2014), therefore codon bias variation might appear in different species of the same genus owing to selection pressure of one gene.

The flower has strong genetic stability in its occurrence pattern, development process, and structure, which is the main criteria for the study of evolution and development of angiosperm and classical taxonomy. Therefore, the floral organ is an ideal model system for analyzing the relationship among development, genes, and evolution (Zenget al. 2001).The current study is mainly focused onPaeonia lactiflora,Arabidopsis thaliana,Zea mays,Petunia×hybrida,Vitis vinifera,Malus domestica, andFragaria ananassa(Jofukuet al. 1994; Suet al. 2005; Songet al. 2009; Ren 2011; Geet al. 2014). The flower-meristem-identity geneAPETALA2(AP2), one of class-A genes, is involved in the establishment of the floral meristem and the forming of sepals and petals(Maeset al. 2001). The study on CUB is of great importance and nucleotide composition dynamics and gene transfer may help to elucidate evolutionary processes at the molecular level, design new transgenes (Carboneet al. 2003) and predict functional conservation in gene expression (Lithwick and Margalit 2005). The study herein aims to analyse codon bias and base composition dynamics inAP2gene codon usage patterns in eight different plant species using GC content, effective number of codons (ENC), relative synonymous codon usage (RSCU) and relative codon usage bias (RCUB). This study will improve our understanding ofAP2gene usage patterns, gene structure and function as well as the evolution of genes in different species.

2. Materials and methods

2.1. Sequence data source

The complete coding sequence (CDS) of the flowermeristem-identity geneAPETALA2(AP2) (ATG as the initiation codon, TAA and TAG or TGA as the termination codon) was identified from NCBI (http://www.ncbi.nlm.nih.gov). The species with a completeAP2gene CDS was selected for the analysis of CUB (Table 1).

2.2. Analysis of base composition and codon preference

The Program CUSP in EMBOSS Software (http://emboss.sourceforge.net/) (Riceet al. 2000), was used to calculate GC1, GC2 and GC3 contents, and GC1, GC2 and GC3 represented the first, second, and third GC base pair contents in codon, respectively. GC12 was used to calculate the average values of GC1 and GC2. CodonW 1.4 (http://codonw.sourceforge.net) (Peden 1999) was used to calculate RSCU, ENC, and frequency of optimal codon (Fop). The T, C, A and G contents at the third base pair in codon shall be expressed as T3, C3, A3 and G3, respectively.

2.3. RSCU

RSCU is the specific value between the actual observation value and theoretical observation values, among whichthe theoretical observation value is the observation value when the synonymous codon usage frequency is the same,namely there is no codon bias. If RSCU=1, there is no codon bias; if RSCU>1, the appearance frequency of codon is higher than the other synonymous codon; conversely, it will be lower (Sharp and Li 1986):

Table 1 The accession number and length of APETALA2 (AP2)gene coding sequences in this study

Where,niis the number of synonymous codons in theith amino acid;xijis the appearance time of thejth codon onith amino acid (Sharp and Li 1986).

2.4. ENC

ENC is the quantitative value of deviation for the difference between codon usage frequency and average frequency of synonymous codon usage, with the scope of 20-61;preference is stronger for those nearer to 20. The value reflects the preference of non-equilibrium use of synonymous codons (Wright 1990; Fuglsang 2004). A lower ENC value indicates codon bias is higher. Therefore, the relative level of endogenous gene is expressed with ENC.

Where, GC3 is the GC content of the third nucleotide of the codon (Wright 1990).

2.5. Fop

Optimal codon refers to those with the highest usage frequency in some specific species. Fop is a common index (Ikemura 1985) used for evaluating gene CUB. Fop refers to the proportion of optimal codons accounting for all synonymous codons (Ikemura 1981), which will alter from 0.36 (genes with the same codon bias) to 1 (genes with relatively strong codon bias) (Stenicoet al. 1994 ).

Where,nirefers to the number oficodons in the gene;Nrefers to the total number of codons in the gene, syn(i)is theith synonymous codons (Lavner and Kotlar 2005)corresponding to amino acid with codonicoding.

