| Litter size is one of the important factors to evaluate the production performance and economic benefits of pig industry.However,litter size is a complex quantitative trait that regulated by multiple micro-effect genes,it has relatively low heritability which is about 0.1.The progress of the traditional breeding based on phenotypic selection is slow.Moreover,the breeding progress could be effectively and rapidly improved using marker-assisted selection(MAS)and genomic selection(GS).Erhualian pig is one of the indigenous pig breeds in the Taihu Lake region,and it is famous for its high prolificacy,featured by 42 litters in one parity.In our previous study,there is a large variation of litter size in this breed,and the number of ovulations and the length of uterine horn were significantly different between extreme high and low prolificacy Erhualian pigs on day 12 and 24 of gestation.Our group previously performed the genome-wide fixation coefficient(Fst)and whole-group association analyses of the litter size in extremely high and low prolificacy Erhualian pigs,and we found that the QTL on chromosome 8 was significantly affected the litter size of Erhualian pigs.However,the real causal genes and functional loci affecting the variation of litter size of Erhualian pigs on this QTL is still unknown.Therefore,it is necessary to identify the causative genes and functional loci of variation for the litter size trait of Erhualian pigs,and using MAS and GS techniques will improve the litter size trait of pigs.Firstly,fine mapping of the QTL on chromosome 8 was carried out by increasing the number of populations and the density of markers in the previous identified regions.And the key genes affecting the variation of litter size of Erhualian pigs were identified.Secondly,we studied the immunohistochemical localization of Ubiquitin C-terminal hydrolase L1(UCHL1)in the ovaries and uterus of Erhualian pigs.Moreover,we analyzed the expression of the UCHL1 gene in ovaries and endometrium of high and low prolificacy Erhualian pigs on day12 and 24 of gestation.And we also analyzed the expression of the UCHL1 gene in ovaries and endometrium of the offspring of high and low prolificacy Erhualian pigs on day 10,96 and 182.Finally,we identified the key functional loci on the candidate UCHL1 gene affecting the litter size of Erhualiar pigs and verified these loci in Meishan and Suhuai pig populations.The main results are as follows:1.Fine mapping of the QTL on chromosome 8 affecting litter size of Erhualian pigsIn this experiment,a total of 55 markers were added in the 500 Kb genomic region of upstream and downstream of rs81399474.Fine mapping of the QTL was performed in 334 Erhualian pigs and 269 Meishan pigs.By integrating the results of the association analysis of a single SNP with the results of the Meta-analysis of Erhualian and Meishan pig populations.We found QTLs for TNB of multiparities and NBA of all parities,which were mapped to 33934355-34114274 bp(179.92 Kb),33934355-34094188 bp(159.83Kb),33884473-33959514(75.04 Kb)and 33934355-33985796 bp(51.44 Kb)on chromosome 8,respectively.In these QTLs,the UCHL1 gene was identified as a key functional candidate gene affecting the litter size of Erhualian pigs.2.Expression analyses of the UCHL1 gene in the ovaries and uterus of Erhualian pigsIn this experiment,we analyzed immunohistochemical localization of UCHL1 in the ovaries and uterus of Erhualian pigs.This study found that the UCHL1 protein was mainly expressed in the cytoplasm of follicular granulosa cells and oocytes in the ovaries of Erhualian pigs.And UCHL1 protein is mainly expressed in the nucleus of uterine gland cells and endometrial stromal cells of Erhualian pigs.We also analyzed the expression of the UCHL1 gene in the ovaries and endometrium of high and low prolificacy Erhualian pigs on day 12 and 24 of gestation,and its offspring on day 10,96 and 182 after birth.In the ovaries of the offspring of Erhualian pigs,the expression of the UCHL1 gene in offspring on day 10 was significantly higher than those on day 96 and 182 after birth(P<0.01),respectively.In the endometrium of Erhualian pigs on day 24 of gestation,the expression of the UCHL1 gene in the high prolificacy Erhualian pigs was significantly higher than low prolificacy ones(P<0.05).In the endometrium of the offspring of Erhualian pigs,the expression of the UCHL1 gene in offspring of Erhualian pigs on day 96 was significantly higher than that of Erhualian pigs on day 182(P<0.01).3.Association analysis between polymorphism of the UCHL1 gene with the litter size traitsWe estimated and ranked the breeding value of the litter size of Erhualian pigs,and selected 20 extremely high and low prolificacy Erhualian pigs for pre-screening SNPs that may affect the litter size of Erhualian pigs.These SNPs were selected in the promoter,coding region,and untranslated region(UTR)of the UCHL1 gene.And through the chi-square test of the genotype distribution of the extremely high and lowyielding Erhualian pigs.Significant SNPs of the chi-square test were verified in the whole Erhualian pig population.Finally,these SNPs that were significantly associated with the litter size of Erhualian pigs were selected and verified in the Meishan and Suhuai pig populations.In our studies,we found that there are nine SNPs in the promoter,coding region,and UTR region of the UCHL1 gene.Our results showed that the polymorphism of the rs339921806 in the promoter and rs342939847 in the 5’UTR of the UCHL1 gene were significantly affected the litter size of Erhualian pigs and Meishan pigs(P<0.05),but was not associated with the litter size of Suhuai pigs.In conclusion,this study identified the UCHL1 gene as a key candidate gene affecting the litter size of Erhualian pigs through the QTL fine mapping on chromosome 8.UCHL1 protein was mainly expressed in the cytoplasm of follicular granulosa cells and oocytes of Erhualian pigs.This protein is preferentially expressed in endometrial stromal cells of Erhualian pigs on day 96 after birth,while it expressed in the uterine gland cell and endometrial stromal cells of Erhualian pigs on day 182 after birth.In the ovaries of the offspring of Erhualian pigs,the expression of UCHL1 gene in the offspring on day 10 was significantly higher than on day 96 and 182 after birth by RTqPCR.The expression of the UCHL1 gene in the high prolificacy Erhualian pigs were significantly higher than low prolificacy ones in endometrium on day 24 of gestation,and the expression of UCHL1 gene in offspring of Erhualian pigs on day 96 after birth was significantly higher than Erhulian on day 182 in the endometrium.The polymorphism of rs339921806 in the promoter and rs342939847 in the 5’UTR of the UCHL1 gene were significantly associated with the litter size of Erhualian and Meishan pig populations. |
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