Genetic diversity of porcine PRLR gene and its relationship to litter size in Large White pigs

. Genetic

The reproductive performance of sows is closely related to the economic success of large-scale pig farms. The factors that can affect an evaluation of the reproductive performance of sows are diverse (Tummaruk et al. 2009). The total number born (TNB) and number born alive (NBA) are the key indicators used to evaluate the reproductive performance of sows (Bakoev et al. 2020). However, low heritability is a reproductive trait of pigs (Zhang et al. 2020;Plaengkaeo et al. 2021). Improving traits with a low heritability with conventional breeding pro-grams is a very slow process. Unlike conventional breeding, molecular mark-assisted selection has triggered faster progress, where a single nucleotide polymorphism (SNP) is the most widely used molecular marker technology (Tian et al. 2015;Xu et al. 2017). SNPs are widespread in animal and plant genomes, and sometimes, mutations in a single nucleotide of specific genes will cause changes in the reproductive performance. Recently, an SNP molecular labelling technology has been widely used to study the quantitative traits of pigs (Wu et al. 2018).
Prolactin (PRL) is a protein hormone secreted by the anterior pituitary gland's eosinophils. Its primary role is to promote the development of an animal's gonads and mammary glands (Schennink et al. 2013). When combined with the corresponding receptor on the target cells (prolactin receptor, PRLR), PRL causes a series of physiological responses (Schennink et al. 2015). The PRLR gene is closely related to the animal's reproductive performance. For example, PRLR knockout mice were found to be infertile due to an embryo implantation failure and showed reproductive disorders such as cycle disorder and a reduced fertilisation rate (Ormandy et al. 1997). Previous studies also found that polymorphisms of the PRLR gene can affect the reproductive performance. In some poultry breeds, the SNP of the exons of the PRLR gene was found to result in significant differences in the egg-laying, growth and development between the different genotypes Liang et al. 2019). Jiang et al. (2005) identified SNP (A9026G) on exon 3 of the chicken PRLR gene, which leads to a nucleotide conversion in the 5'-untranslated region of the PRLR gene. The PRLR gene polymorphism was associated with the number of lambs . Furthermore, it has been shown that porcine PRLR is localised to chromosome 16 that contains ten exons and nine introns, in which the 10 th exon region harbours many mutations and has some associations with the reproductive performance of pigs (Hu et al. 1999).
The Large White pig is also known as the Yorkshire pig. It has the excellent characteristics of a high yield, fast growth rate, high feed utilisation rate, high carcass lean meat percentage, strong adaptability, high feed conversion rate and a high slaughter rate. It is also the world's most famous and widely-distributed dominantly lean pig species. Its related reproductive performance is inseparable from the development of animal husbandry (Alam et al. 2021). Hence, ways to improve the breeding traits of Large White pigs have become a hot topic in the field of animal husbandry. The modern Large White pig has undergone many improvements and different countries have conducted breeding according to their needs. While retaining the characteristics of the Large White pig, on the whole, the animals in different countries now have unique characteristics and form different strains. Therefore, when this study was designed, American, Danish and Canadian Large White pigs were selected to research the effects of different SNP sites in the PRLR gene on the sow reproductive performance and to establish the basis for a molecular marker foundation for sows, in order to increase the litter size.

Sample information and DNA extraction
A total of 201 back hair samples were collected from American Large White pigs (AC, n = 134), Canadian Large White pigs (CD, n = 52) and Danish Large White pigs (DN, n = 15). All of these pigs were selfed progeny of the corresponding breeds and were selected from the farms of Tianren Agriculture and Animal Husbandry Technology Co., Ltd. located in Leshan City, Sichuan Province, China. We recorded the total number born (TNB) and the number of born alive (NBA) of the first and second litter for each sow. Genomic DNA was extracted with the DP316 DNA kit (Tiangen Biochemistry Technology Co., Ltd., Beijing, China) from twenty hairs with follicles of the Large White pigs. The DNA purity was checked on 1% agarose gel electrophoresis and its quality was assessed by spectrophotometry (Thermo Fisher NanoDrop 2000/2000c.

