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作物学报 ›› 2021, Vol. 47 ›› Issue (4): 770-779.doi: 10.3724/SP.J.1006.2021.03031

• 研究简报 • 上一篇    下一篇

玉米杂交种纯度鉴定SNP核心引物的确定及高通量检测方案的建立

王蕊1(), 施龙建1,2(), 田红丽1, 易红梅1, 杨扬1, 葛建镕1, 范亚明1, 任洁1, 王璐1, 陆大雷2, 赵久然1,*(), 王凤格1,*()   

  1. 1北京市农林科学院玉米研究中心 / 玉米DNA指纹及分子育种北京市重点研究室, 北京 100097
    2扬州大学 / 江苏省作物遗传生理国家重点实验室培育点 / 粮食作物现代产业技术协同创新中心, 江苏扬州 225009
  • 收稿日期:2020-06-14 接受日期:2020-10-14 出版日期:2021-04-12 网络出版日期:2020-11-19
  • 通讯作者: 赵久然,王凤格
  • 作者简介:王蕊, E-mail: skywangr@126.com;|施龙建, E-mail: SLJ18752782386@163.com
  • 基金资助:
    “十三五”国家重点研发计划项目(2017YFD0102001)

Identification of SNP core primer and establishment of high throughput detection scheme for purity identification in maize hybrids

WANG Rui1(), SHI Long-Jian1,2(), TIAN Hong-Li1, YI Hong-Mei1, YANG Yang1, GE Jian-Rong1, FAN Ya-Ming1, REN Jie1, WANG Lu1, LU Da-Lei2, ZHAO Jiu-Ran1,*(), WANG Feng-Ge1,*()   

  1. 1Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097, China
    2Jiangsu Key Laboratory of Crop Genetics and Physiology / Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
  • Received:2020-06-14 Accepted:2020-10-14 Published:2021-04-12 Published online:2020-11-19
  • Contact: ZHAO Jiu-Ran,WANG Feng-Ge
  • Supported by:
    13th Five-Year National Key Research and Development Program of China(2017YFD0102001)

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摘要：

纯度是玉米种子质量的重要指标之一, 尤其杂交种自交株是影响田间产量的关键因素。KASP (kompetitive allele specific PCR)技术具有高通量、低成本的优点, 适用于种子纯度检测。本研究基于12套杂交种及其父母本的三联体样本及335份玉米杂交种国家审定标准样品SNP指纹, 从384个SNP基础位点筛选获得60个候选位点, 位点转化为KASP引物的成功率为95%。综合考虑引物双亲互补率、多态性、稳定性和分型效果等多项指标, 最终确定20个引物作为玉米杂交种纯度鉴定的核心引物, 能够有效鉴定99.7%供试样品纯度。对于待测样品京科968通过SNP-DNA指纹数据库查询, 并选择双亲互补型引物进行纯度鉴定。在检测的110个个体中, 共检出1个自交苗和2个异型株, 纯度为97.3%。同时, 基于纯度核心引物对批量样品检测建立高通量纯度检测方案, 具有快捷、准确、高通量和低成本的特点, 为政府监管和企业提供了更多纯度鉴定方案的选择。

关键词: 纯度, SNP, KASP, 玉米杂交种, 核心引物

Abstract:

Purity is one of the most important indexes of maize seed quality, especially for inbred seedlings in hybrids which is a key factor affecting field yield. A new high-throughput and economical technology named KASP (Kompetitive allele specific PCR) is suitable for seed purity detection. This study was based on 12 sets of triplet samples of hybrids with their parents and 335 SNP fingerprint data which was nationally approved as standard samples of maize hybrids. Sixty candidate loci were selected from 384 SNP basic loci, and the success rate of transformation from chip to KASP primers was 95%. Considering comprehensive elements including parental complementary rate, polymorphism and other indicators such as stability and genotyping effect of primers, 20 core primers were finally determined for purity identification of maize hybrids. The results showed these core primers were able to effectively identify the purity of 99.7% of tested samples. The purity of tested sample Jingke 968 was identified by searching SNP-DNA fingerprint database and selecting parental complementary primers. One inbred seedling and two off-types were detected in 110 individuals, and the purity value was 97.3%. Meanwhile, a high-throughput purity detection scheme was established based on the core primers of purity identification for batch samples, which is fast, accurate, high-throughput and low-cost, providing more options of purity identification for government regulatory agencies and enterprises.

