Introduction

Litter size is a complex quantitative trait that is mainly measured as the number of piglets born. However, several other traits, which are more difficult to detect such as ovulation rate, number of corpora lutea, uterine capacity, embryonic, and fetal survival, influence its variability. All these reproductive traits are expressed only in females and after sexual maturity. These biological characteristics and their low heritability limit the effectiveness of selection for prolificacy. Marker-assisted selection (MAS) could be an important tool for genetic improvement of litter size. A few candidate genes for litter size have been already identified in pigs according to their roles in the physiology of reproduction and/or their position within chromosomal regions containing quantitative trait loci (QTL) for reproductive traits (Distl 2007). To use these markers in MAS, it is necessary to verify whether these markers are associated with the traits in the specific population under selection. However, as a preliminary step, it is important to evaluate whether the markers are polymorphic in the investigated populations. Here, we analyzed polymorphisms in 10 candidate genes for litter size in Italian Large White (ITLW) pigs to evaluate their usefulness for association studies.

Materials and methods

Hair samples were collected from 120 ITLW sows from different herds in the Emilia-Romagna and Lombardy regions. The samples were used for the extraction of genomic DNA. Markers in 10 loci identified from the literature were analyzed (Table 1). Single nucleotide polymorphisms (SNPs) were analyzed with a Sequenom Iplex ™ GOLD system that combines primer extension with mass spectrometry (MassARRAY MALDI-TOF MS). A total of 1,200 genotypes were produced. Allelic and genotypic frequencies and the degree of observed and expected heterozygosities were calculated using the POPGENE software, version 1.32 (Yeh et al. 1999) for each marker. The Hardy–Weinberg equilibrium was tested for each polymorphic marker using a chi square test.

Table 1 Genes, porcine chromosome (SSC), and details of the analyzed polymorphisms
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Results

The 10 analyzed genes were chosen by examining the literature for those that were already associated with litter size traits in other pig populations or because they were considered candidates for function and/or chromosomal location. In particular, seven genes map on porcine chromosome (SSC) 8 where QTL for ovulation rate, number of corpora lutea, and uterine capacity were identified (Hu and Reecy 2007). Markers in four out of these seven loci (BMPR1B, GNRHR, MAN2B2, and RNF4) have been previously used in association studies (Campbell et al. 2008; Jang et al. 2001; Niu et al. 2009; Tomas et al. 2006), whereas three (AFP, CLGN, and CXCL10) were considered only for gene mapping in reference families (Kim et al. 2002; 2004). For these three loci, the allelic and genotypic frequencies were not previously obtained in any population (Table 2). Of the analyzed loci, CLGN, encoding a protein essential for sperm fertility, and RFN4, which influences the development of germ cells and oocytes, were not informative in ITLW pigs, as indicated by the presence of only one genotype in the sows. Even the pDAZL gene, which encodes a protein essential for gametogenesis and has a role in embryo implantation and survival (Zhang et al. 2009), was monomorphic. For the remaining loci, both alleles and the three possible genotypes were detected. For the seven polymorphic genes, the observed heterozygosity ranged from 0.173 (AFP) to 0.530 (RBP4) (Table 2). The differences between the observed and expected heterozygosities were minor and not statistically significant. Among the polymorphic genes of SSC8, AFP, which encodes a major fetal protein (Kim et al. 2004), and BMPR1B, which has an important role in the proliferation and differentiation of granulosa cells and oocytes (Tomas et al. 2006), have minor allele frequencies (MAFs) of 0.10 (g.547C) and 0.44 (c.960T), respectively. The CXCL10 gene, which encodes a chemokine of the CXC family with pleiotrophic effects, including a possible role in embryonic development and implantation (Kim et al. 2004), has a MAF of 0.17 for the g.234C allele. For the GNRHR gene, which regulates the secretion of gonadotropins, we analyzed a polymorphism in the 3′-untranslated region (UTR), 92 bp downstream of the stop codon, with possible effects on mRNA stability. The g.1721G allele of GNRHR showed a frequency of 0.25. For the MAN2B2 gene, which encodes a mannosidase involved in glycosylation of hormones that affect ovulation rate, the allele coding for valine (c.1421G) was more frequent than the allele coding for methionine (c.1421A; frequency of 0.11). The remaining two genes, ESR2, coding for one of two estrogen receptors, and RBP4, encoding a retinol-binding protein important in the early stages of pregnancy, showed frequencies close to 0.50.

Table 2 Variability of analyzed loci in a sample of 120 sows of Italian Large White pigs
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Discussion

The objective of this study was to verify whether polymorphisms in 10 candidate genes for litter size segregate in ITLW pigs. Overall, the data obtained allowed the characterization of the genetic structures of these genes in this pig population. Three loci (CLGN, RNF4, and pDAZL) were not informative in the genotyped samples. In particular, for RNF4, the only allele present (g.358C) was shown to result in a favorable effect on the number of piglets born alive in a study by Niu et al. (2009). For pDAZL, the allele g.5999C that in Zhang et al. (2009) was associated with the number of piglets born in Large White pig populations in China was fixed in the ITLW pigs. The remaining seven loci were polymorphic, and their reported allelic frequencies added information on the variability of the analysed genes in pigs. Based on these results, the polymorphisms in the AFP, BMPR1B, CXCL10, ESR2, GNRHR, MAN2B2, and RBP4 genes can be considered suitable markers for association studies of litter size in ITLW pigs. The next step will be to analyze these markers in a larger sample of ITLW sows and evaluate their association with prolificacy.