Synonymous codon usage bias is correlative to intron number and shows disequilibrium among exons in plants
© Qin et al.; licensee BioMed Central Ltd. 2013
Received: 2 July 2012
Accepted: 18 January 2013
Published: 28 January 2013
Evidence has been assembled to suggest synonymous codon usage bias (SCUB) has close relationship with intron. However, the relationship (if any) between SCUB and intron number as well as exon position is at present rather unclear.
To explore this relationship, the sequences of a set of genes containing between zero and nine introns was extracted from the published genome sequences of three algal species, one moss, one fern and six angiosperms (three monocotyledonous species and three dicotyledonous species). In the algal genomes, the frequency of synonymous codons of the form NNG/NNC (codons with G and C at the third position) was positively related to intron number, but that of NNA/NNT was inversely correlated; the opposite was the case in the land plant genomes. The frequency of NNC/NNG was higher and that of NNA/NNT lower in two terminal exons than in the interstitial exons in the land plant genes, but the rule showed to be opposite in the algal genes. SCUB patterns in the interstitial and two terminal exons mirror the different evolutionary relationships between these plant species, while the first exon shows the highest level of conservation is therefore concluded to be the one which experiences the heaviest selection pressure. The phenomenon of SCUB may also be related to DNA methylation induced conversion of CG to AT.
These data provide some evidence of linkage between SCUB, the evolution of introns and DNA methylation, which brings about a new perspective for understanding how genomic variation is created during plant evolution.
KeywordsSynonymous codon usage bias Plant evolution Intron number Exon position DNA methylation
The degeneracy of the nucleotide triplet code, is such that, with the exceptions of Met and Trp, each amino acid residue is encoded by two or more synonymous codons (SCs). SC frequency can vary from one genome to another, and even from one gene to another within a single genome . The resulting variation has been termed “synonymous codon usage bias” (SCUB) and has been identified in prokaryotic organism genomes as well as in those of both animals and plants. The evolution of SCs is proposed to reflect a balance between mutation, genetic drift and natural selection [2, 3].
Evidence has been assembled to suggest a relationship between intron and SCUB (see review by ). The gain/loss of introns is a key component of the evolution of genomes [4, 5], via either transposon insertion  or “reverse splicing” , but also as a by-product of recombinational error . Indel events necessarily entail prior DNA breakage and refusion, processes associated with genomic shock [9, 10], a consequence of which can be the induction of local single nucleotide polymorphisms. Just as is the case for indels, the gain/loss of introns also potentially induces genomic shock and its attendant consequences . The propensity for intron gain/loss is related both to intron number and the intron’s position within the gene , so there is reason to suspect that SCUB may in turn also be related to these variables.
The presumed ancestors of land-based plants, from mosses to angiosperms, are the single celled algae. Polyploidization has been one of the major drivers of genome evolution. The process of genome duplication can result in orthologous genes evolving a different intron content, and in so doing can contribute to the divergence in gene structure between species . For example, DNA replication slippage and repetitive sequence duplication are thought to be the major sources of intron gain [5, 13]; segmental genome duplication can generate a functional intron that could be deleted during RNA editing . Thus, there may well be an association between SCUB and the patterns of plant evolution; but as to whether or not SCUB based on intron number and exon position could shed new light on the evolutionary path of plants has not yet been fully evaluated. Here we have based a study of SCUB on the genome sequences of three species of algae, one of moss, one of fern and six angiosperms (three monocotyledonous species, three dicotyledonous species). Our aim was to identify the correlation, if any, between SCUB and both intron number and exon position.
Intron distribution and gene length
A comparison of the genomes of the three algal species (Ectocarpus fasciculatus, Chlamydomonas reinhardtii, Volvox carteri), the moss (Physcomitrella patens), the fern (Selaginella moellendorffii) and the six angiosperms (Oryza sativa, Zea mays, Sorghum bicolor, Arabidopsis thaliana, Glycine max and Populus trichocarpa) showed that, the number of genes is reduced as the frequency of introns increases (Additional file 1: Figure S1). The proportions of genes containing 0–9 introns ranged from 73.6% in C. reinhardtii to 90.1% in P. trichocarpa.
