Whole-genome Analysis On Structural Variation In Guizhou Local Pig Breeds Based On High-throughput Sequencing Technology

In the long period of evolution and selection process,there derived a variety of pig breeds with diverse of reproductive performance,growth and other traits.It is important to explore such excellent characteristics,including high fecundity,strong resistance and delicious quality in the further development of the pig industry.Previous searches indicate that structural variation(SV)can change the gene expression pattern and result in phenotypic diversity.SVs also have an impact on the pig evolution process.To uncover the influence of SV on gene polymorphism and the genetic mechanisms of phenotypic traits for Guizhou pig breed(GZP),we determined the genome sequence and structure of eighteen GZPs(Xiang pig,Luobo pig,Guanling pig,Qianbei pig,and Kele pig)by next-generation sequencing(NGS)technology based on Illumina sequencing platform.We also downloaded available resequencing data of 36 pigs from the NCBI public database,including eighteen Chinese pigs outside of Guizhou(NPOG)from six breeds in China,eighteen European pigs(EUP)from five breeds.Based on the whole-genome structure comparison using bioinformatics methods,we tried to find the reason for the different phenotypic traits among GZP,NPOG and other breeds,and to screen out important candidate genes affecting litter size traits of Xiang pigs.It might help to reveal the mechanism involved in the litter size difference in Xiang pig.The main results are as follows: 1.Using bioinformatics to analyze SVs in 54 pigsA total of 1215.58 Gb clean reads were generated after genome resequencing for 54 pigs from GZP,NPOG,and EUP breeds.Both of two programs,pindel and soft SV,were used to call SVs and generated a total of 39,166 SVs.All of the randomly selected 81 SVs were confirmed in pig populations using PCR method.Most of SVs were located in the intergenic regions and small proportion of SVs positioned in the coding gene regions.A positive correlation between SVs number and gene transcripts were found out.It suggested that SV might be one of the reasons of the variable splicing of gene primary transcripts.A large number of TE(transposable element)were indentified from SV regions,it was thought to be one of the formation mechanisms of SVs.After PCA analysis using the specific SVs from GZP,NPOG,and EUP three groups,the first vector could distinguish different pig breeds with much diverse hereditary background.2.The specific SVs and associated genes from different pig breedsCompared with the SVs from the genomic sequence data of EUP and NPOG,the SV number in GZP was about 1.9 times higher than that in EUP.Additionally,we identified 1,956 specific SVs encompassing 1,628 protein coding genes in the GZP,in which 93 genes would lose their functions due to the SV interference and related to reproduction ability of pig.The functional analysis revealed that the 1,628 protein coding genes were significantly enriched in the steroid hormone receptor binding,estrogen receptor binding,oxytocin signaling pathway,retinoic acid receptor binding,axon guidance,cholinergic synapse pathways and m TOR signaling pathways.It suggested that SVs might be a factor for the strong adaptive capacity and low reproductive capacity of GZP.These SVs were not distributed throughout all of the chromosomes.A SV hotspot was identified spanning the 20 Mb on chromosome X of GZP,which contained 29 genes and involved in histone modification and reproduction processes.The results might help to infer the SVs function on the distinct phenotypes among GZP and other pig breed.3.Analysis of specific_SV in Guizhou pigsWe performed resequencing to identify SVs of five representative Guizhou pig breeds,yielded 34,159 SVs in the Guizhou pig breed.Among the identified SVs,5,944 SVs were found in the genomes of all five breeds while some SVs with no overlap with any other breeds represented 870 SVs in KL,823 SVs in QB,825 SVs in GL,786 SVs in LB,and 5855 SVs in XP.It was noticeable that the highest number of SVs was in the XP and the lowest in the LB pig breed.In the PCA pattern diagram,the PC1 geographically distinguished GL and LB in KL,QB and XP,whereas the PC2 captured the biological differentiation between XP and KL,QB,LB and GL.These findings revealed XP breed had a phylogenetic distance from the other four Guizhou pig breeds.Go and KEGG analysis revealed that the KL-associated genes were significantly enriched in ATPase activity,LB-associated genes involved in ion channel,QB-associated genes involved in nervous system and glutamate pathway,GLassociated genes enriched in muscle development,and the XP-associated genes were significantly enriched in the reproduction,development,fatty acid biosynthesis,and so on.Furthermore,we identified 51 candidate genes associated with pig growth trait,such as APOD,INO80,IGF2BP3,GSK3 B,AKT3,and MEF2 C.The type DD frequency was higher in XP than the other breeds,and it indicated that the SV might take part in the regulation on growth and development of Xiang pig.4.Identified genes related to the litter size of Xiang pigWe used resequencing to analyze six Xiang pigs with larger litter sizes(XL)and those with smaller litter size(XS),and detected 4,637 and 4,119 specific SVs contained 1,697 and 1,582 genes in XL and XS group,respectively.These genes were mainly enriched in wellknown pathways involved in development and reproduction pathways.Population validation was carried out on 50 SVs using PCR in 144 Xiang pigs(XL=72,XS=72),all of 50 SVs were confirmed by PCR method and 14 SVs were associated with litter size of Xiang pigs.Finally,we identified 14 candidate genes associated with litter size in Xiang pigs,including XPsv23253(ACSL3),XPsv04986(TMEM131),XPsv05574(ADCY3),XPsv06937(HMGCS2),XPsv14754(NAMPT),XPsv02083(JAK2),XPsv06229(RSPO2),XPsv25406(LBP),XPsv10370(LEXM),XPsv12437(KLF3),XPsv14802(PDIA4),XPsv18294(SH3BP5),XPsv26859(WNK3),and XPsv13280(STPG2).In three SVs(XPsv13280,XPsv10370 and XPsv14802),the II genotype had a larger litter size than those with the DD genotype.The other 11 SVs significantly influenced litter size in Xiang pig,in which the DD genotype had a larger litter size than II genotype.These results provide meaningful genomic variation information for the links between genetic variability and reproductive traits in pigs.5.The effect of SV on the expression of PDIA4 geneBase on resequencing data,a short interspersed nuclear element(SINE)polymorphism was identified from the PDIA4 gene in Xiang pig.The population investigation showed the SINE+/+ genotypes of the PDIA4 gene exhibited 1.45 times litter sizes higher than SINE-/-genotype in XP breed,and there were significant association between SINE polymorphism and litter size.In the SINE region,many elements were found out,such as ESE,ESS,ISE,untranslated region(UTR)motifs,C-to-U RNA editing sites using Reg RNA2.0 software,suggesting splicing may be possible.Furthermore,we tested the m RNA and protein expression of PDIA4 gene with three genotypes in the ovaries of Xiang pigs by q PCR and Western Blot.The results confirmed that the expression of PDIA4 with SINE-/-or SINE-/+ genotypes were up-regulated(P<0.05).It indicated that the SINE element suppressed the expression of PDIA4 gene and PDIA4 protein in pig ovary.These results showed that the SINE polymorphism in PDIA4 gene meight be taken as a potential molecular marker for litter size in Xiang pigs.In conclusion,we performed next generation sequencing to identify SVs of five Guizhou pig breeds,constructed SV maps with high resolution,elucidated the distribution characteristics in genome,and identificated some functional elements in the SV regions.Population validation was carried out on SVs related to litter size of pig,and forteen SVs could be taken as molecular markers to improve the litter size of pig.These findings put insight into the genetic characteristics,breed adaptation,divergence and evolution in Guizhou pigs.