玉米自交系InDel标记现场检测技术的研发与应用

doi:10.3864/j.issn.0578-1752.2026.13.002

中国农业科学 ›› 2026, Vol. 59 ›› Issue (13): 2776-2788.doi: 10.3864/j.issn.0578-1752.2026.13.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

玉米自交系InDel标记现场检测技术的研发与应用

王蕊¹(), 胡丽萍¹(), 赵伟¹, 刘志浩¹, 张茗起¹, 卿香玉¹, 许理文¹, 霍永学¹, 葛建镕¹, 田红丽¹, 易红梅¹, 刘亚维¹, 江彬², 吴明生³, 匡猛⁴, 王凤格¹()

¹ 北京市农林科学院玉米研究所/农业农村部农作物DNA指纹创新利用重点实验室（部省共建）/玉米DNA指纹及分子育种北京市重点实验室，北京 100097
² 深圳市纬星软件有限公司，广东深圳 518000
³ 北京市种子管理站，北京 100044
⁴ 三亚中国农业科学院国家南繁研究院，海南三亚 572024

收稿日期:2025-11-24 接受日期:2026-02-20 出版日期:2026-07-01 发布日期:2026-07-01
通信作者:
王凤格，E-mail：gege0106@163.com
联系方式: 王蕊，E-mail：wangrui@baafs.net.cn。胡丽萍，E-mail：hulipinguo@foxmail.com。王蕊和胡丽萍为同等贡献作者。
基金资助:
北京市农林科学院科技创新能力建设专项(KJCX20230301)

Development and Application of InDel Marker Detection Technology for Field Identification of Maize Inbred Lines

WANG Rui¹(), HU LiPing¹(), ZHAO Wei¹, LIU ZhiHao¹, ZHANG MingQi¹, QING XiangYu¹, XU LiWen¹, HUO YongXue¹, GE JianRong¹, TIAN HongLi¹, YI HongMei¹, LIU YaWei¹, JIANG Bin², WU MingSheng³, KUANG Meng⁴, WANG FengGe¹()

¹ Maize Research Institute, Beijing Academy of Agriculture and Forestry Sciences/Key Laboratory for Innovative Application of Crop DNA Fingerprinting, Ministry of Agriculture and Rural Affairs (Jointly Established by the Ministry and the Province)/Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097
² Shenzhen Weixing Software Co., Ltd., Shenzhen 518000, Guangdong
³ Beijing Seed Management Station, Beijing 100044
⁴ National Nanfan Research Institute (Sanya), Chinese Academy of Agricultural Sciences, Sanya 572024, Hainan

Received:2025-11-24 Accepted:2026-02-20 Published:2026-07-01 Online:2026-07-01

摘要/Abstract

摘要：

【目的】品种鉴定是保障农业生产安全和维护市场秩序的关键技术手段。现行作物品种分子鉴定技术主要依赖于实验室专业设备，存在检测周期长、成本较高等局限性。开发玉米品种真实性现场快速检测（maize point-of-genuineness identification，M-POG）试剂盒，旨在推动品种鉴定从实验室向生产一线延伸，填补现有技术中快速检测方案的空白，助力构建快速、高效、可溯源的现代种业检测技术体系。【方法】基于MaizeIDP50K芯片中的优异InDel位点池，利用二分法结合动态规划算法筛选优异候选位点；以9份玉米骨干自交系及其3份近似样品为材料，分别在荧光定量PCR平台和荧光毛细管电泳平台设计显性引物和共显性引物进行评估，利用最优组合算法，筛选获得M-POG核心位点集。基于核心标记显性引物的Ct值，建立M-POG身份条形码转换机制，并利用2 270份玉米自交系核心位点芯片指纹设定现场检测结果判定阈值。以国家标准鉴定方案为对照，评估其一致性和单样品检测效率，以验证该方法的准确性和实用性。【结果】共筛选到80个InDel候选位点，成功设计76对显性引物，得到31个实时荧光定量PCR分型曲线清晰、重复性好、扩增效率均一的优异位点，最终获得16个高区分力M-POG核心位点，在2 270份自交系中品种识别率达95.83%。计算得到指纹转换阈值，当Ct<28时判定为显性态（记为1），当Ct>30时判定为隐性态（记为0），从而获得现场检测DNA条形码。通过2 270份自交系两两比对分析，差异位点数≥4的组数占比98.29%。利用16个核心位点对骨干自交系及近似样品进行比对，90.91%比对结果在本方法和SSR分子标记方法中均被判定为存在差异，说明使用本方案鉴定为有差异的结果可靠，可用于自交系问题品种的快速排查，从而大幅减少需要送往实验室检测的样品数量，其完整检测流程仅需42 min，仅为标准鉴定流程的1/4。【结论】获得16个用于M-POG的InDel核心位点，其品种识别率超过95%；建立了基于InDel现场检测结果的转换机制，确定了品种区分的最小差异位点数判定标准；研发出一套准确性高、速度快的玉米品种现场检测试剂盒，实现作物品种现场快速鉴定。

