应用高分辨溶解曲线(HRM)技术分析猪微卫星多态性
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摘要
为了验证利用高分辨熔解曲线(high-resolution melting curve analysis,HRM)技术对猪微卫星标记进行基因分型的可行性和可靠性,使用非变性聚丙烯酰胺凝胶电泳(PAGE)方法对5个微卫星(SSRs)位点多态性进行检测,再使用HRM技术对这5个微卫星(SSRs)位点进行基因分型测定,最后对1个微卫星的所有基因型进行单克隆测序。结果表明:在样本测试中,使用HRM技术分辨出S0107和SW1953位点均有8种基因型,SW72位点有6种基因型,SW29位点有5种基因型,SW2155位点有4种基因型,其结果与利用非变性聚丙烯酰胺凝胶电泳方法检测的结果完全吻合,并且SW72位点的所有基因型克隆测序结果与HRM检测一致。这些结果提示,HRM分析技术可作为筛选猪微卫星标记多态性和对其进行基因分型的有效手段。
To test the feasibility and reliability of high-resolution melting curve analysis(HRM) on microsntellite genotype of pigs,nondenaturing polyacrylamide gel electrophoresis was conducted to measure the polymorphism of 5 SSR sites,and HRM technology used to test genotypes and all genotypes of one microsatellite sequenced.In the present study,we found that SSR marker S0107,SW1953,SW72,SW29 and SW2155 had 8,8,6,5 and 4 genotypes using HRM analysis,respectively.These HRM results are consistent with the results of PAGE,and sequencing results of all genotype clone at SW72,which indicates that HRM technology can be used as an efficient method to screen polymorphisms of SSR sites in pigs.
To test the feasibility and reliability of high-resolution melting curve analysis(HRM) on microsntellite genotype of pigs,nondenaturing polyacrylamide gel electrophoresis was conducted to measure the polymorphism of 5 SSR sites,and HRM technology used to test genotypes and all genotypes of one microsatellite sequenced.In the present study,we found that SSR marker S0107,SW1953,SW72,SW29 and SW2155 had 8,8,6,5 and 4 genotypes using HRM analysis,respectively.These HRM results are consistent with the results of PAGE,and sequencing results of all genotype clone at SW72,which indicates that HRM technology can be used as an efficient method to screen polymorphisms of SSR sites in pigs.
引文
[1]Akagi H,Yokozeki Y,Inagaki A,et al.Microsatellite DNA markers for rice chromosomes[J].Der Züchter,1996,93(7):1071-1077.
[2]张玲.微卫星DNA标记研究进展及应用[J].安徽农业科学,2007,35(4):972-975,977.
[3]Weitzel J N,Hows J M,Jeffreys A J,et al.Use of a hypervariable minisatellite DNA probe(33.15)for evaluating engraftment two or more years after bone marrow transplantation for aplastic anaemia[J].British Journal of Haematology,1988,70(1):91-97.
[4]Cogan N O,Smith K F,Yamada T,et al.QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass(Lolium perenne L.)[J].Theoretical and Applied Genetics,2005,110(2):364-380.
[5]孙少华,桑滋润,师守坤,等.肉牛杂交优势预测、评估及其应用研究[J].遗传学报,2000,27(7):580-589.
[6]马力鹏,黄涛,卢国昌,等.新疆褐牛近交群体的鉴定研究[J].中国畜牧兽医,2015,42(12):3274-3281.
[7]Lee S Y,Cho G J.Parentage testing of thoroughbred horse in Korea using microsatellite DNA typing[J].Journal of Veterinary Science,2006,7(1):63-67.
[8]陈幼春,马月辉,浦亚斌,等.家养动物选择的基因频率变化与品种聚类和保种的关系[J].畜牧兽医学报,2005,36(11):1236-1240.
[9]Wittwer C T,Reed G H,Gundry C N,et al.High-resolution genotyping by amplicon melting analysis using LCGreen[J].Clinical Chemistry,2003,49(6):853-860.
[10]郭心灵,尤崇革.高分辨率熔解曲线技术常用的仪器和荧光染料[J].分子诊断与治疗杂志,2012,4(1):50-53.
[11]潘登.高分辨率熔解曲线分析技术(HRM)系统的建立及其在基因多态性和突变检测方面的应用[D].上海:复旦大学,2013.
[12]BazsalovicsováE,Králová-HromadováI,Radvánszky J,et al.The origin of the giant liver fluke,fascioloides magna(Trematoda:Fasciolidae)from croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes[J].Parasitology Research,2013,112(7):2661-2666.
[13]Raherison S,Clerc M,Trombert S,et al.Real-time high resolution melting PCR for identification of the swedish variant of chlamydia trachomatis[J].Journal of Microbiological Methods,2009,78(1):101-103
[14]Farmen E,Hultman M T,D'auriac M A,et al.Development of a screening system for the detection of chemically induced DNA methylation alterations in a zebrafish liver cell line[J].Journal of Toxicology and Environmental Healtii Part A,2014,77(9-11):587-599.
