基于高通量测序开发玉米高效KASP分子标记
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摘要
SNP (Single Nucleotide Polymorphism)在基因组中数量多、分布广,适用于大规模、自动化基因型检测。本研究利用205份不同来源的玉米自交系全基因组重测序数据鉴定出一系列多态性高的二态性SNP位点并开发出700个KASP分子标记。其中, 202个在46个玉米代表系中得到验证的KASP标记进一步用于系统进化树构建及群体结构分析。结果显示,开发成功的KASP标记在染色体上分布均匀,平均PIC为0.463,平均MAF为0.451。基于KASP标记位点和总SNP位点的聚类分析结果高度吻合。KASP标记位点与总SNP位点的遗传距离相似性系数高达89.5%,能成功区分玉米的杂种优势群。该KASP标记可在玉米种质资源分析、连锁群构建以及杂种优势群划分等方面发挥重要作用。
SNP(Single Nucleotide Polymorphism) which is abundant and dispersed widely in the genome is suitable for large-scale and automated genotyping. In this study, highly polymorphic bi-allelic SNP loci were screened and 700 KASP(Kompetitive Allele Specific PCR) molecular markers were developed based on resequencing data of 205 diverse maize inbred lines. Among them, 202 KASP markers validated by 46 representative lines were further used for phylogenetic tree construction and genetic structure analysis. The validated KASP markers distributed evenly on 10 chromosomes in maize with an average PIC of 0.463 and an average MAF of 0.451. The phylogenetic tree constructed by KASP markers is highly consistent with that by re-sequencing data. In addition, the genetic similarity coefficient evaluated between KASP loci and the total SNP loci achieved 89.5% which demonstrated the availability of KASP in heterotic group division. These findings suggest that 202 KASP markers play an important role in analysis of germplasm resource, construction of genetic map, and division of heterotic group in maize.
SNP(Single Nucleotide Polymorphism) which is abundant and dispersed widely in the genome is suitable for large-scale and automated genotyping. In this study, highly polymorphic bi-allelic SNP loci were screened and 700 KASP(Kompetitive Allele Specific PCR) molecular markers were developed based on resequencing data of 205 diverse maize inbred lines. Among them, 202 KASP markers validated by 46 representative lines were further used for phylogenetic tree construction and genetic structure analysis. The validated KASP markers distributed evenly on 10 chromosomes in maize with an average PIC of 0.463 and an average MAF of 0.451. The phylogenetic tree constructed by KASP markers is highly consistent with that by re-sequencing data. In addition, the genetic similarity coefficient evaluated between KASP loci and the total SNP loci achieved 89.5% which demonstrated the availability of KASP in heterotic group division. These findings suggest that 202 KASP markers play an important role in analysis of germplasm resource, construction of genetic map, and division of heterotic group in maize.
引文
[1]赵曦,王荣焕,于永涛,王天宇,黎裕.关联分析在玉米遗传学研究中的应用.玉米科学,2008,16(1):52-55.Zhao X,Wang R H,Yu Y T,Wang T Y,Li Y.Application of association analysis in maize genetics.J Maize Sci,2008,16(1):52-55(in Chinese with English abstract).
[2]简银巧.热带玉米全长泛转录组和基因组大小变异及应用.中国农业科学院博士学位论文,北京,2017.Jian Y Q.Variations in Pan-transcriptome and Genome Size in Tropical Maize(Zea mays L.)and Their Applications.PhD Dissertation of Chinese Academy of Agricultural Sciences,Beijing,China,2017(in Chinese with English abstract).
[3]李向拓,毛建昌,吴权明.分子标记在玉米育种中的应用.玉米科学,2004,12(1):26-29.Li X T,Mao J C,Wu Q M.Molecular markers and maize breeding.J Maize Sci,2004,12(1):26-29(in Chinese with English abstract).