Primer design, PCR amplification and SNP identification
The corresponding primers were designed according to the pig PRLR gene sequence (DQ458765.1) using Primer Premier 5.0 software and were synthesised by Chengdu Qingke Zixi Biotechnology Co., Ltd. The primer sequences were 5'-TAAGTAGTGGGATGTTAGGAA-3' and 5'-TGTTGTGGAGAAAGAGGC-3'. PCR was performed in a total volume of 30 μl using the following mixture: 2 × Taq Master Mix 15.0 μl, 1.5 μl of primers, 1.0 μl for the DNA template and ddH 2 O 11.0 μl. The PCR amplification condition was as follows: an initial denaturing step at 94°C for 90 s, followed by 30 cycles of denaturing at 94°C for 20 s, annealing at 60°C for 20 s and extending at 72°C for 60 s, with a final extension step at 72°C for 5 min. The PCR products were sequenced by Chengdu Qingke Zixi Biotechnology Co., Ltd. The sequencing data was analysed with Bioedit software to search for the SNPs.

Data analyses
The Hardy-Weinberg equilibrium (HWE) and the Polymorphism Information Content (PIC) were analysed with SPSS 22.0 software. The allele and genotype frequencies were also calculated. A linkage disequilibrium analysis was performed using Haploview 4.2 software, while a haplotype construction of the SNP locus was performed using Phase 2.0. The association analysis between the SNP locus and the litter size traits was analysed with SAS 8.0 software. The model was designed as follows: where Yij is the reproductive trait recording value;  is the population mean; Gi was the i-th genotype effect; Pj is the parity effect; and eij is the random residual effect. The analysis was expressed as the least squares mean and standard error of the mean (LSM ± SEM), with p < 0.05 indicating a significant difference and p < 0.01 indicating an extremely significant difference. SAS 8.0 software was used for all the statistical analyses.

Results
Polymorphism and the genetic parameters of the PRLR gene The PCR amplification products were sequenced, and the sequencing results were compared with the existing PRLR gene sequences in the gene bank. Nine SNPs were found in the exon region of the PRLR gene (Fig 1). The allele frequency, genotype frequency, PIC and χ 2 text of the nine SNPs polymorphic loci of the PRLR gene in the 201 tested pigs were calculated, and the results are shown in Table 1. According to the genetic parameters, the nine mutation sites in the  PRLR gene were indicated in the Hardy-Weinberg equilibrium by the χ 2 text. A medium polymorphism was found in g.C362T, g.A584G, g.A673T, g.C765T and g.A934G (0.25 < PIC < 0.50), while a low polymorphism was revealed in g.C260G, g.C527G, g.A540G and g.A745G (0.00 < PIC < 0.25).
Linkage disequilibrium relationships and haplotype analysis between the SNPs of the PRLR gene Haploview 4.2 software was used to analyse the linkage disequilibrium (LD) among the nine mutation sites of the PRLR gene in the three Large White pigs lines (Fig. 2). The results showed a strong linkage disequilibrium among the SNP loci in the American Large White pigs. In the Canadian Large White pigs, there was strong linkage disequilibrium among the following loci: g.C360T, g.A584G and g.A673T, g.C765T, and g.A934G. In the Danish Large White pigs, probably due to the small sample size, there was no strong linkage disequilibrium. According to the results of the total samples, it was found that there was a strong linkage imbalance among the other loci, except for the g. A540G loci.
Nine SNPs from the PRLR genes detected from 201 Large White pig individuals were analysed for the haplotype, and the results are shown in Table 2. The detected nine SNPs constituted nine haplotypes in the 201 Large White pigs, including five haplotypes with a frequency above 3%; the three highest frequencies were H2, H1 and H4, at 41.64%, 22.79% and 19.51%, respectively, while the haplotypes with the lowest frequencies were H8 and H9.

Association analysis of the PRLR gene with litter traits
The association analyses of the nine SNPs of the PRLR in different strains of Large White pigs are shown in Table 3. In the analysis of the association between the PRLR gene SNP loci and the litter size of Large White pigs, both the CC genotype at g.C765T and the AA genotype at g.A934G significantly increased the total number born and the number born alive at first parity (p < 0.01), which could also significantly increase the total number born at multiparous parity (p < 0.05). There were no significant differences between the genotypes at the remaining loci.  Table 2 Haplotype frequencies of porcine SNPs in the PRLR gene Haplotype g.C260G g.C362T g.C527G g.A540G g.A584G g.A673T g.A745G g.C765T g.A934G Frequency (%)  Nine SNP loci were used for the haplotype combination and sixteen haplotype combinations were obtained, including eight haplotype combinations with frequencies of more than 3% (Table 4). Furthermore, among the eight haplotype combinations, the highest frequency combination was H1H2, with a frequency of 18.41%. In the sows, H2H7 was the dominant combination of the total number born at first parity (TNB1, 12.07 ± 0.69). Meanwhile, H4H4 was the dominant combination of the number born alive at first parity (NBA1, 11.13 ± 1.00), total number born (TNB2, 12.00 ± 1.28) and the number born alive at multiparous parity (NBA2, 11.60 ± 1.29). H1H1 was the inferior combination of TNB1 (8.44 ± 0.89) and NBA1 (7.44 ± 0.94), and H1H4 was the inferior combination of TNB1 (9.50 ± 0.64) and NBA1 (8.65 ± 0.64).