2.6. ENC plot

ENC plot is drawn from the ENC and GC3 as vertical and horizontal coordinates, respectively, to visualize the usage bias of each gene codon and influence of the base composition on CUB (Wright 1990). The method (Daset al.2006; Jiaet al. 2009; Taoet al. 2009) is used extensively to elucidate the position of the gene using codon bias and base composition.

2.7. Evaluation and analysis of gene expression

Gene expression is evaluated through RCUB, which is defined as relative codon bias of each codon in the gene with influence on overall scoring (Karlin and Mrazek 1996).The RCUB value of eachAP2gene sequence is calculated in accordance with the equation provided by Fox and Erill(2010).

2.8. Clustering based on CUB

Conduct clustering based on the CUBs of eightAP2genes(Daset al. 2006) was calculated using SPSS16.0 (http://www.spss.com/). In the analysis of usage frequency, the gene was referred to as the factor and the relative codon as the variable. The RSCU values of 59 synonymous codons(removing the coded methionine codon AUG and coded tryptophan codon UGG as well as three termination codons)were used for the analysis of CUB.

2.9. Statistical analysis

Both Microsoft Excel and SPSS 16.0 (http://www.spss.com/) were used to conduct the correlation analysis, and the comparison methods were based on Spearman’s rank.The heat map was generated using R Software (https://www.r-project.org/) based on the hierarchical clustering method for analyzing the correlation coefficient of codons,GC3, and RSCU values of different species.

3. Results

3.1. Analysis of codon bias of AP2 gene in different plant species

Codon usage pattern scan provides a foundation for revealing CUB (Hassanet al. 2009). To understand the relationship between codon usage variation inAP2gene sequences and GC contents, a heat map was used to analyze the correlation between codon base composition and GC3 (Fig.1). The results showed that most of the codon ended with GC were positively related with GC3 and most of codon ended with AT were in negatively related with GC preference. However, two codons ended with GC (ACG,TTG) were not negatively related with GC3 and six codons closed with AT (ACA, CCA, CGA, GCA, GGA, CTT) were not positively related with GC preference (P>0.05). It is interesting that two codons AGG and GAG show strong positive relationships with GC3 (P＜0.01,r>0.90); the usage of codon may be under the influence of GC3s preference.

3.2. Preference of AP2 gene codon closed with GC in different plant species

In this study, base composition ofAP2gene CDS is analyzed in different species (Table 2). The average content of base A was the highest (433.4), followed by G (368.9), T (346.6),and C (308.4). The average GC and AT contents were 46.3 and 53.7%, respectively. Overall base composition analysis showed that the usage frequency of AT was higher than that of GC amongAP2gene codons in different plant species.The average content (A3, T3, C3, G3) on the third nucleotide of the codon showed that A3 and T3 contents were higher than G3 and C3. Comparisons were carried out between GC3 (value range of 29.8-54.7% with the average of 40.8%,standard deviation (SD)=0.089) and AT3 values (value range of 45.3-70.2% with the average of 59.2%, SD=0.090). The range of change in average GC content (GC12) on the first and second nucleotide of codons was 44.9-51.8%, with an average value of 49.0% and SD of 0.021. This suggests that the base composition as codon closed with AT was preferred inAP2gene CDS. Moreover, in this study, the method of Lavner and Kotlar (2005) was used to calculate Fop of each amino acid and the high and low frequency codon usages were calculated, respectively. The results revealed 18 optimal codons were closed with A/U inAP2gene of different species; only those codons with UUG and ACC were closed with GC (Fig. 2).

Fig. 1 Heat maps of correlation coefficient of codons with GC3. The red and green colors represent the positive and the negative correlations, respectively, and the black color mean stop codons (TAA, TAG, TGA) or non-degenerate codons (ATG,TGG). -, mean A, T, C, or G randomly.

3.3. Relative codon usage of AP2 gene in different plant species

Synonymous codon usage values of 59 codons ofAP2gene in eight plant species were calculated (except ATG methionine and TGG tryptophan). RSCU>1 indicates a higher usage frequency, and RSCU>1.6 suggests a strong preference. Among the selectedAP2genes, 28 high-frequency codons showed a significant preference for codons closed with A/U (Table 3). Moreover, codons closed with U were preferred in further observations.RSCU cluster analysis (Fig. 3) showed the RSCU value of AGA (exceptMalus domestica), GCU (exceptMalus domestica) and UGU (exceptVitis vinifera) were higher than 1.6 with expression of relatively strong preference amongAP2gene in different species.