[15]殷豪,王彩虹,田义轲,等.利用高分辨率熔解曲线(HRM)分析梨微卫星标记[J].园艺学报,2011,38(8):1601-1606.
[16]Seipp M T,Pattison D,Durtschi J D,et al.Quadruplex genotyping of F5,F2,and MTHFR variants in a single closed tube by high-resolution amplicon melting[J].Clinical Chemistry,2008,54(1):108-115.
[17]Seipp M T,Durtschi J D,Voelkerding K V,et al.Multiplex amplicon genotyping by high-resolution melting.[J].Journal of Biomolecular Techniques,2009,20(3):160-164.
[18]Kristensen L S,Dobrovic A.Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free highresolution melting analysis[J].Cancer Epidemiology Biomarkers and Prevention,2008,17(5):1240-1247.
[19]Norambuena P A,Copeland J A,Krenkova P,et al.Diagnostic method validation:high resolution melting(HRM)of small amplicons genotyping for the most common variants in the MTHFR gene[J].Clinical Biochemistry,2009,42(12):1308-1316.
[20]Tajiri-Utagawa E,Hara M,Takahashi K,et al.Development of a rapid high-tiiroughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses[J].Journal of Clinical Microbiology,2009,47(2):435-440.
[21]Lin J H,Tseng C P,Chen Y J,et al.Rapid differentiation of influenza a virus subtypes and genetic screening for virus variants by high-resolution melting analysis[J].Journal of Clinical Microbiology,2008,46(3):1090-1097.
[22]Nakagawa N,Higashi N,Nakagawa T.Cocirculation of antigenic variants and the vaccine-type virus during the 2004-2005 influenza B virus epidemics in Japan.[J].Journal of Clinical Microbiology,2009,47(2):352-357.
[23]Nakagawa T,Higashi N,Nakagawa N.Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen.[J].Journal of Virological Methods,2008,148(1-2):296-299.
[24]饶丹,嘎利兵嘎,郭小权,等.HRM分析方法中不同扩增片段、染料和仪器的比较[J].江西农业大学学报,2015,37(1):167-175.
[25]赵均良,张少红,刘斌等.应用高分辨率熔解曲线技术分析水稻分子标记基因型[J].中国农业科学,2011,44(18):3701-3708.
[26]尤崇革,李晓军,郜莉娜,等.高分辨熔解技术常用荧光染料的SNP基因分型能力比较[J].临床检验杂志,2011,29(8):575-577.
[27]温立斌,何孔旺,杨汉春,等.应用高分辨熔点曲线分析区分类猪圆环病毒因子P1和猪圆环病毒2型[J].江苏农业学报,2010,26(2):315-319.
[28]Li J S,Wang X M,Dong R X,et al.Evaluation of highresolution melting for gene mapping in rice[J].Plant Molecular Biology Reporter,2011,29(4):979-985.
[29]Mackay J F,Wright C D,Bonfiglioli RG.A new approach to varietal identification in plants by microsatellite high resolution melting analysis:application to the verification of grapevine and olive cultivars[J].Plant Methods,2008,4(1):8.
[30]Wu S B,Wirthensohn M G,Hunt P,et al.High resolution melting analysis of almond SNPs derived from ESTs[J].Theoretical and Applied Genetics,2008,118(1):1-14.
[31]Yang Z J,Fu L,Zhang G W,et al.Identification and association of SNPs in TBCIDI gene with growth traits in two rabbit breeds[J].Asian-Australasian Journal of Animal Sciences,2013,26(11):1529-1535.
[32]陈康,蒋超,袁媛,等.基于熔解曲线分析技术的鹿茸药材分子鉴别[J].中国中药杂志,2015,40(4):619-623.
[2]张玲.微卫星DNA标记研究进展及应用[J].安徽农业科学,2007,35(4):972-975,977.
[3]Weitzel J N,Hows J M,Jeffreys A J,et al.Use of a hypervariable minisatellite DNA probe(33.15)for evaluating engraftment two or more years after bone marrow transplantation for aplastic anaemia[J].British Journal of Haematology,1988,70(1):91-97.
[4]Cogan N O,Smith K F,Yamada T,et al.QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass(Lolium perenne L.)[J].Theoretical and Applied Genetics,2005,110(2):364-380.
[5]孙少华,桑滋润,师守坤,等.肉牛杂交优势预测、评估及其应用研究[J].遗传学报,2000,27(7):580-589.
[6]马力鹏,黄涛,卢国昌,等.新疆褐牛近交群体的鉴定研究[J].中国畜牧兽医,2015,42(12):3274-3281.
[7]Lee S Y,Cho G J.Parentage testing of thoroughbred horse in Korea using microsatellite DNA typing[J].Journal of Veterinary Science,2006,7(1):63-67.
[8]陈幼春,马月辉,浦亚斌,等.家养动物选择的基因频率变化与品种聚类和保种的关系[J].畜牧兽医学报,2005,36(11):1236-1240.
