Identification, Evolution And Function Of Silkworm MITEs As Well As Evolutionary Dynamics Of Silkworm TEs

Miniature inverted-repeat transposable elements (MITEs) were originally discovered in maize genome by Bureau and Wessler. And they are widespread in the eukaryotic genomes. Silkworm, Bombyx mori, is a model insect for the order Lepidoptera. The draft genome sequences of silkworm were released by Mita et al. and Xia et al., respectively. Recently, a new assembly has been completed. Analyses of the silkworm genome sequence suggested that-40%of the genome is composed of the known TEs. Although silkworm (Bombyx mori) has a large amount of and a variety of transposable elements, the genome-wide information of the silkworm MITEs is unknown.Transposable elements (TEs) constitute a substantial amount of eukaryotic organisms and content varied in different organisms. Thus knowledge of the evolutionary dynamics of TEs is very important for understanding the evolution of eukaryotic genomes. Previous studies revealed that transposition, natural selection, demography and mating system play significant roles in the TE evolutionary dynamics. However, all these lines of cognition mainly came from research on a few model organisms such as Drosophila and Arabidopsis and previous studies on the evolutionary dynamics of TEs focused exclusively on natural populations of individual model species. Yet there has been no study concerning the evolutionary dynamics of TEs in domesticated species. Howbeit, undoubtedly, this kind of research has very important implications for understanding both the genetics of domestication of higher organisms and the evolution of eukaryotic genomes.MITEs are widespread in the eukaryotic genomes. At present types, contents, distribution and transpose mechanism of MITEs have been explored in depth. And we have a clear idea of these characters. However, the function of MITEs was remained unknown. In this study, identification, classification, distribution and evolution analysis of silkworm MITEs were performed using software of MUST with a series of methods and software of bioinformatics; Evolution dynamics of silkworm TEs were estimated by transposon display, theories and methods of population genetics and bioinformatics; Using transgene technology, technology of cell transfection and most basic techniques of molecular biology to detect whether BmMITE-2might regulate expression of its neighboring genes. The major results were as follow:1. Types, contents and charaters of silkworm MITEsMUST, a program designed to detect MITE elements, was first used to search the silkworm genome sequence. With MUST, we mined143333MITE candidates in the silkworm genome, which were grouped into1350families. Then, we filtered out pseudo-MITEs from predicted ones by Perl scripts. By this way, number of the MITE families reduced to17. These17families include5785intact MITEs and were designated as BmMITE-1to BmMITE-17, which constitute-1.86Mb (0.4%) of the silkworm genome. TSD lengths of all MITEs range from2to9bp, TIR lengths from8to59bp, and full lengths of complete MITEs from210to567bp. Based on the nucleotide composition of TSD,3MITE families (BmMITE-4,5,6) belong to Tourist-like family,3families (BmMITE-2,3,8) belong to Stowaway-like family, based on the nucleotide composition of TSD and TIR,4families (BmMITE-13,14,15,16) belong to Pegasus-like family, and the remaining7families (BmMITE-1,7,9,10,11,12,17) were novel.2. Expansion of silkworm MITEsTo estimat expansion of silkworm MITEs, estimates of insertion date, network analysis and sliding window analysis were performed, respectivly. Results of insertion date showed that insertion date varies greatly among members of each family as well as among families, which ranges from0to4million years ago (mya). The major expansion events of17families might happen within2mya. Strikingly, BmMITE-2might be dramatically expanded during a period from0mya to1mya and accumulated up to2173copies during this short period; Results of network analysis indicated that8(BmMITE-4, BmMITE-6, BmMITE-7, BmMITE-11, BmMITE-14, BmMITE-15, BmMITE-16and BmMITE-17) of17MITE families presented the topologies that these MITE families might experience old population expansions. The topologies of rest9 families indicated that these families might undergo recent expansions. These observations are basically consistent with the insertion time estimates; Results of sliding window analysis suggested that regional variation pattern varies greatly among these17silkworm MITE families. Almost all families have highly conserved TIRs, however,6families (BmMITE-4, BmMITE-5, BmMITE-9, BmMITE-12. BmMITE-14, and BmMITE-15) have only one conserved TIR. Because TIRs must be first recognized by corresponding transposase for transposition, Thus these results could be reflected that most