The correlation between SCUB frequency and intron number
SCUB frequency is variable within exonic sequence
The mean ratios of NNAs, NNTs to NNCs, NNGs within the first exon were comparable among genes with 2–10 exons in either algal or land genomes; in comparison with the first exon, these ratios in the subsequent exons were higher in the land plant but lower in the algal genes (Figures 3, Additional file 5: Figure S4). In the final exon, the ratios were conserved among the algal genes, but were positively correlated with intron number among the land plant genes; this correlation was weakest among the angiosperm species. In the interstitial exons, the ratios were conserved among the algal genes, but were variable among the land plants, particularly in genes having a larger number of introns. Heterogeneity between exons was also reflected by the frequencies of NNA, NNT, NNC and NNG (Figure 4), which were relatively well conserved in the first exon across all the test species. Conservation was good in the final exon among the algal species; the frequency of NNC and NNG was positively correlated with intron number in the moss, fern and monocotyledonous angiosperm species, but that of NNA and NNT was negatively correlated; among the dicotyledonous species, the frequency of NNC and NNG was well conserved, but that of NNA was reduced and that of NNT was increased in genes carrying a larger number of introns.
The role of DNA methylation in the formation of SCs
The role of methylation in SCUB is also revealed by frequencies of SCs within a certain amino acid (Additional file 2: Table S1). For the residues Ala, Pro, Ser and Thr, each of which is encoded by more than two SCs each with a C in its middle position, the NCG frequency declined more sharply than that of NCC as the intron number increased, while the NCA frequency rose more obviously. For Arg, Gly, Leu and Val (codons without a C in the middle position), the frequencies of NNCs were clearly lower than those of NNGs, while those of NNTs was higher than those of NNAs. A comparison between the pairs of residues Asn vs Lys, Asp vs Glu and Gln vs His (the first two nucleotides of the SCs lacking C at the second position are the same in each pair) showed that the frequencies of NNCs and NNTs had more distinguishable alteration than NNGs and NNAs, respectively. A similar analysis of asymmetric methylation, based on the codons CHG and CHH (H = A, C or T) was carried out by assessing the frequencies of N|NN and NNN, and a more obvious alternation in C|NN and NNG frequencies than in others was found based on both intron number and exon position (data not shown). Unlike for the land plant genomes, in the algal genomes the frequencies of NCG and NC|G, and of NCA and NT|G were not different from those of NNN and NN|N, and were uncorrelated with both intron number and exon position (Figure 5A,B, 6A,B, Additional file 8: Figure S7, Additional file 9: Figure S8, Additional file 10: Figure S9).
Plants are clustered with respect to SCUB based on intron number and exon position
Intron evolution is a strong driver of SCUB in land plants
Intron loss is a major feature of eukaryotic evolution [16, 17]. Changes in the intron structure can induce mutations in adjacent exons, forming either SCs or non-synonymous codons that lead to a bias towards lower GC content . The present analysis has suggested that genes with fewer introns tend to show a heightened frequency of NNC and NNG and a concomitant lowered frequency of NNA and NNT codons. Genes with fewer introns are thought to be favored by selection, and to evolve more slowly , with the result that the GC content of the exonic fraction of the genome has tended to have risen over time . Thus it is possible that SCUB is directed to GC preference in genes with less introns that occur lower frequency of single-nucleotide substitution induced by intron evolution.
The single nucleotide changes induced by indel formation can occur over a distance of several hundred bases from the site of the indel itself , and the substitution level is negatively correlated with its distance to the indel . Thus, the gain or loss of an intron is only likely to induce single nucleotide change in the flanking exons. Since intron gain/loss takes place preferentially at the 3′ terminus of eukaryotic genes [22, 23], the implication is that GC enrichment at the 5′ terminus of exons is not likely to be greatly affected by intron evolution. The first exon does in fact tend to be the most enriched with respect to NNC and NNG, at least in land plant genes, and the frequency of these codons occurs is largely independent of how many exons are present. Thus it is the first exon which experiences the most intense selection pressure, and it is therefore this exon which remains most highly conserved. We have demonstrated that the frequency of NNC and NNG codons in the final exon appears to be higher than in the interstitial exons. If it is the case that indels tend to occur most readily in regions where the GC content is relatively low [21, 24] and that their effect is to reduce GC content , then this represents a contradiction with the proposal that intron evolution (and especially intron loss) is most rapid at the 3′ terminus of genes . The present analysis suggests rather that intron evolution is most rapid in the interstitial exons, consistent with the observation that a large proportion of intron loss is experienced in the middle of gene sequences .
SCUB allows insights into plant evolution
The algae arose long before the appearance of land plants, and had already been exposed to a long period of selection which would have tended to favor GC enrichment . Our analysis of three algal genomes has shown a marked SC bias of NNC and NNG over NNA and NNT (Figures 2, 4). A possible inference from this observation is that algal genomes have become very stable and that intron evolution now is very much slowed. Polyploidization has been a ubiquitous process in the evolution of higher plants. It induces a range of genomic shock associated events, such as gene loss and single nucleotide changes . The latter are heavily biased towards A and T . A salient property of enlarged genomes is that they provide buffering against selection pressure [17, 28], and such a reduction favors the enrichment of the genome’s GC content. These two processes together could account for the observed shift in SCUB from NNC and NNG to NNA and NNT in land plant genes, a shift which is most pronounced in the dicotyledonous species (Figure 2).