[4]卢柏山,史亚兴,宋伟,徐丽,赵久然.利用SNP标记划分甜玉米自交系的杂种优势类群.玉米科学,2015,23(1):58-62.Lu B S,Shi Y X,Song W,Xu L,Zhao J R.Heterotic grouping of sweet corn inbred lines by SNP markers.J Maize Sci,2015,23(1):58-62(in Chinese with English abstract).
[5]吴金凤,宋伟,王蕊,田红丽,李雪,王风格,赵久然,蔚荣海.利用SNP标记对51份玉米自交系进行类群划分.玉米科学,2014,22(5):29-34.Wu J F,Song W,Wang R,Tian H L,Li X,Wang F G,Zhao J R,Wei R H.Heterotic grouping of 51 maize inbred lines by SNPmarkers.J Maize Sci,2014,22(5):29-34(in Chinese with English abstract).
[6]Jiao Y,Zhao H,Ren L,Song W,Zeng B,Guo J,Wang B,Liu Z,Chen J,Li W,Zhang M,Xie S,Lai J.Genome-wide genetic changes during modern breeding of maize.Nat Genet,2012,46:812-815.
[7]Sun S L,Zhou Y S,Chen J,Shi J P,Zhao H M,Zhao H N,Song W B,Zhang M,Cui Y,Dong X M,Liu H,Ma X X,Jiao Y P,Wang B,Wei X H,Stein J C,Glaubitz J C,Lu F,Yu G L,Liang CZ,Fengler K,Li B L,Rafalski A,Schnable P S,Ware D H,Buckler E S,Lai J S.Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.Nat Genet,2018,50:1289-1295.
[8]宁洽,刘文国,杨伟光,路明.SNP标记在玉米研究上的应用进展.玉米科学,2017,25(1):57-61.Ning Q,Liu W G,Yang W G,Lu M.Progress and application of SNP markers in maize.J Maize Sci,2017,25(1):57-61(in Chinese with English abstract).
[9]Gaudet M,Fara A G,Beritognolo I,Sabatti M.Allele-specific PCR in SNP genotyping.Method Mol Biol,2009,578:415-424.
[10]Huang X Q,Roder M S.Development of SNP assays for genotyping the puroindoline b gene for grain hardness in wheat using pyrosequencing.J Agric Food Chem,2005,53:2070-2075.
[11]Tabone T,Mather D E,Hayden M J.Temperature switch PCR(TSP):robust assay design for reliable amplification and genotyping of SNPs.BMC Genomics,2009,10:580.
[12]Rasheed A,Wen W,Gao F,Zhai S N,Jin H,Liu J D,Guo Q,Zhang Y J,Dreisigacker S,Xia X C,He Z H.Development and validation of KASP assays for genes underpinning key economic traits in bread wheat.Theor Appl Genet,2016,129:1843-1860.
[13]赵久然,李春辉,宋伟,王元东,张如养,王继东,王凤格,田红丽,王蕊.基于SNP芯片揭示中国玉米育种种质的遗传多样性与群体遗传结构.中国农业科学,2018,51:626-634.Zhao J R,Li C H,Song W,Wang Y D,Zhang R Y,Wang J D,Wang F G,Tian H L,Wang R.Genetic diversity and population structure of important Chinese maize breeding germplasm revealed by SNP-chips.Sci Agric Sin,2018,51:626-634(in Chinese with English abstract).
[14]王文斌,徐淑兔,高杰,张兴华,郭东伟,李向阳,薛吉全.基于SNP标记的玉米自交系遗传多样性分析.玉米科学,2015,23(2):41-45.Wang W B,Xu S T,Gao J,Zhang X H,Guo D W,Li X Y,Xue JQ.Analysis of genetic diversity of maize inbred lines based on SNP markers.J Maize Sci,2015,23(2):41-45(in Chinese with English abstract).
[15]Thomson M J.High-throughput SNP genotyping to accelerate crop improvement.Plant Breed Biotechnol,2014,2:195-212.
