Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica

   

Development and application of functional insertion and deletion (indel) markers associated with maize Waxy gene compatible with dual-platform

ZHU Wei-Jia1,2,**,WANG Rui1,**,XUE Ying-Jie1,TIAN Hong-Li1,FAN Ya-Ming1,WANG Lu1,LI Song1,XU Li1,LU Bai-Shan1,SHI Ya-Xing1,YI Hong-Mei1,LU Da-Lei2,YANG Yang1,*,WANG Feng-Ge1,*   

  1. 1 Maize Research Institute, Beijing Academy of Agricultural and Forestry Sciences / Key Laboratory of Crop DNA Fingerprinting Innovation and Utilization (Co-construction by Ministry and Province) / Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097, China; 2 Yangzhou University /Jiangsu Key Laboratory of Crop Genetics and Physiology / Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou 225009, Jiangsu, China
  • Received:2025-02-25 Revised:2025-06-01 Accepted:2025-06-01 Online:2025-06-13 Published:2025-06-13
  • Supported by:
    The study was supported by the Construction of Scientific and Technological Innovation Capacity of Beijing Academy of Agriculture and Forestry Sciences (KJCX20230303, KJCX20230301).

PDF

0

Abstract:

To enable rapid identification of variation types associated with the Waxy gene in waxy maize and to support its application in modern waxy maize breeding, we developed functional markers targeting four common InDel variations of the Waxy gene: wx-D7, wx-D10, wx-124, and wx-hAT. The specificity and effectiveness of these markers were validated across multiple molecular detection platforms, including in common maize, waxy maize, sweet maize, and sweet-waxy maize. Results showed that the four Waxy-associated functional markers enabled specific genotyping on both the KASP and fluorescence capillary electrophoresis platforms. These markers effectively distinguished common maize from waxy maize and identified the Waxy gene variation types in waxy maize lines. The waxy phenotype could be inferred based on the Waxy gene haplotype when specific functional markers were detected in inbred lines. Maize germplasms lacking these four waxy variations exhibited either non-waxy phenotypes or rare waxy variants. For hybrid maize samples, four possible genotypic combinations were observed based on Waxy haplotype analysis: recessive homozygous, recessive allele heterozygous, waxy/non-waxy heterozygous, and dominant homozygous genotypes. Notably, over 85% of waxy maize carried the wx-D7 variation, indicating that wx-D7 is the predominant allele used in modern waxy maize breeding in China. Additionally, we found that multiple Waxy gene variations, such as D7/D10, coexisted in waxy maize hybrids, while only a single variation type was present in waxy inbred lines. This suggests that the aggregation of different Waxy variations may contribute to genetic improvement in waxy maize breeding. In summary, we developed a set of functional markers for the Waxy gene that are compatible with multiple molecular detection platforms, providing an efficient tool for the identification and screening of waxy maize germplasm.

Key words: waxy maize, Waxy gene, functional marker, indel, KASP, fluorescence capillary electrophoresis

[1] Huang L, Sreenivasulu N, Liu Q. Waxy editing: old meets new. Trends Plant Sci, 2020, 25: 963–966.

[2] Yang Y, Zhou L H, Feng L H, Jiang J Y, Huang L C, Liu Q, Zhang Y D, Zhang C Q, Liu Q Q. Deciphering the role of Waxy gene mutations in enhancing rice grain quality. Foods, 2024, 13: 1624.

[3] Yang J, Wang J, Fan F J, Zhu J Y, Chen T, Wang C L, Zheng T Q, Zhang J, Zhong W G, Xu J L. Development of AS-PCR marker based on a key mutation confirmed by resequencing of Wx-mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding. Plant Breed, 2013, 132: 595–603.

[4] Nakamura T, Yamamori M, Hirano H, Hidaka S, Nagamine T. Production of waxy (amylose-free) wheats. Mol Gen Genet, 1995, 248: 253–259.

[5] 韩蕾. 糯玉米距离分析、杂种优势及特殊配合力的关系. 吉林农业大学硕士学位论文, 吉林长春, 2006.
Han L. Relationship among Distance Analysis and Heterosis and Specific Combining Ability in Waxy Corn. MS Thesis of Jilin Agricultural University, Changchun, Jilin, China, 2006 (in Chinese with English abstract).

[6] 杨明花, 嵇闯, 崔亚坤, 刘强, 彭云承, 赵文明, 孟庆长, 张美景, 陈艳萍. 鲜食糯玉米货架期苞叶相关性状的配合力及其遗传效应分析. 玉米科学, 2023, 31(6): 10–16.
Yang M H, Ji C, Cui Y K, Liu Q, Peng Y C, Zhao W M, Meng Q C, Zhang M J, Chen Y P. Analysis on combining ability and genetic effect of bract related traits related characters of fresh waxy corn. J Maize Sci, 2023, 31(6): 10–16 (in Chinese with English abstract).

[7] 李芳芳, 刘松涛, 么大轩, 刘云婷, 代亮, 段会军. 一个糯玉米突变体的遗传鉴定. 河北农业大学学报, 2018, 41(1): 6–10.
Li F F, Liu S T, Yao D X, Liu Y T, Dai L, Duan H J. Genetic identification of a waxy maize mutant. J Hebei Agric Univ, 2018, 41(1): 6–10 (in Chinese with English abstract).

[8] Wessler S R. The maize transposable Ds1 element is alternatively spliced from exon sequences. Mol Cell Biol, 1991, 11: 6192–6196.

