보고서 정보
주관연구기관 |
제주대학교 Cheju National University |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2015-03 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201500010520 |
DB 구축일자 |
2015-07-11
|
초록
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Ⅳ. 연구개발결과
1세부과제명 : 흑돼지 육성 집단 조성 및 육종체계 설정
- 차세대 염기서열 분석 방법(Next Generation Sequencing)을 사용하여 제주 재래 흑돼지의전장 유전체 (whole genome) 데이터를 생산
- 미토콘드리아 지놈서열(mitochondrial genome sequence)로 재해독하여 수행
- 공개 데이터베이로부터 버크셔, 듀록, 랜드레이스, 요크셔, 야생 멧돼지의 미토콘드리아 서열을 이용하여 제주 재래 돼지의 계통적 유연관계 분석
- NGS기술인 reseque
Ⅳ. 연구개발결과
1세부과제명 : 흑돼지 육성 집단 조성 및 육종체계 설정
- 차세대 염기서열 분석 방법(Next Generation Sequencing)을 사용하여 제주 재래 흑돼지의전장 유전체 (whole genome) 데이터를 생산
- 미토콘드리아 지놈서열(mitochondrial genome sequence)로 재해독하여 수행
- 공개 데이터베이로부터 버크셔, 듀록, 랜드레이스, 요크셔, 야생 멧돼지의 미토콘드리아 서열을 이용하여 제주 재래 돼지의 계통적 유연관계 분석
- NGS기술인 resequencing기법을 통해 전장 유전체를 이용하여 미토콘드리아 서열을 분석
- 제주 재래 돼지 계통 분석: 제주 재래 돼지가 속한 가지의 계통 관계를 보면 요크셔과 제일가까운 유연 관계를 갖고 있고 멧돼지와 그 다음으로 가까운 관계를 갖고 있는 것으로 확인됨.
- Outgroup에 있는 품종들을 보면 버크셔가 먼저 가지가 갈라지고 랜드레이스와 듀록과 가까운 관계를 형성하는 것을 볼 수 있음.
- Neighbor-joining 방법과 Bayesian 방법으로 계통수를 보면 제주 재래 돼지는 요크셔와 가장 가까운 계통으로 확인돼 다산성이 높은 요크셔와의 교잡으로 개량된 재래 돼지로 추정됨
2세부과제명 : 흑돼지 성장과 육질 촉진 및 저해 관련 전사체 개발
- 육질 전사체 프로파일링과 육질 파일링을 통한 표현형 상관관계 확립
- DB 완성을 통한 RNA-DEG분석을 완료
- 각 성장 단계별 그리고 제주재래흑돼지와 버크셔간의 육질 차이 및 맛 관련 유전자 선별및 차별화 분석 완료
- 표현형과 유전자형의 상관관계 유전자 발현을 통해 규명
- 세포내 유전자 조절에 의한 선발된 육질마커 및 성장조절 마커의 발현 검증
- 세포(근섬유 세포주:마우스 C2C12 세포주)를 통하여 육질조절 유전자의 과발현을 통한 조절기작 규명 완료
- 육질관련 SNP 마커 선별 및 이 마커에 단백질 결합분석을 통한 기능성 규명 및 활용 가능성 제시
1협동과제명 : 육질·맛 우수 흑돼지 조기선발 유전체육종가 프로그램 구축
- GBLUP을 이용한 육질·맛 유전체육종가(GEBV) 추정 모델 개발
- 다산 흑돼지농장 소재 1,200두에 대한 24개 맛·육질 표현형 및 대용량 SNP 자료수집
- 유전체(대용량SNP)정보를 활용한 G matrix(혈연관계) 및 맛·육질 GEBV 추정
- 근친도를 고려한 교배조합 작성 및 유전체육종가(GEBV) 정확도 평가
- 16개 형질의 맛·육질 GEBV 정확도 평균은 0.59로 나타남
- G matrix 요소(개체들간 혈연관계 정도)를 고려한 교배 조합 작성 정보를 제공하여 줌
- 맛과 육질과 관련된 후보 종모돈/종빈돈을 조기 선발하는 GEBV 정보를 제공하여 줌
- 추가적인 표현형 및 유전자형 자료 수집으로 GEBV 정확도 향상이 필요함
2협동과제명 : 돼지 생산 · 이력 추적 시스템 도입을 위한 DNA 마커 개발
- 돼지 이력 추적제 적용을 위한 현 돼지고기 판매경로의 확보
- 묶음번호와 가공장/판매장의 Match Rate 분석
- 정보가 없는 시료도 가공장/판매장 샘플의 도축일자 추정하여 Match 분석시 하나의 묶음번호를 제외한 나머지 시료는 추적이 가능하였음
- 96SNP 마커를 이용한 돼지 이력 추적에 대한 가능성 확인
- 제주 흑돼지의 이력 추적 가능 시스템 구축을 위한 연구
- 도축 일자별 가공장, 판매장, 식당에서 무작위로 각각 3개 개체씩 6그룹 샘플링하여 Match Rate 실시
- 하나의 샘플에서만 매치율이 92%가 나타나고, 나머지 샘플에 대해서는 90%이상의 매치율이나타나지않음
- 제주 지역 흑돼지에 대한 이력 추적관리 시스템도입 필요성 대두
Abstract
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In the 1980s, Korean native black pigs from Jeju Island (Jeju black pigs) served as representative sample of Korean native black pigs, and efforts were made to help the species rebound from the brink of extinction, which occurred as a result of the introduction of Western pig breeds. Geographical se
In the 1980s, Korean native black pigs from Jeju Island (Jeju black pigs) served as representative sample of Korean native black pigs, and efforts were made to help the species rebound from the brink of extinction, which occurred as a result of the introduction of Western pig breeds. Geographical separation of Jeju Island from the Korean peninsula has allowed Jeju black pigs not only to acquire unique characteristics but also to retain merits of rare Korean native black pigs. To further analyze the Jeju black pig genome, we performed whole-genome re-sequencing (average read depth of 14×) of 8 Jeju black pig and 6 Korean pigs (which live on the Korean peninsula) to compare and identify putative signatures of positive selection in Jeju black pig, the true and pure Korean native black pigs. The candidate genes potentially under positive selection in Jeju black pig support previous reports of high marbling