Cloning of Wheat TaSOD-B1 Gene and Development and Verification of KASP Marker

doi:10.7668/hbnxb.20195519

Abstract

Abstract:

Cloning the wheat grain superoxide dismutase(SOD)gene and developing competitive allele-specific PCR(KASP)markers related to SOD activity are of great significance for breeding wheat varieties with high SOD activity.According to the gene ID,specific primers were designed to clone the gDNA sequence of TaSOD-B1 gene.The single nucleotide polymorphism(SNP)loci were obtained by comparing the wheat genome genetic variation and Ensembl Plants database,and the KASP markers closely related to the SOD activity of wheat were developed.The practicability of the markers was verified by the correlation analysis between SOD activity and genotypes of 287 winter wheat varieties(lines).The TaSOD-B1 gene fragment of Chinese spring variety was amplified by six pairs of specific primers,and the TaSOD-B1 gene on chromosome 5B was obtained by splicing.The full length of the gene was 6 491 bp,including an open reading frame(ORF)of 1 650 bp,which encoded a total of 549 amino acids.The predicted molecular weight was 60.80 ku,the gene was composed of 12 exons and 11 introns.The intron conformed to the typical GT-AG structure.The KASP marker was developed based on the 44th base of the first exon of the TaSOD-B1 gene,and was verified by sequencing.The results showed that the genotype of the allelic variation type TaSOD-B1a was AA,which was associated with high SOD activity,and was labeled with the fluorescent gene FAM(shown as blue).The genotype of TaSOD-B1b was GG,which was associated with low SOD activity and was marked with the fluorescent gene HEX(shown as red).The detection of 287 winter wheat varieties(lines)at home and abroad showed that the SOD activity of different genotypes was significantly different.Based on the TaSOD-B1 sequence,a set of KASP markers related to SOD activity was successfully developed and could be used for genetic improvement of SOD activity.

Key words: Common wheat, Superoxide dismutase, Gene cloning, KASP marker

CLC Number:

S336生物技术育种方法

HOU Peike, CHENG Yukun, WANG Jiqing, SUN Ling, WANG Jianpeng, GENG Hongwei. Cloning of Wheat TaSOD-B1 Gene and Development and Verification of KASP Marker[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 1-8. doi: 10.7668/hbnxb.20195519.

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References 39

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引物名称 Primer	引物序列(5'—3') Sequence of primer (5'—3')	PCR产物/bp PCR product	退火温度/℃ Tm
5B1	F: AAAGGCCGCATGTTGAGAGA	1 121	58
	R: GGGATGACAGAGTTCGGTT
5B2	F: ACGCGTCTATATACATCCGATT	1 231	61
	R: CTTTAGAGATTGTAGAAAACTT
5B3	F: GCCATTATCGTGCCTGTG	932	61
	R: TGTCCTCGCAAAATGTCG
5B4	F: AGACCCTGAAACTCGCTGA	1 001	58
	R: CGACTAATGGCAGTTGCGAA
5B5	F: AAAAAGGTTCATGCCGATAAAGTTC	2 123	58
	R: GCCATTATCGTGCCTGTG
5B6	F: CCACAGACAACACAGGACCTT	948	58
	R: CGACCTTGCATTTTTGCCCTT

引物名称 Primer	引物序列(5'—3') Sequence of primer (5'—3')	PCR产物/bp PCR product	退火温度/℃ Tm
5B1	F: AAAGGCCGCATGTTGAGAGA	1 121	58
	R: GGGATGACAGAGTTCGGTT
5B2	F: ACGCGTCTATATACATCCGATT	1 231	61
	R: CTTTAGAGATTGTAGAAAACTT
5B3	F: GCCATTATCGTGCCTGTG	932	61
	R: TGTCCTCGCAAAATGTCG
5B4	F: AGACCCTGAAACTCGCTGA	1 001	58
	R: CGACTAATGGCAGTTGCGAA
5B5	F: AAAAAGGTTCATGCCGATAAAGTTC	2 123	58
	R: GCCATTATCGTGCCTGTG
5B6	F: CCACAGACAACACAGGACCTT	948	58
	R: CGACCTTGCATTTTTGCCCTT

环境 Environment	等位变异 Genotype	数量/份 Number	SOD活性均值/(U/g) Mean SOD activity	标准差/(U/g) Standard deviation	变化范围/(U/g) Range
Ⅰ	TaSOD-B1a	13	1 816.56a	54.82	1 729.22~1 930.84
	TaSOD-B1b	35	1 788.87a	46.70	1 667.57~1 988.86
Ⅱ	TaSOD-B1a	15	1 826.63a	82.69	1 548.95~1 890.39
	TaSOD-B1b	5	1 786.30a	42.88	1 721.47~1 837.62
Ⅲ	TaSOD-B1a	21	1 777.94a	39.04	1 711.34~1 847.39
	TaSOD-B1b	95	1 769.88a	58.10	1 558.24~1 937.09
Ⅳ	TaSOD-B1a	4	1 854.92a	63.20	1 793.81~1 937.56
	TaSOD-B1b	34	1 749.11a	112.61	1 354.13~1 899.57
Ⅴ	TaSOD-B1a	4	1 766.94a	109.56	1 655.08~1 907.58
	TaSOD-B1b	61	1 790.28a	76.52	1 478.07~1 923.75
全部品种	TaSOD-B1a	57	1 804.19a	66.83	1 548.95~1 937.56
All varieties	TaSOD-B1b	230	1 776.96b	74.03	1 354.13~1 988.86

环境 Environment	等位变异 Genotype	数量/份 Number	SOD活性均值/(U/g) Mean SOD activity	标准差/(U/g) Standard deviation	变化范围/(U/g) Range
Ⅰ	TaSOD-B1a	13	1 816.56a	54.82	1 729.22~1 930.84
	TaSOD-B1b	35	1 788.87a	46.70	1 667.57~1 988.86
Ⅱ	TaSOD-B1a	15	1 826.63a	82.69	1 548.95~1 890.39
	TaSOD-B1b	5	1 786.30a	42.88	1 721.47~1 837.62
Ⅲ	TaSOD-B1a	21	1 777.94a	39.04	1 711.34~1 847.39
	TaSOD-B1b	95	1 769.88a	58.10	1 558.24~1 937.09
Ⅳ	TaSOD-B1a	4	1 854.92a	63.20	1 793.81~1 937.56
	TaSOD-B1b	34	1 749.11a	112.61	1 354.13~1 899.57
Ⅴ	TaSOD-B1a	4	1 766.94a	109.56	1 655.08~1 907.58
	TaSOD-B1b	61	1 790.28a	76.52	1 478.07~1 923.75
全部品种	TaSOD-B1a	57	1 804.19a	66.83	1 548.95~1 937.56
All varieties	TaSOD-B1b	230	1 776.96b	74.03	1 354.13~1 988.86

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