Table 2 Nucleotide composition analysis in the coding sequences (CDS) of APETALA2 (AP2) gene1)

3.4. System relationship of codon usage patterns of AP2 gene in different plant species

Fig. 2 Overall frequency of optimal and non-optimal codon used in APETALA2 (AP2) genes among different species. Red color represents optimal used codons with corresponding amino acid. Bars mean SD.

Table 3 Overall relative synonymous codon usage (RSCU) for APETALA2 (AP2) gene among eight plant species

In this study, adjacent evolutionary tree (neighbor joining(NJ)) (Fig. 4) was established based on the RSCU ofAP2gene in different plant species kimura 2-parameter (K2P).The results showed thatAP2genes with closer proximity between species had significantly similar codon usage mode, among which theAP2gene ofMalus domesticais similar to that ofVitis vinifera,AP2gene ofPaeonia lactiflorais similar to that ofPaeonia suffruticosa, andAP2ofSolanum lycopersicumis similar to that ofPetunia×hybrida.In general, genes with similar function have similar usage mode (Tatarinovaet al. 2010). Therefore, the usage mode of theAP2gene codon have a certain similarity among eight different species in the study.

Fig. 3 Clustering of relative synonymous codon usage (RSCU) values of each codon among APETALA2 (AP2) gene across plants. Each rectangular box on the map represents the RSCU value of a codon (in rows) corresponding to the AP2 gene across plant species (in columns). The intensity of color coding indicates different RSCU values: intensity towards blue RSCU＜1.6 and red RSCU>1.6.

3.5. lnfluence from selection pressure of AP2 gene in different plant species

ENC values of 56-61 (average value 59.37±1.69) showed that there was a relatively small variation in the usage ofAP2gene codon among eight different plant species.However, the scope of GC3 values ranged from 0.298 to 0.547 (average value 0.408±0.090). ENC plot showed that there was a significant positive relationship between ENC and GC3s (Pearsonr=0.71,P＜0.05) (Fig. 5). The results showed a GC3 skew, which changing scope was from –0.101 to 0.122. Results showed a lower Fop value and higher ENC value as the codon usage ofAP2gene in different species with moderate preference (Granthamet al.1981) (Table 4).

Gene RCUB value can be used to measure the effective index of gene expression, which is mainly under the influence of gene base composition preference (Wright 1990). RCUB value ofAP2in different specifies was from 0.130 to 0.198, with an average of 0.174±0.021 (Fig. 6).RCUB values indicated thatAP2gene with lower expression level had lower codon preference.

Fig. 4 Phylogenetic analysis of the Kimura 2-parameter (K2P) distances of the selected coding sequences (CDSs) among APETALA2(AP2) genes of different plant species. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) is shown next to the branches. The analysis involved eight CDSs. All positions containing gaps and missing data were eliminated. Evolutionary analyses were performed with MEGA5.

4. Discussion

Fig. 5 Effective number of codon (ENC) vs. GC3 values for APETALA2 (AP2) gene. The dots represent ENC and GC3 values of the coding sequences (CDS) for AP2 gene across different plant species.

