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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (12): 3025-3034.doi: 10.3724/SP.J.1006.2024.41009

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• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Development of functional markers of wheat grain size related gene TaCYP78A17

ZHANG Tian-Xing1,2(), LI Meng1,2, WU Lin-Nan2, ZHAO Hui-Xian2, HU Sheng-Wu1,*(), MA Meng2,*()   

  1. 1Collage of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China
    2College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi, China
  • Received:2024-02-05 Accepted:2024-08-15 Online:2024-12-12 Published:2024-09-02
  • Contact: *E-mail: swhu83251@nwafu.edu.cn; E-mail: mengma5@nwsuaf.edu.cn
  • Supported by:
    Key Research and Development Project of Shaanxi Province(2021NY-079);National Natural Science Foundation of China(32072059);National Natural Science Foundation of China(32072003)

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Abstract:

Grain size is a crucial factor influencing wheat yield. To develop functional markers related to wheat grain size, we cloned a potential grain size-related gene, TaCYP78A17, and conducted systematic evolution, expression pattern, allelic variation, and functional marker development studies. The results revealed that TaCYP78A17 belongs to the wheat cytochrome P450 CYP78A family and is highly expressed in wheat spikes and grains. Six SNPs and two InDels were identified in the TaCYP78A17-Ap among thirty common wheat varieties. A functional marker, InDel-A17, was developed based on InDel 4 and InDel 8, classifying the thirty wheat varieties into three haplotypes: TaCYP78A17-Ap-HapI, TaCYP78A17-Ap-HapII, and TaCYP78A17-Ap-HapIII. This functional marker was validated in 323 wheat varieties, demonstrating its ability to effectively distinguish the three haplotypes. Phenotypic investigations indicated that wheat with the TaCYP78A17-Ap-HapI haplotype exhibited significantly higher thousand-grain weight and larger seed size compared to those with TaCYP78A17-Ap-HapII or TaCYP78A17-Ap-HapIII. These findings hold promise for the application of molecular marker-assisted selection in wheat breeding.

Key words: wheat, grain size, TaCYP78A17, functional marker

Table 1

Grain weight information of thirty wheat varieties"

大粒小麦品种名称
Variety names of large-grain wheat		 百粒重
100-grain weight (g)		 小粒小麦品种名称
Variety names of small-grain wheat		 百粒重
100-grain weight (g)
西农981 Xinong 981 5.29 西农2000 Xinong 2000 4.03
陕512 Shaan 512 5.34 陕159 Shaan 159 4.36
陕优225-12 Shaanyou 225-12 5.51 US50 3.99
中优9507 Zhongyou 9507 5.73 早优504 Zaoyou 504 4.15
荔高6号Ligao 6 4.90 Suneca 3.86
郑麦9405 Zhengmai 9405 5.42 Bodallin 3.61
US34 4.92 Ciano 4.16
小偃6号Xiaoyan 6 4.84 紫麦 Zimai 4.14
闫麦8911 Yanmai 8911 4.79 高优503 Gaoyou 503 4.30
晋麦47 Jinmai 47 5.39 小偃81 Xiaoyan 81 4.20
长武134 Changwu 134 5.21 矮早781 Aizao 781 4.11
郑麦9023 Zhengmai 9023 5.39 京771 Jing 771 4.53
西农1043 Xinong 1043 6.35 US83 4.01
兰考大粒Lankaodali 4.96 小偃22 Xiaoyan 22 4.12
绵阳19 Mianyang 19 5.76 普冰143 Pubing 143 4.59

Table 2

Primers used for TaCYP78A17 amplification"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 引物用途
Function of primers
TaCYP78A17-Ap-F
TaCYP78A17-Ap-R		 TGCTGATTCGTGTACAATGGGCTAGGATCTT
GCCGGAGCGAAGATTCACCG		 TaCYP78A17-A启动子扩增
Amplification of TaCYP78A17-A promoter
TaCYP78A17-Bp-F
TaCYP78A17-Bp-R		 TGCAGTAGCTGCCTTAGAGCATCTTCAACAG
GAGTCGCCTTGGATTCTTCGCTT		 TaCYP78A17-B启动子扩增
Amplification of TaCYP78A17-B promoter
TaCYP78A17-Dp-F
TaCYP78A17-Dp-R		 CTGGTGGGAAAGACGTAAATTAGTGCATAAGGA
AGACCGGAGCGAAGATTCACCGT		 TaCYP78A17-D启动子扩增
Amplification of TaCYP78A17-D promoter
TaCYP78A17-A-F
TaCYP78A17-A-R		 TCCAGAGCTGGTGGGTGCTTCCCCTG
TCGTGAAGCACGGGGTCACGTCACG		 TaCYP78A17-A编码区扩增
Amplification of TaCYP78A17-A coding region
TaCYP78A17-B-F
TaCYP78A17-B-R		 AGCACACACCCCACCAAAGTCCGG
AGCACGGGGTCACGGGCTACGAG		 TaCYP78A17-B编码区扩增
Amplification of TaCYP78A17-B coding region
TaCYP78A17-D-F
TaCYP78A17-D-R		 GTAGATCATATATGCGAGTGAGTTCCTAGG
CGTACACCATGTGGAACCTCACTG		 TaCYP78A17-D编码区扩增
Amplification of TaCYP78A17-D coding region

Fig. 1

Amplification of TaCYP78A17-A/B/D coding regions and promoters TaCYP78A17-Ap, TaCYP78A17-Bp, TaCYP78A17-Dp: promoters of TaCYP78A17-A, TaCYP78A17-B and TaCYP78A17-D genes; TaCYP78A17-A, TaCYP78A17-B, TaCYP78A17-D: coding regions of TaCYP78A17-A, TaCYP78A17-B, and TaCYP78A17-D genes."

