제주흑돼지와 랜드레이스 교배 2세대의 도체형질과 IDH3B 유전자형의 상관관계

Association between Genotypes of the Isocitrate Dehydrogenase 3, beta subunit (IDH3B) Gene and Carcass Traits in an F₂ Crossbred Population of Landrace × Jeju (Korea) Black Pigs

Abstract

본 연구에서는 제주흑돼지와 랜드레이스 품종 사이에서 생산된 F₂ 교배 집단의 경제형질과 isocitrate dehydrogenase 3, beta subunit (IDH3B) 유전자의 유전적 다형성의 상관관계를 시험하였다. IDH3B 유전자의 프로모터 지역에서 304-bp 삽입/결실 돌연변이를 기준으로 전체 1,105 F₂들에 유전자형 분석에 이용하였다. 기초축군과 F₁, F₂에서 세 가지 유전자형(AA, AB, BB)이 모두 발견되었다. 통계적 상관 분석결과에서 도체중(CW), 세 지점(4-5번 흉추 사이, 11-12번 흉추 사이, 13번 흉추-1번 요추 사이)에서 측정한 등지방두께와 도체장(CL)의 수준은 유전자형애 따른 유의적인 차이를 나타내었지만(p<0.05), 육색(MC), 등심단면적(EMA)과 근내지방도(MARB)은 유의적인 차이를 나타내지 않았다(p>0.05). IDH3B 동형접합자 BB를 보유한 F₂ 돼지들은 도체중이 더 무겁고(80.790±0.725 kg), 도체장은 더 짧은(101.875±0.336 cm) 양상을 보였다(p<0.05). 또한, IDH3B 유전자형 BB인 개체들은 IDH3B AA나 AB 유전자형에 비해 4-5번 흉추 사이, 11-12번 흉추 사이에서 측정된 등지방두께도 더 두꺼운 수준을 나타내었다(p<0.05). 이상의 결과들은 IDH3B의 유전적 다양성이 제주흑돼지와 랜드레이스와 관련된 교배육종 체계의 산육능력 향상을 위한 유전적 분자 표지인자로 활용될 수 있음을 나타내었다.

This study tested the association between genetic polymorphisms of the isocitrate dehydrogenase 3, beta subunit (IDH3B) gene and economic traits in an F₂ crossbred population of Landrace × Jeju (South Korea) Black pigs. A 304-bp insertion/deletion mutation in promoter region was screened for determining genotypes of the IDH3B gene in a total of 1,105 F₂ pigs. Three genotypes (AA, AB, and BB) were identified in the founder, F₁, and F₂ populations. Association analysis showed significant differences in carcass weights (CW), backfat thicknesses in three positions of the body (4^th-5^th ribs, BF5; 11^th-12^th ribs, BF12; 13^th rib-1^st lumbar, BFL), and carcass lengths (CL) (p<0.05), but not in meat color (MC), eye muscle area (EMA), or marbling scores (MARB) (p>0.05). The F₂ IDH3B BB homozygotes showed heavier CW (80.790±0.725 kg) and shorter CL (101.875±0.336 cm) than the other genotypes (p<0.05). In addition, the BF levels between the 4^th - 5^th and 11^th - 12^th vertebrae were thicker in the carcasses of pigs with the IDH3B BB genotype than with the other genotypes (p<0.05). These results suggested that genetic variations in the IDH3B gene may serve as molecular genetic markers for improving the Landrace × Jeju Black pig crossbreeding systems.

Introduction

The Jeju Black pig (JBP), one of native pig breed in South Korea, has been raised on the Jeju Island. It has unique genetic properties differing from those of the mainland pig populations because it is raised on this island that has been isolated for more than a thousand years. The JBP has uniformly black coat color, and shows low growth rate and high level of backfat thickness comparing to those of Western commercial pig breeds. In the carcass grading system of South Korea, the level of backfat thickness is regarded as one of the major factor for determining the quantitative grades of the pork meat. However, it has excellent meat quality characteristics such as white colored fat, rich meat juice, red meat color, and good marbling [1, 3, 10]. In spite of its commercial weaknesses in meat productivity, JBP and its related crossbred populations are preferred by breeders choosing the JBP as final sire in crossbreeding production system in pig industry due to their higher meat quality and disease tolerance.