CUB of different organisms is related to many genomic features such as gene length, GC content, recombination rate, gene expression level, and genetic code (Karlin and Mrazek 1996; Duret 2000; Urrutia and Hurst 2003;Roymondalet al. 2009; Palidworet al. 2010). However,this study also showed that the usage pattern of codons may be under the influence of mutation pressure, translation selection, secondary protein structure, replication and selective transcription, protein hydrophobicity and environment (Buttet al. 2014). The codon usage in prokaryotic and eukaryotic organisms are mainly under the joint influence of mutational bias and selection (Gouy and Gautier 1982; Sharpet al. 2005; Bragget al. 2012). The codon usage in multicellular eukaryotes such asDrosophila melanogasterandCaenorhabditis elegansare mainly under the influence of selection (Shieldset al. 1988; Stenicoet al.1994; Vicarioet al. 2007). The codon usage in viruses such as Parvoviridae are mainly under the joint influence from mutation pressure and natural selection (Shiet al. 2013).Study on single cell and multicellular organisms found that the amount and expression level of tRNA gene increased as the quantity of optimal codons increased, which suggesting that there is a positive relationship between optimal codons and tRNA abundance (Ikemura 1981; Akashi 1995; Duret 2000). However, plant species such asArabidopsis thaliana,Oryza sativa,Zea maysand herbaceous peony are mainly under the influence of gene base composition and gene expression level (Chiapelloet al. 1998; Liuet al.2004; Morton and Wright 2007; Liuet al. 2010; Wuet al.2015). The appearance frequency of different codons in genes of differen plant species is obviously different and the ratio of different codons in the same amino acid is different, among which codon preference CUB is under the influence of different composition (Wonget al. 2002). Base composition is one of the fundamental features of genomic DNA, the usage frequency of different nucleotides is decided jointly by the balance of mutation and reverse mutation. In general, base composition is thought to be the main factor used in different organisms under natural selection and mutation pressure (Ikemura 1981; Li 1987; Xuet al. 2011).Furthermore, GC content is thought to reflect the overall trend of mutation, the change of the third codon nucleotide will not generally result in the alteration of the coding amino acid. Therefore, there is little selection pressure on the base mutation on the third nucleotide and GC3 is also an important criterion for the analysis of codon usage mode.This study analyzed the compositional dynamics ofAP2gene codon in different plant species; there was an obvious correlation between codon and GC3 andAP2gene codon preference in species may be influenced heavily by base composition, especially GC3 content. The ENC figure(ENCvs. GC3s) revealed that most of the ENC values were significantly correlated with GC3s with a smaller GC3 skew,which further indicates that the third base GC3 composition GC3 plays an important role in the codon usage preference.(Yang and Nielsen 2008). Furthermore, the larger the ENC value, the lower the gene expression level. This study found that all theAP2ENC values in eight plant species weregreater than 50. And the higher values of ENC indicate lowerAP2gene expression in species, which might be related toAP2gene function mechanism. There was a difference in codon preference in different plant species and different genes in the same species. In the result of this study, AT frequency in codon was higher than GC, and most codons ended with AT were the characteristics of gemini plant and in agreement with the results in Wuet al. (2015). Transgenic research requires the heterologous expression of gene and the difference in codon usage among different species is relatively large. Comparison and clustering ofAP2gene codons in different plant species revealed similarities in codon usage characteristics betweenMalus domesticaandVitis vinifera,Paeonia lactifloraandPaeonia suffruticosaas well asSolanum lycopersicumandPetunia×hybrida. Codon preference is gradually formed in the process of biological evolution with an adaptation to the environment of the host genome. This study revealed the usage characteristics and similarity ofAP2gene codons in different plant species and confirmed the optimal codons (AGA, GCU and UGU) inAP2genes, which may guide further study ofAP2gene function.Therefore, our results provide insights into the study of the synonymous codon usage patterns ofAP2in different plant species, and was a theoretical basis for the transformation of codons and improvements in the expression level ofAP2.

Table 4 Codon usage bias indices for APETALA2 (AP2) gene across plant species1)

Fig. 6 Distribution of relative synonymous codon usage (RSCU) for APETALA2 (AP2) gene across eight plant species. Bars mean SD.

5. Conclusion

In this study we found that the codon usage ofAP2gene was influenced by GC bias, especially GC3. Our analysis of base composition and optimal codon frequency indicated thatAP2gene coding sequences prefer to ended with AT in the nine selected species. In the analysis ofAP2gene,AGA, GCU, and UGU had relatively high RSCU value among all selected species, indicating that this is the optimal codons. Additionally, the codon usage characteristics were similarities betweenM. domesticaandV. vinifera, betweenP. lactifloraandP. suffruticosa, and betweenS. lycopersicumandPetunia×hybrida. Low Fop and ENC values with higher AT content mean moderate preferential codon usage in theAP2gene among different species.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31372097), and the Agricultural Science&Technology Independent Innovation Fund of Jiangsu Province, China (CX(13)2014).

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