Advanced
Association between Genotypes of the Isocitrate Dehydrogenase 3, beta subunit (IDH3B) Gene and Carcass Traits in an F<sub>2</sub> Crossbred Population of Landrace × Jeju (Korea) Black Pigs
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
Journal of Life Science. 2016. Apr, 26(4): 414-418
Copyright © 2016, Korean Society of Life Science
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : February 12, 2016
  • Accepted : March 12, 2016
  • Published : April 30, 2016
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
About the Authors
용준, 강
동기, 정
인철, 조
choic4753@korea.kr
상현, 한
choic4753@korea.kr

Abstract
This study tested the association between genetic polymorphisms of the isocitrate dehydrogenase 3, beta subunit ( IDH3B ) gene and economic traits in an F 2 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 F 2 pigs. Three genotypes ( AA, AB , and BB ) were identified in the founder, F 1 , and F 2 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 F 2 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.
Keywords
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 F 2 population between Landrace and JBP.
Materials and Methods
- Animals and genomic DNA preparation
The F 2 population was produced via reciprocal inter-crosses between Landrace and the Jeju Black pig [3] . Muscle and blood samples were prepared from the F 2 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 F 2 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] .
Primers for amplificationIDH3Bpromoter region
PPT Slide
Lager Image
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 (4 th -5 th ribs, BF5; 11 th -12 th ribs, BF12; 13 th rib-1 st 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, F 1 and F 2 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.
Genotype distribution of 304-bp insertion/deletion polymorphisms in the promoter region ofIDH3Bgene in founder, F1and F2populations
PPT Slide
Lager Image
* , Ho, He, and PIC indicate the values of observed heterozygosity, expected heterozygosity, and polymorphic information content, respectively.
- Association between the genotypes ofIDH3Bpromoter 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 F 2 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).
Mean and SE of traits in pig F2population
PPT Slide
Lager Image
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 F 2 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 F 2 animals possessing IDH3B A/A and A/B genotypes showed longer CL than those of B/B genotypes ( p <0.05). In addition, the F 2 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 ofIDH3Bgene 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 F 2 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.
Acknowledgements
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ010123042016)” Rural Development Administration, Republic of Korea.
References
Cho I. C. , Kim S. G. , Kim Y. K. , Kang Y. J. , Yang S. N. , Park Y. S. , Cho W. M. , Cho S. R. , Kim N. Y. , Chae H. S. , Seong P. N. , Park B. Y. , Lee J. H. , Lee J. B. , Yoo C. K. , Han S. H. , Ko M. S. 2011 Association between Numerical Variations of Vertebrae and Carcass Traits in Jeju Native Black Pigs, Landrace Pigs, and Crossbred F2 Population J. Life Sci. 23 854 - 862
Cho I. C. , Park H. B. , Yoo C. K. , Lee G. J. , Lim H. T. , Lee J. B. , Jung E. J. , Ko M. S. , Lee J. H. , Jeon J. T. 2011 QTL analysis of white blood cell, platelet and red blood cell-related traits in an F2 intercross between Landrace and Korean native pigs Anim. Genet. 42 621 - 626    DOI : 10.1111/j.1365-2052.2011.02204.x
Cho I. C. , Yoo C. K. , Lee J. B. , Jung E. J. , Han S. H. , Lee S. S. , Ko M. S. , Lim H. T. , Park H. B. 2015 Genome-wide QTL analysis of meat quality-related traits in a large F2 intercross between Landrace and Korean native pigs Genet. Sel. Evol. 47 7 -    DOI : 10.1186/s12711-014-0080-6
Cohen P. F. , Colman R. F. 1972 Diphosphopyridine nucleotide dependent isocitrate dehydrogenase from pig heart. Charactgerization of the active substrate and modes of regulation Biochemistry 11 1501 - 1508    DOI : 10.1021/bi00758a027
Hathaway J. A. , Atkinson D. E. 1963 The effect of adenylic acid on yeast nicotinamide adenine dinucleotide isocitrate dehydrogenase, a possible metabolic control mechanism J. Biol. Chem. 238 2875 - 2881
Hartong D. T. , Dange M. , McGee T. L. , Berson E. L. , Dryja T. P. , Colman R. F. 2008 Insights from retinitis pigmentosa into the roles of isocitrate dehydrogenases in the Krebs cycle Nature Genet. 40 1230 - 1234    DOI : 10.1038/ng.223
Huh T. L. , Kim Y. O. , Oh I. U. , Song B. J. , Inazawa J. 1996 Assignment of the human mitochondrial NAD+-specific isocitrate dehydrogenase alpha subunit (IDH3A) gene to 15q25.1 → q25.2by in situ hybridization Genomics 32 295 - 296    DOI : 10.1006/geno.1996.0120
Kalinowski S. T. , Taper M. L. , Marshall T. C. 2007 Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment Mol. Ecol. 16 1099 - 1106    DOI : 10.1111/j.1365-294X.2007.03089.x
MacDonald M. J. , Brown L. J. , Longacre M. J. , Stoker S. W. , Kendrick M. A. 2013 Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion Biochim. Biophys. Acta 1830 5104 - 5111    DOI : 10.1016/j.bbagen.2013.07.013
Park J. C. , Kim Y. H. , Jung H. J. , Park B. Y. , Lee J. I. , Moon H. K. 2005 Comparison of meat quality and physicochemical characteristics of pork between Korean native black pigs (KNBP) and Landrace by market weight J. Anim. Sci. Technol. 47 91 - 98    DOI : 10.5187/JAST.2005.47.1.091
Ren Z. , Liu W. , Zheng R. , Zuo B. , Xu D. , Lei M. , Li F. , Li J. , Ni D. , Xiong Y. 2012 A 304 bp insertion/deletion mutation in promoter region induces the increase of porcine IDH3β gene expression Mol. Biol. Rep. 39 1419 - 1426    DOI : 10.1007/s11033-011-0876-1
1999 SAS program package. SAS/STAT software for PC. Release 8.0.1 SAS Institute Inc Cary, NC, USA
Sambrook J. , Fritsch E. F. , Manniatis T. 1989 Molecular cloning: a laboratory manual 2nd ed. Cold Spring Harbor Laboratory Press New York Isolation of high-molecular-weight DNA from mammalian cells 9.14 - 9.23
Zeng A. P. , Deckwer W. D. 1994 Pathway analysis of oxygen utilization and tricarboxylic acid cycle activity in Saccharomyces cerevisiae growing on glucose J. Biotechnol. 37 67 - 77    DOI : 10.1016/0168-1656(94)90204-6