[9]Wittwer C T,Reed G H,Gundry C N,et al.High-resolution genotyping by amplicon melting analysis using LCGreen[J].Clinical Chemistry,2003,49(6):853-860.
[10]郭心灵,尤崇革.高分辨率熔解曲线技术常用的仪器和荧光染料[J].分子诊断与治疗杂志,2012,4(1):50-53.
[11]潘登.高分辨率熔解曲线分析技术(HRM)系统的建立及其在基因多态性和突变检测方面的应用[D].上海:复旦大学,2013.
[12]BazsalovicsováE,Králová-HromadováI,Radvánszky J,et al.The origin of the giant liver fluke,fascioloides magna(Trematoda:Fasciolidae)from croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes[J].Parasitology Research,2013,112(7):2661-2666.
[13]Raherison S,Clerc M,Trombert S,et al.Real-time high resolution melting PCR for identification of the swedish variant of chlamydia trachomatis[J].Journal of Microbiological Methods,2009,78(1):101-103
[14]Farmen E,Hultman M T,D'auriac M A,et al.Development of a screening system for the detection of chemically induced DNA methylation alterations in a zebrafish liver cell line[J].Journal of Toxicology and Environmental Healtii Part A,2014,77(9-11):587-599.
[15]殷豪,王彩虹,田义轲,等.利用高分辨率熔解曲线(HRM)分析梨微卫星标记[J].园艺学报,2011,38(8):1601-1606.
[16]Seipp M T,Pattison D,Durtschi J D,et al.Quadruplex genotyping of F5,F2,and MTHFR variants in a single closed tube by high-resolution amplicon melting[J].Clinical Chemistry,2008,54(1):108-115.
[17]Seipp M T,Durtschi J D,Voelkerding K V,et al.Multiplex amplicon genotyping by high-resolution melting.[J].Journal of Biomolecular Techniques,2009,20(3):160-164.
[18]Kristensen L S,Dobrovic A.Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free highresolution melting analysis[J].Cancer Epidemiology Biomarkers and Prevention,2008,17(5):1240-1247.
[19]Norambuena P A,Copeland J A,Krenkova P,et al.Diagnostic method validation:high resolution melting(HRM)of small amplicons genotyping for the most common variants in the MTHFR gene[J].Clinical Biochemistry,2009,42(12):1308-1316.
[20]Tajiri-Utagawa E,Hara M,Takahashi K,et al.Development of a rapid high-tiiroughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses[J].Journal of Clinical Microbiology,2009,47(2):435-440.
[21]Lin J H,Tseng C P,Chen Y J,et al.Rapid differentiation of influenza a virus subtypes and genetic screening for virus variants by high-resolution melting analysis[J].Journal of Clinical Microbiology,2008,46(3):1090-1097.
[22]Nakagawa N,Higashi N,Nakagawa T.Cocirculation of antigenic variants and the vaccine-type virus during the 2004-2005 influenza B virus epidemics in Japan.[J].Journal of Clinical Microbiology,2009,47(2):352-357.
[23]Nakagawa T,Higashi N,Nakagawa N.Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen.[J].Journal of Virological Methods,2008,148(1-2):296-299.
[24]饶丹,嘎利兵嘎,郭小权,等.HRM分析方法中不同扩增片段、染料和仪器的比较[J].江西农业大学学报,2015,37(1):167-175.
[25]赵均良,张少红,刘斌等.应用高分辨率熔解曲线技术分析水稻分子标记基因型[J].中国农业科学,2011,44(18):3701-3708.
[26]尤崇革,李晓军,郜莉娜,等.高分辨熔解技术常用荧光染料的SNP基因分型能力比较[J].临床检验杂志,2011,29(8):575-577.
[27]温立斌,何孔旺,杨汉春,等.应用高分辨熔点曲线分析区分类猪圆环病毒因子P1和猪圆环病毒2型[J].江苏农业学报,2010,26(2):315-319.
[28]Li J S,Wang X M,Dong R X,et al.Evaluation of highresolution melting for gene mapping in rice[J].Plant Molecular Biology Reporter,2011,29(4):979-985.
[29]Mackay J F,Wright C D,Bonfiglioli RG.A new approach to varietal identification in plants by microsatellite high resolution melting analysis:application to the verification of grapevine and olive cultivars[J].Plant Methods,2008,4(1):8.
[30]Wu S B,Wirthensohn M G,Hunt P,et al.High resolution melting analysis of almond SNPs derived from ESTs[J].Theoretical and Applied Genetics,2008,118(1):1-14.
[31]Yang Z J,Fu L,Zhang G W,et al.Identification and association of SNPs in TBCIDI gene with growth traits in two rabbit breeds[J].Asian-Australasian Journal of Animal Sciences,2013,26(11):1529-1535.
[32]陈康,蒋超,袁媛,等.基于熔解曲线分析技术的鹿茸药材分子鉴别[J].中国中药杂志,2015,40(4):619-623.
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