关键词: 品种鉴定, 玉米, InDel标记, 现场检测, 核酸检测技术, 实时荧光定量PCR

Abstract:

【Objective】Variety identification is vital for the security of agricultural production and integrity of seed markets. Current molecular approaches for identification of crop varieties predominantly require specialized laboratory equipment, resulting in relatively slow turnaround times and high costs. The Maize Point-of-Genuineness (M-POG) identification kit aims to extend variety identification from the laboratory to the field, address the lack of rapid on-site detection methods, and support the establishment of a rapid, efficient, and traceable molecular detection system for the modern seed industry. 【Method】Candidate InDel loci from the MaizeIDP50K chip were screened using a stepwise dichotomous partitioning method in combination with a dynamic optimization algorithm. Nine maize elite inbred lines and three closely related maize varieties were used to design dominant and co-dominant primers, which were evaluated by conducting quantitative real-time PCR and fluorescence capillary electrophoresis. An optimal combination algorithm was used to screen and determine the core set of loci. Based on the cycle threshold (Ct) value for the dominant primer for the core markers, a barcode conversion mechanism suitable for field detection was established. The detection thresholds were set using the core loci fingerprints of 2 270 maize inbred lines. The consistency and single sample detection efficiency were evaluated by comparison with the traditional method for variety identification using simple sequence repeat (SSR) markers, thereby verifying the accuracy and practicability of the M-POG method. 【Result】Eighty InDel candidate loci from the chip were screened for which 76 pairs of dominant primers were designed. Among these loci, 31 high quality loci showed clear qPCR amplification curves, high repeatability, and uniform amplification efficiency. Ultimately, 16 highly discriminative core loci were identified, achieving a variety recognition rate of 95.83% across the 2 270 inbred lines. The fingerprint detection threshold for each locus was calculated; loci with Ct<28 were classified as dominant type (coded as 1), and those with Ct>30 were classified as recessive type (coded as 0), enabling generation of a DNA fingerprinting for M-POG detection. Pairwise comparisons among the 2 270 inbred lines revealed that 98.29% of the pairs of lines differed at four or more loci. A comparative analysis between the M-POG method and the SSR molecular marker method, conducted using elite inbred lines and closely related varieties, showed that the samples were consistently discriminated by both methods in 90.91% of the comparisons. This result demonstrated the reliability of the M-POG method. Thus, the M-POG approach enables efficient screening of genetically dissimilar inbred lines, thereby substantially reducing the number of samples requiring further laboratory analysis. The complete field detection process required only 42 min, approximately one-quarter of the time required by standard identification procedures. 【Conclusion】Sixteen core InDel loci were selected for the M-POG method, achieving a variety recognition rate exceeding 95%. Based on the InDel on-site detection results, the minimum number of differential loci required for variety discrimination was determined. Development of the M-POG kit enables rapid and accurate identification of maize inbred lines in the field.

Key words: variety identification, maize, InDel marker, field test, nucleic acid detection technology, quantitative real-time PCR

王蕊, 胡丽萍, 赵伟, 刘志浩, 张茗起, 卿香玉, 许理文, 霍永学, 葛建镕, 田红丽, 易红梅, 刘亚维, 江彬, 吴明生, 匡猛, 王凤格. 玉米自交系InDel标记现场检测技术的研发与应用[J]. 中国农业科学, 2026, 59(13): 2776-2788.

WANG Rui, HU LiPing, ZHAO Wei, LIU ZhiHao, ZHANG MingQi, QING XiangYu, XU LiWen, HUO YongXue, GE JianRong, TIAN HongLi, YI HongMei, LIU YaWei, JIANG Bin, WU MingSheng, KUANG Meng, WANG FengGe. Development and Application of InDel Marker Detection Technology for Field Identification of Maize Inbred Lines[J]. Scientia Agricultura Sinica, 2026, 59(13): 2776-2788.

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样品 Sample	C-KT12			C-KT16			C-KT20
样品 Sample	共显性- 等位基因 Co-dominant alleles	显性-Ct值 Dominant-Ct value	现场检测DNA指纹 On-site DNA fingerprinting	共显性- 等位基因 Co-dominant alleles	显性-Ct值 Dominant-Ct value	现场检测 DNA指纹 On-site DNA fingerprinting	共显性- 等位基因 Co-dominant alleles	显性-Ct值 Dominant-Ct value	现场检测 DNA指纹 On-site DNA fingerprinting
京92 Jing92	334/334	35.0	0	154/154	24.5	1	257/257	24.6	1
京724 Jing724	334/334	35.0	0	154/154	24.1	1	253/253	35.0	0
LX9801	338/338	24.7	1	154/154	24.9	1	257/257	25.4	1
黄早四 HuangZaoSi	338/338	25.1	1	146/146	32.2	0	257/257	25.6	1
郑58 Zheng58	338/338	23.2	1	154/154	24.3	1	257/257	24.6	1
综3 Zong3	338/338	24.4	1	154/154	24.8	1	253/253	35.0	0
齐319 Qi319	338/338	24.3	1	154/154	25.3	1	253/253	33.1	0
掖478 Ye478	338/338	22.7	1	146/146	32.5	0	257/257	23.5	1
昌7-2 Chang7-2	334/334	35.0	0	154/154	24.5	1	257/257	24.1	1
样品A Sample A	338/338	24.6	1	154/154	24.2	1	257/257	24.3	1
样品B Sample B	338/338	23.7	1	146/146	33.1	0	257/257	24.5	1
样品C Sample C	338/338	24.1	1	154/154	23.9	1	253/253	34.7	0

