Potential of eight mutations for marker-assisted breeding in Chinese Lulai black pigs

Abstract Molecular marker-assisted selection (MAS) provides an efficient tool for pig breeding. In this study, according to the literature, we selected eight effective or causal mutations from eight functional genes, including five causal mutations in PHKG1 (rs330928088), MUC13 (rs319699771), IGF2 (g.3072 G>A), VRTN (g.20311_20312ins291), and MYH3 (XM_013981330.2:g.-1805_-1810del) genes, and three effective mutations in LIPE (rs328830166), LEPR (rs45435518), and MC4R (rs81219178) genes, to investigate their potential breeding effect in 418 Lulai pigs. The linear model was used to analyse the association between mutations and intramuscular fat (IMF) content, average backfat thickness, and muscle moisture %. The results revealed that amongst the three effective mutations, only the mutation in the LEPR gene, which affects IMF deposition, was significantly associated with IMF content. However, the other molecular markers were not significantly associated with the affected traits reported in previous studies, and these mutations are ineffective for MAS in the Lulai black pig population. Therefore, causal mutations in PHKG1, IGF2, and VRTN genes, and an effective mutation in LEPR gene could be used as effective breeding makers for MAS in Lulai pigs. These results can provide helpful information for further breeding in Lulai black pigs. Résumé La sélection assistée par marqueur moléculaire (MAS — « molecular marker-assisted selection ») offre un outil efficace pour la reproduction des porcs. Dans cette étude, selon la littérature, nous avons choisi huit mutations efficaces et causales provenant de huit gènes fonctionnels, incluant cinq mutations causales dans les gènes PHKG1 (rs330928088), MUC13 (rs319699771), IGF2 (g.3072G>A), VRTN (g.20311_20312ins291) et MYH3 (XM_013981330.2:g.-1805_-1810del), et trois mutations efficaces dans les gènes LIPE (rs328830166), LEPR (rs45435518) et MC4R (rs81219178), afin d’étudier les effets potentiels de celles-ci sur la reproduction de 418 porcs Lulai. Le modèle linéaire a été utilisé pour analyser l’association entre les mutations et la teneur en gras intramusculaire (IMF — « intramuscular fat »), l’épaisseur moyenne du gras dorsal (ABT — « average backfat thickness »), et le pourcentage d’humidité dans le muscle (MMP — « muscle moisture percent »). Les résultats ont révélé que parmi les trois mutations efficaces, seule la mutation dans le gène LEPR, qui a un effet sur le dépôt d’IMF, était associée de façon significative à la teneur en IMF. Par contre, les autres marqueurs moléculaires n’étaient pas associés de façon significative avec les caractéristiques affectées rapportées dans les études préalables, et ces mutations sont non efficaces pour la MAS dans la population de porcs noirs Lulai. Donc, les mutations causales dans les gènes PHKG1, IGF2 et VRTN, et une mutation efficace dans le gène LEPR pourraient être utilisées comme marqueurs efficaces de reproduction pour la MAS chez les porcs Lulai. Ces résultats pourraient offrir de l’information utile pour la reproduction ultérieure chez les porcs noirs Lulai. [Traduit par la Rédaction]


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, markerassisted selection (MAS) has become a rapid and simple method for molecular breeding (Chen et al. 2019;. 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) , 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.

Ethics statement
All the methods during slaughter were carried out following the national standards (GB/T 17236-2019) from the State Administration for Market Regulation of the People's Republic of China. The Ethical Committee approved all procedures of animal handling of Qingdao Agricultural University.

Animals
In this study, 418 Lulai black pigs, including 267 males and 151 females, were used. All pigs were fed in the breeding pig farm of Jinan Laiwu Pig Breeding Co., Ltd. Ear tissues of each pig were collected in tubes containing 75% ethanol and stored at −20 • C for DNA extraction.

Phenotypic determination and genomic DNA extraction
Lulai black pigs were slaughtered when their body weight (BW) reached approximately 90 kg. Pigs were fasted for 24 h before slaughter, then BW was measured. After slaughtering, the BF thicknesses of the dorsal line shoulder, the BF thicknesses of the thoracolumbar junction, and the BF thicknesses of the last lumbar vertebrae were measured using Vernier callipers. The average of three values was regarded as the result. The longissimus dorsi (LD) muscle tissues at the last rib were collected to measure IMF content and muscle moisture % (MMP) of LD. Before the measurements, the peripheral fascia was separated. Then, the LD was minced using a meat grinder and oven-dried at 65 • C to a constant weight in a dryer. Finally, the moisture content was calculated by dividing the weight difference before and after mass by fresh weight. The IMF content was analysed using the Soxhlet extraction method (Wang et al. 2019).
Collected ear tissues were used to isolate genomic DNA using TIANamp Genomic DNA Kit from TIAGEN Biotech (Beijing, China) following the manufacturer's instructions. DNA quality and concentrations were checked using SpectraMax QuickDrop Micro-Volume Spectrophotometer (Molecular Device, USA). The DNA samples with an A260/A280 ratio between 1.8 and 2.0 and an A260/A230 ratio greater than 2.0 were qualified DNA. The DNA concentration in multiple samples was diluted to a range of 20−50 ng/μL by ddH 2 O and the samples were used for a subsequent test.
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 ddH 2 O, 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.

Statistical analyses
The total number of animals analysed here was 418. Allelic and genotypic frequencies were calculated by genetic equilibrium law. Polymorphic information content (PIC), observed heterozygosity (HO), and expected heterozygosity (HE) were computed by using where P i and P j are the frequencies of the i-th and j-th alleles, respectively, and k is the number of alleles. A PIC value from 0 to 0.25 indicates low polymorphism, a PIC value from 0.25 to 0.5 indicates intermediate polymorphism, and a PIC value from 0.5 to 1 indicates high polymorphism.
Normality distribution test was performed on the data using SPSS software (Version 26). Correlation function within the R package "Performance Analytics" was used to calculate the correlation between IMF, MMP, and average backfat thickness (ABT) and to generate the correlation scatter plot. And the sex and weight were adjusted when calculating the phenotypic correlation. Association analyses were done using the linear model in PLINK software (Purcell et al. 2007). Sex and weight were included as fixed effects. The full model is y = αSNP + xb + e. y denotes the phenotypic values. α is the marker effects for each SNP. x is design matrices for the fixed effects, and b is the sex and weight fixed-effect vectors. e is the residual error. Excel was used to calculate the FDR (False Positive Rate) value of the statistical results, so as to perform multiple tests on the p value. In general, FDR = Q value = adjusted p value. The calculation formula is Q value = p × (m/k), where m is the number of tests and k is the ranking of p value of this test amongst all tests. The individuals with significant differences were analysed by ANOVA (Analysis of Variance) using the SPSS (Version 26).

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.

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 nega-tive 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.

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.
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. 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 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.]  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.

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