Crops ›› 2025, Vol. 41 ›› Issue (4): 41-48.doi: 10.16035/j.issn.1001-7283.2025.04.005

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Cloning of the du1 Novel Allelic Variant and the Development of Its Molecular Markers

Shi Yaxing1(), Liu Junling1(), Zhu Guichuan1,2, He Zhongyou3, Liu Hui1, Fan Yanli1, Xu Li1, Lu Baishan1(), Zhao Jiuran1, Luo Meijie1()   

  1. 1Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097, China
    2Beijing University of Agriculture, Beijing 102206, China
    3Hainan Lüchuan Seed Co., Ltd., Haikou 571100, Hainan, China
  • Received:2024-07-08 Revised:2024-09-10 Online:2025-08-15 Published:2025-08-12

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Abstract:

The “Shuang Tian Nuo” maize is a new type of maize germplasm, whose endosperm characteristics are jointly controlled by the waxy gene and the sweet gene. However, the sweet regulation gene has not yet been located and cloned. The results showed that the soluble sugar content of the naturally air-dried grains of “Shuang Tian Nuo” maize XT was 62.82 mg/g, significantly higher than the 19.32 mg/g of ordinary maize, but lower than the 110.9 mg/g of sugary (su1) maize. Using BSR-seq technology and based on the wrinkled grain phenotype, the sweet regulatory gene of XT maize was mapped to the 24-68 Mb interval on chromosome 10. By comparing the full-length DNA sequence of the candidate Du1 gene in XT maize and ordinary maize B73, a 5839 bp Gypsy-like LTR retrotransposon insertion was found after the 1455 bp position in the third exon of the du1 gene in XT maize. cDNA sequence analysis revealed that the transposon insertion sequence in the du1 gene of XT maize was transcribed in large segments, resulting in abnormal transcripts. So it was determined as the key gene for sugary quality regulation. This gene is a new allelic variant of the du1 gene, and the KASP molecular marker developed for its mutation site can efficiently distinguish between du1du1, du1Du1, and Du1Du1 three genotypes.

Key words: Maize, New du1 allelic variation, BSR-seq, Retrotransposon insertion, KASP molecular marker

Table 1

The primers for du1 gene DNA amplification"

引物名称Primer name 5′→3′
DU3F CGTTTGCTGGGAAGTGGTTC
DU3R AGTCTTGGCGCATAGTTGCT
DU4F GCTCCAAGAAGAGCGAACAC
DU4R ATAGCCAGCAACGGATTGGATT
DU5F TCCAATCCGTTGCTGGCTAT
DU5R GCCACAGCTGATAGATCACGA
DU6F CGTGATCTATCAGCTGTGGC
DU6R AGCAGGGCAAACGGTATTCT
DU7F GACCATTTGGGCCATTCATCAC
DU7R AGCACGTGAAAGACTAGTGAC
DU8F GGCACCAATCGCAAAGGTTA
DU8R GAGGGCTTCCCTTGCTGTATTT
DU9F GCCTTTCCTATTTGGCAGCAC
DU9R TTTTCCTTGTGTAGCCAGGCA
DU10F GCGAAGTTGCACTGTTAGCTT
DU10R TGTCAGGCGAGTGACGATTC
DU11F GATGTCCCCGTCGTAGGAAT
DU11R TACATTGGCCGTTGGTTGGAT
DU12F CAACCAACGGCCAATGTAGT
DU12R GGGTGAGAAGACAAAGATACCCT

Table 2

The primers for du1 gene cDNA amplification and KASP molecular marker assay"

引物名称Primer name 5′→3′
cDNA扩增cDNA amplification
DU3F CGTTTGCTGGGAAGTGGTTC
DU4R ATAGCCAGCAACGGATTGGATT
DX18F ATAGTTCACTTCCCTGAGCC
DX6R TCAACGCTGCCATCATAGGT
DX16F CAAGTCCCGTCATATGTGCT
DX4R AGATGAATGCGGTGGTCGTAG
DX10F CTACGACCACCGCATTCATCT
DX2R TCAAGGTACCATCCTGGAGCT
DX6F TTGAGGTTGAGTATCGTCCG
DX1R GTAGTAGTCTGCCATTCTGGAC
DUX5F CTGCACGACGTATTTCATGTGG
DUX5R CCATTCTGTATGCCTGCTTAGG
DU6F CGTGATCTATCAGCTGTGGC
DU9R TTTTCCTTGTGTAGCCAGGCA
DX17F GTCACTAGTCTTTCACGTGCTG
DU11R TACATTGGCCGTTGGTTGGAT
KASP分子标记KASP molecular marker
AlleleX AAGAGAACCAACAATGGACTGGTG
AlleleY AAGAGAACCAACAATGGACTGGTC
Common1 TCCAATCCATCACCAGTTACGATAAACTA
Common2 TCCGTGGTAGGGATCGTATTCCTAT

Fig.1

Phenotypic appearance and soluble sugar contents of XT grains (a) Naturally dried grain morphology. (b) Observation diagram of grain transmission light. (c) Grain cross-section diagram. (d) Grain longitudinal section diagram. Scale bar=1 cm.“*”represents P < 0.05,“****”represents P < 0.0001."

Table 3

Total sugar, total starch and amylose contents in naturally dried maize grains"

编号
Number		 样品名称（玉米类型）
Sample name (maize type)		 可溶性糖含量
Soluble sugar content (mg/g)		 总淀粉含量
Total starch content (mg/g)		 直链淀粉含量
Amylose content (mg/g)		 直链淀粉比例
Amylose ratio (%)
1 京724（普通玉米） 19.32±0.11d 537.20±3.00b 153.20±1.11b 29.50±0.18a
2 京2416（普通玉米） － 676.00±4.18a 172.80±1.33a 26.44±0.15b
3 SH251（超甜玉米） 192.80±2.59a[14] 229.60±3.38h 50.21±1.47c 22.62±0.47c
4 T9（普甜玉米） 154.30±2.63b[14] 524.40±2.96c 45.53±1.07d 8.98±0.19d
5 219M（糯玉米） － 502.20±3.79d 8.51±1.11f 1.75±0.22f
6 XT（爽甜糯玉米） 62.82±0.11c 460.60±1.24e 21.25±0.41e 4.78±0.08e
7 XT选系1 － 330.50±2.27g 7.66±0.72f 2.39±0.21f
8 XT选系2 － 387.00±1.07f 6.38±0.72f 1.70±0.19f
9 XT选系3 － 457.10±2.50e 6.80±0.84f 1.54±0.18f

Fig.2

BSR-seq mapping results for the sweetness regulating gene in XT"

Fig.3

Comparison analysis of Du1 gene DNA sequences between XT and B73 (a) Discrepancy in the PCR amplification products between XT maize and its three inbred lines (XT1, XT2, XT3) compared to ordinary maize B73 utilizing the Du5F/Du5R primer pair."

Fig.4

Comparison analysis of Du1 gene cDNA amplification products between XT and B73 (a) The location of cDNA amplification primers on the du1 gene. (b) Comparison analysis of the differences in cDNA segment amplification products."

Fig.5

Illustration of genotyping assay utilizing the du1-KASP molecular marker Black dots represent the blank control, red dots represent the homozygous genotype without insertion mutation, blue dots represent the homozygous genotype with insertion mutation, and green dots represent heterozygous genotypes."

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