引物编号 Primer number	位点名称 Name of the loci	染色体 Chr.	染色体位置 Chromosome position (bp)	InDel变异 InDel variation	MAF	引物序列 Primer sequence (5′-3′)
KT01	YJ02939	1	278976599	-/CGCG	0.49	F:CCTTGCTTCCGTGCCGCGCG
KT01	YJ02939	1	278976599	-/CGCG	0.49	R:CCGCTTTCCTTTAATTTGTCAGGAT
KT02	YJ45925	2	24653642	-/ATGA	0.44	F:TTCAAATGTGGTGACCCACCCATGA
KT02	YJ45925	2	24653642	-/ATGA	0.44	R:GACCAAACACGCTTAATCTAGAAGT
KT03	YJ08646	2	88426353	-/AGAAACAGTC	0.49	F:CACATTAAGTATGCTGACTGTTTCT
KT03	YJ08646	2	88426353	-/AGAAACAGTC	0.49	R:GAAGATCTTGAGACACTGCAAA
KT04	YJ09625	3	172717920	-/ACACAACT	0.45	F:TAGTGCAGTTCCACCTTTCATCTTGTGTAAGTTGT
KT04	YJ09625	3	172717920	-/ACACAACT	0.45	R:AGTGTAAAATCTAGAGCCCACTACT
KT05	YJ12830	3	124035057	-/TTG	0.49	F:TAGCCTAACATTGTGGCGATTCAA
KT05	YJ12830	3	124035057	-/TTG	0.49	R:CCGTAATTCCTTAAAAACGTACCCA
KT06	YJ13716	4	192970579	-/AGTTAG	0.49	F:TGAGCAGCATTATTGCACTCTAACT
KT06	YJ13716	4	192970579	-/AGTTAG	0.49	R:CATTATCAAGGGTTTCTACAGGGGA
KT07	YJ61195	5	14612889	-/TGAC	0.42	F:CTTTGGATCAGTCATAGGGGTCA
KT07	YJ61195	5	14612889	-/TGAC	0.42	R:AGAAAAGTGAACATGGAGAGTGC
KT08	YJ61195	5	15209041	-/TTGATG	0.49	F:TGAAGCGAGAGGATCAACATCAA
KT08	YJ61195	5	15209041	-/TTGATG	0.49	R:CTCTTTCTGGTTTTAAAGACGTGCT
KT09	YJ64216	6	159829453	-/ACAGT	0.43	F:GGGTCTTGGATCGTAATGTACTGT
KT09	YJ64216	6	159829453	-/ACAGT	0.43	R:CTCCCAGCTAGCTCCATTGG
KT10	YJ21432	6	3240073	-/TTG	0.40	F:CACACCAGCGTGACAACCAA
KT10	YJ21432	6	3240073	-/TTG	0.40	R:CTTCCTCAACCAATTCCACACTTAT
KT11	YJ22927	6	138858575	-/AAT	0.49	F:AGTATATGTAGGGGGAATCCACTTATT
KT11	YJ22927	6	138858575	-/AAT	0.49	R:GTGTAATCGTATAGACATCGACCCA
KT12	YJ22725	6	132187454	-/ATGCACTG	0.49	F:GAGCTTGCCGTGCATCAGTG
KT12	YJ22725	6	132187454	-/ATGCACTG	0.49	R:GGCATAGGAGAGTGTACATAGCTAG
KT13	YJ25969	7	151285049	-/CGGAG	0.45	F:CTAAACCATGGAATCGGAGGCTC
KT13	YJ25969	7	151285049	-/CGGAG	0.45	R:TAGTCAAAAAGGACGACAGCATG
KT14	YJ29113	8	159528845	-/TTGCTTG	0.43	F:GAATGAAGCCGCTAAACCAAGCAA
KT14	YJ29113	8	159528845	-/TTGCTTG	0.43	R:AAGTAGAGAACAAACACTAGAGGGG
KT15	YJ31022	9	113082501	-/TGT	0.48	F:CTACGAAGCTGAATGTCAACAACA
KT15	YJ31022	9	113082501	-/TGT	0.48	R:ATGGAGTAGTGTTCGATCCATCG
KT16	YJ34143	10	145370739	-/ATA	0.49	F:AGGTAGGACTGGAGGTTCAAATTAT
KT16	YJ34143	10	145370739	-/ATA	0.49	R:GGCAAACATGCATAGATCTGATGAA

玉米自交系InDel标记现场检测技术的研发与应用

Development and Application of InDel Marker Detection Technology for Field Identification of Maize Inbred Lines

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