Discussion
Recently, molecular markers have been widely used to advance quantitative, functional and evolutionary genomics (Jiang et al. 2016). More and more SNPs are being used to explore the litter traits in pigs . For example, Wang et al. (2018) detected 41 suggestive significant SNPs associated with six reproductive traits in Large White pigs. Through a genome-wide association study of four reproductive traits in a Duroc pig herd, Zhang et al. (2019) detected 20 SNPs that were potentially associated with these traits of interest. The SNP loci identified in each experiment could lay the foundation for using molecular markers to increase litter size in sows. In addition, genetic variations in the functional genes could increase the number of litter births, TNB and the NBA (Laliotis et al. 2017). In this study, a correlation was sought between the associated polymorphism loci in the PRLR gene and the litter traits in Large White pigs.
Many studies have found that the PRLR gene plays an essential regulatory role in animal reproduction; for example, the prolactin receptor can maintain the corpus luteum and plays an important role in maternal pregnancy and foetal development (Zi et al. 2012) mRNA expression of prolactin receptor (PRLR. A number of polymorphisms have been identified in the porcine PRLR gene. Tomás et al. (2006) sequenced the complete coding region of the porcine PRLR gene and found 6 nonconservative SNPs. Skrzypczak et al. (2015) found three genotypes (AA, AT and TT) of PRLR by digestion with the NcoI restriction enzyme. Terman (2005) used the restriction enzyme Alu I to detect PRLR gene polymorphisms in Polish pigs and identified two alleles. Furthermore, Drogemuller et al. (2001) found that the PRLR gene polymorphism was associated with the reproductive trait of the live litter size in sows. In this study, nine SNPs were detected in exon 10 of the PRLR gene of Large White pigs (g.C260g, g.C362T, g.C527g, g.a540g, g.A584G, g.A673T and g.A745g). This will provide more reference sites for the molecular labelling of the PRLR gene.
Related studies have shown that the litter size is affected by various factors, such as the season (Mayorga et al. 2019;Caamaño et al. 2021), semen quality (Peña et al. 2005;Belstra et al. 2020), gilts (nulliparous) and multiparous sows (Peltoniemi et al. 2016). Genetic variations of the PRLR gene in pigs can also cause changes in the litter performance. Researchers have studied different pig breeds successively and found that, in some studies of foreign breeding pigs and local pig breeds in China, the A allele is related to an excellent reproductive performance (Van Rens et al. 2003). Linville found that the PRLR gene affected the ovulation number of sows and the number of piglets produced in the first litter (Linville et al. 2001), and it was found that the number of individuals with the BB genotype of the PRLR gene was significantly higher than that of other genotypes (Mikhaĭlov et al. 2014). Isler found that the PRLR gene affects the average number of uterine horn foetuses in AA, AB and BB sows, and showed that AA > AB > BB, among which the type B gene is the favourable gene (Isler et al. 2002). In our study, the CC genotype at the g.C765T loci and the AA genotype at the g.A934G loci very significantly increased the TNB and NBA at first parity, as well as the TNB at multiparous parity in Large White pigs. In the haplotype construction and haplotype combination association analysis, H2H7 was the dominant combination of TNB at first parity (12.07 ± 0.69). On the other hand, H4H4 was the dominant combination of NBA at first parity (11.13 ± 1.00), TNB at multiparous parity (12.00 ± 1.28) and NBA at multiparous parity (11.60 ± 1.29). However, due to the limited sample size in this study, further investigation is required.

Conclusions
In conclusion, we found nine porcine SNPs in the PRLR gene, which confirmed the existence of its polymorphism. The correlation between the PRLR polymorphisms and the litter traits of the Large White pigs was verified. The g.C362T and g.A584G loci had significant effects on the litter traits (p < 0.05), while the g.C765T and g.A934G loci were also correlated with litter traits (p < 0.01). Therefore, the polymorphisms of PRLR were correlated with litter traits that have a selective value for the genetic breeding improvement of Large White pigs.