Both the divergence of the gymnosperms and angiosperms from the ferns, and that of the angiosperms from the gymnosperms involved whole genome duplication events . The dependence of SCUB pattern on intron number is comparable between the fern and the monocotyledonous species (Figure 2), so does not reflect a major effect of either of these genome-wide events. The marked preference for NNA and NNT among the dicotyledonous species is suggestive of the influence of polyploidization events occurring post the divergence of the monocotyledonous and dicotyledonous species [29–36]. SCUB based on exon position mirrors very closely the important events which have driven plant evolution (Figure 7). The cluster pattern of algal and land plant species based on either the first, last or interstitial exons (Additional file 11: Figure S10) both resembles the presumed chronology of plant evolution, and suggests a degree of SCUB heterogeneity.
DNA methylation contributes to SCUB during intron evolution
The formation of indels contributes to the level of DNA methylation . The DNA methylation induced conversion of CG to AT is thought to be a potent agent of naturally occurring mutagenesis . The present data has shown that changes in the frequencies of both NNC and NNG dependent on either intron number or exon position are well correlated with those of, respectively, NCG and NC|G (Figure 56). The implication is that the increased rate of intron evolution associated with genes having a higher number of introns drives up the likelihood of DNA methylation and therefore generates a bias towards NNA and NNT. This bias is more recognizable in the interstitial exons than in the two outermost ones, so implies that intron evolution is favored in the interstitial region of genes. DNA methylation thus is likely to be a major driver of SCUB during intron evolution.
SCUB is correlated with intron number and is non-homogeneous across all exons. The pattern of its heterogeneity differs from plant species to plant species. It has also been shown that DNA methylation is likely a major driver of SCUB. These inferences provide a new perspective for understanding how genomic variation is created during plant evolution. As yet it is unclear whether or not animal genomes behave in the same way as plant genomes appear to.
The genome sequences of O. sativa and A. thaliana were downloaded from http://www.ncbi.nlm.nih.gov/genome, that of S. moellendorffii from http://genome.jgi-psf.org/Selmo1/Selmo1.download.ftp.html, E. fasciculatus from https://bioinformatics.psb.ugent.be/webtools/bogas/. and other species from http://www.plantgdb.org/XGDB/phplib/download.php.
The intron/exon structure of the O. sativa and A. thaliana genes was obtained from the CDS annotation, while E. fasciculatus genes were identified from their cds file and their structure was inferred from the relevant gff3 file. For the other species, gene sequences were extracted from the appropriate nucleotide fa files, and their structure from the relevant gff3 files. For genes which encoded more than one transcript, the intron structure was inferred from the sequence of the primary transcript. ATG triplets were taken as start codons, and TAA, TGA and TAG as stop codons . The codon separated by a intron between the first and the second nucleotides was acted as the condon of the intron’s 3′-adjacent exon, while that separated between the second and the third nucleotides belonged to the 5′-adjacent exon. These analyses were performed using a customized Pearl script.
Calculation of SCUB frequency
Calculations were based on 59 (of the possible 64) codons, encoding 18 amino acids; the five not considered comprised the three stop codons, ATG (Met) and TGG (Trp). The SC frequency of a given residue was defined as the ratio between the number of a given SC to the number of all SCs for that particular amino acid. The SC frequency based on the third position nucleotide (NNA, NNT, NNC and NNG) was given by the ratio of the number of SCs having a given nucleotide to the total number of 59 codons. The SC frequency of the second/third nucleotide combinations (NNN) and the third nucleotide/first nucleotide of the following codon (NN|N) was defined as the number of a certain combination to the total number of 59 codons.
Cluster and PC analysis
SC frequencies were subjected to both a cluster analysis based on the joining tree method implemented within the STATISTICA software package (V6.0, StatSoft) and a PC analysis based on the varimax method implemented within the SAS software package (V8.0, SAS Institute Inc.). Scatter plot diagrams were generated from the coefficients given by the first two PCs.
Synonymous codon usage bias
NNC, NNG, NNT: Synonymous codons with A, C, G and T at the third position
the synonymous codon combinations based on the second-third nucleotides
the synonymous codon combinations based on the third nucleotide of the codon and the first nucleotide of the next codon.
This work was supported by the Natural Science Foundation of China (31171175), the Excellent Young Scientist Award Foundation of Shandong Province (BS2009SW023), the Major Program of the Natural Science Foundation of China (31030053), the College Innovation Foundation of Jinan City (200906021).
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