Key words: purity, SNP, KASP, maize hybrid, core primers

表1

玉米三联体样品信息表"

编号
No.		 杂交种
Hybrid		 母本
Maternal parent		 父本
Paternal parent
1 先玉335 Xianyu 335 PH6WC PH4CV
2 郑单958 Zhengdan 958 郑58 Zheng 58 昌7-2 Chang 7-2
3 农大108 Nongda 108 X178 黄C Huang C
4 鲁单981 Ludan 981 齐319 Qi 319 Lx 9801
5 沈单16 Shendan 16 K12 沈137 Shen 137
6 东单60 Dongdan 60 A801 丹598 Dan 598
7 丹玉39 Danyu 39 C8605-2 丹598 Dan 598
8 中单2号 Zhongdan 2 Mo17 自330 Zi 330
9 掖单13 Yedan 13 掖478 Ye 478 丹340 Dan 340
10 SC704 B73 Mo17
11 京科糯2000 Jingkenuo 2000 京糯6 Jingnuo 6 白糯6 Bainuo 6
12 农华101 Nonghua 101 NH60 S121

表2

玉米杂交种纯度鉴定候选引物表"

引物编号
Primer ID		 引物名称
Primer name		 等位变异
Allelic variation		 引物序列
Primer sequence (5'-3')
MSNP01 MG002 A/G F1: GGAACTGTGCGTAGTTCCTGAGA
F2: GAACTGTGCGTAGTTCCTGAGG
R: TGATCGAGGAGGACCCCTGCAA
MSNP02 MG013 A/G F1: CCGGAGCTGGCTTCTGAATCAA
F2: CGGAGCTGGCTTCTGAATCAG
R: ACATCCCCATCCTGGGGAGGAA
MSNP03 MG016 T/C F1: GAGCGTCAACGACAAAGCCAAGT
F2: AGCGTCAACGACAAAGCCAAGC
R: CCTGTTAGGTTGTAAGAATTGAGCCTTAT
MSNP04 MG035 A/G F1: GGAGATGCTCTACAAGTTTGTCA
F2: GGAGATGCTCTACAAGTTTGTCG
R: CCTTCGAGGAGGCCAAGGACTT
MSNP05 MG046 A/G F1: CCATGGTTTTAAGGAACTATCGAAAGA
F2: CATGGTTTTAAGGAACTATCGAAAGG
R: ATCAATGTACTCCCATAAGCAGCAACTTT
MSNP06 MG049 C/T F1: CCGTATCATTTAGTGCATCAGAACC
F2: CCGTATCATTTAGTGCATCAGAACT
R: GAGAAATGCAATGCATCAGATCCGGAT
MSNP07 MG055 A/G F1: CATCGAATGGCTGATCATGTTGCAT
F2: ATCGAATGGCTGATCATGTTGCAC
R: CCATTCCCTGTATGACAGACACGAT
MSNP08 MG059 C/T F1: GCCCCTGCATTGTTTGCAGC
F2: CTGCCCCTGCATTGTTTGCAGT
R: GCCACTGCAAATCCAAAGAATCCGTA
MSNP09 MG064 G/T F1: GACTAAACACACTCACTTTCCTGC
F2: CTGACTAAACACACTCACTTTCCTGA
R: AATCGGTGATGAGTGAGTGTCTAACTAAA
MSNP10 MG066 A/C F1: AGGATCACAATCCATCTGCTGCAAA
F2: GATCACAATCCATCTGCTGCAAC
R: CCTGCAGTTGCTACTGATAGTTCTCAA
MSNP11 MG067 A/G F1: ATGTTTCTCAGGACGGTAATAGTGAT
F2: GTTTCTCAGGACGGTAATAGTGAC
R: CCCATCCATTCCACATATTCGGCAA
MSNP12 MG072 A/C F1: GAGTTGTGCTCTGATCCACCCT
F2: AGTTGTGCTCTGATCCACCCG
R: TATTGCGGCATTAAACAAGGGAAAGGAAA
MSNP13 MG088 C/T F1: CGGCCTCCATGCTTGATGATG
F2: CCGGCCTCCATGCTTGATGATA
R: GTCGGTCGAGTCAAAATTCATTTTGGAT
MSNP14 MG092 C/T F1: ACGTCTGACTTGCACGAAACGG
F2: GACGTCTGACTTGCACGAAACGA
R: CTCCGGAGATGATCTCCCGTAATTT
MSNP15 MG094 G/T F1: ATTCGAACTGCTGCCTTGACTAATC
F2: ATTCGAACTGCTGCCTTGACTAATA
R: GCAGCAGTGACTATCCTTCTGAAGAA
MSNP16 MG101 A/C F1: CAATTCCTCCCCTGCAATTTCAACAA
F2: AATTCCTCCCCTGCAATTTCAACAC
R: CAGCCTGGTCGTTGCTTCTGTAATT
MSNP17 MG106 C/T F1: CCAATCAAGGCGGCAACATACC
