The Evolutionary Difference Between Spermatophyte ITS2 Pseudogene And Functional Gene Reveals The Pattern Of Base Compensation Substitution

As a widely used phylogenetic marker,ITS2 is one of the most abundant sequences in the GenBank.These rich available data greatly facilitate studies of the function,structure and evolution of this region.Recent research have already made the secondary structure and function of ITS2 increasingly clear,laying a solid foundation for research of their evolution of base pairs.ITS2 function action based on their secondary structure in vivo.Although ITS2 sequence invariably evolve,its secondary structure can still be maintained under the mechanism of compensatory base changes(CBC)between paired bases.CBC sudstitution indicates that when one of the paired bases changes,the other side of the pairing will change accordingly to compensate for the prior base mutations.Therefore,CBC hypothesis challenges the traditional view of base mutation pattern as independent and random.Verification of CBC hypothesis from different perspective is necessaary.Previous studies on CBC have mainly limited to functional genes,providing little evidence for CBC.The neutrally evolved pseudogenes in the ITS2 mutigene family provide natural materials for further exploration of the pattern of base-pair substitution in ITS2.This study screened all published ITS sequences of seed plants by searching the GenBank database and related papers in the Web of Science database,and a total of 63 genera containing ITS pseudogenes were obtained.After further screening,488 sequences of 10 orders,11 families,16 genera were finally retained for subsequent calculation and analysis:(1)Two comparable prediction software of secondary structure,LocARNA and 4SALE,were used to predict the structure of functional genes and pseudogenes,and compare their differences in secondary structures and minimum free energy;(2)ITS2 Sequences were partitioned into paired and unpaired regions based on the common secondary structure and compare the differences in basic information such as base composition,conversion ratio and mutation site;(3)The program RNAconvert were used to convert the sequence-structure matrix into the specific coded characters,in which number of CBC and hemi-CBC sites were counted;(4)The PHASE program were performed with the optimal evolution model to calculate the base pair substitution rate matrix in each genus,and to compare the rate and frequency differences of each base pair in CBC process;(5)The calculation results obtained by two different structural prediction methods were statistically analyzed to compared their differences.The main conclusions are as follows:(1)Compared with functional genes,pseudogenes have shorter paired regions,longer unpaired regions,significantly higher mutation contents and relatively higher minimum free energy,indicating that they tend to become unstable during evolution.By contrast,the ability of functional genes to maintain a conservative structure indicates that they are subject to stronger evolutionary constraints than pseudogenes.(2)When the base pairing changes,CBC is an important mechanism to maintain structural stability.The higher Watson-Crick base pair frequency,the faster conversion to GU(UG)pairing intermediates when mutations occur in functional genes.It indicates that the functional sequences have obvious evolutionary directionto ensure its secondary structure remaining unchanged when base mutations occurrs.This observation provides strong evidence to verify the real existence of the CBC process from the perspective of pseudogenes.In contract,the analysis of AC(CA)paired intermediates showed no such obvious result.The most likely explanation is that the AC(CA)base pair itself is a base pair with a faster mutation rate,their difference is not obvious compared with rapidly evolving pseudogenes.In addition,AC(CA)base pairs are relatively rare,and there are not so many evolutionary models on their sequence characteristics.It likely makes PHASE program identify AC(CA)as a common type of mismatch,resulting in not so much CBC as the GU(UG)base pair.(3)LocARNA and 4SALE software were used to predict the common structure separately in sequence composition,partition characteristics,optimal evolution model,GU(UG)base pair frequency,variable rate as well as overall CBC rate.The results showed that different software has little effect on the results,making the conclusions more reliable.In summary,this study confirmed the authenticity of the CBC hypothesis from the perspective of pseudogenes by comparing the base pairing and evolution rate of ITS2 functional genes in the context of secondary structure.It also provides new datasetsfor improving RNA evolution models.However,our available pseudogene data are still limited presently,further study is necessary to expand the sample and test in other functional RNA.