Identification And Characterization Of Two LTR Retrotransposon Families In Tetraploid Cottons

Retrotransposons comprise of a major fraction of plant genomes,accounting for more than half of the total DNA content.Their transposition,mediated by RNA,produces a new copy in the genomes via a “copy-and-paste” pattern,this proliferation mechanism makes retrotransposons be the most abundant transposable elements in plants.Their insertion into or near genes may alter the expression of target genes.A large number of studies have shown that the retrotransposons play an important role in the evolution of plant genomes,and have an important effect on the plant genomes size,composition and gene structure and functions.In recent years,retrotransposons have became an important tool to study gene cloning and expression,biological diversity and species evolution,they are also important to study sequence composition,copy number and transcription expression of plant genes.Cotton(Gossypium)is an important economic crop,and cotton fibers are the most widely used natural fibers in the textile industry,which occupies a pivotal position in the national economy.The completion of genome sequencing of diploid cottons G.raimondii,G.arboretum and tetraploid cottons G.hirsutum,G.barbadense,provided an excellent opportunity for the comprehensive identification of the retrotransposons and analysis of their evolution in the cotton genomes.At present,most of the analyses of cotton retrotransposons are concentrated in classes or superfamily level,whereas the identification and detailed analysis of certain retrotransposon family is rarely reported.This study was focused on two LTR retrotransposon families in tetraploid cottons(DOCL and REL retrotransposon family).On the basis of comprehensive identification of their family members,the classification,distribution,insertion position bias of DOCL and REL retrotransposon family and their influences on expression of target genes were analyzed in detail.The main results are as followed:1 Two LTR retrotransposon families were significantly amplified in tetraploid cottonsBased on the molecular characterization of GhDOCL,DOCL retrotransposons were identified from the fully sequenced cotton genomes.The DOCL retrotransposon family contained 157,112 and 73 members from G.hirsutum,G.barbadense and G.raimondii,respectively.Used GbRE1 as probe sequence,92 and 43 REL members were identified from G.hirsutum and G.barbadense,respectively.There was no DOCL retrotransposons identified in G.arboretum,while neither G.arboretum nor G.raimondii contained REL retrotransposons.Apart from this,quantitative-PCR analysis showed that the copy number of DOCL retrotransposons in tetraploid cottons was significantly higher than that in G.raimondii.These results indicated that DOCL and REL retrotransposon families are significantly amplified in tetraploid cottons.2 Phylogenetic analyses of LTR retrotransposon familiesThe phylogenetic relationship of two LTR retrotransposon families in tetraploid cottons was analyzed by MEGA 6.0 software.The DOCL retrotransposon family consisted of nine groups,the G1-G4 and G9 were significantly amplified after the emergence of tetraploid cottons;while the G5-G8 were formed in the D genome before the formation of tetraploid cottons,and they were not significantly amplified in tetraploid cottons.The REL retrotransposon family consisted of two groups and began to amplify after the emergence of tetraploid cottons.3 Distribution of LTR retrotransposons in cotton genomesDOCL and REL retrotransposons were distributed relatively evenly in different chromosomes,and LTR retrotransposons in the same family or group clustering in a particular chromosomal region were not detected,indicated that the amplification of the two retrotransposon families in the genomes was relatively random.According to the insertion position of LTR retrotransposons and their flanking sequences,the allelic insertion sites of DOCL retrotransposons in different cotton species were identified,and the insertion position of LTR retrotransposons in different tetraploid cottons materials were verified by PCR amplification.The results showed that the insertion position of most of the DOCL retrotransposons differed in cotton species,suggesting that their amplifications in different cotton species were relatively independent.The protein coding properties of flanking sequences of LTR retrotransposons were analyzed by BLASTX.The results showed that most of the DOCL and REL retrotransposons inserted in genic regions.4 The relationship between LTR retrotransposons and protein coding geneCombined with the PCR detection and the BLASTX analysis results,we selected DOCL and REL retrotransposons,inserted in the protein-coding regions,to analyze the expression level of target genes in G.hirsutum or G.barbadense.The results showed that the insertion of retrotransposons significantly reduced or eliminated the expression of 12 target genes(8 DOCL-and 4 REL-targeted genes)in certain tissues of cottons.In summary,there existed cotton LTR retrotransposon families,like DOCL and REL retrotransposon families,which significantly amplified in tetraploid cottons.The insertion of retrotransposons affected the expression level of target genes.Our data also indicated that these newly-amplified retrotransposon families are important driving force for the evolution of tetraploid cottons.