F2: ACCAATCAAGGCGGCAACATACT
R: GCGTTCATGTTCATGGAAGGCCAAA
MSNP18 MG111 A/G F1: ATTCTGAATGTAAAACTTAACATGCTGCTA
F2: CTGAATGTAAAACTTAACATGCTGCTG
R: AAGTCCTTCCAACTTTCAGCATAAGCAAA
MSNP19 MG134 A/C F1: GGTTACACGACCAAATGAGTACCAT
F2: GTTACACGACCAAATGAGTACCAG
R: TAGGCAGAGCAGCCATTGACAAGTA
MSNP20 MG135 G/T F1: CGCATCCTAATAACATAATTACTCACG
F2: ACGCATCCTAATAACATAATTACTCACT
R: GCGAAACGGGGTGTTAGATAGAGTT
引物编号
Primer ID		 引物名称
Primer name		 等位变异
Allelic variation		 引物序列
Primer sequence (5'-3')
MSNP21 MG143 G/T F1: TATCACTTGTGGATCTATATCTGTG
F2: GCTTATCACTTGTGGATCTATATCTGTT
R: TGAACCCAAAGCCTCGGTGTTCTTT
MSNP22 MG148 G/T F1: AGCTTAGCAGAGCTGCATCTG
F2: CTAGCTTAGCAGAGCTGCATCTT
R: CCCACGTCACCTAGATAAGCCAAAA
MSNP23 MG155 A/G F1: ATACAGTGAAACAGCTTGCACTGGA
F2: CAGTGAAACAGCTTGCACTGGG
R: TTAATTTTTGGAAGAGCTTGCGTTGGGAA
MSNP24 MG157 A/G F1: AGTTACCTGTCATCGATCTCTGGAT
F2: ACCTGTCATCGATCTCTGGAC
R: AAAGCCCTCTGACAATGCTCCAGTA
MSNP25 MG175 C/T F1: GATATTTCTGCAACTAAACATGGCAAG
F2: GGATATTTCTGCAACTAAACATGGCAAA
R: ATACTGGGGTTGTGGGGATAGGATT
MSNP26 MG181 A/G F1: CCTCTGTAAGCGCAGTACTGGT
F2: CTCTGTAAGCGCAGTACTGGC
R: AAATTGCTATGCAAACAGGTTCTGGAGTA
MSNP27 MG186 C/T F1: GAGCTAGTAAATATTGTTGTTGTTCCTC
F2: AGAGCTAGTAAATATTGTTGTTGTTCCTT
R: CGCCGACGGGACGACGGAT
MSNP28 MG191 A/G F1: CATAAACAGTAGGTTTATCGCTGACATAA
F2: AAACAGTAGGTTTATCGCTGACATAG
R: GTGATAACCGATGCAAAATGCTGCTTAAT
MSNP29 MG195 C/T F1: CCAAAGGATAGCACATCTTGGTG
F2: GTCCAAAGGATAGCACATCTTGGTA
R: TGTCAACCGCATCCTGGCAGATAAT
MSNP30 MG207 A/G F1: ACTTCTCCATCCTCTTCCAACATATTA
F2: CTTCTCCATCCTCTTCCAACATATTG
R: AGCTGTCCACCATCAGTACTGGAAT
MSNP31 MG215 T/C F1: AGATGGCATTGTGATCTGTGCACA
F2: GATGGCATTGTGATCTGTGCACG
R: AGCCGAAGGATTGATCCTCCTCAT
MSNP32 MG216 G/T F1: GACGACGACTCCATCGTGACC
F2: GACGACGACTCCATCGTGACA
R: TCAACCCATGGCTGCTCACATGTAA
MSNP33 MG221 G/T F1: GGCATTCTGATTTGACAGCCCAC
F2: GGCATTCTGATTTGACAGCCCAA
R: TCCTGATTCTGTACTTGATTGGACCAAA
MSNP34 MG230 C/T F1: GCAGCTGAGAAACAATTGCAAAGTG
F2: GCAGCTGAGAAACAATTGCAAAGTA
R: GTACTCTCAGATGGTTTTGTGACATCAA
MSNP35 MG236 C/T F1: GTGCTCGAACGAATCGACCAG
F2: CGTGCTCGAACGAATCGACCAA
R: CATCCATGGCGAAGCTCATGAACAA
MSNP36 MG240 G/T F1: GAAACATGAATGCCCTAAATCCTTCG
F2: AGAAACATGAATGCCCTAAATCCTTCT
R: ATTATGTTCACCAAGTATCCAGATGGCAT
MSNP37 MG262 G/T F1: GTAGCGTGTCTCTACGCTCTG
F2: ATGTAGCGTGTCTCTACGCTCTT
R: CAGCGCGTTACGACGAACTCCAA
MSNP38 MG270 A/C F1: GGTTCCATGGCTACCTGACAAGT
F2: GTTCCATGGCTACCTGACAAGG
R: TAGGAGCTAGCCAAGAGCCTACTA
