Identification Of L1 Insertion Polymorphism In Pig LncRNA Genes And Its Association With Production Performance

Retrotransposons,as major genomic parasites of mammals,account for 30-45%of mammalian genomes.They are classified into three major groups:long interspersed nuclear elements(LINEs),short interspersed nuclear elements(SINEs),and long terminal repeats(LTR).L1 in LINE are the most abundant class of transposable elements in mammals,accounting for 16.59%of the genome in pigs(Our annotated data),18.78%in mice,and 16.89%in humans.Because of their ubiquitous distribution,high copy number,and high number of insertion polymorphisms,L1s are believed to be suitable for developing new molecular markers in mammals.At the same time,studies have shown that retrotransposons make a strong contribution to LncRNA sequences.Over two thirds of mature LncRNA sequences(75 and 68%of human and mouse,respectively)have at least a partiai(?)transposon msertion in their sequence,Here wetry to identify the insertion polymorphisms of LI within LncRNA genes,and conduct association analysis with growth performance in pig to develop new useful makers for pig molecular breeding.At the same time,we explored the effect of transposon insertion on the regulation of target genes by IncRNA.The experiment includes the following parts:1.We obtained 1553 L1 insertion site genomic sequence of pig LncRNA genes by RepeatMasker,and obtain a sequence of 800 bp extending from both sides of the insertion sites from the genome.Then each sequence was subjected to online Blast alignment of the genome sequencing data in the NCBI and WGS libraries,and the polymorphism of each insertion site was predicted based on the results.At last,266 suspected L1 insertion polymorphism sites were obtained.Then we designed primers and performed PCR validation on 8 varieties of Duroc,Landrace,Yorkshire,Erhualian,Meishan,Bama,Tibetan and Wild Boar.Finally,we obtained 114 polymorphic insertion sites.L1A-D has 28,6,14 and 66 polymorphic insertion sites,respectively.The Blast results of LID showed that the determined number of polymorphic sites accounts for 39.52%of the total number of LID sites in LncRNA gene,and the proportion of polymorphism in PCR verification is 81.44%.It is the highest polymorphic rate in L1 family.2.Based on the 114 polymorphic sites,the chimeric transcripts between L1 and LncRNAs were identified by BLAST,and a total of 40 fusion transcripts between L1 and LncRNA were obtained,corresponding to 28 LncRNA genes.Annotation of these fusion transcripts revealed that 22(55%)of the LncRNAs consist entirely of the L1 retrotransposon sequence.3.The polymorphisms of L1-LNC-276-F10 were detected in slaughter pigs of three breed hybrids,and the expression of target genes(ERAP1 and ERAP2)of L1-LNC-276 were evaluated by RT-qPCR in major organs of these pigs.The results showed that the expression levels of ERAP1 and ERAP2 genes were significantly higher in the homozygous individuals with non L1 insertion(L1-/-)than individuals with heterozygous and homozygous L1 insertions(L1+/-and L1+/+).4.Correlation analysis were conducted between the growth traits and reproduction traits with genotypes in Yorkshire population(462)by using 2 molecular markers(L1-LNC-49 and L1-LNC-253-F1)based on the obtained 114 L1 insertion polymorphic sites.The results showed that pigs without L1 insertion(L1-/-)had a significantly higher backfat thickness(11.30 ± 2.70)than the pigs with L1 insertion(L1+/-and L1+/+)(10.68±2.53)(P<0.05);and the pigs without the L1 insertion(L1-/-)had a significantly higher alive litter size(11.45 ± 2.77)than the pigs with the L1 insertion(L1+/-and L1+/+)(10.33±3.22)(P<0.05)...