of these MITEs remianed actively and expansion in recently.3. Distributions of MITEs on chromosomes and Estimation of MITE distance to the nearest geneResults were as follow:all MITE families are widely distributed on all28silkworm chromosomes. Then, we examined whether MITEs were randomly distributed among28silkworm chromosomes using χ2test. The null hypothesis was rejected (P<0.01), suggesting that the distribution of MITEs is nonrandom in the silkworm genome. And observed values were significantly higher than expected ones in the7silkworm chromosomes (chromosome2,16,20,24,26-28). Furthermore, we examined whether insertion site of each MITE preferentially is in or close to genes. The results indicated that3794(66%) of the5785predicted MITEs inserted into gene regions. Of3794MiTEs inserted into gene regions,962(25%) were located in5’flanking regions of the closest genes,60(2%) in exons,1427(38%) in introns and1343(35%) in3’flanking regions of the closest genes, respectively. It appears that silkworm MITEs preferentially inserted into introns,3’-flanking regions and5’-flanking regions rather than exons.4. TE-display data and population polymorphism of7major silkworm TEs in the domesticated silkworm and wild silkworm populationsWe investigated the distribution and diversity of seven TE families (Bml, Pao, CR1, Jockey, Bmmarl, BmMITE-7and BmMITE-2) in the corresponding populations using TE-display technique. With these TE band data, we calculated six initial statistics (MPBF:mean polymorphic band frequency; var(f):variance of polymorphic band frequencies; S:the number of observed TE bands; Sx:the number of unique of TE bands in a pairwise comparison; MTB:mean TE bands in each individual of each population; Sf. the number of within-population fixed TE bands) for each TE in each silkworm population. Finally, results were as follow:(1) almost all the domestication silkworm populations have higher MPBF, MTB and Sf values than wild silkworm population;(2) there are eleven MTB values in the domesticated silkworm significant higher than those in wild silkworm;(3) there are twenty three Sf values in the domesticated silkworm significant higher than those in wild silkworm. These results suggested that these TE families experienced expansions and widely fixed in the domesticated silkworm.5. Factors of TEs population polymorphism in the domesticated silkworm and wild silkworm populationsIn order to detected reasons of TEs experienced expansions and widely fixed in the domesticated silkworm other than wild silkworm, maximum likelihood estimate and coalescent simulation were performed. Results of maximum likelihood estimate were as follow:(1) all95%credible intervals of Nes do not overlap zero and the Nes values of five TE families (Bml, Jockey, CR1, Bmmarl, BmMITE-2) in each population are positive;(2) Almost all of Nes values in the domesticated silkworm are significant higher than those in wild silkworm. These results indicated that these TEs experienced strong positive selection in the domesticated silkworm and these TEs experienced weak positive selection in the wild silkworm. However, it should be noted that the maximum likelihood approach does not take into account demographic history (Lockton et al.2008). To further detect whether there is positive selection on these TE families during silkworm domestication, the selection pressure of these TEs were estimated by coalescent simulation that take into account demographic history. And results showed that these TEs did experience positive selection in the domesticated silkworm population. However, they were under neutrality in the wild silkworm population.6. BmMITE-2regulates expression of its neighboring genesAccording to above results, BmMITE-2prefers to insert in silkworm gene regions and may experience positive selection during silkworm domestication. Whether BmMITE-2might regulate expression of its neighboring genes? To answer this question, we detected BmMITE-2effected on expression of its neighboring genes using transgene technology, technology of cell transfection and most basic techniques of molecular biology. Finally, the results indicated that BmMITE-2can up-regulate expression of its neighboring genes when BmMITE-2inserted into5’terminal of genes; In contrast, BmMITE-2can down-regulate expression of its neighboring genes when BmMITE-2inserted into3’terminal of genes.According to the above results, we can conclude:There are abundent and varied MITEs in the silkworm genome, most these MITEs are experienced burst expantion; Most TEs are experienced strong positive selection in the domesticated silkworm popuplation; BmMITE-2can up-regulate expression of its neighboring genes when BmMITE-2inserted into5’terminal of genes, BmMITE-2can down-regulate expression of its neighboring genes when BmMITE-2inserted into3’terminal of genes.