MSNP39 MG271 A/G F1: CAGCGACCTCAAGAAGTTGAAGTAA
F2: AGCGACCTCAAGAAGTTGAAGTAG
R: GTACGACATGCAGTTTGACATCAAGTAT
MSNP40 MG277 A/G F1: GAAGCTACTATTAGCAATGATCTATATGAT
F2: AAGCTACTATTAGCAATGATCTATATGAC
R: ACAGGATTGATAAACATTACCTGCAGGAA
MSNP41 MG278 A/G F1: GATCGTTGTCTTCACAAATGAAGAATAGT
F2: CGTTGTCTTCACAAATGAAGAATAGC
R: GCGAGATATTGAAAGCTAGTGGTGCTA
引物编号
Primer ID.		 引物名称
Primer name		 等位变异
Allelic variation		 引物序列
Primer sequence (5'-3')
MSNP42 MG279 A/G F1: AACGTATGAGATGAACTCACCAGAAA
F2: ACGTATGAGATGAACTCACCAGAAG
R: CTCCGCCGCTGGTGGAGCTA
MSNP43 MG288 A/C F1: GAACTAACTGAGTGTTAAAGGAGCTTAT
F2: AACTAACTGAGTGTTAAAGGAGCTTAG
R: CCTTGACACAACCGCTCTCCTTAAA
MSNP44 MG292 A/G F1: AACCATTCCCTTCATACTTCTTCTCT
F2: ACCATTCCCTTCATACTTCTTCTCC
R: GGGAGTATCTTTTAGGAAGATGTACAGAT
MSNP45 MG293 A/C F1: GACCTGAAATGCTTGGCGAGTCA
F2: ACCTGAAATGCTTGGCGAGTCC
R: GCAGGAGCCTTAGCGTGGCTAT
MSNP46 MG298 A/G F1: CAATCCAAAGCAGAAAGAAGTTGTTCT
F2: AATCCAAAGCAGAAAGAAGTTGTTCC
R: CCAAAACAGTGAAGTGACCGCCAT
MSNP47 MG304 C/T F1: CAAAGTGGTGTAAATGGATGGATCG
F2: CAAAGTGGTGTAAATGGATGGATCA
R: TTGGACACTCCAGGGGATCCTATA
MSNP48 MG311 C/T F1: CCTCAGATCTCATCTATGCTGCC
F2: CCTCAGATCTCATCTATGCTGCT
R: CGTTTCCACATTTTCTGAAGGTTTCACAA
MSNP49 MG328 A/G F1: GCCTCACACATCCATATACGTAGAA
F2: CCTCACACATCCATATACGTAGAG
R: CTTCCATGCATCGCCCTATGGATAT
MSNP50 MG331 C/T F1: AACACTCATGTCTGCTCCAGGG
F2: AAACACTCATGTCTGCTCCAGGA
R: TCGATGTTTTCGATCCCAAGTTCAACATT
MSNP51 MG334 G/T F1: CCACTTCTGCTCGTATGATCTTC
F2: GCCACTTCTGCTCGTATGATCTTA
R: TCCCGTAATCATCTGCTCGTCTGTA
MSNP52 MG347 A/G F1: AACACGAGCTGGTTGATGGATTAGT
F2: CACGAGCTGGTTGATGGATTAGC
R: GCCTCTGGTACGTTAGTTTGCAGTT
MSNP53 MG349 A/G F1: TACTGACCGAGCGATGCTGCT
F2: CTGACCGAGCGATGCTGCC
R: CGCTGATGGTCACAGAAACATCGTT
MSNP54 MG353 A/G F1: ATACCCTCTCCACCAGTTGTTGAT
F2: ACCCTCTCCACCAGTTGTTGAC
R: TCGCAGGGAGGCGTCGTTCAA
MSNP55 MG356 A/C F1: AAAAGTGCAGTTCCTTGCTGTTCATTT
F2: AAGTGCAGTTCCTTGCTGTTCATTG
R: CCCAATGAGCAAAAAGAATAGCACCAAA
MSNP56 MG361 C/T F1: GAAGCAATCCTTCCGGAGGAATG
F2: GAAGCAATCCTTCCGGAGGAATA
R: GAATGTGCAGATTGGATTTGAGGGATAAA
MSNP57 MG364 A/G F1: TGTTCCGAATAGCAAGTGATCTCTTT
F2: GTTCCGAATAGCAAGTGATCTCTTC
R: GGGAAACCTGCAGAATGCTGTTGAT
MSNP58 MG369 A/G F1: GGTTGACATGAGACTTGCAGAGA
F2: GGTTGACATGAGACTTGCAGAGG
R: TCGGGAAGCCATACTTCACATGCAT
MSNP59 MG371 G/T F1: CAAGTGCGCAGCAAGCCAAAAG
F2: AAACAAGTGCGCAGCAAGCCAAAAT
R: CCGTTCTTAAGCGCTCCATCCTTTT
MSNP60 MG382 C/T F1: CACGAAGCTCTCGCGCTCTTC
F2: CACGAAGCTCTCGCGCTCTTT
R: GGCATGGAGCCCCTATCCTTGAT