Fig. 2

Conserved domain and evolutionary analysis of TaCYP78A17 (a): TACYP78A17-A/B/D conserved domain analysis, yellow for hydrophobic region, blue for oxygen binding site, and red for heme binding site. (b): phylogenetic tree analysis of TaCYP78A17 (protein sequence derived from TaCYP78A17-A) and CYP78A family proteins in major plants."

Fig. 3

TaCYP78A17 gene expression pattern S: stem; L: leaf; FL: flag leaf; YS5: 5 mm young panicles; YS15: 15 mm young panicles; GR5: grain 5 days post-flowering; GR10: grain 10 days post-flowering; GR15: grain 15 days post-flowering; GR20: grain 20 days post-flowering."

Fig. 4

Analysis of nucleotide polymorphism and haplotypes in TaCYP78A17-Ap (a): schematic illustration of the structure and polymorphic sites of the TaCYP78A17-A promoter, 1-8 represent allelic variation sites (SNP/InDel). (b): analysis of haplotypes and allelic variations in TaCYP78A17-Ap."

Fig. 5

Development of InDel molecular marker for TaCYP78A17-Ap"

Table 3

Genotypes of thirty wheat accessions"

小麦品种信息
Wheat accessions		 TaCYP78A17序列基因型
Genotypes of TaCYP78A17		 小麦品种信息
Wheat accessions		 TaCYP78A17序列基因型
Genotypes of TaCYP78A17
西农981 Xinong 981 TaCYP78A17-Ap-HapI 西农2000 Xinong 2000 TaCYP78A17-Ap-HapI
陕512 Shaan 512 TaCYP78A17-Ap-HapI 陕159 Shaan 159 TaCYP78A17-Ap-HapI
陕优225-12 Shaanyou 225-12 TaCYP78A17-Ap-HapI US50 TaCYP78A17-Ap-HapIII
中优9507 Zhongyou 9507 TaCYP78A17-Ap-HapIII 早优504 Zaoyou 504 TaCYP78A17-Ap-HapI
荔高6号Ligao 6 TaCYP78A17-Ap-HapII Suneca TaCYP78A17-Ap-HapII
郑麦9405 Zhengmai 9405 TaCYP78A17-Ap-HapIII Bodallin TaCYP78A17-Ap-HapII
US34 TaCYP78A17-Ap-HapII Ciano TaCYP78A17-Ap-HapII
小偃6号Xiaoyan 6 TaCYP78A17-Ap-HapI 紫麦 Zimai TaCYP78A17-Ap-HapI
闫麦8911 Yanmai 8911 TaCYP78A17-Ap-HapIII 高优503 Gaoyou 503 TaCYP78A17-Ap-HapIII
晋麦47 Jinmai 47 TaCYP78A17-Ap-HapI 小偃81 Xiaoyan 81 TaCYP78A17-Ap-HapIII
长武134 Changwu 134 TaCYP78A17-Ap-HapI 矮早781 Aizao 781 TaCYP78A17-Ap-HapII
郑麦9023 Zhengmai 9023 TaCYP78A17-Ap-HapII 京771 Jing 771 TaCYP78A17-Ap-HapIII
西农1043 Xinong 1043 TaCYP78A17-Ap-HapI US83 TaCYP78A17-Ap-HapIII
兰考大粒 Lankaodali TaCYP78A17-Ap-HapI 小偃22 Xiaoyan 22 TaCYP78A17-Ap-HapIII
绵阳19 Mianyang 19 TaCYP78A17-Ap-HapI 普冰143 Pubing 143 TaCYP78A17-Ap-HapIII

Fig. 6

Distribution of three haplotypes of TaCYP78A17-Ap in a natural wheat population and their effects on grain weight (a): proportions of population materials with three haplotypes (TaCYP78A17-Ap-HapI, TaCYP78A17-Ap-HapII, and TaCYP78A17-Ap-HapIII) of TaCYP78A17-Ap in 323 modern wheat cultivars; Miss: wheat samples for which the haplotype could not be identified. (b): statistical analysis of thousand grain weight (n = 10) for population materials with TaCYP78A17-Ap-HapI, TaCYP78A17-Ap-HapII and TaCYP78A17- Ap-HapIII haplotypes. (c): statistical analysis of grain length, width, and thickness (n = 60) for population materials with TaCYP78A17-Ap- HapI, TaCYP78A17-Ap-HapII and TaCYP78A17-Ap-HapIII haplotypes. Data are shown as mean ±SE; significance of differences was performed using t-test (*: P < 0.05, **: P < 0.01)."

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