Identification And Analysis Of Retrocopies In Polyploidy Wheat Genome

RNA-mediated retroposition is one of the important mechanisms of gene duplication.Studies have found that retroposition is of great significance for genome evolution,is an important source of new genes,and has a profound impact on the phenotype of individual organisms.In recent years,with the development of sequencing technology,genome data of species have been published one after another.Genome-wide analysis of retrocopy have been carried out in many species,and a large number of functional retrogenes have been excavated,providing a new perspective for the generation of new genes and biological evolution.However,it was still unknown whether polyploidy plays an important role in the generation and evolution of retrocopy in polyploid species,and there was no fast and accurate process tool for annotating retrocopy,which severely limited the research on the function and evolution of retrocopy.In this study,hexaploid wheat(Triticum aestivum L.),a typical allopolyploid plant,was used as the research object.Firstly,we developed a comprehensive and one-step retrocopy identification tool that was widely applicable to various species,then mined retrocopies in the genomes of wheat and its ancestral species based on this,the identified retrocopies were further analyzed from three aspects:sequence structure characteristics,origin and evolution,and functional significance.This study was the first exploration of retrocopies in polyploid wheat and its ancestors,which will lay a foundation for understanding the evolutionary mechanism of retrocopies in other polyploid species and provide reference for subsequent studies.The main findings are as follows:1.Development of RetroScan,an easy-to-use pipeline for retrocopy annotationIn order to accurately and rapidly annotate the retrocopies in wheat genomes,we integrated bioinformatics software such as LAST,BEDtools,ClustalW2,KaKs_Calculator,HISAT2,StringTie,and SAMtools with Python and Shell scripts,developed an easy-to-use tool for retrocopy annotation and named it RetroScan.The tool could not only annotate retrocopy in the genome based on sequence similarity and intron loss,but also analyze synonymous and nonsynonymous substitutions between the retrocopy and parental gene.When the user provides transcriptome data,the expression of retrocopies and parental genes in each tissue could also be calculated.Compared with other identification processes,RetroScan could dig up more retrocopies in the genome,and could effectively reduce false positive results,and performed well in terms of ease of installation,speed of operation,and accuracy of annotation,and was a reliable tool for annotating and analyzing retrocopies.2.Identification of retrocopies in the genomes of common wheat and its ancestorsWe applied RetroScan to the identification of wheat retrocopies,and customized the identification process according to the characteristics of the wheat genome.4638 retrocopies and 2139 parental genes were identified in the genomes of Triticum aestivum(AABBDD)and its ancestral species Triticum dicoccoides(AABB),Triticum urartu(AA),Aegilops tauschii(DD)and outgroup barley(Hordeum vulgare).The number of retrocopy in hexaploid wheat was 1999,which was significantly more than that of its ancestral species and outgroup species.At the same time,it was found that most retrocopy sequences were truncated at both the 5’ and 3’ ends,and the upstream and downstream repeat sequences were mainly composed of LTR-retrotransposon,indicating that in wheat and its ancestral species,LTR-retrotransposon was the main driving force for the production of the retrocopies.3.The origin and evolution of wheat retrocopiesWe discussed the origin and evolution of wheat retrocopies by combining synonymous substitutions rate and orthologous analysis.Firstly,the Ks distribution curve showed that all of wheat and its ancestral species experienced two outbreaks of retroposition events in the evolutionary process,and the two outbreaks could be traced back to very ancient lineage differentiation events.Phylogenetic analysis of retrocopies in hexaploid wheat showed that most of them existed in the ancestral species and outgroup species,and only 5 retrocopies were unique to hexaploid wheat.These results indicated that the independent polyploid events of wheat had no significant effect on the generation of retrocopy,and wheat retrocopies were mainly derived from the ancient retroposition events.However,it was worth noting that although hexaploidy did not promote the mass production of new retrocopies,it caused the repeated retention of the same retrocopy,which was also a characteristic of retrocopy in polyploid species.We further compared the sequence structure of these orthologous retrocopies and found that some retrocopies underwent intronization,chimerization and other structural evolution after hexaploidization,forming new gene structures.Moreover,the chimerization rate of hexaploid wheat retrocopies was significantly higher than that of rice and primates.It was also found that the expression pattern of retrocopies changed.These results suggested that hexaploidy may accelerate the structural evolution of retrocopy,and also proved that retroposition events are important sources of new genes in wheat.4.The significance of retrotransposon events for the wheat genomeKa/Ks distribution showed that most retrocopies in hexaploid wheat were strongly functionally restricted and had potential functionality.Among the 1999 retrocopies of wheat,a total of 1656 retrocopies contributed coding regions to 1653 protein-coding genes(Retrocopy genes)and participated in the coding of proteins.Furthermore,the tissue expression patterns of most retrocopy genes were not correlated with their parental genes.The results of GO enrichment analysis showed that although the retrocopy genes participated in similar biological processes with the parental genes,it assumed different molecular functions.The results suggested that retrocopy may have evolved a different functional pattern from the parental gene after undergoing general sequence truncation and acquiring new coding sequences.In addition,part retrocopies could also be transcribed from antisense chain to lncRNA,and could form dimers with mRNA and pre-mRNA of its parental gene,which might be related to the combination of miRNA and alternative splicing of the parental gene transcript.In conclusion,this study developed RetroScan,a retrocopy annotation tool suitable for multiple species for the first time,which provided convenience and reference for subsequent related research.And conducted an in-depth analysis of retrocopies of polyploid wheat and its ancestral species,found that polyploidization significantly affected the structural evolution of retrocopies,and that the function of retrocopies were different from their parental genes.This study was the first exploration of retrocopies in polyploid species,which laid the foundation for subsequent related research.