Pig Mobilome Annotation,Activity Analysis And Its Effects On Genome,Transcriptome And Functional Genes

In the past several decades,the genome annotation revealed that transposable elements(TEs)or transposons and their recognizable remnants,also refered as the mobilome,distribute extensively in prokaryote and eukaryote organisms,and played a key role in the genome and genetic evolution.Mobilome has became a research hotspot in the post-genome era.However,the annotations of pig mobilome are very roughly,and the published data are not ab initio results,a lot of information was missed.And the diversity,evolution,transcription and transposition activity of pig mobilome and their effects on functional genes remain largely unknown.At the same time,very few reports are avaliable about the development of transposon insertion polymorphism(TIP)markers in livestock including pigs,which is far behind that in humans,mice and plants.Based on this background,we conducted the following studies in this dissertion:1.We performed de novo detection of retrotransposons in pig genime using multiple pipelines.We defined the classification of LINEs,SINEs,and ERVs at the family and subfamily levels,highlighted the evolution dynamics and insertion age,and built a new pig repeat library.2.Based on the new pig repeat library,we re-annotated the pig mobilome,and investigated the impact of mobilome on the transcriptome and genome.3.According to the RepeatMasker annotation results,we investigated the intersection between retrotransposons and host genes,including protein-encoding and IncRNA genes,4.The transcriptional activity,promoter activity and retrotransposition activity of young retrotransposons were examined.5.Taking SINE as an example,the effect of transposon insertion polymorphisms on the structural variations of gene exons were systematically analyzed.6.Taking the GHR gene,an important candidate gene for growth and development,as an example,the effects of transposon insertion polymorphism on its structural variation and its association with phenotype were evaluated.The main results are as follows:1.A total of 5,937 L1 sequences were identified from the pig genome,which were divided into four families(L1A,LIB,L1C,and LID)and 53 subfamilies.Structural analysis indicated that pig L1 has a typical mammalian LI structure(5’UTR,ORF1,IGR,ORF2 and 3’UTR)with a total length ranging from 5837 bp to 8822 bp,and the length of the 5’ UTR varies greatly,between 551 bp and 3254 bp,and most subfamilies have longer IGRs(390 bp to 529 bp).The two ORFs are relatively conservative,about 900 bp and 3800 bp,respectively.They present different evolutionary patterns,each family dominates a certain period of evolutionary activities,combined with age analysis,showing that four families are younger than other LINE members,and LID is the youngest family,and L1D1-7 represents the youngest seven subfamilies.A total of 98 copies with intact L1 structures and two ORFs with coding ability were identified in the LID family,which may have transposition activity.2.According to the length,similarity and structural characteristics of the SINEs,they were divided into three families(SINEA,SINEB and SINEC)and 25 subfamilies,all belonging to the tRNA origin.The lengths of the three SINE families are significantly different.The length of SINEA is about 250 bp(excluding polyA tail),while SINEB and SINEC are relatively short,about 200 bp and 120 bp,respectively.SINE presents three amplification waves,each corresponding to a SINE family.Age analysis shows that SINEA is the youngest family,and SINEA1,SINEA2,SINEA3 represent the youngest three INE subfamilies.3.ERVs are the major member of the LTR retrotransposon and divided into 18 families.Most of the ERV families are relatively old and have lost retrotransposition activity,and most of them are distributed between 8.5 kb and 11 kb.While ERV6 has an outbreak trend in nearly 10 million years,insertion age analysis shows that ERV6 is the youngest family and is further divided into two subfamilies(ERV6A and ERV6B),each contain a copy that can encode a long peptides containing gag,pol and env,and may still active.4.The genome was re-annotated by Repeatmasker with the new repeat library.The results indicated that the retrotransposon accounted for 37.13%(929.09 MB)of the genome.LINEs,LTRs and SINEs accounted for 18.52%,7.56%and 11.05%of the genome,respectively.DNA transposons are relatively rare,accounting for only 1.99%of the genome.The youngest seven L1 subfamilies(L1D1-7)account for only 0.17%of the genome.The youngest three