IDH3B encodes the beta subunit of nicotinamide adenine dinucleotide (NAD)-specific isocitrate dehydrogenase (IDH) which catalyzes the oxidative decarboxylation of isocitrate into alpha-ketoglutarate in the Krebs cycle in mitochondrion. The Krebs cycle is a central pathway of oxidative phospholylation metabolism, where fatty acid was catabolized into Acetyl-CoA during ATP production by the respiratory complexes [4-6, 14]. Recently, Ren et al. [11] documented that the IDH3B transcript have been found as differential expression in intercross population between Large White and Meishan, and an insertion/deletion mutation in promoter region of IDH3B gene might induce the increase of gene expression which highly related with the levels of backfat thickness according to their genotypes. However, the geno-type effects of IDH3B gene had been not evaluated in JBP and related populations. In order to look for the effect of genetic variations of IDH3B gene on the phenotypic differences especially in backfat thickness, this study examined the association between the genotypes and carcass traits in the crossbred F2 population between Landrace and JBP.

 

Materials and Methods

Animals and genomic DNA preparation

The F2 population was produced via reciprocal inter-crosses between Landrace and the Jeju Black pig [3]. Muscle and blood samples were prepared from the F2 progeny for the genomic DNA isolation. Genomic DNA was isolated from the blood and tissue samples with a slightly modified sucrose-proteinase K method [13] and used as a template for polymerase chain reaction (PCR). The association test used the 1,105 F2 animals. All carcass traits as well as other additional traits were measured, including eye muscle area and marbling scores, in accordance with legal grading standard parameters endorsed by professional meat-quality graders of the Animal Products Grading Service of Korea. The study was conducted in accordance (approval number 2015-0023) with recommendations described in “The Guide for the Care and Use of Laboratory Animals’’ published by the Institutional Animal Care and Use Committee of the Jeju National University, Republic of Korea.

PCR amplification and genotyping

A reference nucleotide sequences (Sscrofa10.2:17:37445726:37451909:-1) were obtained from the Pig Genome Project in ENSEMBL database (http://asia.ensembl.org) and used for PCR primer design. PCR primers for amplification of promoter region of IDH3B gene were given in Table 1. PCR was performed using 25 ul of reaction mixture including 100 ng of DNA, 1.0 nmole of each primer, and 2.5 units of i-Taq DNA polymerase (Intron Biotechnology, South Korea). PCR conditions included initial heating at 95℃ for 5 min, 35 cycles of 45 s for denaturation at 94℃, 30 s for annealing at 60˚C, and 60 s for extension at 72℃, followed by a 5 min extension at 72℃. The PCR products were separated on agarose gels and visualized by UV-illumination. After purification of PCR products, three primary PCR products from each homozygote for IDH3B A/A and B/B were directly sequenced using a MegaBACE 1000 automated sequencer (Amersham-Pharmacia, USA). Each genotype was determined based on the differences in lengths due to the insertion/deletion patterns of 304-bp fragment in promoter region. Alleles A and B were defined as 648-bp and 344-bp PCR amplicons on the gels, respectively, according to those previously reported by Ren et al. [11].

Table 1.The length of PCR products for each allele A and B shows 648-bp and 344-bp, respectively.

Data analysis

We calculated allele and genotype frequencies of the IDH3B gene using the CERVUS 3.0.3 program [8]. Phenotypic traits included carcass weight (CW), carcass body length (CL), backfat thicknesses (4th-5th ribs, BF5; 11th-12th ribs, BF12; 13th rib-1st lumbar, BFL), meat color (MC), eye muscle area (EMA), total meat muscle area (TMA, M. longissimus dorsi + M. iliocostalis) and marbling scores (MARB). Carcass data were collected within 24 hours postmortem. The association between genotypes and phenotypic traits were evaluated using the least squares method of General Linear Model procedure, SAS version 8.0 [12]. We used the following model for analysis of the IDH3B promoter polymorphism effect:

Yij= μ + Genotypei + eij

where Yij signifies observed traits, μ is the population mean, Genotypei represents the IDH3B genotypes (A/A, A/B, and B/B), and eij is the random error. We used Duncan’s multiple range test from the General Linear Model procedure to separate means, and we considered significance at p<0.05.

 

Results and Discussion

Using the DNA sequencing for promoter region of IDH3B gene in founder animals, 304-bp insertion/deletion mutation was also found as like Ren et al. [11]. There were no additional mutations found in the 304-bp fragment spanning promoter sequences in Landrace and the JBP populations. The polymorphism of the IDH3B 304-bp insertion/deletion was genotyped in the founder, F1 and F2 populations (Table 2). In the founder population, IDH3B 304-bp fragment inserted allele, B is remarkably frequent in JBP (0.974) but 304-bp fragment deleted allele A is more frequent in Landrace (0.853) showing similar pattern comparing to those previously reported by Ren et al. [11] which reported a higher frequency in IDH3B allele A in Western pig breeds, but allele B in Asian indigenous pig breeds of China. In addition, as like JBP, the Chinese indigenous pig, Meishan possessing higher frequency of allele B and thicker levels of backfat at buttocks than those of Large White. The Landrace used in this study, is a Western pig breed popular for lean meat, thinner backfat, fast growth high productivity, showed higher frequency of allele A in genotyping results. In this point of view, we concluded that the genotypic distribution of IDH3B, at least in part, coincides with those of phenotypic characteristics of backfat thickness both pig breeds.

