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作物学报 ›› 2024, Vol. 50 ›› Issue (12): 3025-3034.doi: 10.3724/SP.J.1006.2024.41009

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦籽粒大小相关基因TaCYP78A17的功能标记开发

张天星1,2(), 李梦1,2, 吴林楠2, 赵惠贤2, 胡胜武1,*(), 马猛2,*()   

  1. 1西北农林科技大学农学院, 陕西杨凌 712100
    2西北农林科技大学生命科学学院, 陕西杨凌 712100
  • 收稿日期:2024-02-05 接受日期:2024-08-15 出版日期:2024-12-12 网络出版日期:2024-09-02
  • 通讯作者: *胡胜武, E-mail: swhu83251@nwafu.edu.cn; 马猛, E-mail: mengma5@nwsuaf.edu.cn
  • 作者简介:E-mail: Tianxing-Zhang0805@163.com
  • 基金资助:
    陕西省重点研发项目(2021NY-079);国家自然科学基金项目(32072059);国家自然科学基金项目(32072003)

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 Published:2024-12-12 Published online: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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摘要：

籽粒大小是影响小麦产量的重要因素之一。为了开发小麦籽粒大小相关的功能标记, 本研究克隆得到一个潜在籽粒大小相关基因TaCYP78A17, 并对其进行了系统进化、表达模式、等位变异分析和功能标记开发。结果表明,TaCYP78A17基因是小麦细胞色素P450 CYP78A家族的一员, 且在小麦幼穗和籽粒中高表达; 在30份普通小麦品种中发现TaCYP78A17-Ap存在6个SNP和2个InDel; 根据InDel 4和InDel 8开发功能标记InDel-A17, 可将30份普通小麦品种分为TaCYP78A17-Ap-HapI, TaCYP78A17-Ap-HapII和TaCYP78A17-Ap-HapIII三种单倍型; 利用该功能标记在323份普通小麦品种中进行验证, 发现该功能标记可以有效区分上述3种单倍型; 表型调查发现具有TaCYP78A17-Ap-HapI单倍型的小麦其千粒重和籽粒大小显著高于具有TaCYP78A17-Ap-HapII或TaCYP78A17- Ap-HapIII单倍型的小麦。研究结果有望应用于小麦分子标记辅助选择育种。

关键词: 小麦, 籽粒大小, TaCYP78A17, 功能标记

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

表1

30份小麦品种粒重信息"

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

表2

用于TaCYP78A17扩增的引物"

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

图1

TaCYP78A17-A/B/D基因编码区和启动子扩增 TaCYP78A17-Ap、TaCYP78A17-Bp、TaCYP78A17-Dp: TaCYP78A17-A、TaCYP78A17-B和TaCYP78A17-D基因的启动子; TaCYP78A17-A、TaCYP78A17-B、TaCYP78A17-D: TaCYP78A17- A、TaCYP78A17-B和TaCYP78A17-D基因的编码区。"

图2

TaCYP78A17保守结构域和进化分析 (a): TaCYP78A17-A/B/D编码蛋白的序列比对和保守结构域分析, 黄色标记为疏水区, 蓝色标记为氧结合位点, 红色标记为亚铁血红素结合位点。(b): TaCYP78A17 (由TaCYP78A17-A推导的蛋白序列)和主要植物CYP78A家族蛋白的系统进化树分析。"

图3

TaCYP78A17基因的表达模式分析 S: 茎; L: 叶; FL: 旗叶; YS5: 5 mm的幼穗; YS15: 15 mm的幼穗; GR5: 花后5 d的籽粒; GR10: 花后10 d的籽粒; GR15: 花后15 d的籽粒; GR20: 花后20 d的籽粒。"

图4

TaCYP78A17-Ap核苷酸多态性和单倍型分析 (a): TaCYP78A17-A启动子的结构和多态性位点示意图, 1~8表示等位变异位点(SNP/InDel)。(b): TaCYP78A17-Ap单倍型和等位变异分析。"

图5

TaCYP78A17-Ap的InDel分子标记开发"

表3

30份小麦品种基因型"

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

图6

TaCYP78A17-Ap三种单倍型在自然小麦群体中的分布及对粒重的影响 (a): 323份现代育成小麦品种中分别具有TaCYP78A17-Ap三种单倍型(TaCYP78A17-Ap-HapI、TaCYP78A17-Ap-HapII和TaCYP78A17-Ap-HapIII)群体材料所占的比例; Miss: 未成功鉴定单倍型的小麦。(b): 统计分析分别具有TaCYP78A17-Ap-HapI、TaCYP78A17-Ap-HapII和TaCYP78A17-Ap-HapIII单倍型群体材料的千粒重(n = 10)。(c): 统计分析分别具有TaCYP78A17-Ap-HapI、TaCYP78A17-Ap-HapII和TaCYP78A17-Ap-HapIII单倍型群体材料的籽粒长度、宽度和厚度(n = 60)。所有数据为平均值±标准误表示; 差异显著性分析采用t检验(*: P < 0.05, **: P < 0.01)。"

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