Identification of marker in processed meat products of different species by using multiplex PCR

강호찬,김은호,김지영,Cheol-Hyun Myung,Jae-Bong Lee,Chae-Kyoung Yoo,임현태 한국동물유전육종학회 2021 한국동물유전육종학회지 Vol.5 No.4

A processed meat product is a food that changes the character or shape of meat, and it is difficult to identify the raw meat because the original shape is destroyed. False record and disguised sales of raw meat can negatively affect not only the safety of consumers but also the income of producers. Therefore, this study was conducted to establish a multiplex PCR (polymerase chain reaction) method to simultaneously identify cattle (Bos taurus), pigs (Sus scrofa), and chickens (Gallus gallus), which are mainly used as raw meat in the manufacture of processed meat products. As a result of species-specific PCR of the raw meat DNA to confirm the cross-reactivity, species-specific amplification products of cattle 384 bp, pigs 249 bp, and chickens 721 bp were identified, and no other non-specific amplification appeared. Based on this, in order to evaluate the applicability in the market, multiplex PCR was performed by classifying 52 processed meat products on sale by 5 types (Ham, Tteokgalbi, Pattie, Can ham, Sausage). As a result, the indicated ingredients were detected in all products except Can ham No. 7 among 52 processed meat, and specific reactions were observed in chickens that were not marked in No. 1, No. 4 of Tteokgalbi. In particular, among the pigs and chickens marked on Can ham No. 7, only pigs showed a specific reaction, confirming the possibility of false record. If this study is used to identify the types of raw meat used in processed meat products, it is expected that it will contribute to improving awareness and promoting consumption of processed meat by reducing cases of illegal distribution and restoring consumer confidence in processed meat. 본 연구는 육가공품에서 종 식별 마커의 multiplex PCR 방법에 대한 적용 가능성을 확인하고 실시하였다. 육가공품에서 가장많이 사용되는 소, 돼지, 닭의 종 식별 마커를 선별하여 원료육과 가열육에서 추출한 DNA를 활용하여 PCR을 실시하였다. 그 결과, 원료육과 가열육에서 소, 돼지, 닭의 종 식별 마커가 교차반응 없이 모두 식별이 가능하였고, 검출한계 실험은 원료육에서 소, 돼지, 닭이 최대 0.01 ng/㎕, 가열육에서는 소는 최대 0.01 ng/㎕, 돼지와 닭은 최대 0.1 ng/㎕으로 가열로 인한 DNA 파괴를 고려하더라도 육가공품에서 충분히 종 식별이 가능할 것으로 판단된다. 이후, 5종류로 나눈 52개의 육가공품에 multiplex PCR를 실시하였다. 표기된 성분과 종 식별 PCR 결과, 햄류, 패티류, 소시지류에서 표기된 성분과 multiplex PCR의 종 식별 결과가 동일하였으나, 떡갈비 1번, 4번의 제품이 표기된 성분 외에 닭의 특이적 반응이 검출됨으로써 의도적 또는 비의도적으로 닭고기가 포함되었으며, 캔 햄 7번의 제품에서 표기된 돼지, 닭 중 돼지만 특이적 반응이 검출되어 제품의 원재료명과 표기가 다른 것을 확인하였다. 본 실험을 통해 육가공품 제조 시 실제로 표기되지 않은 축종, 특히 가장 값이 저렴한 닭고기를 사용할 가능성이 있음을확인하였으며, 허위기재의 가능성 또한 확인하였다. 육가공품 원료육 표기의 정확도 향상 및 육가공품에 대한 소비촉진을 위해육가공품에 대한 원료육의 종 식별뿐만 아니라 원료육 성분비 판별을 위한 추가연구가 필요할 것으로 판단된다

전장유전체 관련성 분석을 통한 순종 랜드레이스 생시체중 형질의 위치상 후보유전자 탐색

강호찬,이재봉,유채경,최태정,임현태 경상국립대학교 농업생명과학연구원 2018 농업생명과학연구 Vol.52 No.3

Genetic diversity and relationship analyses of the Korea native black goat line using microsatellite markers

강호찬,김관우,김은호,명철현,이정규,임현태 충남대학교 농업과학연구소 2021 Korean Journal of Agricultural Science Vol.48 No.4

The aim of this study was to analyze the genetic diversity and distance of the Korean native black goat line. Thus far, this Korean native black goat line has not been studied intensively, especially in genetic diversity and relationship studies in comparison with other breeds. In total, eleven microsatellite (MS) markers were used to evaluate alleles from 391 Korean native black goats and foreign hybrid animals. The genetic diversity index was evaluated based on the allele distributions. Four Korean native black goat lines showed expected ranges of observed heterozygosity, expected heterozygosity, and polymorphism information content (PIC) values for use in genetic diversity research (0.509 - 0.643, 0.434 - 0.623 and 0.356 - 0.567). Lines from the Korean native black goat and foreign hybrid were clearly separated according to principal coordinates analysis (PCoA), phylogenetic tree and tended to be clustered in each Korean native black goat line. Thus, this study can be used for analyzing the genetic relationships between Korean native black goats and foreign breeds for line preservation and for fundamental information to determine breed improvement strategies.

