Genome-wide Annotation And Evolutionary Analysis Of Long Terminal Repeat Retrotransposons In Pear(Pyrus Bretschneideri Rehd.)

As the most abundant repetitive sequences,LTR-retrotransposons usually enrich in pericentromeric regions of plant chromosomes.The replication,insertion and recombination of LTR retrotransposons(LTR-RTs)always play a great role in the plant genome diversity.The structure,activity and copy number of LTR-RTs are significantly different in different plant species and even closely related species,so the LTR-RTs can be used to develop a variety of molecular markers for species identification and genetic diversity analysis.According to the insertion time of orthologous LTR-RT,we can also investigate the evolutionary history events between related species.In present study,the LTR-RTs database of 'Dangshansuli' pear genome has been built through deep annotation,structural characterization,and family classification based on series of bioinformatics methods.As results,the abundance,distribution pattern,and proliferation timeframe of LTR-RTs have been deeply characterized,LTR-RTs with new structure types have been discovered,and the evolutionary genome divergence between Oriental and Occidental pear have also been analyzed according to the evolutionary history of orthologous LTR-RTs and sequence divergence of orthologous single copy genes.In addition,the LTR-RTs database of the pear genome will also facilitates the genetic diversity and evolutionary history analysis of pear germplasm resources.The main results are as follows:1.Annotation of LTR-RTs from 'Dangshansuli' Pear GenomeTotally,we have identified 7247 LTR-RTs with clear boundaries in the well assembled Oriental pear genome(P.bretschneideri Rehd.).Overall,3221(44.4%)are intact elements with TSDs(IT),578(8.0%)are intact element without TSDs(InT),2896(40.0%)are solo LTRs with TSDs(ST),and 552(7.6%)are solo LTRs with no TSDs(SnT).According to the ' 80-80-80' rule of family classification,all the annotated LTR-RTs can be separated into 148 families,including 115 Copia-like families(2675 elements),21 Gypsy-like families(1914 elements),9 TRIM families(2580 elements)and 3 LARD families(78 elements).Out of the 148 families,the Pbr148 family with 2411 copies(only intact elements and solo LTRs),also named Cassandra belonging to TRIM super-family,is the largest one.Notably,approximately 90%of these elements were randomly distributed on the persudo-chromosomes.We also confirmed that the Maximus lineage of Copia super family has been lost in the Rosaceae species.Further analysis reveals that the amplification timeframe of elements varies between different super-families and lineages,and the Copia-like elements were the most activated and highly burst in the last 1 Mys.2.Comparative Genomic Analysis of Cassandra in Five Rosaceae speciesCassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature(TRIM).Although Cassandra TRIM elements have been found in almost all vascular plants,detailed investigations on the nature,abundance,amplification timeframe,and evolution have not been performed in an individual genome.We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species,including apple,peach,mei,and woodland strawberry.Our data reveal several interesting findings for this particular retrotransposon family:(1)a large number of the intact copies contain three,four,or five long terminal repeats(-20%in pear);(2)intact copies and solo LTRs with or without target site duplications(TSDs)are both common(approximately 80%vs 20%)in each genome;(3)the elements exhibit an overall unbiased distribution among the chromosomes;(4)the elements are most successfully amplified in pear(5032 copies,including truncated elemetns);(5)the evolutionary relationships of these elements vary among different lineages,species,and evolutionary time.These results indicate that Cassandra retrotransposons contain more complex structures(elements with multiple LTRs)than what we have known previously,and that frequent inter-element unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes.Thus this study provides insights into the property,propensity,and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons,and enhances our understanding of the structural variation,evolutionary history,and transposition process of LTR-RTs in plants.3.Evolutionary Genome Divergence Event between Oriental and Occidental PearOut of 6117 LTR-RTs and solo LTRs with TSDs from the well assembled Oriental pear genome,1194(19.5%)orthologous LTR-RTs including 656(20.4%)intact elements and 538(18.6%)solo LTRs with TSDs from the 'Bartlett' genome have been identified through the orthologous searching method.In addition,a total of 774 orthologous single copy genes between the two Pyrus species genomes as well as 299 orthologs among the two pear genomes and the Arabidopsis thaliana reference genome have also been identified.We investigated and compared the rates of nucleotide substitution between two long LTRs of individual orthologous LTR-RTs and the rates of synonymous substitution(Ks)and non-synonymous substitution(Ka)between orthologous single genes between the two Pyrus species genomes.The results showed that two genomes have experienced symmetric evolutionary process after their splitting from the same ancestor-0.77-1.66 Mya,and also confirmed the nucleotide substitution rate of LTR-RTs is almost twofold of coding sequences.Overall,this is the first report uncovering the landscape of LTR-RTs and regarding the evolutionary relationship of Pyrus species at the genome-wide level,which will also support genomic resources for genetic diversity analysis between various Pyrus species and provide a foundation to unravel the genome evolutionary process between Oriental and Occidental pear genomes.