[16]Wang S C,Wong D B,Forrest K,Allen A,Chao S M,Huang B E,Maccaferri M,Salvi S,Milner S G,Cattivelli L,Mastrangelo A M,Whan A,Stephen S,Barker G,Wieseke R,Plieske J,Lillemo M,Mather D,Appels R,Dolferus R,Brown-Guedira G,Korol A,Akhunova A R,Feuillet C,Salse J,Morgante M,Pozniak C,Luo M C,Dvorak J,Morell M,Dubcovsky J,Ganal M,Tuberosa R,Lawley C,Mikoulitch I,Cavanagh C,Edwards K J,Hayden M,Akhunov E.Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array.Plant Biotechnol,2014,12:787-796.
[17]Semagn K,Babu R,Hearne S,Olsen M.Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR(KASP):overview of the technology and its application in crop improvement.Mol Breed,2014,33:1-14.
[18]Kumpatla S P,Buyyarapu R,Abdurakhmonov I Y,Mammadov JA.Genomics-assisted plant breeding in the 21st century:technological advances and progress.In:Abdurakhmonov I ed.Plant Breeding.Rijeka,Croatia:InTech,2012.pp 131-184.https://www.intechopen.com/books/plant-breeding/genomics-assisted-plantbreeding-in-the-21st-century-technological-advances-and-progress.
[19]吕远大,李坦,石丽,张晓林,赵涵.基于全基因组重测序信息开发玉米H99自交系特异分子标记.作物学报,2014,40:191-197.Lyu Y D,Li T,Shi L,Zhang X L,Zhao H.Next-generation sequencing for molecular marker development in maize inbred H99.Acta Agron Sin,2014,40:191-197(in Chinese with English abstract).
[20]周玲,梁帅强,林峰,吕远大.玉米二态性InDel位点的鉴定和分子标记开发.江苏农业学报,2016,32:1223-1231.Zhou L,Liang S Q,Lin F,Lyu Y D.Bi-allelic InDel loci detection and molecular marker development in maize.Jiangsu J Agric Sci,2016,32:1223-1231(in Chinese with English abstract).
[21]Lyu Y,Liu Y,Zhao H.m InDel:a high-throughput and efficient pipeline for genome-wide InDel marker development.BMC Genomics,2016,17:290.
[22]周玲,梁帅强,吕远大,张体付,戴惠学,赵松涛,赵涵.中国黄淮海地区玉米杂种优势候选位点的鉴定.玉米科学,2017,25(1):15-23.Zhou L,Liang S Q,Lyu Y D,Zhang T F,Dai H X,Zhao S T,Zhao H.Identification of candidate loci associated with maize heterosis in Huang-Huai-Hai region of China.J Maize Sci,2017,25(1):15-23(in Chinese with English abstract).
[23]Saitou N.The neighbor-joining method:a new method for reconstructing phylogenetic trees.Mol Biol Evol,1987,4:406-425.
[24]何中虎,阿韦斯·拉希德,卢家玲,夏先春.基于KASP技术检测小麦功能基因的成套引物及其应用.CN105112546A[P].2015.He Z H,Aves Rashid,Lu J L,Xia X C.Complete set of primers for detecting wheat functional genes and their application based on KASP technology.CN105112546A[P].2015(in Chinese).
[25]余四斌,袁志阳,孙文强,熊银,龚蓉.基于KASP技术用于水稻产量基因分型的引物组合及其应用.CN 106939349 A[P].2017.Yu S B,Yuan Z Y,Sun W Q,Xiong Y,Gong R.Based primer composition and its application technique for rice yield based on KASP genotyping.CN 106939349 A[P].2017(in Chinese).
[2]简银巧.热带玉米全长泛转录组和基因组大小变异及应用.中国农业科学院博士学位论文,北京,2017.Jian Y Q.Variations in Pan-transcriptome and Genome Size in Tropical Maize(Zea mays L.)and Their Applications.PhD Dissertation of Chinese Academy of Agricultural Sciences,Beijing,China,2017(in Chinese with English abstract).
