基于单核苷酸多态性鉴别小梅山猪及其制品
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摘要
小梅山猪(Sus scrofa)是太湖流域独特的地方猪品种,具有肉质鲜美等特点,市场上存在小梅山猪及其肉制品的假冒问题,而传统鉴别技术在实际应用中具有一定的局限性。本研究利用第3代分子标记—单核苷酸多态性(single nucleotide polymorphism, SNP)开发小梅山猪及其制品的分子标记鉴定方法,将小梅山猪与太湖流域其他6个品种及常见西方品种猪进行鉴别区分。对于太湖流域7个品种(品系)(中梅山猪、小梅山猪、二花脸猪、米猪、嘉兴黑猪、枫泾猪和沙乌头猪)和西方5个引进猪品种(杜洛克、长白猪、大白猪、皮特兰、巴克夏)进行简化基因组测序(genotyping by genome reducing and sequencing, GGRS),筛选出小梅山猪与其他猪种具有差异的5个SNP位点,采用Sanger测序进行验证。结果表明,上述5个SNPs在小梅山猪中存在较高等位基因突变率,利用5个候选位点的组合可达到鉴定概率99.35%、准确率100%,无假阳性错判。本研究为小梅山猪遗传资源保护、假冒伪劣品种辨别提供了参考依据。
Small Meishan pig(Sus scrofa) is a unique local pig breed in the Taihu River Basin which has the characteristics of delicious meat. There is the problem of counterfeiting meat products in the market, while the traditional identification technology has some limitations in practical application. The present study is to develop a new method to distinguish Small Meishan pig from other pig breeds or strains of Taihu Basin and common western pig breeds by using the third generation molecular marker called Single Nucleotide Polymorphism(SNP). The data of genotyping by genome reducing and sequencing(GGRS) of 7 local breeds(strains) in Taihu Basin(Middle Meishan pig, Small Meishan pig, Erhualian pig, Mi pig, Jiaxinghei pig,Fengjing pig and Shawutou pig) and 5 imported pig breeds(Duroc, Landrace, Yorkshire, Piltland, and Baxia)was used. Five SNP sites only in Small Meishan pig were selected and Sanger sequencing was used to verify the results. It was showed that the 5 SNP sites had high mutation rate in Small Meishan pig. By using the combination of 5 candidate sites, the identification probability could be up to 99.35% and the accuracy was100% with no false positive identification. The present study provides a basic principle for the protection ofgenetic resources and identification of fake and inferior pork products in Small Meishan pig.
Small Meishan pig(Sus scrofa) is a unique local pig breed in the Taihu River Basin which has the characteristics of delicious meat. There is the problem of counterfeiting meat products in the market, while the traditional identification technology has some limitations in practical application. The present study is to develop a new method to distinguish Small Meishan pig from other pig breeds or strains of Taihu Basin and common western pig breeds by using the third generation molecular marker called Single Nucleotide Polymorphism(SNP). The data of genotyping by genome reducing and sequencing(GGRS) of 7 local breeds(strains) in Taihu Basin(Middle Meishan pig, Small Meishan pig, Erhualian pig, Mi pig, Jiaxinghei pig,Fengjing pig and Shawutou pig) and 5 imported pig breeds(Duroc, Landrace, Yorkshire, Piltland, and Baxia)was used. Five SNP sites only in Small Meishan pig were selected and Sanger sequencing was used to verify the results. It was showed that the 5 SNP sites had high mutation rate in Small Meishan pig. By using the combination of 5 candidate sites, the identification probability could be up to 99.35% and the accuracy was100% with no false positive identification. The present study provides a basic principle for the protection ofgenetic resources and identification of fake and inferior pork products in Small Meishan pig.
引文
国家畜禽遗传资源委员会组.2011.中国畜禽遗传资源志猪志[M].北京:中国农业出版社,pp.231-235(Group of the National Livestock and Poultry Genetic Resources Commission.2011.Animal Genetic Resources in China Pigs[M].China Agricultural Publishing House,Beijing,China,pp.231-235.)
史子学,赵晓明,唐赛涌,等.2016.应用ISSR-PCR技术鉴别梅山猪肉及其肉制品的研究[J].猪业学,33(04):132-133(Shi Z X,Zhao X M,Tang S Y,et al.2016.Study on the identification of Meishan pig and its meat products by ISSR-PCR technique[J].Pig Science,33(04)132-133)
唐立群,肖层林,王伟平.2012.SNP分子标记的研究及其应用进展[J].中国农学通报,28(12):154-158.(Tang L QXiao C L,Wang W P.2012.Advances in the research and application of SNP molecular marker[J].China Agronomy Bulletin,2012,28(12):154-158.)
唐赛涌,王勃,陆林根.2013.上海地区梅山猪的历史发展与保种现状[J].中国猪业,2013(s1):69-71.(Tang S YWang B,Lu L G.2013.Historical development and conservation status of Meishan pigs in Shanghai[J].Chinese Pig Industry,2013(s1):69-71.)
