| The transposable element(TE)is a kind of DNA sequence that can move in the biological genome,which has an important influence on the evolution of genes and the stability of the genome.The LTR retrotransposon is the most abundant one.The retrotransposon family in tea plant accounts for 67.21%of the genome,which is an important reason for the huge genome of tea plant,but its related research is extremely inadequate,far behind other species.In this study,the transposon of tea plant was excavated,identified,classified and annotated on the whole genome scale by means of the published tea plant genomic,and its characteristics were analyzed.Based on this,the first tea tree transposon database was constructed.At the same time,RT sequences of different types of retrotransposon were isolated,and IRAP(inter retrotransposon amplified polymorphism)system based on retrotransposon was developed,which was applied to the analysis of genetic diversity of 32 tea cultivars.The main results are as follows:1.Genome-wide identification of TEs in Camellia sinensis.In order to make a complete and accurate identification of Camellia sinensis transposon,a combination of methods were used,including Denovo,homology-based and structure-based.There were 45125 transposons identified in the Camellia sinensis genome and were classified into 12 superfamilies and 1117 families according to the 80-80-80 rule proposed by Wicker.2.Construction of the Camellia sinensis transposon database.Based on the above identified transposon sequences,Camellia sinensis transposon database(CsTEdb,http://genedenovoweb.ticp.net:81/CSTEdb/index.php)was constructed to provide users with browsing,searching,querying and comparison.Compared with the comprehensive tea plant information archive(TPIA),CsTEdb focused on Camellia sinensis transposons information,provided more accurate and comprehensive information on Camellia sinensis transposon,and integrated related analysis tools of transposon to provide research foundation on Camellia sinensis transposon.3.Characterization of the Camellia sinensis transposons.(1)Genomic annotation.A total of 2.59GB sequence was annotated as transposon-related sequences,accounting for 83.55%of the Camellia sinensis genome(3.1G).The LTR retrotransposon accounted for the highest proportion(77.78%).This is consistent with some other reported species.(2)Distribution of transposons.The 693 scaffolds above scaffold N50 were selected for analysis.The results showed that the distribution of transposons was significantly negatively correlated with the distribution of genes.The transposons were concentrated in the region with fewer genes,and the transposons were less distributed in the gene-rich region.(3)Correlation between transposons and gene expression.The criterion for the expression activity of the gene was that the RPKM value was greater than or equal to 1 in one of the 8 tissues sequenced at least.The transcriptionally active genes in 693 scaffolds accounted for 82.24%(15183/18461),and there was also a negative correlation between transposons and expression-active genes.(4)GO functional annotation and KEGG pathway analysis of transposon-related genes.The results indicated that the biological processes involved in these genes were mainly concentrated in metabolic process,cellular process,and single-organism process.The cellular component was mainly concentrated in the cell,membrane,and organelle.The major molecular function terms were mainly concentrated in binding and catalytic activity.KEGG pathway analysis showed that transposon-related genes are involved in at least 132 pathways,indicating that transposons play an important role in many biological processes of tea plants.(5)Identification and phylogenetic analysis of full-length LTR transposon.A typical,well-structured LTR transposon CsRE1 was selected for feature analysis.CsRE1 was 17597 bp in length and contained five enzymes required for transposition and LTR,PBS,and PPT.It belonged to the Ty3-gypsy family and was inserted into the Camellia sinensis genome for about 841,000 years.It was a younger transposon with potential transposition activity.The LTR region contained multiple regulatory elements that respond to drought,low temperature,ABA and so on.Five domains shared high homology with known retrotransposons of other species.(6)Identification and analysis of Nested-LTR.Analysis of the relative position of LTR retrotransposons revealed that 87.84%of the total LTR retrotransposons formed complex Nested-LTRs,covering the Camellia sinensis genome 39.97%.These Nested-LTRs with multi-level nesting have an important impact on the amplification and evolution of the host genome.4.Cloning and characterization of RT sequence.The RT sequences of three different types of retrotransposons,Ty1-copia,Ty3-gypsy and LINE,were amplified using degenerate primers.The sequence lengths were 260-267bp,417-433bp,526-593bp,respectively.All kinds of RT sequences are highly heterogeneous,with different degrees of frameshift mutations,stop codon mutations,etc.At the same time,some amino acid regions were conserved highly,which was constructed with RT sequences from different sources.It was found that RT sequences in Camellia sinensis evolved closely with some similar genera and even distant species from the phylogenetic tree.It was speculated that lateral transfer also occurred,in addition to vertical transmission during evolution.5.Establishing of IRAP system and carrying out the analysis of genetic diversity in Camellia sinensis.Based on the isolated RT sequences and LTRs sequence,specific primers were designed and IRAP system was established in 32 tea cultivars.A total of 179 polymorphic locis were detected,the percentage of polymorphism was 89.05%,(Ne)was 1.4523,(H)was 0.2717,and(I)was 0.4165.The genetic diversity of each tea area was in South China>Jiangnan>Southwest>Jiangbei.According to GS,32 tea cultivars were clustered into 5 groups(0.7195 is the threshold)by UPGMA method.The clustering results show that most of the varieties from the same tea area are often clustered because of their close geographical location.Those Camellia sinensis varieties with different geographical origins but similar genetic backgrounds are also clustered together.It is feasible to analyze the genetic diversity of Camellia sinensiss by IRAP. |
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