[3]李向拓,毛建昌,吴权明.分子标记在玉米育种中的应用.玉米科学,2004,12(1):26-29.Li X T,Mao J C,Wu Q M.Molecular markers and maize breeding.J Maize Sci,2004,12(1):26-29(in Chinese with English abstract).
[4]卢柏山,史亚兴,宋伟,徐丽,赵久然.利用SNP标记划分甜玉米自交系的杂种优势类群.玉米科学,2015,23(1):58-62.Lu B S,Shi Y X,Song W,Xu L,Zhao J R.Heterotic grouping of sweet corn inbred lines by SNP markers.J Maize Sci,2015,23(1):58-62(in Chinese with English abstract).
[5]吴金凤,宋伟,王蕊,田红丽,李雪,王风格,赵久然,蔚荣海.利用SNP标记对51份玉米自交系进行类群划分.玉米科学,2014,22(5):29-34.Wu J F,Song W,Wang R,Tian H L,Li X,Wang F G,Zhao J R,Wei R H.Heterotic grouping of 51 maize inbred lines by SNPmarkers.J Maize Sci,2014,22(5):29-34(in Chinese with English abstract).
[6]Jiao Y,Zhao H,Ren L,Song W,Zeng B,Guo J,Wang B,Liu Z,Chen J,Li W,Zhang M,Xie S,Lai J.Genome-wide genetic changes during modern breeding of maize.Nat Genet,2012,46:812-815.
[7]Sun S L,Zhou Y S,Chen J,Shi J P,Zhao H M,Zhao H N,Song W B,Zhang M,Cui Y,Dong X M,Liu H,Ma X X,Jiao Y P,Wang B,Wei X H,Stein J C,Glaubitz J C,Lu F,Yu G L,Liang CZ,Fengler K,Li B L,Rafalski A,Schnable P S,Ware D H,Buckler E S,Lai J S.Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.Nat Genet,2018,50:1289-1295.
[8]宁洽,刘文国,杨伟光,路明.SNP标记在玉米研究上的应用进展.玉米科学,2017,25(1):57-61.Ning Q,Liu W G,Yang W G,Lu M.Progress and application of SNP markers in maize.J Maize Sci,2017,25(1):57-61(in Chinese with English abstract).
[9]Gaudet M,Fara A G,Beritognolo I,Sabatti M.Allele-specific PCR in SNP genotyping.Method Mol Biol,2009,578:415-424.
[10]Huang X Q,Roder M S.Development of SNP assays for genotyping the puroindoline b gene for grain hardness in wheat using pyrosequencing.J Agric Food Chem,2005,53:2070-2075.
[11]Tabone T,Mather D E,Hayden M J.Temperature switch PCR(TSP):robust assay design for reliable amplification and genotyping of SNPs.BMC Genomics,2009,10:580.
[12]Rasheed A,Wen W,Gao F,Zhai S N,Jin H,Liu J D,Guo Q,Zhang Y J,Dreisigacker S,Xia X C,He Z H.Development and validation of KASP assays for genes underpinning key economic traits in bread wheat.Theor Appl Genet,2016,129:1843-1860.
[13]赵久然,李春辉,宋伟,王元东,张如养,王继东,王凤格,田红丽,王蕊.基于SNP芯片揭示中国玉米育种种质的遗传多样性与群体遗传结构.中国农业科学,2018,51:626-634.Zhao J R,Li C H,Song W,Wang Y D,Zhang R Y,Wang J D,Wang F G,Tian H L,Wang R.Genetic diversity and population structure of important Chinese maize breeding germplasm revealed by SNP-chips.Sci Agric Sin,2018,51:626-634(in Chinese with English abstract).
[14]王文斌,徐淑兔,高杰,张兴华,郭东伟,李向阳,薛吉全.基于SNP标记的玉米自交系遗传多样性分析.玉米科学,2015,23(2):41-45.Wang W B,Xu S T,Gao J,Zhang X H,Guo D W,Li X Y,Xue JQ.Analysis of genetic diversity of maize inbred lines based on SNP markers.J Maize Sci,2015,23(2):41-45(in Chinese with English abstract).
