Verification Of The QTL And Analysis Of Candidate Gene CLSTN2 For Litter Size Of Erhualian Pig On Chromosome 13

As one of the important economic traits in pig production,litter size is directly related to the production efficiency.Researchers have focused on the improvement of litter size of pig.However,litter size is a quantitative trait controlled by complex polygenes,and the traditional breeding method has made very slow progress.With the emerge of DNA sequencing technology,MAS（Marker assisted selection）and GS（Genome selection）,the analyses of the genetic mechanism of litter size variation has become more and more important in molecular breeding process.Erhualian pig has kept the record of the world’s highest prolificacy of pig,which is a good material to study the mechanism of litter size.Our preliminary investigation found that litter size of the population of Erhualian pig has shown a large variation,and there are significant differences in ovulation number and uterine horn length between high and low prolificacy population.Genome-wide association study（GWAS）found that there is a QTL interval affecting litters of Erhualian pig on chromosome 13（Chromosome 13,chr13）.By increasing the marking density and the experimental sample（Erhualian and Meishan pig）to fine mapping,a QTL of 118.13 Kb affecting the total number born（TNB）was found,which was located at the 1st intron of Calsyntenin 2（CLSTN2）gene on porcine Chromosome 13.And we also found another QTL of 333.34 Kb affecting the number born alive（NBA）,which located in part of the 5’UTR and 2nd intron of CLSTN2 gene.On one hand,by using GWAS and fine mapping,our previous research has found a few SNPs loci that significantly associated with litter size of Erhualian and Meishan pigs,then we asked whether the SNPs can be applied to the breeding of the French and Canadian Yorkshire pigs.And these results will provide a basis for further QTL fine mapping related to litter size.On the other hand,in order to identify the main functional site associated with litter size of Erhualian pig,the CLSTN2 gene within the QTL was selected for further study as a candidate gene affecting litter size of Erhualian pig on chromosome 13.The results are as follows:1.The verification of QTL associated with the Erhualian’s litter size in Canadian Yorkshire pigIn French Yorkshire pigs which have the consanguinity of Taihu lake region pig breeds,and Canadian Yorkshire pigs with higher litter size collected by our team,7 SNPs that were significantly related to litter size of Erhualian and Meishan pig populations were verified,including the chr13₈0629229、chr13₈0658363、chr13₈0677615、chr13₈0866737、chr13₈0977115、chr13₈1010955 and chr13₈1168033 loci.Then we evaluated the CV（Coefficient of variation）of litter size of the 2 pig populations,we found that there was a large variation of litter size in these two pig populations and the value of CV was greater than 20%,meaning that there is a great space for the selection and improvement of litter size.Firstly,according to the estimated breeding value（EBV）,the extremely high and low prolificacy pigs were selected to verify the effect of 7 SNPs on litter size.Then,the whole population association verification is performed for significant loci.It turned out that among the 7 loci,it was found that the chr13₈0629229 locus had a significant related trends with French Yorkshire pig（P<0.1）.There are 3 SNP loci（chr13₈0629228,chr13₈0977105 and chr13₈1010955）significantly correlated with the litter size in the Canadian Yorkshire pig（P<0.05）.In summary,the chr13₈0629228 locus which located in the 1st intron of the CLSTN2 gene can affect litter size of Canadian Yorkshire pig.Therefore,we verified the existence of QTL that affected litter size on chromosome 13.2.The expression of two transcripts of the CLSTN2 gene in the uterus and ovary between extremely high and low prolificacy Erhualian sowsOur previous RNA-seq results showed that the CLSTN2 gene mRNA（published on the NCBI website,the full length is 14625 bp）was hardly expressed in endometrium of the high and low prolificacy Erhualian pigs at the day 12 pregnant stage.However,we noticed that CLSTN2 gene expressed one transcript（～120 bp length）in the 10th intron of this gene,and it did not overlap with the 14625 bp transcript of CLSTN2 gene.Therefore,we named this new transcript as CLSTN2-intronl0-RNA.Using RT-qPCR,we detected the expression of CLSTN2 gene in the uterus and ovary of Erhualian sows at pregnant stage of day 12 and 24.We found that the full length transcript of CLSTN2 gene was expressed in the uterus and ovary,but the relative expression level was quite low（the Ct value is～30）.Moreover,the expression of CLSTN2 gene between high and low prolificacy Erhualian sows was not significant in the ovary and uterus.RT-PCR method was used to verify that the CLSTN2-intron10-RNA was not an alternative splicing exon of the CLSTN2 gene mRNA,they are independent transcripts.3.Validation of DNA sequence encoding the CLSTN2-intronl0-RNA transcript and identification of the processing pseudogene of RPLP1 geneWe found that the sequence of CLSTN2-intron10-RNA transcript is 98%similar with part of RPLP1 gene by sequence alignment.In order to rule out the misalignment errors in the RNA-seq,and prove the existence of the CLSTN2-intronl 0-RNA transcript,we designed primers to amplify all the DNA sequences between the 10th and 11th exon of the CLSTN2 gene based on reference genome of Sus scrofa 11.1.By sequence alignment,we proved that there was a DNA sequence coding the CLSTN2-intronl0-RNA on the 10th intron of CLSTN2 gene in both Erhualian pigs and foreign pig populations.In addition,bioinformatics analysis revealed the presence of a pseudogene of RPLP1 gene on the 10th intron of the CLSTN2 gene,which had 93%similarity with the RPLP1 mRNA.The full-length of this RPLP1 pseudogene was 524 bp,which is 15 bp less than the RPLP1 full length mRNA.This RPLP1 pseudogene was not fully expressed and only about 120 bp transcript（CLSTN2-intronl0-RNA）was expressed.We speculated that the RPLP1 gene pseudogene could not transcribe the full length of RPLP1 mRNA,which is likely due to the absent of the original promoter sequence during the transposition of the pseudogenes.However,we speculated that the new core promoter region,transcription start site and transcription factor binding site were formed on the new CLSTN2-intron10-RNA transcript,which lead to its transcription.4.Study of the SNPs loci related to litter size on the upstream of CLSTN2-intron10-RNA transcriptFirstly,8 SNPs loci were identified in 600 bp DNA sequence upstream of the CLSTN2-intron10-RNA transcript.chr13₈1429230 and chr13₈1429652 loci were extremely significantly related to litter size of Erhualian pigs at P<0.01,and the other 3 loci,chr13₈1429549,cHr13₈1429641 and chr13₈1429686,were found related to litter size of Erhualian pigs at P<0.05.In addition,these 5 loci did not affect the core promoter and transcription initiation sites of the CLSTN2-intron10-RNA transcript,but they may change the type and location of some transcription binding factors using bioinformatics analysis.In conclusion,the French and Canadian Yorkshire pigs were used to verify the existence of QTL that affects litter size on chromosome 13.We found that the CLSTN2 gene is located in this QTL,and there are two transcripts of CLSTN2 gene.The expression of classical full-length transcript of CLSTN2 gene was extremely low and there was no expression difference between high and low prolificacy Erhualian sows in uterus and ovary tissues.Moreover,we found that there is a processing pseudogene of RPLP1 gene on the 10th intron of the CLSTN2 gene.This pseudogene only expressed about 120 bp transcript,and we named it as CLSTN2-intron10-RNA.More importantly,we found that there were 5 SNPs on the promoter region of CLSTN2-intron10-RNA transcript were significantly associated with litter size of Erhualian pigs.