杨晓玲,施苏华,唐恬.新一代测序技术的发展及应用前景[J].生物技术通报,2010(10):76-81.(Yang X L,Shi SH,Tang T.2010.Development and application prospect of new generation sequencing technology[J].Biotechnology Bulletin,2010(10):76-81.)
Alves E,Castellanos C,Ovilo C,et al.2002.Differentiation of the raw material of the Iberian pig meat industry based on the use of amplified fragment length polymorphism[J].Meat Science,61(2):157.
Dai M,Thompson R C,Maher C,et al.2010.NGSQC:Crossplatform quality analysis pipeline for deep sequencing data[J].BMC Genomics,11(Suppl4):1-9.
Li H,Durbin R.2010.Fast and accurate long-read alignment with Burrows-Wheeler transform[J].Bioinformatics,26(5):589-95.
Li H,Handsaker B,Wysoker A,et al.2009.The sequence alignment/map format and SAMtools[J].Bioinformatics,25(16):2078-2079.
Sasazaki S,Itoh K,Arimitsu S,et al.2004.Development of breed identification markers derived from AFLP in beef cattle[J].Meat Science,67(2):275-280.
Suekawa Y,Aihara H,Araki M,et al.2010.Development of breed identification markers based on a bovine 50KSNP array[J].Meat Science,85(2):285-288.
Wang Z,Chen Q,Liao R,et al.2017.Genome-wide genetic variation discovery in Chinese Taihu pig breeds using next generation sequencing[J].Animal Genetics,48(1):38-47.
Wang Z,Chen Q,Yang Y,et al.2015a.Genetic diversity and population structure of six Chinese indigenous pig breeds in the Taihu Lake region revealed by sequencing data[J].Animal Genetics,46(6):697-701.
Wang Z,Chen Q,Yang Y,et al.2015b.A genome-wide scan for selection signatures in Yorkshire and Landrace pigs based on sequencing data[J].Animal Genetics,45(6):808-816.
Yang Y,Wang Q,Chen Q,et al.2014.A new genotype imputation method with tolerance to high missing rate and rare variants[J].PLoS One,9(6):e101025.
史子学,赵晓明,唐赛涌,等.2016.应用ISSR-PCR技术鉴别梅山猪肉及其肉制品的研究[J].猪业学,33(04):132-133(Shi Z X,Zhao X M,Tang S Y,et al.2016.Study on the identification of Meishan pig and its meat products by ISSR-PCR technique[J].Pig Science,33(04)132-133)
唐立群,肖层林,王伟平.2012.SNP分子标记的研究及其应用进展[J].中国农学通报,28(12):154-158.(Tang L QXiao C L,Wang W P.2012.Advances in the research and application of SNP molecular marker[J].China Agronomy Bulletin,2012,28(12):154-158.)
唐赛涌,王勃,陆林根.2013.上海地区梅山猪的历史发展与保种现状[J].中国猪业,2013(s1):69-71.(Tang S YWang B,Lu L G.2013.Historical development and conservation status of Meishan pigs in Shanghai[J].Chinese Pig Industry,2013(s1):69-71.)
杨晓玲,施苏华,唐恬.新一代测序技术的发展及应用前景[J].生物技术通报,2010(10):76-81.(Yang X L,Shi SH,Tang T.2010.Development and application prospect of new generation sequencing technology[J].Biotechnology Bulletin,2010(10):76-81.)
Alves E,Castellanos C,Ovilo C,et al.2002.Differentiation of the raw material of the Iberian pig meat industry based on the use of amplified fragment length polymorphism[J].Meat Science,61(2):157.
Dai M,Thompson R C,Maher C,et al.2010.NGSQC:Crossplatform quality analysis pipeline for deep sequencing data[J].BMC Genomics,11(Suppl4):1-9.
Li H,Durbin R.2010.Fast and accurate long-read alignment with Burrows-Wheeler transform[J].Bioinformatics,26(5):589-95.
Li H,Handsaker B,Wysoker A,et al.2009.The sequence alignment/map format and SAMtools[J].Bioinformatics,25(16):2078-2079.
Sasazaki S,Itoh K,Arimitsu S,et al.2004.Development of breed identification markers derived from AFLP in beef cattle[J].Meat Science,67(2):275-280.
Suekawa Y,Aihara H,Araki M,et al.2010.Development of breed identification markers based on a bovine 50KSNP array[J].Meat Science,85(2):285-288.
Wang Z,Chen Q,Liao R,et al.2017.Genome-wide genetic variation discovery in Chinese Taihu pig breeds using next generation sequencing[J].Animal Genetics,48(1):38-47.
Wang Z,Chen Q,Yang Y,et al.2015a.Genetic diversity and population structure of six Chinese indigenous pig breeds in the Taihu Lake region revealed by sequencing data[J].Animal Genetics,46(6):697-701.
Wang Z,Chen Q,Yang Y,et al.2015b.A genome-wide scan for selection signatures in Yorkshire and Landrace pigs based on sequencing data[J].Animal Genetics,45(6):808-816.
Yang Y,Wang Q,Chen Q,et al.2014.A new genotype imputation method with tolerance to high missing rate and rare variants[J].PLoS One,9(6):e101025.