This experiment was reviewed and approved by the Institutional Animal Care and Use Committee of Rajamangala University of Technology Isan (No.48/2564).

A random population from a total of 91 blood samples of Kradon pigs (39 castrated males and 52 females) were collected from the swine farm unit of the Department of Animal Science, Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon Campus. All pigs were raised under an open-house system in cement pens of 2×4 m and were handled with identical procedures. The pigs received clean water from a nipple drinker and were fed with a diet of not less than 20% crude protein (CP). Their birth and weaning weights were recorded. Blood samples were collected from the jugular vein by needles No.18G×1 in for a volume of 5 to 10 mL of collected blood and added to a 15 mL tube containing 0.5 M ethylenediaminetetraacetic acid. These samples were centrifuged at 4,200 rpm for 5 min to precipitate the white blood cells and then stored at −4°C until they were used for DNA extraction.

The buffy coat or white blood cells was prepared from 50 μL of whole blood added to 1.5 mL microtubes after which 1,000 μL 0.9% NaCl was added to the tubes, and then mixed and vortexed for 5 to 10 s. After centrifugation at 10,000 rpm for 5 min, the supernatant was discarded. Genomic DNA was extracted by a guanidine hydrochloride protocol modified procedure developed by Goodwin et al [14]. Fifty microliters of white blood cells were divided into 1.5 mL microtubes, and then 70 μL 20% SDS, 50 μL 7.5 M Na-acetate, 25 μL 1% Proteinase K, and 625 μL 5 M Guanidine HCl were added to the tubes and mixed by flipping the microtubes up and down 2 to 3 times and vortexed for 5 to 10 s. They were then incubated overnight at 65°C in a waterbath. These microtubes were centrifuged at 10,000 rpm for 5 min after which 500 μL of supernatant was aspirated into the new tube. Five-hundred microliters of cold absolute isopropanol was then added to this mixture, The microtubes were flipped up and down, and DNA precipitation was noted. The microtubes were then centrifugated at 10,000 rpm for 5 min after which the supernatant was discarded while the DNA precipitate was isolated. The DNA was washed by adding 500 μL of 75% ethanol and then centrifuged at 10,000 rpm for 1 min after which the supernatant was discarded twice, while the DNA was isolated. The DNA was dried at room temperature for 30 to 60 min after which 25 μL of TE buffer was added, and the DNA was incubated in a waterbath at 37°C overnight to dissolve the DNA. One microliter of the DNA sample was used to determine the DNA quality using a NanoDrop2000 spectrophotometer (Nano Drop Thermo Scientific, Wilmington, DE, USA) at 260 and 280 nm wavelengths. The DNA concentration was adjusted to 50 ng/μL and stored at −20°C for use in subsequent amplification.

The primers for the IGF2 and MC4R genes were designed from published sequences. The IGF2 primer sequence has been reported by Vykoukalová et al [15] and the MC4R primer sequence was reported by Dvorakova et al [16]. The IGF2 and MC4R fragments were amplified using polymerase chain reaction (PCR), 10 μL contained 4.1 μL distill water, 1 μL dNTP (1 mM; Fermentas, Hanover, MD, USA), 0.8 μL MgCl₂ (25 mM), 1 μL 10× PCR buffer, 1 μL of 3 μM each primer (forward and reverse primer), 0.1 μL 5 U Taq DNA polymerase (MBI Fermentas, USA), and 1 μL 50 ng genomics DNA. Primers and are listed in Table 1. A T-Professional Standard Thermo cycler (Biometra GmbH, Gottingen, Germany) was used for amplification under specific conditions: i) 95°C for 3 min, ii) 30 cycles at 95°C for 30 s, iii) annealing for 30 s and 72°C for 45 s, and iv) a final extension at 72°C for 5 min. The products were evaluated using a 2% agarose gel electrophoresis.

