두록 돼지의 등지방두께와 연관된 렙틴수용체 유전자의 신규 SNP 마커 A Novel Single Nucleotide Polymorphism of the Leptin Receptor Gene Associated with Backfat Thickness in Duroc Pigs원문보기
돼지에게 있어서 지방 형질은 가장 중요한 경제형질 중 하나다. 돼지의 렙틴수용체 유전자(LEPR)는 염색체 상의 위치와 그 생리 활성 측면에서 돼지 6번 염색체 상의 지방형질 관련 양적형질좌위(QTL)에 대한 주요 후보유전자로 알려져 있다. 본 연구에서는 LEPR 유전자의 구조 변이와 돼지 경제형질과의 연관성을 분석하였다. 이를 위하여 돼지 LEPR 유전자를 포함하고 있는 박테리아 인공 염색체(BAC) 클론에 대한 샷건 염기서열 해독을 수행하여 114 kb 크기의 유전체 서열을 확보하였다. 그리고 전사개시 코돈으로부터 1.2 kb 상위 영역에서 여러 전사인자 결합부위를 발견하였다. 또한 LEPR 유전자 엑손 영역의 6개 SNP와 5’ 조절영역의 18개 SNP에 대해 550두의 두록 개체를 대상으로 연관성 분석을 수행하였다. 이들 SNP 중, −790C/G만이 등지방두께와 정육율 형질과 유의적으로 연관되어 있었으며, 2개의 미스센스 다형성 SNP를 포함하여 다른 SNP에서는 어떤 형질과도 연관성을 보이지 않았다. 결론적으로 −790C/G SNP는 두록 돼지에서 지방과 정육형질을 유전적으로 개량하는데 유용한 마커로 활용될 수 있을 것이다.
돼지에게 있어서 지방 형질은 가장 중요한 경제형질 중 하나다. 돼지의 렙틴수용체 유전자(LEPR)는 염색체 상의 위치와 그 생리 활성 측면에서 돼지 6번 염색체 상의 지방형질 관련 양적형질좌위(QTL)에 대한 주요 후보유전자로 알려져 있다. 본 연구에서는 LEPR 유전자의 구조 변이와 돼지 경제형질과의 연관성을 분석하였다. 이를 위하여 돼지 LEPR 유전자를 포함하고 있는 박테리아 인공 염색체(BAC) 클론에 대한 샷건 염기서열 해독을 수행하여 114 kb 크기의 유전체 서열을 확보하였다. 그리고 전사개시 코돈으로부터 1.2 kb 상위 영역에서 여러 전사인자 결합부위를 발견하였다. 또한 LEPR 유전자 엑손 영역의 6개 SNP와 5’ 조절영역의 18개 SNP에 대해 550두의 두록 개체를 대상으로 연관성 분석을 수행하였다. 이들 SNP 중, −790C/G만이 등지방두께와 정육율 형질과 유의적으로 연관되어 있었으며, 2개의 미스센스 다형성 SNP를 포함하여 다른 SNP에서는 어떤 형질과도 연관성을 보이지 않았다. 결론적으로 −790C/G SNP는 두록 돼지에서 지방과 정육형질을 유전적으로 개량하는데 유용한 마커로 활용될 수 있을 것이다.
Fatness is one of the most important economic traits in pigs. The leptin receptor (LEPR) gene may be a potential candidate for the fatness quantitative trait locus (QTL) on porcine chromosome 6, due to its position and physiological role. Thus, this study was carried out to evaluate the associations...
Fatness is one of the most important economic traits in pigs. The leptin receptor (LEPR) gene may be a potential candidate for the fatness quantitative trait locus (QTL) on porcine chromosome 6, due to its position and physiological role. Thus, this study was carried out to evaluate the associations between structural variants in the LEPR gene and economic traits in pigs. We obtained an approximately 114-kb sequence containing the complete genomic DNA of the porcine LEPR gene, using shotgun sequencing of a bacterial artificial chromosome clone. We report the complete genomic structure of the porcine LEPR gene. Dozens of transcription factor-binding sites were found in the 1.2 kb upstream region from the transcription start point. An association study was performed with 550 Duroc pigs for 24 single-nucleotide polymorphisms (SNPs), including 6 SNPs within exons and 18 SNPs within the putative 5‘ regulatory region of the porcine LEPR gene. Among them, one SNP (−790C/G) was significantly associated with backfat thickness and lean meat percentage, whereas the others, including two SNPs with missense polymorphisms, had no effect on any phenotype. These results suggest that SNP −790C/G may be a useful marker for genetic improvements of fatness and leanness in Duroc pigs.
Fatness is one of the most important economic traits in pigs. The leptin receptor (LEPR) gene may be a potential candidate for the fatness quantitative trait locus (QTL) on porcine chromosome 6, due to its position and physiological role. Thus, this study was carried out to evaluate the associations between structural variants in the LEPR gene and economic traits in pigs. We obtained an approximately 114-kb sequence containing the complete genomic DNA of the porcine LEPR gene, using shotgun sequencing of a bacterial artificial chromosome clone. We report the complete genomic structure of the porcine LEPR gene. Dozens of transcription factor-binding sites were found in the 1.2 kb upstream region from the transcription start point. An association study was performed with 550 Duroc pigs for 24 single-nucleotide polymorphisms (SNPs), including 6 SNPs within exons and 18 SNPs within the putative 5‘ regulatory region of the porcine LEPR gene. Among them, one SNP (−790C/G) was significantly associated with backfat thickness and lean meat percentage, whereas the others, including two SNPs with missense polymorphisms, had no effect on any phenotype. These results suggest that SNP −790C/G may be a useful marker for genetic improvements of fatness and leanness in Duroc pigs.
