Molecular Evolution Of The Chloroplast Gene Rps12 In Ferns

As the second largest group of vascular plants,ferns play an important role in biological evolution and ecosystems.The chloroplast genome has the characteristics of small genome,high copy number and evolutionary conservation.Complete chloroplast genomic sequences can provide unique information in unveiling the mechanisms or features of cp DNA evolution at both gene and genomic levels.RPS12(Ribosomal Protein Small Subunit 12)plays an important role in the initiation of ribosomal 30 S small subunit formation,which is encoded by the chloroplast rps12 gene in plants.In different plants,the location of rps12 gene in chloroplast genome is different.The 5'-rps12(exon1)is located in the large single copy region,while the 3'-rps12(exon2-3)is located in the large single copy region or the inverted repeat region.The inverted repeat region has a different substitution rate for their genes from those in the large single copy region due to its double-copy nature.By comparing the evolution rates of different parts of the rps12 gene(exon1 and exon2-3),the difference in evolution rates between the inverted repeat region and the single copy region can be explored.At present,there are few studies on the molecular evolution of the fern rps12 gene.Analysis of the adaptive evolution of this gene has important reference value for studying the changes of gene structure and functional variation.The rps12 gene sequence of 68 fern species and 2 lycophyte species was selected in this study.Using Hy Phy software,we have analyzed the molecular evolution rate of the rps12 coding sequences,exon 1 and exon2-3,as well as the selection pressure(positive selection and negative selection)they have experienced.The PAML software has been used to identify the positive selection of amino acid sites using the branch model,site model,and branch site model.The results are as follow:(1)The evolution rate of exon 1 in plants with exon2-3 located in the inverted repeat region(IR-61)is 3-6 times t of that in exon2-3.For plants with both exon1 and exon2-3located in large single-copy region(LSC-9),their evolution rate of exon1 was not different from that of exon2-3.The evolution rate of the rps12 coding sequence of LSC-9 is 3-5times that of IR-61.The evolution rate of exon2-3 of LSC-9 is 4-10 times of that in IR-61.That is,when genes(or gene fragments)are located in the inverted repeat region,their evolution rate is decelerated.It is speculated that the inverted repeat region has a low evolution rate due to its double-copy nature,and its frequency of gene conversion is high,which may repair the mutation site.It is also found that the GC content of the third position of the codon of IR-61 is significantly higher than that of LSC-9,suggesting that there is a GC-bias for the gene conversion in the inverted repeat region.(2)Using Hy Phy,three positive selection sites were detected under a Bayesian factor greater than 1 and 116 negative selection sites were detected under a Bayesian factor greater than 20.The positive selection of amino acid sites was also analyzed by running PAML.The branch model did not detect any positively selected branch,the site model detected 1 positively selected site,while the branch-site model detected no positively selected sites.There are a total of four positively selected sites detected via the two methods.They all are located in the general structural region of RPS12(irregular region or?-helix region).Of the 116 negatively selected sites,60 are kept consistent in the RPS12 protein sequence alignment of 75 plants,6 sites are found to locate in the ?-helix region,and 36 sites locate in the ?-sheet region.The sites in RPS12 that interact with S8,S17,16 S r RNA,or 23 S r RNA are all found to be negatively selected.We speculate that the rps12 gene has been stabilized during the evolution of ferns,and strong negative selection pressure may help to maintain the functional and structural stability of rps12.