[15]Thomson M J.High-throughput SNP genotyping to accelerate crop improvement.Plant Breed Biotechnol,2014,2:195-212.
[16]Wang S C,Wong D B,Forrest K,Allen A,Chao S M,Huang B E,Maccaferri M,Salvi S,Milner S G,Cattivelli L,Mastrangelo A M,Whan A,Stephen S,Barker G,Wieseke R,Plieske J,Lillemo M,Mather D,Appels R,Dolferus R,Brown-Guedira G,Korol A,Akhunova A R,Feuillet C,Salse J,Morgante M,Pozniak C,Luo M C,Dvorak J,Morell M,Dubcovsky J,Ganal M,Tuberosa R,Lawley C,Mikoulitch I,Cavanagh C,Edwards K J,Hayden M,Akhunov E.Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array.Plant Biotechnol,2014,12:787-796.
[17]Semagn K,Babu R,Hearne S,Olsen M.Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR(KASP):overview of the technology and its application in crop improvement.Mol Breed,2014,33:1-14.
[18]Kumpatla S P,Buyyarapu R,Abdurakhmonov I Y,Mammadov JA.Genomics-assisted plant breeding in the 21st century:technological advances and progress.In:Abdurakhmonov I ed.Plant Breeding.Rijeka,Croatia:InTech,2012.pp 131-184.https://www.intechopen.com/books/plant-breeding/genomics-assisted-plantbreeding-in-the-21st-century-technological-advances-and-progress.
[19]吕远大,李坦,石丽,张晓林,赵涵.基于全基因组重测序信息开发玉米H99自交系特异分子标记.作物学报,2014,40:191-197.Lyu Y D,Li T,Shi L,Zhang X L,Zhao H.Next-generation sequencing for molecular marker development in maize inbred H99.Acta Agron Sin,2014,40:191-197(in Chinese with English abstract).
[20]周玲,梁帅强,林峰,吕远大.玉米二态性InDel位点的鉴定和分子标记开发.江苏农业学报,2016,32:1223-1231.Zhou L,Liang S Q,Lin F,Lyu Y D.Bi-allelic InDel loci detection and molecular marker development in maize.Jiangsu J Agric Sci,2016,32:1223-1231(in Chinese with English abstract).
[21]Lyu Y,Liu Y,Zhao H.m InDel:a high-throughput and efficient pipeline for genome-wide InDel marker development.BMC Genomics,2016,17:290.
[22]周玲,梁帅强,吕远大,张体付,戴惠学,赵松涛,赵涵.中国黄淮海地区玉米杂种优势候选位点的鉴定.玉米科学,2017,25(1):15-23.Zhou L,Liang S Q,Lyu Y D,Zhang T F,Dai H X,Zhao S T,Zhao H.Identification of candidate loci associated with maize heterosis in Huang-Huai-Hai region of China.J Maize Sci,2017,25(1):15-23(in Chinese with English abstract).
[23]Saitou N.The neighbor-joining method:a new method for reconstructing phylogenetic trees.Mol Biol Evol,1987,4:406-425.
[24]何中虎,阿韦斯·拉希德,卢家玲,夏先春.基于KASP技术检测小麦功能基因的成套引物及其应用.CN105112546A[P].2015.He Z H,Aves Rashid,Lu J L,Xia X C.Complete set of primers for detecting wheat functional genes and their application based on KASP technology.CN105112546A[P].2015(in Chinese).
[25]余四斌,袁志阳,孙文强,熊银,龚蓉.基于KASP技术用于水稻产量基因分型的引物组合及其应用.CN 106939349 A[P].2017.Yu S B,Yuan Z Y,Sun W Q,Xiong Y,Gong R.Based primer composition and its application technique for rice yield based on KASP genotyping.CN 106939349 A[P].2017(in Chinese).