- Genome-wide analysis on the maize genome reveals weak selection on synonymous mutations. - Background: Synonymous mutations are able to change the tAI (tRNA adaptation index) of a codon and consequently affect the local translation rate. - Intuitively, one may hypothesize that those synonymous mutations which increase the tAI values are favored by natural selection.. - The first supporting evidence is that the tAI- increasing synonymous mutations have higher fixed-to-polymorphic ratios than the tAI-decreasing ones. - Conclusion: The synonymous mutations in the maize genome are not strictly neutral. - The tAI-increasing mutations are positively selected while those tAI-decreasing ones undergo purifying selection. - As understood by the broad researchers, synonymous mutations do not change the amino acid (AA) se- quences. - For instance, a few synonymous mutations occurring in the proper place could affect mRNA spli- cing [4, 5]. - Another impact of synonymous mutations is the change in tRNA adaptation index (tAI) [6], a termin- ology which described the tRNA availability of a codon.. - It is intuitive to consider that the change in tAI caused by synonymous mutations should undergo selection force. - A study in the ancient gymnosperm species Gingko biloba found higher frequency for A/T ending codons than G/. - C ending codons, but meanwhile it found that the highly expressed genes and those genes involved in environ- mental adaptation tend to use C/G ending codons, sug- gesting that the Gingko genome is dominated by natural. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Similarly, in angiosperms, it was found that the G/C ending codons were optimal (e.g. - Another study in four non-grass monocot species also proposed that the G/C ending codons are optimal and that this preference is not likely caused by mutation biases [11]. - Nevertheless, a recent study on four cotton species found the pattern that the pyrimidine-enriched codons (especially those ending with T) have higher fre- quency in the CDSs [12]. - Meanwhile, it was also de- clared that the GC3 (GC content at the 3rd codon position) method was not always suitable for evolution- ary comparisons at different scales [13] although CAI is positively correlated with GC3 [11]. - In this study, we are going to test our assumption in the maize (Zea mays) genome. - Our results would reveal the weak selection force act- ing on synonymous mutations caused by tAI-changing.. - Our current work on synonymous mutations could be appealing to the geneticists, evolutionary biologists as well as phytologists.. - To en- sure that the orthologous sites in wheat or carrot are not polymorphic, we downloaded RNA-seq data of roots generated from the corresponding species and mapped the RNA-seq reads to the reference CDS and discarded all the potential polymorphic sites (Methods).. - Synonymous mutations that increase or decrease the tAI value. - We defined the tAI values for each sense codon in maize (Fig. - Within each AA, any single base synonymous mutations that change a low-tAI codon to a high-tAI codon are defined as mutations that increase tAI (e.g. - Note that the tAI defined by us is also termed “AA tAI” in some literature [19]. - Fixed to polymorphic ratios reveal the weak selection on synonymous mutations. - It is well established that the nonsynonymous or non- sense mutations are overall deleterious and while the synonymous mutations are regarded as neutral. - Accordingly, we observed that the fixed to polymorphic ratios of nonsynonymous or nonsense mutations are significantly lower than that of synonymous mutations (Fig. - We wonder whether we could see differences in fixed to polymorphic ratios be- tween the two categories of synonymous mutations.. - This result means that the synonymous mutations that. - increase the tAI have significantly higher fixed to poly- morphic ratio (115.0) than those mutations that decrease the tAI (68.7) (Fig. - 2, Fisher’s exact test), suggesting an overall positive selection for synonymous mutations that increase the tAI or purifying selection on synonymous. - mutations that decrease the tAI. - We should emphasize that the absolute values of the fixed to polymorphic ra- tios might not be precise because if some polymorphic mutations are omitted due to the limited coverage of RNA-seq data, then this fixed to polymorphic ratio. - a Phylogeny of the plant species used in this study. - b tAI of the sense codons in maize. - c A diagram telling the readers our definition of synonymous mutations that increase or decrease the tAI. - These categories include synonymous, nonsynonymous and nonsense mutations as well as the synonymous mutations that increase or decrease the tAI. - Derived allele frequency (DAF) spectrum supports the positive selection on synonymous mutations that increase the tAI. - Similar to our analyses in the pre- vious section, we listed and plotted the DAF spectrum of synonymous (tAI up and tAI down, respectively), nonsy- nonymous and nonsense mutations (Fig. - We first veri- fied the known trend that the derived allele frequencies of mutations exhibit nonsense <. - Next, within the synonymous mutations, we found that the tAI-up mutations have significantly higher DAF than the tAI-down mutations (Fig. - 3, Wil- coxon rank sum tests), supporting the positive selection for synonymous mutations that increase the tAI and puri- fying selection on mutations that decrease the tAI.. - Here we also first verified the known pattern that the MAF of mutations ex- hibited nonsense <. - 4, Wilcoxon rank sum tests) due to the purifying selection on nonsynonymous and nonsense mutations. - Next, within the synonymous mutations, we found that the tAI-up mu- tations have significantly higher MAF than the tAI-down mutations (Fig. - 4, Wilcoxon rank sum tests), supporting the advantage of synonymous mutations that increase the tAI and the slightly deleterious effect of mutations that de- crease the tAI.. - The advantage of RNA-seq is that the data contain in- formation of expression level for each gene. - While tAI measures the tRNA accessibility of a codon, CAI directly measures the