Paragraph: Codon usage bias (CUB) is an important evolutionary feature in a genome which provides important information for studying organism evolution, gene function and exogenous gene expression. The CUB and its shaping factors in the nuclear genomes of four sequenced cotton species, G. arboreum (A 2 ), G. raimondii (D 5 ), G. hirsutum (AD 1 ) and G. barbadense (AD 2 ) were analyzed in the present study. The effective number of codons (ENC) analysis showed the CUB was weak in these four species and the four subgenomes of the two tetraploids. Codon composition analysis revealed these four species preferred to use pyrimidinerich codons more frequently than purine-rich codons. Correlation analysis indicated that the base content at the third position of codons affect the degree of codon preference. PR2-bias plot and ENC-plot analyses revealed that the CUB patterns in these genomes and subgenomes were influenced by combined effects of translational selection, directional mutation and other factors. The translational selection (P2) analysis results, together with the non-significant correlation between GC12 and GC3, further revealed that translational selection played the dominant role over mutation pressure in the codon usage bias. Through relative synonymous codon usage (RSCU) analysis, we detected 25 high frequency codons preferred to end with T or A, and 31 low frequency codons inclined to end with C or G in these four species and four subgenomes. Finally, 19 to 26 optimal codons with 19 common ones were determined for each species and subgenomes, which preferred to end with A or T. We concluded that the codon usage bias was weak and the translation selection was the main shaping factor in nuclear genes of these four cotton genomes and four subgenomes. OPEN ACCESS Citation: Wang L, Xing H, Yuan Y, Wang X, Saeed M, Tao J, et al. (2018) Genome-wide analysis of codon usage bias in four sequenced cotton species. PLoS ONE 13(3): e0194372. https://doi. codon known as synonymous codons. The synonymous codons are not used at equal frequencies in coding sequences in many organisms [1]. This phenomenon called 'synonymous codon usage bias (SCUB)' reflects non-uniform usage of synonymous codons encoding the same amino acid during the translation of genes to proteins [2,3]. The degree of SCUB divergence varies greatly among different species and genes [4-7]. SCUB was affected by many factors, including directional mutation, neutral selection [8], GC content, synonymous substitution rate [9], tRNA abundance [10], selection for efficient translation initiation [11], codon hydropathy and DNA replication initiation site [12], gene length [13] and expression level [14] , etc. Of these factors, directional mutation and neutral selection are the two main ones, with varying relative importance in different species [15] [16][17]. The codon usage patterns always represent balance between the directional mutation and the neutral selection that leads to translational efficiency of genes [8] [9] [10] [11] . SCUB patterns were also associated with phylogenetic relationship among given species. Distant phylogenetic species usually has greater variations in codon usage bias [18,19]. Information on the SCUB patterns can provide significant insights pertaining to the prediction, classification, and molecular evolution of genes, design of highly expressed genes and cloning vectors and reveal about the host-pathogen coevolution and adaptation of pathogens to specific hosts [20,21]. Clustering results based on relative synonymous codon usage (RSCU) values could provide useful reference for phylogenetic relationship analysis [18,19,22]. Cotton (Gossypium spp.) is the main source of renewable textile fibers and is also grown to produce vegetable oil and high protein meals for humans and livestock [23]. The genus Gossypium includes around 46 diploid (2n = 2x = 26) and 6 tetraploid (2n = 4x = 52) and 1 purported species [24][25][26], including four commercial ones, G. arboreum (A 2 ), G. herbaceum (A 1 ), G. hirsutum (AD 1 ) and G. barbadense (AD 2 ). It has been proposed that all diploid cotton species may have evolved from a common ancestor and allopolyploid cotton may have appeared through hybridization and subsequent polyploidization events between the A-and D-subgenome progenitors. The D-genome species G. raimondii (D 5 ) and the A-genome species much like modern G. arboreum (A 2 ) and G. herbaceum (A 2 ) are the donor species for the D and A chromosome groups of the tetraploid cotton species, respectively [27][28]. Recently, the nuclear genome sequences of G. arboreum, G. raimondii, G. hirsutum and G. barbadense, and chloroplast and mitochondrial genome sequences of G. hirsutum have successively been released [29][30][31][32][33][34][35][36][37][38], which has advanced the understanding of cotton genomics and genetics, and made it possible to investigate SCUB patterns in cotton nuclear and organelle genomes. But until now, SCUB analysis has been performed only in chloroplast genome of G. hirsutum [36]. The purpose of this study was to analyze the SCUB patterns of the four sequenced cotton species and explore the key factors influencing codon choice. 
 Title: Genome-wide analysis of codon usage bias in four sequenced cotton species
Output: True