score, rare occurrence of pale, soft, exudative (PSE) meat, but low growth rate and carcass weight compared to Western breeds. Several candidate genes potentially under positive selection were involved in fatty acid transport and may have contributed to the unique characteristics of meat quality in JBP. Jeju black pigs can offer a unique opportunity to investigate the true genetic resource of once endangered Korean native black pigs. Further genome-wide analyses of Jeju black pigs on a larger population scale are required in order to define a conservation strategy and improvement of native pig resources. Pork is a major source of animal protein for humans. The subcutaneous, intermuscular and the intramuscular fat are the factors responsible for meat quality. RNA-seq is rapidly adopted for the profiling of the transcriptomes in the studies related to gene regulation. The discovery of differentially expressed genes (DEGs) between adult animals of Jeju Native Pig (JNP) and Berkshire breeds are of particular interest for the current study. RNA-seq was used to investigate the transcriptome profiling in the fat tissue. Sequence reads were obtained from Ilumina HiSeq2000 and mapped to the pig genome using Tophat2. Total 153 DEGs were identified and 71 among the annotated genes, have BLAST matches in the non- redundant database. Metabolic, immune response and protein binding are enriched pathways in the fat tissue. In our study, biological adhesion, cellular, developmental and multicellular organismal processes in fat were up-regulated in JNP as compare to Berkshire. Multicellular organismal process,developmental process, embryonic morphogenesis and skeletal system development were the most significantly enriched terms in fat of JNP and Berkshire breeds (p = 1.17E-04, 0.044, 3.47E-04 and 4.48E-04 respectively). COL10A1, COL11A2, PDK4 and PNPLA3 genes responsible for skeletal system morphogenesis and body growth were down regulated in JNP. This study is the first statistical analysis for the detection of DEGs from RNA-seq data generated from fat tissue sample. This analysis can be used as stepping stone to understand the difference in the genetic mechanisms that might influence the identification of novel transcripts, sequence polymorphisms, isoforms and noncoding RNAs. RNA-seq is being rapidly adopted for the profiling of the transcriptomes in different areas of biology, especially in the studies related to gene regulation. The discovery of differentially expressed genes (DEGs) between adult animals of Jeju Native Pig (JNP) and Berkshire breeds of Sus scrofa, is of particular interest for the current study. For the better understanding of the gene expression profiles of the liver and longissimus dorsi muscle, DEGs were identified via RNA-seq.Sequence reads were obtained from Illumina HiSeq2000 and mapped to the pig reference genome (Sscrofa10.2) using Tophat2. We identified 169 and 39 DEGs in the liver and muscle of JNP respectively, by comparison with Berkshire breed. Out of all identified genes, 41 genes in the liver and 9 genes in the muscle have given significant expression. Gene ontology (GO) terms of developmental process and KEGG pathway analysis showed that metabolic, immune response and protein binding were commonly enriched pathways in the two tissues. Further the heat map analysis by ArrayStar has shown the different levels of expression in JNP with respect to the Berkshire breed. The validation through real time PCR and western blotting also confirmed the differential expression of genes in both breeds. Genes pertaining to metabolic process and inflammatory