Analysis of the relationship between the end weight trait and the gene ADGRL2 in purebred landrace pigs using a Genome-wide association study

강호찬,김희성,이재봉,유채경,최태정,임현태 충남대학교 농업과학연구소 2018 Korean Journal of Agricultural Science Vol.45 No.2

The overall consumption of meat is increasing as the level of national income increases. The end weight is a trait closely associated with dressed meat. Genome-wide association study (GWAS) is an effective method of analyzing genetic variation and gene identification associated with a number of natural alternative traits because it can detect variations. So this paper did a GWAS analysis to identity the location on the genome related to the end weight in purebred landrace pigs and to explore the relevant candidate gene. This study identified a significant single nucleotide poly morphism (SNP) marker in chromosome 6 (ASGA0029422, p = 1.22 x 10-6). Adhesion G protein-coupled receptor L2 (ADGRL2 ) was found to be the candidate gene at the identified SNP marker location. ADGRL2 genes have been found to be associated with cell development in relation to the external and internal environment of a cell. In addition, genotype and statistical analyses were done on nine variations on the exon of ADGRL2 . The results show that the SNP marker (ASGA0029422, p = 1.32 × 10-6) was significant, but the significance of the nine variations on the ADGRL2 exon was not verified. However, by performing further experiments and functional studies on other SNPs showing possible genetic ADGRL-Exon mutations, objects with high associations of high-end weights can be selected.

순종 랜드레이스의 10주령 체중 형질에 영향을 미치는 후보유전자 탐색을 위한 전장유전체 분석

강호찬,이재봉,유채경,최태정,임현태 강원대학교 동물자원공동연구소 2019 동물자원연구 Vol.30 No.3

According to Livestock Inspection Standards, the piglets enter the feedlot at approximately 30 kg, and the inspection starts after the preliminary feeding period. The reason for applying the preliminary feeding period is to select inspection piglets with no diseases after the complete growth of the internal organs until 10 weeks of age. Furthermore, the age of 10 week is the time when the muscle fibers grow to their maximum size and the piglets are prepared for fat deposition at the later fattening period. In the study, a genome-wide association study (GWAS) was performed through the mlma command of the genome-wide complex trait analysis (GCTA) program with 703 purebred Landrace population, and the candidate genes associated with the weight of 10 week were searched. The GWAS identified 3 single nucleotide polymorphism (SNP) markers, which have a significant genome-wide suggestive level, on chromosome 6 (DIAS0002615; p value=1.62×10-6, MARC0083933; p value=4.94×10-6, ASGA0028717; p value=5.40×10-6). The 2 genes (Ubiquitin protein ligase E3 component n-recognin 4; UBR4, WD and tetratricopeptide repeats 1; WDTC1) in which these 3 SNP markers are located are positional candidate genes of the weight of 10 week of the purebred Landrace population. 2 candidate genes have been reported to be associated with fattening. Therefore, the positional candidate genes in this study, UBR4 and WDTC1, are expected to be usable as genes for traits associated with the weight of 10 week weight and fattening through additional experimental research with other population 가축검정기준(Livestock Inspection Standards)에 따르면 약 30 kg에 입식을 하여 예비사육기간 이후 검정을 시작한다. 이는 10주령 시기까지 체내 기관의 온전한 성장과 질병에 문제가 없는 검정자돈을 선발하기 위함이며, 또한 10주령 시기는 근섬유의 크기가 최대치로 증가하고 이후 비육시기의 지방 침착을 위해 준비하는 시기이다. 이에 본 논문은 순종 랜드레이스 집단 703두와 genome-wide complex trait analysis(GCTA) 프로그램의 mlma command를 통한 전장유전체 연관성 분석(genome-wide association study, GWAS)을 수행하여 10주령 체중과 연관된 후보유전자를 탐색하였다. 전장유전체 연관성 분석 결과 genome-wide suggestive level의 유의성을 가지는 single nucleotide polymorphism(SNP) marker가 6번 염색체에서 3개 동정되었다(DIAS0002615; p value=1.62×10-6, MARC 0083933; p value=4.94×10-6, ASGA0028717; p value=5.40× 10-6). 이 3개의 SNP marker가 위치한 2개의 유전자(Ubiquitin protein ligase E3 component n-recognin 4; UBR4, WD and tetratricopeptide repeats 1; WDTC1)들은 순종 랜드레이스 집단의 10주령 체중의 위치상 후보 유전자이며, 두 개의 후보유전자는 비육과 연관된 유전자로 보고되어 있다. 따라서 본 연구의 위치상 후보유전자인 UBR4, WDTC1은 타 집단과 추가적인 실험 연구를 통해 10주령 체중과 비육 연관 형질의 유전자로서 이용이 가능할 것으로 생각된다.