SINE subfamilies(SINEA1-3)account for only 0.63%,and the youngest two ERV subfamilies(ERV6A and ERV6B)account for only 0.02%of the genome.5.Bioinformatics analysis showed that most protein coding and IncRNA(>80%)genes contained retrotransposon insertions,and about half of protein coding genes(44.30%)and one-fourth(24.13%)of IncRNA genes contained the youngest retrotransposon insertions.Nearly half of protein coding genes(43.78%)could generate chimeric transcripts with retrotransposons.Significant distribution bias of retrotransposon composition,location,and orientation in IncRNA and protein coding genes,and their transcripts,were observed.6.The retrotransposition activity of young L1s in the pig genome was verified by retrotransposition assay.Experiments have shown that retrotransposition can occur in HeLa cells regardless of whether the 5’UTR is used as a promoter or a CMV promoter,but the transposition activity is significantly lower than that of human L1(p<0.01).When the IGR of pig L1 was replaced by the IGR of human L1,the number of surviving cells was significantly increased,suggesting that the retrotransposition activity became higher(p<0.05).7.The promoter activity of L1 5’UTR and ERV LTR were tested by the dual luciferase reporter system.Three sense 5’UTRs from L1D1,L1D2 and L1D7 and one antisense 5’UTR from L1D2 showed weak promoter activity in different cell lines.The sense LTR of ERV6B showed higher prpmoter activity,while the sense LTR of ERV6A and the antisens LTR of ERV6B showed moderate promoter activity,and the antisense LTR of ERV6A showed no promoter activity in this study.8.Sense and antisense transcripts of three classes of young retrotransposons were identified in multiple pig tissues and cell lines.Similar transcriptional patterns appeared in pig tissues and cell lines and all have antisense transcripts,while different transcriptional patterns are exhibited in the reproductive organs,the sense transcription of L1 ORF1,ORF2,ERV gag、pol and env,and the antisense transcription of ERV LTR in the testis were inhibited,but the antisense transcription product of the L1 5’UTR was clearly detected.In addition,both the sense and antisense transcripts of SINE were detected in the ovary,but neither were detected in the testis.Higher antisense transcription levels of ERV6 LTR were observed in the brain and cerebellum.9.The total number of insertion sites of SINEA,SINEB,SINEC in the genome was 1204691,and a total of 24,424 insertion sites were identified in the exon region of the gene,corresponding to about 30%of the genes.Among them,792 were identified in the coding region(CDS),and 23232 were located in non-coding regions.By comparing the polymorphic frequencies of SINE insertion sites of different ages,it was found that the younger subfamilies tend to show higher polymorphic frequency.The polymorphic insertion sites of the youngest subfamilies(SINEA1-3)in the exon region was analyzed by Blast and experimental verification.Finally,151 polymorphic insertion sites were obtained and most of them distributed in the untranslated region.10.The mobilome annotation revealed that 155 transposon insertions were found in the the GHR genic and flanking region,and most of them were retrotransposons(152/98.06%),corresponding to 25.63%of the sequences.Among them,the SINEs were the most abundant component,and 68 of them were from the youngest family SINEA,accounting for 5.64%of the sequence.34 large structure variations were obtained by alignment,and they all correspond to retrotransposons.Some of the structural variations derived from retrotransposons were selected for experimental verification,and 5 TIPS were obtained,one of them was detected in the Large White pig population.Correlation analysis with corrected backfat traits showed that individuals with homozygous transposon insertion genotype(SINE+/+)have significantly lower thickness than individuals with genotype(SINE-/-).Totally,we performed a systematic de novo detection,classification,evolution analysis of mobilome in pigs,and evaluated the transcriptional activity,promoter activity and retrotransposition activity of young retrotransposons.We also evaluated the impact of mobilome on the transcriptome,genome,and functional genes,and developed the TIP markers and evaluated their application.These results provided experimental and theoretical basis for comprehensive and profound understanding of the pig mobilome,and it also provides an important reference for the development of new molecular markers for fine maping the QTL of important economic traits.