Introduction

Breeders in China have devoted themselves to the genetic improvement of pigs for a long time and have achieved remarkable achievement through steadily advancing in the last decade (Palombo et al. 2021). However, the improvement of breeding efficiency and accuracy is still a challenge. With the development of molecular marker techniques, marker-assisted selection (MAS) has become a rapid and simple method for molecular breeding (Chen et al. 2019; Yang et al. 2020). It uses the linkage between phenotypes of a certain trait and molecular markers in the genomic. By selecting target genotype, individuals with target traits were quickly and accurately selected. It is regarded as one of the effective means of improving breeding efficiency.

In pigs, some causal genes or tightly linked genes affecting crucial traits and used in practical production have been reported in previous studies, such as PHKG1 (rs330928088) (Ma et al. 2014), MUC13 (rs319699771) (Zhang et al. 2008), IGF2 (g.3072G>A) (Burgos et al. 2012), VRTN (g.20311_20312ins291) (Ren et al. 2012a; Fan et al. 2013), MYH3 (XM_013981330.2:g.-1805_-1810del) (Cho et al. 2019), LIPE (rs328830166) (Zhao et al. 2009), LEPR (rs45435518) (Ros-Freixedes et al. 2016), and MC4R (rs81219178) (Schroyen et al. 2015). The mutations in these genes could be used as markers for MAS to accelerate the breeding progress in pigs.

Amongst the genes mentioned above, PHKG1 is a vital candidate gene affecting pork water-holding capacity, pH value, pork colour, and glycolysis (Zappaterra et al. 2019). C to A substitution at position 8283 in intron 9 of PHKG1 gene will lead to the decrease of the rate of glycogenolysis in muscle tissue. However, excessive glycogen could induce the accumulation of lactate, which leads to the decrease of final pH value and the increase of drip loss (Ma et al. 2014; Florowski et al. 2017; Liu et al. 2019b). MUC13 is a major gene that affects diarrhoea in neonatal and young pigs. The mutation rs319699771 in MUC13 can influence the adhesion of enterotoxigenic Escherichia coli f4ac to the small intestinal villous of pigs. Amongst the genotypes, individuals with GG genotype are resistant ones, and individuals with AG and AA genotypes are susceptible ones (Ren et al. 2012b; Shi et al. 2017; Liu et al. 2019a). IGF2 is a causal gene affecting the lean meat rate in pigs. Base change of G to A in intron 3 of IGF2 gene can increase muscle production and reduce backfat (BF) deposition (Van Laere et al. 2003). The indel g.20311_20312ins291 in VRTN gene is the causative mutation affecting the thoracic vertebrae number in pigs, and individuals with this mutation have more than one thoracic vertebra than the wild type (Fan et al. 2013; Yang et al. 2016). MYH3 gene encodes the embryonic heavy chain myosin, which is mainly used to control the traction and slide of muscles (Knight and Molloy 2000; Maheshwari et al. 2017; Cope et al. 2020). Recently, Cho et al. (2019) reported that the causal mutation XM 013981330.2:g.-1805-1810del, which is a 6 bp deletion variant in the promoter region of MYH3 gene, could affect intramuscular fat (IMF) content and red flesh colour (a*) in pigs. The protein encoded by the hormone-sensitive lipase (LIPE) gene is one of the lipolytic enzyme. The gene plays a crucial role in controlling lipid deposition (Lampidonis et al. 2011; Piórkowska et al. 2018; Al-Thuwaini et al. 2020). Several studies have suggested that the mutation (rs328830166) of LIPE is significantly correlated with IMF content in the crossbred pigs (Duroc × Shanzhu pigs), and the IMF content of individuals with allele A was higher (Xue et al. 2015). LEPR gene encoding proteins binding to leptin protein could regulate several physiological processes including body energy balance and fat metabolism (Zhang et al. 2016). Individuals with the TT genotypes of rs45435518 in LEPR have more saturated fatty acids (SFA) than ones with wild type. In addition, genome-wide association studies have confirmed that LEPR gene is one of the major loci that affect IMF content and fatty acid composition in Duroc (Rodriguez et al. 2010; Galve et al. 2012; Ros-Freixedes et al. 2016). MC4R gene is one of the candidate genes affecting growth, BF thickness, and meat quality. When a G/A substitution at position 298 of MC4R gene occurs, it will significantly increase BF thickness, carcass weight, moisture content, and the content of SFA (Fontanesi et al. 2013; Hirose et al. 2014; Choi et al. 2016).