图1

测试引物分型效果图 A, B, C, F为CTAB法分型效果; D, E为快提法分型效果。A, D为引物MG279, 在CTAB法和快提法均具有清晰、紧密的分型效果; B, E为引物MG304, 该位点使用CTAB法能够正常分型, 但快提法无法正常分型; C为引物MG262, KASP引物分型效果为单态; F为引物MG067, 分型效果较散且数据缺失率较高。图中每1个点代表1个反应孔的结果, 其中蓝色和红色点分别代表2种纯合基因型, 绿色点代表双亲互补基因型, 粉色点代表未采集基因分型, 黑色点代表空白对照。"

表3

玉米杂交种纯度鉴定SNP核心引物信息表"

引物编号
Primer ID		 引物名称
Primer name		 染色体
Chromosome		 染色体位置
Chromosome position		 双亲互补率
Parental complementary
rate (%)		 最小等位
基因频率
MAF		 多态信息
含量
PIC
MSNP01 MG002 1 8974336 51.1 0.50 0.38
MSNP06 MG049 1 289408285 58.0 0.46 0.37
MSNP09 MG064 2 109563976 55.8 0.34 0.35
MSNP12 MG072 2 186339948 53.6 0.47 0.37
MSNP14 MG092 3 27775731 62.3 0.47 0.37
MSNP16 MG101 3 118083714 65.6 0.49 0.38
MSNP20 MG135 4 20077010 51.6 0.39 0.36
MSNP22 MG148 4 128874466 46.9 0.33 0.35
MSNP25 MG175 5 13402375 53.6 0.45 0.37
MSNP30 MG207 5 204879476 53.7 0.39 0.36
MSNP31 MG215 6 39822979 62.4 0.50 0.38
MSNP36 MG240 6 141476300 51.1 0.43 0.37
MSNP38 MG270 7 126677146 47.5 0.33 0.35
MSNP42 MG279 7 155821714 50.3 0.42 0.37
MSNP45 MG293 8 20978845 51.4 0.45 0.37
MSNP48 MG311 8 118299376 56.5 0.36 0.35
MSNP52 MG347 9 104695670 60.4 0.42 0.37
MSNP53 MG349 9 127197714 61.5 0.50 0.38
MSNP56 MG361 10 39960289 57.6 0.48 0.38
MSNP58 MG369 10 109396730 53.3 0.50 0.38

图2

样品双亲互补基因型信息统计图 A为样品双亲互补基因型比率比较图, 横坐标为按照核心引物的样品双亲互补基因型比率从低到高的样品排序, 纵坐标为样品双亲互补基因型比率; B为样品双亲互补基因型比率柱状统计图, 柱状图表示样品双亲互补基因型比率每增加10%区间的样品数。"

图3

待测样品京科968纯度鉴定分型图(快速提取法, 引物MG072) 图中每1个点代表1个单株的检测结果, 其中蓝色和红色点分别代表2种纯合基因型, 绿色点代表双亲互补基因型, 黑色点代表空白对照, 三角形点代表亲本自交系样品。"

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