Analysis of gut microbiota in the Feces of Korean native black goat using 16S rRNA sequencing

강호찬,명철현,김지영,송상현,정우철,김승창,진대혁,임현태 한국동물유전육종학회 2024 한국동물유전육종학회지 Vol.8 No.3

Despite the recent surge in consumer demand for native goats as an alternative meat source, driven by shifting perceptions of health foods, there is a notable lack of prior research on this livestock breed. This study aimed to characterize the gut microbiota of native goat populations to enhance our understanding of the species and to provide fundamental data for improving productivity. Fecal samples collected from January to April 2024 were analyzed using 16S rRNA sequencing to assess the gut microbiota composition. The analysis revealed that the phyla Bacillota and Bacteroidota were the most abundant, playing key roles in fiber degradation and short-chain fatty acid production, which are crucial for the physiological and functional health of the animals. The gut microbiota composition was found to be similar to that of other ruminants. Interestingly, individuals with lower abundances of Bacillota and Bacteroidota showed relatively higher levels of Bacteroidales, suggesting that their digestive efficiency was not compromised. The gut microbiota of deceased animals also displayed a similar composition to that of healthy individuals, with no specific associations with particular diseases identified. The findings from this study provide foundational data on the gut microbiota of native goats, which could be further elaborated through additional research.

Genome-wide association study identifies positional candidate genes affecting back fat thickness trait in pigs

이재봉,강호찬,김은호,김윤주,유채경,최태정,임현태 충남대학교 농업과학연구소 2018 Korean Journal of Agricultural Science Vol.45 No.4

This study was done to search for positional candidate genes associated with the back fat thickness trait using a Genome-Wide Association Study (GWAS) in purebred Yorkshires (N = 1755). Genotype and phenotype analyses were done for 1,642 samples. As a result of the associations with back fat thickness using the Gemma program (ver. 0.93), when the genome-wide suggestive threshold was determined using the Bonferroni method (p = 1.61 × 10-5), the single nucleotide polymorphism (SNP) markers with suggestive significance were identified in 1 SNP marker on chromosome 2 (MARC0053928; p = 3.65 × 10-6), 2 SNP markers on chromosome 14 (ALGA0083078; p = 7.85 × 10-6, INRA0048453; p = 1.27 × 10-5), and 1 SNP marker on chromosome 18 (ALGA0120564; p = 1.44 × 10-5). We could select positional candidate genes (KCNQ1, DOCK1, LOC106506151, and LOC110257583), located close to the SNP markers. Among these, we identified a potassium voltage-gated channel subfamily Q member gene (KCNQ1) and the dedicator of cytokinesis 1 (DOCK1) gene associated with obesity and Type-2 diabetes. The SNPs and haplotypes of the KCNQ1 and DOCK1 genes can contribute to understanding the genetic structure of back fat thickness. Additionally, it may provide basic data regarding marker assisted selection for a meat quality trait in pigs.

Identification of marker in processed meat products of different species by using Real-time PCR

김은호,강호찬,김지영,Cheol-Hyun Myung,Jae-Bong Lee,Chae-Kyoung Yoo,임현태 한국동물유전육종학회 2021 한국동물유전육종학회지 Vol.5 No.4