China is rich in pig species resources and has many local pigs. Most local pigs have high fecundity and the characteristics of strong stress resistance, and especially the meat quality is significantly better than foreign pig breeds (Cheng-yi et al. 2003). Lulai black pig is a new variety with the comprehensive advantages of local pigs and foreign pigs from the cross between Laiwu pig (representative of North China black pig) and Yorkshire pig after six generations of breeding (Chen et al. 2017; Wang et al. 2019). Known for its high IMF content, it is the best animal model to study fat deposition and animal breeding. It was released by the National livestock and Poultry Genetic Resources Management Committee in 2006. And it was announced by the Ministry of Agriculture of the People's Republic of China and issued the certificate as a new variety of Lulai black pig. In this study, Lulai black pigs were used to analyse the frequency distribution of the abovementioned eight effective or causal mutation sites in the abovementioned eight genes by restricted fragment length polymorphism (RFLP) analysis or sequencing. Moreover, we investigated the correlation between the effective mutation sites and the meat quality traits in the Lulai black pig population. The aim of the present study was to provide a theoretical basis for Lulai black pig breeding about the disease resistance, meat quality, and growth performance. In addition, the insights provided are valuable for the application and improvement of MAS in pig breeding.

Materials and methods

Primer design and PCR (Polymerase Chain Reaction) amplification

The primers containing IGF2 (g.3072G>A), VRTN (g.20311_20312ins291), and MYH3 (XM_013981330.2:g.-1805_-1810del) mutation sites were designed using Premier 5.0 based on the IGF2, MYH3, and VRTN sequence of GenBank. The primers are described in Table 1. All primers were synthesized by the TSINGKE Company (Qingdao, China).

Table 1.

Primer sequences and annealing temperature.

The multiplex PCR reaction of MYH3 and VRTN, performed in a 25 µL total volume, contained 12.5 µL of Dream Taq™ Green PCR Master Mix (2×), 8.5 µL of ddH₂O, 1 µL of Primer F and Primer R, and 2 µL of genomic DNA. The PCR programme was as follows: 94 °C for 5 min, 35 cycles (94 °C for 30 s, 61.5 °C for 30 s, and 72 °C for 1 min), 72 °C for 10 min, and hold at 4 °C. The multiplex PCR reaction of IGF2, performed in a 25 µL total volume, contained 0.25 µL of Ex Taq DNA Polymerase (5 U/µL), 12.5 µL of 2× GC Reaction Buffer, 5 µL of GC Enhancer Buffer (5 mol/L), 1 µL of dNTP Mix (10 mmol/L each), 1 µL of Primer F and Primer R, 2.25 µL of RNase free water, and 2 µL of genomic DNA. The PCR programme was as follows: 94 °C for 1 min, 35 cycles (98 °C for 10 s, 63.2 °C for 30 s, and 72 °C for 30 min), 72 °C for 5 min, and hold at 4 °C. PCR products obtained were electrophoresed on 1.5% agarose gel, and the gel running conditions were 180 V for 35 min. PCR products with bright and single bands were used for subsequent tests.

Genotyping

The PCR products of IGF2 were sent to TSINGKE Company (Qingdao, China) for Sanger sequencing. The sequencing results were analysed by using DNAMAN 8.0.8 (Lynnon Corporation, Canada) and Chromas 2.6.5 (Technelysium Pty, Ltd., Ireland) to identify mutations. The VRTN (g.20311_20312ins291) genotypes results were determined by electropherograms. The mutant (QQ) genotype generates a band of 411 bp, whereas the wild type (qq) genotypes a band of 120 bp and two bands for heterozygous (Qq) genotype (Fig. 1). Genotyping of the MYH3 gene was performed by PCR–RFLP. The multiplex PCR reaction of HpyCH4Ⅳ in a 25 µL total volume contained 10 µL of PCR product, 0.5 µL of HpyCH4Ⅳ, 2.5 µL of 10× NEBuffer, and 12 µL of ddH₂O. The reaction was incubated for 15 min at 37 °C. The genotypes were determined by electrophoresis on a 3% agarose gel (one band: 143 bp for wild type (qq) genotype; two bands: 91 and 46 bp for the mutant (QQ) genotype; and three bands: 143, 91, and 46 bp for heterozygous (Qq) genotype) (Fig. 2). The remaining five sites were genotyped by Tianhao Biotechnology Company (Shanghai, China) through SNaPshot, including PHKG1 (rs330928088), MUC13 (rs319699771), LIPE (rs328830166), LEPR (rs45435518), and MC4R (rs81219178).

Fig. 1.

The electrophoretogram of the PCR amplification of VRTN gene.

Fig. 2.

The electrophoretogram of the PCR amplification of MYH3 gene.

Results

Phenotypic measurement results of Lulai black pig and descriptive statistics

The results of measurements for traits of 418 Lulai black pigs are shown in Table 2. The means of ABT and MMP of the Lulai black pig population were 38.50 ± 5.66 mm and 71.15 ± 2.83%, respectively, and the means of overall IMF content were 5.02 ± 3.10%. MMP had the smallest coefficient of variation (CV; 3.98%), and IMF was the most remarkable (61.94%) in the population. That suggested that interindividual variation in IMF content was wide.