and immune system are more enriched in Berkshire breed. This comparative transcriptome analysis of two tissues suggests a subset of novel marker genes which expressed differently between the JNP and Berkshire. Jeju native livestock species are very unique and Jeju native pig (JNP) keeps special place in food animals. The pork from JNP is juicy, have better marbling quality, more tender meat and JNP are resistant to diseases as compare to the western breeds. Major snags with JNP are small litter size, lower feed efficiency, less body growth rate and low meat fat ratio. It has been documented that myosin Binding Protein- H(MYBPH) and Myogenic Factor 6 (MYF6) are most abundantly expressed myogenic factors in adult muscles. Therefore, the current study was conducted with the aim to find that would MYBPH and MYF6 be differentially expressed in JNP and Berkshire. Adult pigs at the age of 6 months were used for the study. Animals were reared and slaughtered according to the animal bioethics committee of Jeju National University. After slaughtering "loin eye muscle" were collected and were stored immediately in dry ice which were later stored at -80℃. TRIzolTM reagent was used for the RNA extraction and first strand cDNA was synthesized using SupersciptR-III for RT-PCR. Expressions of MYBPH and MYF6 in loin eye muscle were confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR).Analysis of data with "Image-J"has shown that MYBPH is almost 30% more expressive in JNP as compare to Berkshire and MYF6 is almost equally expressive in both breeds. It is suggested that JNP has different expression pattern for the gene controlling meat quality and body growth rate. There is a need to establish projects that will monitor livestock development, will set standard in livestock production, and will ensured safety of consumers with the advancement of knowledge and utilization of modern technologies. The project aims to increase public awareness and understanding livestock production and products. Moreover, many DNA markers are used in the field to trace animal products point origin or farm source up to the market selling point but this process was hindered by the use of expensive equipment, location and time. Inspection of meat products by various inspection bodies utilizing less expensive method will be advantageous.Target SNP was selected for tracing pork, and pork SNP to maximize number of DNA marker traceability effect. A total of 384 analysis was done using 5 breeds namely Jeju Native Black pig (19 heads),Landrace (17 heads), Yorkshire (168 heads), Berkshire (84 heads) and Duroc (96 heads) to detect target allele. Animals undergo first selection, then a minor allele frequency of equal to or above 40 % was set for the second selection. Second selection resulted to 133 types of SNP marker of the Jeju Native Black pig. Out of 133 types of SNP, 50 types DNA marker were set as target for the analysis. This method was able to identify breed and trace 96 DNA SNP marker at minimum cost. The match rate analysis was conducted between slaughter site and market shops resulted to limited information.An estimation of date of slaughtered was applied to the analysis that match 96 SNP of swine. Thus, pork traceability was possible using SNP marker. Match rate analysis between market shop and slaughter site were done randomly on six group. Result showed that match rate is less than 90 % for 5 groups and 1 group is more than 90 % match. The need for the introduction of traceability systems are emerging in Jeju Native black pig.
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