产仔数的高低是评价猪场的生产水平和经济效益的重要指标之一，然而产仔数是由微效多基因调控的复杂数量性状，遗传力很低（约为0.1）。基于表型选择的传统选育进展缓慢，利用分子标记辅助选择（Marker assisted selection，MAS）和全基因组选择（Genomic selection，GS）技术，可以有效快速提高产仔数的选育进展。二花脸猪作为太湖流域地方猪种之一，以高繁殖力著称，单胎产仔高达42头锚点。锚点本团队前期研究发现该品种内存在较大的产仔数变异，高、低产组妊娠12天和24天二花脸猪排卵数和子宫角长度也存在显著差异；并且前期通过极端高、低产二花脸猪的遗传分化系数（Genome-wide fixation coefficient，Fst）分析和全群关联性分析，在8号染色体上鉴定到显著影响其产仔数变异的数量性状基因座（Quantitative trait loci，QTL），但该QTL内的因果基因和功能位点未知。因此，鉴别二花脸猪产仔数变异的因果基因和功能位点，利用MAS和GS技术提高其产仔性能优势十分必要。

本研究首先通过增加目标区域的标记密度和群体量进行QTL精细定位，在8号染色体上精准鉴定影响二花脸猪产仔数变异的关键基因。其次选择精细定位区间内的泛素羧基末端水解酶-1（Ubiquitin C-terminal hydrolase 1，UCHL1）基因作为候选因果基因进行研究。在二花脸猪卵巢和子宫组织中对UCHL1进行免疫组化定位研究；并选择极端、高低产组妊娠12天和24天二花脸猪的卵巢和子宫内膜样品以及高、低产组子代10、96和182天二花脸猪的卵巢和子宫内膜样品进行UCHL1基因表达量的差异分析。锚点最后，在候选UCHL1基因上鉴定影响二花脸猪的总产仔数（Total number born，TNB）和产活仔数（Number born alive，NBA）的关键功能位点，并在梅山猪和苏淮猪群体中验证。本研究主要结果如下：

1. 猪8号染色体影响二花脸猪产仔数性状的QTL精细定位

本章试验在rs81399474位点上下游各500Kb基因组区域内共增加55个标记位点，在334头二花脸猪和269头梅山猪群体中进行精细定位。通过单标记关联分析以及二花脸猪与梅山猪群体的Meta分析，将影响二花脸猪经产胎次TNB、经产胎次NBA、全部胎次TNB和全部胎次NBA的QTL分别精细定位锚点于8号染色体上33934355-34114274 bp（179.92 Kb）、33934355-34094188 bp锚点锚点（159.83 Kb）、锚点33884473-33959514 bp锚点锚点（75.04 Kb）和33934355-33985796 bp（51.44 Kb）范围内。在上述QTL区间内鉴定到UCHL1基因是影响二花脸猪产仔数的功能候选基因。

2. UCHL1基因在二花脸猪卵巢和子宫组织中的表达分析

本章试验对UCHL1蛋白在二花脸猪卵巢和子宫组织进行免疫组化定位发现，在卵巢中，该蛋白主要定位于二花脸猪的各级卵泡颗粒细胞和卵母细胞的胞质；在子宫中，主要定位于二花脸猪的子宫腺体细胞核和子宫内膜间质细胞。通过对极端、高低产组妊娠12天和24天二花脸猪以及极端、高低产组子代10、96和182天二花脸猪的卵巢和子宫内膜样品进行UCHL1基因表达量的差异分析。发现在二花脸猪子代10、96和182天母猪卵巢中，UCHL1基因在10天卵巢的表达量极显著高于96和182天的表达量（P < 0.01）。在妊娠24天二花脸猪的子宫内膜中，高产组的基因表达量显著高于低产组（P < 0.05）；在二花脸猪96和182天母猪子宫内膜中，基因在96天的表达量极显著高于182天的表达量（P < 0.01）。

3. UCHL1基因多态性与产仔数的关联性分析

本章试验通过估计并排序二花脸猪产仔数的育种值，选择极端各20头高、低产二花脸猪，在UCHL1基因的启动子区、编码区和非翻译区（Untranslated region，UTR）初步筛选可能影响产仔数的位点；并通过极端高、低产群体基因型分布的初步显著性检验，极端群体检验显著的位点在二花脸猪全群中验证；最后挑选显著影响二花脸猪产仔数关联的位点在梅山猪和苏淮猪群体中进行验证。本研究发现UCHL1基因的启动子区、编码区和非翻译区共发现9个SNP位点；其中，UCHL1基因启动子区rs339921806和5’UTR区rs342939847位点与二花脸群体的产仔数显著关联；经梅山猪和苏淮猪群体验证发现rs339921806和rs342939847位点多态性与梅山猪的产仔数显著关联，但均不影响苏淮猪产仔数。

综上所述，本研究通过增加标记密度和群体量，成功地在8号染色体上精细定位到影响二花脸猪产仔数的QTL，并在QTL区间内鉴别到UCHL1基因为关键位置功能候选基因。通过免疫组化试验发现UCHL1蛋白主要表达于二花脸猪的各级卵泡颗粒细胞和卵母细胞的胞质；该蛋白仅在96天二花脸猪的子宫内膜间质细胞表达，在182天二花脸猪的子宫腺体细胞核和子宫内膜间质细胞都有表达。通过定量PCR发现该基因在10天卵巢的表达量极显著高于96天和182天的表达量；并且该基因在高产组妊娠24天子宫内膜的表达量显著高于低产组；该基因在96天子宫内膜表达量极显著高于182天的表达量。通过关联性分析发现，UCHL1基因启动子区rs339921806和5’UTR区rs342939847位点多态性与二花脸猪和梅山猪群体的产仔数显著关联。

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 1 (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 Erhualian 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 pigs

In 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, NBA of multiparities, TNB of all parities and NBA of all parities, which were mapped to 33934355 - 34114274 bp (179.92 Kb), 33934355 - 34094188 bp (159.83 Kb), 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

pigs

In 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 traits

We 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 low-yielding 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 RT-qPCR. 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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