Carcass weight is one of economically important traits for beef cattle  . The detection of genes affecting carcass weight and the establishment of an effective marker-assisted selection technique based on the genomic information are an important goal for the genetic improvement of beef production.
Setoguchi et al.  identified the c.1326T>G single nucleotide polymorphism (SNP) in the non-SMC condensin I complex, subunit G (NCAPG) gene, which changes the amino acid Ile442 to Met442 in the encoded protein, as a candidate causative variation for a bovine carcass weight quantitative trait locus (QTL) on chromosome 6. In addition, Karim et al.  identified the stature quantitative trait nucleotide (QTN) in the pleiomorphic adenoma gene 1 (PLAG1)-coiled- coil-helix-coiled-coil-helix domain containing 7 (CHCHD7) intergenic region, as the causative variation for another carcass weight QTL on chromosome 14. Recently, Hoshiba et al.  compared the effects on growth-related traits of the c.1326T>G SNP and the FJX_250879 SNP that was approximately 50-kb centromeric from the stature QTN and nearly linkage disequilibrium with the QTN on chromosome 14, using Japanese Black steers. As a result, they detected a genetic interaction between the c.1326T>G and FJX_250879 SNPs for body and rump lengths.
Because the two SNPs have relatively large effects on carcass weight, it is important to examine whether a genetic interaction for carcass weight is present between them. Thus, I performed multiple marker analysis for association with carcass weight, using the model including the main effects of the two SNPs and their interaction in Japanese Black beef cattle.
2. Materials and Methods
2.1. Samples and Data
Two hundred eighteen paternal half-sib Japanese Black steers were produced from 53 sires (1 - 16 steers per sire) in Niigata prefecture. Hair root specimens of the progeny steers were provided from Niigata Agricultural Research Institute Livestock Research Center, and were used for genotyping the SNPs. DNA samples were prepared from the materials using DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The carcass weight was measured according to the Japanese meat grading system by certified graders from the Japan Meat Grading Association (Tokyo, Japan)  . The records of carcass weight for the progeny steers were obtained from the Niigata Prefectural Headquarters, National Federation of Agricultural Cooperative Association (Niigata, Japan).
2.2. SNP Genotyping
The c.1326T>G SNP was genotyped by the PCR-restriction fragment length polymorphism method as described previously  . The FJX_250879 SNP was genotyped by direct sequencing of the PCR products according to Nishimura et al.  . Primers used in this study were shown in Table 1. In the Japanese Black population, the C-allele of FJX_250879 increases carcass weight and corresponds to the Q (superior) allele of the stature QTN  .
2.3. Association Analysis
I performed association analysis with multiple marker model of the two SNPs, c.1326T>G and FJX_250879, in which carcass weight records were used as the
Table 1. PCR primers used in this study.
dependent variable. The model included the two SNPs and their gene to gene (inter-allelic) interaction effect between the SNPs, slaughter years and months, and fattening farms as the fixed effects, and the sires as the random effect. The slaughter age of the animals was included as a covariate in linear regression. The analysis was performed by the MIXED procedure of the SAS program (SAS Institute, Inc., Cary, NC).
3. Results and Discussion
Using the multiple marker association analysis, I analyzed the main effects of the two SNPs, c.1326T>G and FJX_250879, and their gene to gene interaction effect for carcass weight. The main effects of c.1326T>G and FJX_250879 were statistically significant (P = 0.0272 for c.1326T>G and P = 0.0235 for FJX_250879) (Table 2). The least-squares mean of the GG homozygote of c.1326T>G was significantly higher than the mean of the TT homozygote, and the mean of the heterozygote was intermediate. The mean of the CC homozygote of FJX_250879 was significantly higher than that of the GG, and the mean of the heterozygote was intermediate. These results correspond with the previous studies   where it was concluded that the favorable alleles for carcass weight were G at c.1326T>G and C at FJX_250879. The interaction between c.1326T>G and FJX_250879 was statistically significant for carcass weight (P = 0.0147) (Table 2). The combination of TT at c.1326T>G and GG at FJX_250879 gave a marked decrease.
In the multiple marker analysis of this study, c.1326T>G and FJX_250879 had significant main effects on carcass weight with the same favorable tendency of alleles as previous studies   . Further, I detected a significant epistatic interaction between c.1326T>G and FJX_250879 for carcass weight. These findings suggest that c.1326T>G and FJX_250879, respectively, affect carcass weight both directly and through its interaction with FJX_250879 and c.1326T>G.
The maximum difference between the largest and smallest combined genotypic effects of the two SNPs was expected as 101.96 in carcass weight scale (= 33.10 − [−68.86]; see below) based on Table 2. The largest effect came from the combination of GG at c.1326T>G (5.40) and CC at FJX_250879 (10.72), and the interaction between GG at c.1326T>G and CC at FJX_250879 (16.98) (i.e. 5.40 + 10.72 + 16.98 = 33.10). The smallest effect came from the respective combination of TT (−7.45) and GG (−14.78), and the interaction between TT at c.1326T>G and GG at FJX_250879 (−46.63) (i.e. [−7.45] + [−14.78] + [−46.63] = [−68.86]).
Table 2. Main and interaction effects of the two single nucleotide polymorphisms (SNPs) for carcass weight in the multiple marker analysis using 218 animals.
1Number of genotyped animals. 2Least-squares mean (LSM) ± SE of carcass weight. LSMs are shown as the deviation from the general mean of carcass weight. a,b,cStatistically significant among means with different superscripts (P < 0.05). p value was shown in the parentheses.
An epistatic interaction is measured as the departure of the combined effects of two or more genes from the sum of their individual effects  . This phenomenon is thought to play a significant role in evolution   . Matsuhashi et al.  recently clarified multiple marker effects of four gene polymorphisms on the fatty acid composition and several carcass traits in Japanese Black cattle, but detected no epistatic interactions. However, I detected a possible epistatic interaction for carcass weight between NCAPG (c.1326T>G) and PLAG1 (FJX_250879), the strongest candidate gene for the carcass weight QTL on chromosome 14. Although the functional relevance of such an interaction is still unknown, NCAPG is involved in arginine metabolism  and PLAG1 in regulation of the expression of growth factors, including insulin-like growth factor 2  . An interaction between them could play an important role in the variation of carcass weight in beef cattle. For confirmation of the presence of the interaction, other replication studies are recommended using other Japanese Black populations. In addition, a further investigation on detection of an epistatic interaction among three or more genes affecting the variation of carcass weight would be required for effective marker-assisted selection.
In conclusion, I have demonstrated significant associations and interaction of two SNPs, referred to as c.1326T>G and FJX_250879, for carcass weight in Japanese Black population. For the effective genetic improvement of beef production in Wagyu population, the role of interactions among SNP markers under marker-assisted selection should be considered.
I would like to give my sincere thanks to Prof. Takahisa Yamada of Niigata University for his valuable guidance of this work.
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