In order to secure consumer safety against false record and inaccurate information on processed meat products, it is necessary to establish a detection method for raw meat used in processed meat products. This study performed a quantitative analysis of raw meat of cattle (Bos taurus), pigs (Sus scrofa), and chickens (Gallus gallus) used in the production of processed meat products using the Real-time PCR method, which can confirm gene expression and detection amount in real time. For the species-specific primer design, the sequence of the D-loop and Cytochrome b (Cyt-b) gene in mitochondrial DNA (mtDNA), which are widely used for species identification due to its high mutation rate, were used. As a result of the species-specific real-time PCR, the cycle quantification (Cq) value expressed according to the species was found to be high in the order of pigs, cattle, and chickens, and no cross-reaction appeared. Based on the results of confirming species specificity, 52 processed meat products on sale were classified into 5 types (Ham, Tteokgalbi, Pattie, Can ham, Sausage), and real-time PCR was performed. All ingredients label in the processed meat products except for Can ham No. 7 were detected, and those that did not match the label were No. 2 and No. 5 in Ham, No. 2, No. 6, No. 16 in Sausage, and No. 1 and 4 in Tteokgalbi. A total of 7 products showed a specific reaction of chickens. In the case of products with different detection results from the labeled ingredients, intentional or unintentional mixing could be suspected through the difference in Cq values of the main ingredient and unlabeled ingredient. If a quantitative analysis method for raw meat is established and commercialized by optimizing these Real-time PCR conditions, it is thought that consumers' awareness and safety will be improved. 이번 연구를 통하여 육가공품에서의 종 식별 마커의 적용 가능성을 확인하였으며, 총 5종류 52개의 육가공품을 대상으로 Realtime PCR을 수행한 결과로 캔 햄 7번을 제외하고 표기된 성분이 모두 검출이 되었으며, 제품 표기와 다른 성분이 있는 제품 7가지를 확인하였다. 이를 통해 미표기된 원료육이 혼합될 가능성을 확인할 수 있었다. 현재 식품 공정에 관한 법령의 경우 육가공품의 제조 기준과 포장재 표기 사항 등의 내용은 기재되어 있지만, 원료 성분을 확인할 분석방법에 관한 규정은 없는 실정이다. 따라서, 이러한 검사법을 통해 최종 육가공품의 원료 종류 및 함량을 분석할 수 있는 기술이 필요한 것으로 보이며, 소비자 관점에서 동일한 유형의 육가공품에 대하여 사용한 원료육의 품질구분이 어렵다는 점을 고려했을 때 고기 함량 표기에 세부사항까지표기할 수 있도록 하는 규제가 필요한 것으로 사료된다.

Causal inference using genome-wide association study results of Hanwoo carcass traits: Mendelian randomization analysis

김지영,강호찬,명철현,임현태 한국동물유전육종학회 2024 한국동물유전육종학회지 Vol.8 No.3

Setting breeding objectives to enhance carcass traits in Hanwoo is essential for increasing the profitability of the Hanwoo industry and ensuring that consumers receive high-quality meat. Key carcass traits, including carcass weight (CWT), eye muscle area (EMA), back-fat thickness (BFT), and marbling score (MS), are genetically correlated. Understanding the causal relationships among these traits is vital for comprehending the complex biological systems of Hanwoo. This study analyzed data from 392 Hanwoo using a Genome-Wide Association Study (GWAS) to identify genetic variants. These variants were subsequently employed as instrumental variables in a Mendelian Randomization (MR) analysis to infer causality. With CWT as the exposure variable and MS as the outcome variable, the selected instrumental variables were validated for their assumptions through tests for heterogeneity and pleiotropy. The MR analysis revealed that, except for the MR-Egger model, significant positive relationships were observed across all models, indicating that an increase in CWT causally influences an increase in MS, beyond mere genetic correlations. It was confirmed that a 1 kg increase in CWT results in an approximate 0.01-point increase in MS. Thus, this study underscores the importance of utilizing genetic variants identified through GWAS as instrumental variables for inferring causal relationships between CWT and MS via MR analysis. These findings can contribute to developing effective breeding strategies and improve our understanding of the biological mechanisms related to carcass traits.

Genome-wide association study for ethological traits of purebred Landrace and Yorkshire populations

최태정,강호찬,이재봉,유채경,김은호,임신재,임현태 경상국립대학교 농업생명과학연구원 2020 농업생명과학연구 Vol.54 No.2

Some behaviors of pigs that are not expressed in the wild state or are observed in a small minority of individuals after groups of pigs are mixed have been reported to indicate poor welfare. A GWAS analysis was performed by measuring the frequency and duration of the four ethological traits and using the mlma command provided by the genome-wide complex trait analysis (GCTA). The positional candidate genes on significantly identified single nucleotide polymorphism (SNP) markers were identified by using the dbSNP provided by the National Center for Biotechnology Information (NCBI). When the GWAS analysis was applied the 43,565 (of the purebred Landrace population) and 41,700 (of the purebred Yorkshire population) SNP markers, 1, 2, and 1 significant SNP markers were identified for the traits of feeding frequency (LOC110262254), locomotion time (LOC110260361), and locomotion frequency (LOC110260361) of the purebred Landrace population, respectively. Meanwhile, 1 and 7 significant SNP markers were identified for the traits of drinking time (LOC110260090) and feeding frequency (MAP3K19; LOC110257013; ACMSD; TMEM163; RAB3GAP1) of the purebred Yorkshire population, respectively. The results of this study may suggest that the GWAS analysis of the ethological traits of purebred Landrace and Yorkshire populations could be used to perform a GWAS analysis on non-economic traits, and the results can thus be provided as basic data for GWAS analyses of other non-economic traits in the future.