Study On Helitron Distribution In Brassicaceae And Its Relationship With The Evolution Of Brassica Napus

Helitron,a rolling-circle DNA transposon,was first reported in Arabidopsis thaliana,rice and nematodes in 2001,and commonly found in eukaryotes.Helitron has played an important role in plant evolution.The Brassicaceae is a medium-sized family of flowering plants and is also suitable materials for researches on bioinformatics,comparative genomics because many genuses genomic sequences have been published in the Brassicaceae family,including a younger species,Brassica napus.In this study,we developed a new tool for rapid predictions of Helitron,analyzed the distribution of Helitron in plant genomes,and explored the effects of Helitron on species evolution.The main results were as follows: 1.EAHelitron is a fast and efficient program to predict Helitrons.EAHelitron(Easy to Annotate Helitrons),was developed by Perl using the regular expression engine and its embedded-code construct to search the Helitrons.In the prediction testing of Arabidopsis thaliana TAIR 10 genome,EAHelitron increased the maximum speed of the prediction process by 99.3 times with a lower false positive rate(5.91%).Meantime,EAHelitron predicted 665 Helitrons in TAIR 10,of which 499(75%)Helitrons were supported by other software.EAHelitron also had the ability to discover new Helitrons.In a comparative study of polymorphisms among 18 A.thaliana ecotypes,EAHelitron predicted 166 new Helitrons,of which 41 newly discovered Helitrons have been proved to represent the polymorphisms in those ecotypes.This indicated that EAHelitron had the ability to find genuine new Helitrons.2.Helitron's bias distribution hidden the evolution process in Brassicaceae.EAHelitron predicted 49,213 Helitrons on 16 genomes of the 13 Brassicaceae species.In the average,Helitron insertion of intergenic regions accounted for about 93% of the total Helitron of each species.The chi-square test proved that the distribution of Helitron was not random and Helitron preferred to insert in the intergenic regions.The Helitron distribution in the chromosomes revealed that the Helitron bias was concentrated near the centromeres in these Brassicaceae species.GO and KEGG enrichment analysis of Helitroninserted genes implied that,some Helitron insertions might be selected by domestication.3.Helitron polymorphism and association analysis in 18 A.thaliana ecotypes.Analysis of the Helitron distribution of 18 Arabidopsis ecotypes worldwide showed that 508 Helitron insertions had polymorphisms.Hierarchical clustering of Helitron polymorphism,showed that these ecotypes could be clustered into three subgroups,including Western Europe,South-Central Europe and East-Central Europe.We found 13 Helitron polymorphism locations associated with flowering-time phenotypes using Apriori correlation analysis,including two loci links to two known flowing-time related genes.The Helitron polymorphism might have potential as a molecular marker for association analysis.4.Whole genome Helitron density as a character for distinguishing plant species.Using EAHelitron,104,653 Helitrons were identified in 53 published plant genomes,including a wide range of monocots and eudicots.Whole genome Helitron density,(the number of 3' termini of Helitrons/genome size),was potentially a more accurate genomic characteristic.Either Helitron counts(38–13,968)or Helitron densities(0.23–26.0)greatly varied in these plants.Helitron density had the stability to a certain degree in the genomes of one species.Correlation analysis revealed that genome size and Helitron density were two independent genomic features.Simulations have shown that Helitron density can be used to identify plant species in linear discriminant analysis.5.Helitron insertion affected gene expression differences between Darmor and Zhongshuang 11 in B.napus.There were differences in Helitron insertions in the promoter region of about 1,850 orthologous genes in Darmor and ZS 11 of B.napus.According to the analysis of RNAseq,50.4% of the promoter different genes with the Helitron insertion were differentially expressed,which was much higher than the proportion of differentially expressed genes(41.2%).Given that data,the promoter difference caused by Helitrons or other transposons was one of the reasons that affected the difference in gene expression.6.Helitron insertion did not affect evolution rate but reduced expression level of Helitron-inserted genes in B.napus.Ka,Ks and Ka/Ks of Helitron-inserted genes were not different from other genes.This indicated that Helitron insertion and point mutation were independent from each other.The length and exon number of Helitron-inserted genes were longer and larger(1,237 bp and 6.5)than the average(1001 bp and 4.9),but the expression level was significantly lower than other gene groups(5.5).Helitron insertion may result in reduced gene expression.In this study,we characterized the distribution of Helitron and evaluated its possible effects on the evolution of B.napus through tool development,data mining,gene expression and evolution rate analysis.It has certain practical significance for accelerating the study of transposon and evolution in plant genome.