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문제 정의
Therefore, this study was carried out to evaluate the por- cine LEPR gene as a positional candidate controlling the QTL for growth and fat deposition traits on SSC6. In addition, we report the complete genomic structure containing the 5′ regulatory region of the porcine LEPR gene.
This study was conducted to evaluate the influence of variations in the porcine LEPR gene on production traits in pigs. This is the first report providing evidence for the effect of one SNP in the 5' regulatory region on production traits in a Duroc population. Moreover, we revealed the complete genomic sequences including the 5' regulatory re- gion in the porcine LEPR gene.
Association studies yield significant results when an SNP within a candidate gene is a causal variation or in linkage disequilibrium with it [20]. This study was conducted to evaluate the influence of variations in the porcine LEPR gene on production traits in pigs. This is the first report providing evidence for the effect of one SNP in the 5' regulatory region on production traits in a Duroc population.
제안 방법
Data were analyzed with the general linear model proce- dure using SAS (SAS Institute, Cary, NC, USA) to test the effect of each genotype on performance traits. Mean differ- ences were established based on the least squares means comparison.
Genomic DNAs were extracted with the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). PCR reactions were per- formed in a 25 μl final volume containing 25 ng template DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.5 mM MgCl2, 0.2 μM each primer, 100 μM each dNTP, and one unit Taq DNA polymerase (GeNet Bio) for genotyping of the 18 SNPs in the promoter region and the 6 SNPs in the exon region (Table 2). Thermal cycling parameters were defined as fol- lows: pre-denaturation at 95℃ for 5 min, followed by 35 cycles of 95℃ for 30 s, annealing temperature for 30 s (Table 1), 72℃ for 1 min, and then a final step at 72℃ for 10 min using a PTC 225 Peltier Thermal Cycler (MJ Research).
2 kb up- stream and 11 exon regions were designed based on the BAC clone sequence obtained (Table 1). The PCR reaction was performed in a 50 μL final volume containing 50 ng template DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.5 mM MgCl2, 0.2 μM each primer, 100 µM each dNTP, and one unit Taq DNA polymerase (GeNet Bio, Korea). Reaction profiles in- cluded a 5 min denaturation step at 94℃ followed by 35 cycles each consisting of 30 s at 94℃, 30 s at the annealing temperature (Table 1), 1 min at 72℃, and then a final 10 min extension step at 72℃ using a PTC-225 Peltier Thermal Cycler (MJ Research, Waltham MA, USA).
4 professional program. The puta- tive promoter sequence of the porcine LEPR gene was aligned with the human (AC097063) and mouse (AL929373) sequences using the ClustalW2 program (http://www.ebi. ac.uk/Tools/msa/clustalw2/) to investigate consensus se- quences within the promoter regions among species.
We screened a BAC clone (KNP_645H8) containing the LEPR-CA microsatellite marker and obtained an approx- imate 114 kb sequence (GenBank acc. no. FN673752) using the shotgun sequencing method. The complete genomic structure of the LEPR gene including a putative promoter region was revealed by comparison with the porcine cDNA sequence (AF092422).
대상 데이터
screening method. A BAC clone containing the LEPR gene was screened with LEPR-CA STS (UniSTS: 253565, Forward: 5‘-TTCCAGAAACATAAGACACGCG-3‘, Reverse: 5’- GACCAATTCTAAATTTCAACCAGAGG-3‘). A shotgun library of the screened BAC clone, KNP_645H8, was con- structed using the pUC19 plasmid vector (Qbiogene, Irvine, CA, USA).
The traits ana- lyzed in this study were average daily weight gain, feed effi- ciency, backfat thickness, and lean meat percentage. Blood samples were collected from 550 Duroc pigs at the Pig Breeding Stock Evaluation Center of the Korean Swine Association in Korea for the association test. Genomic DNAs were extracted with the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA).
Yorkshire. Eleven pairs of primers covering 1.2 kb up- stream and 11 exon regions were designed based on the BAC clone sequence obtained (Table 1). The PCR reaction was performed in a 50 μL final volume containing 50 ng template DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.
The DNA sequences were assembled with Phred and Phrap software (University of Washington). The assembled sequence was deposited into GenBank of NCBI (FN673752).
이론/모형
A bacterial artificial chromosome (BAC) clone containing the LEPR gene was obtained from the Korean native pig (KNP) BAC library [10] using a polymerase chain reaction (PCR) screening method. A BAC clone containing the LEPR gene was screened with LEPR-CA STS (UniSTS: 253565, Forward: 5‘-TTCCAGAAACATAAGACACGCG-3‘, Reverse: 5’- GACCAATTCTAAATTTCAACCAGAGG-3‘).
성능/효과
the regulatory region for the association study. Only one SNP at the -790C/G polymorphism on the regulatory region in the LEPR gene was significantly associated with pro- duction traits such as backfat thickness (p<0.001) and lean meat percentage (p<0.003), but had no significant effect on average daily weight gain or feed efficiency (Table 5). That is, backfat thickness was higher and lean meat percentage lower in the individual of the genotype GG rather than in that of the genotype CC.
investigate the allelic frequencies of SNPs. The traits ana- lyzed in this study were average daily weight gain, feed effi- ciency, backfat thickness, and lean meat percentage. Blood samples were collected from 550 Duroc pigs at the Pig Breeding Stock Evaluation Center of the Korean Swine Association in Korea for the association test.
후속연구
A significant phenotypic effect may have been ob- served if a causal mutation in the LEPR promoter region occurred or if the mutation was closely linked with a causal mutation. Therefore, further studies are needed to determine whether these results are due to a polymorphic site that is critical for transcription or linkage disequilibrium with a causal mutation.
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