Table 2.

Statistical description of carcass and meat quality traits in Lulai black pig population.

Phenotype correlation analysis of Lulai black pigs

The degree of phenotypic correlation amongst traits of Lulai black pigs is shown in Figs. 3 and 4. Q–Q plots are approximately linear. And tests for normality indicated that three traits followed an approximately normal distribution. The correlation was strongest for IMF and MMP, showing a negative correlation, with a correlation coefficient of −0.87. However, there is a weak correlation between IMF and ABT, with a correlation coefficient of 0.22. As the ABT and MMP changed, the results revealed that a corresponding increase/decrease was seen in IMF content.

Fig. 3.

Examining plots of normal distribution. (A) Normal Q–Q plot of ABT; (B) normal Q–Q plot of MMP; and (C) normal Q–Q plot of IMF.

Fig. 4.

Phenotypic correlation coefficients between carcass and meat quality traits in Lulai black pig population. | r | ≤ 0.3, weak correlation; 0.3 < | r | ≤ 0.4, moderate correlation; and | r | > 0.4, strong correlation. ^**p < 0.05; ^***p < 0.01. [Colour online.]

Genetic analysis of effective/causal mutations of Lulai black pigs

The allele frequency, genotype frequency, and genetic diversity parameters (HO, HE, and PIC) of the effective/causal sites in the PHKG1, VRTN, MYH3, LIPE, and other genes are summarized in Table 3. The frequencies of favourable alleles were higher in mutation sites of causal genes PHKG1, VRTN, and MYH3, means 0.927 (C), 0.854 (q), and 0.523 (Q), respectively. And the population lacks individuals with homozygous genotype from VRTN unfavourable alleles. The frequencies of favourable alleles were lower in MUC13 and IGF2, means 0.394 (G) and 0.075 (A), respectively. The favourable homozygous genotype of the IGF2 gene was only 5 cases in 418 Lulai black pigs. For other candidate genes, the frequencies of favourable alleles were higher in LIPE, mean 0.737 (A); the frequencies of favourable alleles were lower in LEPR and MC4R, means 0.275 (T) and 0.122 (A), respectively. The favourable homozygous genotype of the MC4R gene was only one case.

Table 3.

Genetic parameters of eight causal or effective mutations in 12 genes of Lulai black pigs.

In the Lulai black pig population, the homozygosity of all effective/causal mutation sites was higher than the heterozygosity. The effective/causal mutations of MUC13, MYH3, LIPE, and LEPR genes were moderately polymorphic, with 0.364, 0.375, 0.313, and 0.319 PIC values. The rest of the sites belonged to a low polymorphic locus (0 < PIC < 0.25). The values of HE and PIC of the PHKG1, IGF2, and MC4R were low due to the small number of homozygous mutants.

Association analysis between Lulai black pigs’ effective mutation sites and ABT, MMP, and IMF

In the population, we performed association analysis between effective mutation sites and ABT, MMP, and IMF. Association analysis results of effective mutation sites LIPE (rs328830166), LEPR (rs45435518), and MC4R (rs81219178) with ABT, MMP, and IMF are shown in Table 4. The results revealed that the effective mutation site of the LEPR gene was significantly associated with MMP and IMF (p < 0.05) with sex and baseline weight as fixed effects, and IMF of the CC genotype (5.32% ± 0.22%) was significantly higher than that of the TT genotype (4.29% ± 0.42%). However, the effective mutation sites of the LIPE and MC4R genes had no significant association with ABT, MMP, and IMF.

Table 4.

Association analysis of eight effective/causal mutations in eight genes with ABT, MMP, and IMF of Lulai black pigs.

In addition, amongst the five causal genes, Huang et al. (2021) discovered that the XM_013981330.2:g.-1805_-1810del mutation in MYH3 was not a causal mutation. Therefore, in this study, the association analyses of genotypes with meat quality traits were conducted. It was found that the mutant genotype reported in MYH3 was not significantly associated with IMF.

Discussion

Improving meat quality has become the aim of pig farming and the primary goal of pig genetic breeding programmes (Huang et al. 2021). In this study, we analysed the phenotypic data of IMF, MMP, and ABT of Lulai black pigs. The results revealed that the value of the CV% of MMP was deficient and stable in the group, and that of IMF was considerably high, which may be due to a large separation of IMF phenotypes in the Laiwu pig population (Chen et al. 2017; Wang et al. 2020). The ancestor of Lulai black pigs may not have been intensively selected for the IMF during the breeding process.