The PCR products of IGF2 and MC4R genes were digested in a total volume of 10 μL, containing 6.7 μL of distilled water, 2 μL PCR products, 1 μL cut smart buffer, and 0.3 μL each of restriction enzymes, and the reactions were digested overnight at 37°C for BcnI, and 65°C for TaqI (New England Biolabs, Ipswich, MA, USA). The digested PCR products of IGF2 and MC4R were separated on 2% agarose gel electrophoresis at 100 V for 30 min, and RFLP fragments were visualized using Gel Star (Lonza, Rockland, ME, USA). The IGF2 gene pattern were three genotypes pattern with GG, GC, and CC. GG genotype DNA banding sizes were 308 and 28 bp, GC genotypes were 308, 208, 100, and 28 bp, and CC genotype pattern were 208, 100, and 28 bp. Additionally, MC4R genotyping with three patterns demonstrated the presence of GG, AG, and AA. The GG pattern was represented with 156 and 70 bp, GA genotype was 226, 156 and 70 bp, and AA genotype was 226 bp.

The genotype patterns of IGF2 and MC4R genes were used for calculating the gene and genotype frequencies, observed heterozygosity (H_O), expected heterozygosity (H_E), and unbiased expected heterozygosity (H_U) following Nei’s method [17]. The polymorphic information content (PIC) was assessed according to the method by Botstein et al [18]. The Hardy–Weinberg Equilibrium of the IGF2 and M4CR genes was tested using the GENEPOP software (version 4.2) available online: http://genepop.curtin.edu.au/. The investigation of the association between the polymorphism of genotype pattern and both birth and weaning weight traits was carried out with the general linear model procedure using the SAS procedure (Statistical Analysis System 9.0; SAS Institute, Cary, NC, USA) by the following statistical linear model for birth weight trait according to Y_ijk = μ+Sex_i+D_j+G_k+ɛ_ijk, and weaning weight trait according to Y_ijk = μ+Sex_i+D_j+W_k+G_l+ɛ_ijk in which Y_ijk represents the phenotypic values; μ is the overall mean; Sex_i represents the sex effect; D_j is the maternal effect; W_k is the covariate of birth weight effect; G_k and G_l are genotype effects; and ɛ_ijk represents the random error.

Genotype patterns of the genes were studied from the amplified gene fragments that were digested with the restriction enzymes, and it was found that the size of the IGF2 and MC4R gene fragments matched the size of the reference reports. IGF2 gene variations in Kradon pigs found with the three genotypes were GG, GC, and CC (Figure 1A). The polymorphism of MC4R was found in only the GG genotype with fragment sizes of 156 and 70 bp (Figure 1B).

Our study of polymorphism of the IGF2 gene analyzed the allele and genotype frequencies, and it was found that the GG genotype frequency was the highest (0.516) followed by the GC genotype frequency (0.374), and that the CC genotype frequency was the lowest (0.110) as shown in Table 2 with an allele frequency G and C of 0.703 and 0.297, respectively.

The HWE analysis is reported in Table 2. The results showed the IGF2 genotype followed the null hypothesis that stated that Kradon pigs might not be affected by gene forces because this initial population was maintained in a relatively closed area without a systematic breeding program. Thus, migration and selection factors may not significantly affect the HWE of the IGF2 gene. In contrast, MC4R gene could not undergo HWE analysis because it is a non-diverse allele with only GG genotype.

An evaluation of the MC4R gene was not feasible because only one genotype pattern was found. Therefore, the association between the genotype pattern and both their birth and weaning weight traits could not be analyzed. However, the association of IGF2 with birth and weaning weight traits was analyzed. The results of the study found that the genotype pattern was not associated with birth weight traits (p>0.05). Regardless of the genotype of the Thai native pigs, no influence on the birth weight of piglets was found. However, a correlation between the IGF2 gene pattern and weaning weight traits was found (Table 3). Pigs with the CC genotype had the highest weaning weight, and pigs with the GG genotype had the lowest (p<0.001).