relative abundance of syn- onymous codons in the genome. - Since both tAI and CAI are frequently used in the studies of codon usage bias, we wonder whether our observed patterns on tAI hold true if we look at CAI.. - We compared the tAI versus CAI values within syn- onymous codons (Fig. - This suggests that the majority of syn- onymous mutations that increase tAI would also in- crease CAI, and vice versa. - We divided all synonymous mutations into two groups: CAI-up and CAI-down. - We found that the CAI-up group had significantly higher DAF as well as MAF than the CAI-down group (Fig. - As we listed in Additional file 2: Table S1, the syn- onymous mutations that increase the tAI are usually NNA/T-to-NNG/C mutations. - This pattern also exists for CAI because the GC-ending synonymous codons often have higher frequencies in the genome. - We surmise that the link between tAI and RNA sec- ondary structure might be mediated by the GC content because so far, no evidence shows the direct causality re- lationship between tAI and RNA folding. - Together with our observations above, the genome-wide GC content might have impact on the tAI patterns.. - To discuss how gBGC could affect the tAI and our conclusion, we divided the genes into two halves accord- ing to GC contents and see whether our observed pat- terns on tAI still exist. - We guess it is possible that the genes with higher GC content are fa- vored (compared to those with lower GC content) and therefore the strength of natural selection is more effi- cient in these genes.. - The pattern has been observed in the human genome [25]. - We show our data that the median distance between the nearby tAI- up and tAI-down mutations is less than 90 bp (Add- itional file 1: Figure S7). - If recombination bias does play a role, it should equally affect the tAI-up and tAI-down mutations, which means that the tAI-up mutations near the hotspots should have higher allele frequencies than the tAI-up mutations far from recombination hotspots. - a The relationship between tAI and CAI. - b DAF and MAF spectrum of synonymous mutations that increase or decrease the CAI. - This result denies the possibility that the observed differ- ence between “up” and “down” mutations is caused by recombination hotspots.. - Literally, “synonymous” means “the same”. - But a bunch of studies already demonstrated that some synonymous mutations affected mRNA splicing [4, 5, 26]. - Here we further broadened people’s understanding on the selec- tion force acting on synonymous mutations. - Synonym- ous mutations potentially change the tAI of a codon, and the changed tAI alters the translation rate of the codon. - This chain reaction finally exposes synonymous mutations to natural selection.. - 6 Relative contribution of each feature to the allele frequency spectrum of synonymous mutations. - Together with our current work (“context independent” selection pattern), it seems that the mRNA translation or splicing process is the main as- pect that a synonymous mutation could impact. - For example, two derived mutation sites, T-to-C at site1 and G-to-A at site2, mean that in the population there are T and C alleles at site1, and G and A alleles at site2. - We retrieved all A-to-T or T-to-A mutations, the tAI-up mutations have a me- dian DAF of 0.388 and those tAI-down mutations have a median DAF of 0.301 (p-value = 2.9e-4, Wilcoxon rank sum test). - Among all C-to-G or G-to-C mutations, the tAI-up mutations have a median DAF of 0.407 and those tAI-down mutations have a median DAF of 0.303 (p- value = 6.3e-8, Wilcoxon rank sum test). - The A-T switches and C-G switches ensure that the comparison between the bi-allelic site is fair and not affect by the biased gene conversion. - Our observations reveal the selection force acting on synonymous mutations in the maize genome. - We mapped the reads in the RNA-seq data to the refer- ence sequences (coding sequences) using aligner Bow- tie2 [29]. - Next, we should define the ancestral state of each position in maize CDS and decide whether a site should be included in the downstream variation analyses.. - We also excluded any polymorphic sites in the wheat and carrot CDS by mapping the RNA- seq data to their corresponding reference CDS. - The Zea mays sites analyzed in our study have no polymorphisms in the orthologous sites in wheat and carrot.. - Calculation of tAI and CAI. - Al- though there are only 52 types of anticodons in maize, there are as many as 1198 tRNA loci in the genome. - Next, for each CDS, we calculated the fraction of bases that are lo- cated in the structured regions (denoted as “struc- tured. - The statistical analyses were performed in the R environ- ment (http://www.R-project.org. - Inference of ancestral state of the mutations. - Take the case (of polymorphic mutations) shown in the graph as an example, the orthologous sites in two outgroups are C, and the reference genome of maize is also C, while in the RNA-seq data, both C and another nucleotide are detected. - These mutation categories include nonsynonymous and nonsense mutations as well as the synonymous mutations that increase or decrease the tAI. - a Spearman correlation between tAI and GC content of genes. - Spearman correlation between tAI and the percentage of RNA structured regions of genes. - These mutation categories include non- synonymous and nonsense mutations as well as the synonymous mutations that increase or decrease the tAI. - tAI-up ” and “ tAI-down ” synonymous mutations within each gene. - These genes are ranked by the median distance of the “ tAI-up ” and “ tAI-down ” mutations within them. - The list of single base synonymous mutations that increase the tAI.. - During this hard time, we thank the numerous anonymous doctors and health workers who endangered their own lives in the war against SARS-CoV-2. - Synonymous mutations frequently act as driver mutations in human cancers. - Parsing the synonymous mutations in the maize genome:. - Biased gene conversion affects patterns of codon usage and amino acid usage in the Saccharomyces sensu stricto Group of Yeasts
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