In addition, to screen the molecular markers that were valuable for meat quality trait improvement, this test analysed the genotype distribution and genetic variation of eight effective/causal mutation sites, including PHKG1 (rs330928088), MUC13 (rs319699771), and MYH3 (XM_013981330.2:g.-1805_-1810del), and others, in the Lulai black pig population. The study further explored the association between genotypes at the effective mutation sites and meat quality traits. In the five causal mutations, the PHKG1 gene mutation is a loss-of-function mutation, resulting in muscle glycogen degradation defects, forming sour meat, reducing pork quality, and bringing significant economic losses for pork production (Ma et al. 2014). The unfavourable mutant allele was fixed in the Duroc and pigs with Duroc kinship (Liu et al. 2019b). The mapping population used in this study was Lulai black pig, a new hybrid of Laiwu pigs and Yorkshire pigs. Thus, the unfavourable allelic frequency of the A allele was considerably low, and the number of “Sour Meat” alleles, AA genotypes, was smaller. Still, 12% of Lulai black pigs with unfavourable alleles (AA and AC) were observed, phasing out during the follow-up breeding. MUC13 disease resistance gene has been extensively used for identifying susceptible and resistant individuals with piglet diarrhoea (Ren et al. 2012b). The results revealed that the frequency of the favourable gene G was lower than A in the Lulai black pig population, and the number of GG genotypes was smaller. Therefore, in the breeding process of Lulai black pigs, individuals with the AA genotypes should be appropriately eliminated. Individuals with the G allele could be selected from generation to generation to establish a core group of the pig individuals with homozygous resistant alleles. The process can effectively reduce the diarrhoea rate of piglets. A causal mutation in IGF2 (g.3072G>A) is paternally expressed. The mutant AA genotype could increase lean meat production by 3–4% (Van Laere et al. 2003), improving remarkable economic benefits. Studies indicate that these gene polymorphisms were distributed in Landrace, Large White breed, and Erhualian pig. The A allele was the superior allele in the Landrace and Large White breed (Yang et al. 2006). The study revealed that the frequency of the favourable allele A was only 0.075 in the Lulai black pig population, responsible for the characteristics of low lean meat. A method in which paternal AA type and maternal GG type can be cultivated in breeding exists, increasing the frequency of the A alleles and improving the shortboard with a low lean meat rate of cultivated varieties with high consanguinity. In addition, our results revealed extremely high vertebra number alleles VRTN (g.20311_20312ins291) in this population. No homozygous genotyped individuals with unfavourable alleles of the VRTN gene were observed. Vertebra numbers are an essential economic trait in a pig that can influence carcass length meat production, and its heritability was remarkably high (Borchers et al. 2004). Chinese and Western purebred pigs, such as Laiwu pigs and Landrace, harbour beneficial alleles, and the frequency is higher in Western pigs. Several studies indicate that mutation leading to changes in vertebra numbers originated from Chinese native pigs (Fan et al. 2013; Yang et al. 2016). Results of this trial provided a basis for the favourable mutant allele derived from Chinese native pigs.

Studies have indicated that the causal mutation affects IMF in pigs, a 6 bp deletion variant in the porcine MYH3 promoter region. Still, the results of this study revealed that the mutation was not significantly associated with IMF content. Thus, the result could not support that the mutation occurred at a causal mutation site, affecting the IMF content of Lulai black pigs. The result is consistent with previous studies (Huang et al. 2021). The finding of the causal mutation for the QTL (Quantitative Trait Locus) on SSC8 affecting IMF content by Cho et al. (2019) must be further studied.

For other essential three candidate genes, the results of our study revealed that individuals with the CC genotype of the LEPR (rs45435518) gene had higher IMF content, further confirming that the mutation in the LEPR gene (rs328830166) was an effective mutation affecting the IMF content. However, its specific mechanism of action needs further research in Lulai black pig population. Although the MC4R and the LIPE genes were candidate genes associated with meat quality traits and lipid deposition control, the association between MC4R (rs81219178) and LIPE (rs328830166) mutation genotypes and ABT, MMP, and IMF meat quality traits exhibited no significant level in Lulai black pigs. Therefore, the MC4R and LIPE effective mutations could not be used as a molecular marker for improving the ABT, MMP, and IMF meat quality traits of Lulai black pigs.

Conclusions

In this study, the eight effective/causal mutation sites affecting fat deposition, meat quality, growth, and disease resistance traits were detected and analysed in 418 Lulai pigs. The results indicated that the eight effective/causal mutation sites were all genetically polymorphic in Lulai black pig population. The causal mutation of MYH3 (XM_013981330.2:g.-1805_-1810del) was not significantly associated with IMF content (p > 0.05). In contrast, amongst the three effective mutation sites, the effective mutation of the LEPR gene was significantly associated with IMF content in Lulai black pigs (p < 0.05). With overall evaluation, the causal mutation sites of PHKG1 (rs330928088), MUC13 (rs319699771), IGF2 (g.3072G>A), and VRTN (g.20311_20312ins291), and the effective mutation site of LEPR (rs45435518) have critical breeding values to Lulai black pigs.