Table 2.* , Ho, He, and PIC indicate the values of observed heterozygosity, expected heterozygosity, and polymorphic information content, respectively.

Association between the genotypes of IDH3B promoter and carcass traits

Table 3 shows the results of the association analyses between the insertion/deletion mutation of promoter region of IDH3B and the phenotypic traits recorded from the F2 population. For the IDH3B genotypes, we measured carcass traits and several additional traits including CBL, BF12, BFL, and MMA. Among the carcass traits tested in this study, the five traits, (i.e., CW, CL, BF5, BF12, and BFL) were showed statistical associations according to genotypes of IDH3B polymorphisms (p<0.05). However, all other traits, MC, MARB, EMA, and TMA were not showed the statistical association with the IDH3B genotypes (p>0.05).

Table 3.1, all abbreviations of each trait are given in the Materials and Methods section. 2, LS Mean±SE values in the same row are significantly different at 5% (*), 1% (**) and 0.1% (***) significance thresholds, respectively. n.s. indicates not significant.

The F2 animals carrying the IDH3B B/B homozygotes showed relatively heavier body weights for carcass weights (80.790±0.725 kg) than those of the other genotypes (77.807±0.825 kg and 78.954±0.534 kg) (p<0.05). The F2 animals possessing IDH3B A/A and A/B genotypes showed longer CL than those of B/B genotypes (p<0.05). In addition, the F2 progeny carrying the IDH3B B/B showed significantly larger levels of BF5, BF12, and BFL than those of A/A and A/B (p<0.05).

Putative roles of IDH3B gene related to carcass in pigs

Genetic variations provide important bases to explain and analyze phenotypic variation. The statistical associations of the IDH3B promoter polymorphisms with CW, CL, and BFs have been found in this study, suggested that the IDH3B gene may be involved at least in the fat deposition in subcutaneous fat tissues without relation with intramuscular fat deposition in M. longissimus dorsi, and may affect significantly higher growth rates related to the CW and CBL in an intercross population between Landrace and JBP. The levels of MARB representing the levels of intramuscular fat deposition in M. longissimus dorsi did not show the significant association between the IDH3B genotypes and phenotypic differences, whereas the levels of BFs showed significant association. These results proposed that the genetic differences of IDH3B gene might be involved only in fat deposition and adipocyte differentiation in the subcutaneous tissues but not in M. longissimus dorsi. Ren et al. [11] have also described the significant association between the IDH3B genotypes and the traits of backfat thickness at buttocks. These findings suggested that IDH3B gene may play an important role in fat deposition in tissue-specific manner. Especially, the F2 animals carried IDH3B B/B genotype showed similar patterns of higher levels of BFs and CW, which suggested that the difference of CW might be due to the differences in backfat deposition. On the other hand, the levels of CL among IDH3B genotypes were not coincide with those of BFs, indicating that the traits CL and BFs were probably independently affected by IDH3B genotypes.

IDH3B, a key enzyme in Krebs cycle is required for energy synthesis in mitochondrion in the cells [4, 5, 7]. In human, the loss of this enzyme by the homozygous frame-shift mutation is only reported in the cases of retinitis pigmentosa in the eye [6]. Therefore, MacDonald et al. [9] suggested that it may be possible that the lack of the IDH3 enzymatic activities in mitochondrion might be compensated by the cytoplasmic activities of IDH1 for oxidation of isocitrate and NADH reduction. On the other hand, IDH3B has been proposed as a candidate gene for backfat thickness in pig [11]. There were no direct molecular evidences have been reported to reveal the molecular biological backgrounds related to cause the differences of those economic traits, however we hypothesized that IDH3B would effect on the weights, body lengths and the levels of backfat thickness via regulation of IDH3B protein and efficiency of the Krebs cycle.

Our study provides the hypothesis that the IDH3B genotypes may be involved in phenotypic variations of adipogenesis or fat deposition of the pork meat, and productivity related to body weights and body lengths, at least in part through IDH3B pathways. We found different effects of this genetic marker associated with economically important traits, but the molecular function of IDH3B in the backfat deposition has not been clearly defined until now. Further biochemical and molecular biological experiments will provide more explainable information for the molecular mechanisms of genotype-related functional differences between both IDH3B genotypes.