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

Abstract

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

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

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

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