[9] 田孟良, 黄玉碧, 谭功燮, 刘永建, 荣廷昭. 西南糯玉米地方品种waxy基因序列多态性分析. 作物学报, 2008, 34: 729–736.
Tian M L, Huang Y B, Tan G X, Liu Y J, Rong T Z. Sequence polymorphism of waxy genes in landraces of waxy maize from Southwest China. Acta Agron Sin, 2008, 34: 729–736 (in Chinese with English abstract).

[10] Okagaki R J, Neuffer M G, Wessler S R. A deletion common to two independently derived waxy mutations of maize. Genetics, 1991, 128: 425–431.

[11] 武晓阳, 隆文杰, 陈丹, 周国雁, 杜娟, 伍少云, 蔡青. 云南糯玉米地方品种糯性等位基因wx-xuanwei的分子特征. 江西农业学报, 2020, 32(3): 35–41.
Wu X Y, Long W J, Chen D, Zhou G Y, Du J, Wu S Y, Cai Q. Molecular characteristics of waxy allele wx-xuanwei in Yunnan waxy maize landraces. Acta Agric Jiangxi, 2020, 32(3): 35–41 (in Chinese with English abstract).

[12] Luo M J, Shi Y X, Yang Y, Zhao Y X, Zhang Y X, Shi Y M, Kong M S, Li C H, Feng Z, Fan Y L, et al. Sequence polymorphism of the waxy gene in waxy maize accessions and characterization of a new waxy allele. Sci Rep, 2020, 10: 15851.

[13] 姚坚强, 鲍坚东, 朱金庆, 桂毅杰, 沈秋芳, 胡伟民, 樊龙江. 中国糯玉米wx基因种质资源遗传多样性. 作物学报, 2013, 39: 43–49.
Yao J Q, Bao J D, Zhu J Q, Gui Y J, Shen Q F, Hu W M, Fan L J. Genetic diversity of waxy gene in Chinese glutinous maize. Acta Agron Sin, 2013, 39: 43–49 (in Chinese with English abstract).

[14] Fukunaga K, Kawase M, Kato K. Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype. Mol Genet Genom, 2002, 268: 214–222.

[15] Fan L J, Quan L Y, Leng X D, Guo X Y, Hu W M, Ruan S L, Ma H S, Zeng M Q. Molecular evidence for post-domestication selection in the Waxy gene of Chinese waxy maize. Mol Breed, 2008, 22: 329–338.

[16] 石建斌, 周红, 王宁, 许庆华, 乔文青, 严根土. 棉花SSR标记种质资源纯度鉴定及遗传多样性分析. 生物技术通报, 2018, 34(7): 138–146.
Shi J B, Zhou H, Wang N, Xu Q H, Qiao W Q, Yan G T. Purity identification and genetic diversity analysis of cotton germplasm resources using SSR markers. Biotechnol Bull, 2018, 34(7): 138–146 (in Chinese with English abstract).

[17] 徐辰武, 徐扬, 焦宇馨, 李成, 于广宁. 玉米糯性基因的KASP分子标记的开发方法及应用. 中国专利: CN202210282537.6. [2022-06-10].

Xu C W, Xu Y, Jiao Y X, Li C, Yu G N. The development method and application of KASP molecular markers for the waxy gene in maize. Chinese Patent: CN202210282537.6. [2022-06-10] (in Chinese). 

[18] 袁文娅, 赵晓雷, 周旭梅, 王磊, 彭勃, 王奕. waxy基因功能标记开发及在糯玉米育种中的应用. 作物杂志, 2020, (4): 99–106.
Yuan W Y, Zhao X L, Zhou X M, Wang L, Peng B, Wang Y. The development of waxy gene function marker and its application in waxy maize breeding. Crops, 2020, (4): 99–106 (in Chinese with English abstract).

[19] 宋伟, 王凤格, 易红梅, 李翔, 赵久然. 功能标记及在品种鉴定和辅助育种中的应用前景. 分子植物育种, 2009, 7: 612–618.
Song W, Wang F G, Yi H M, Li X, Zhao J R. Functional markers and their potential application in varieties identification and MAS breeding. Mol Plant Breed, 2009, 7: 612–618 (in Chinese with English abstract).

[20] 任佳丽. 转基因玉米标准物质-质粒阳性物质构建与检测方法的建立. 新疆农业大学硕士学位论文, 新疆乌鲁木齐, 2021.
Ren J L. Construction of Transgenic Maize Standard Substance-plasmid Positive Substance and Establishment of Detection Method for Transgenic Maize. MS Thesis of Xinjiang Agricultural University, Urumqi, Xinjiang, China, 2021 (in Chinese with English abstract).

[21] 梁紫越, 刘志浩, 马世鹏, 赵怡锟, 许理文, 康定明, 王凤格. 基于双平台的InDel标记玉米杂交种纯度鉴定方法. 玉米科学, 2022, 30(3): 32–39.
Liang Z Y, Liu Z H, Ma S P, Zhao Y K, Xu L W, Kang D M, Wang F G. Using indel markers for purity identification method of hybrid seeds in maize based on dual platforms. J Maize Sci, 2022, 30(3): 32–39 (in Chinese with English abstract).

[22] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016, 33: 1870–1874.

[23] Rasheed A, Wen W E, Gao F M, Zhai S N, Jin H, Liu J D, Guo Q, Zhang Y J, Dreisigacker S, Xia X C, et al. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theor Appl Genet, 2016, 129: 1843–1860.

[24] 王蕊, 施龙建, 田红丽, 易红梅, 杨扬, 葛建镕, 范亚明, 任洁, 王璐, 陆大雷, 等. 玉米杂交种纯度鉴定SNP核心引物的确定及高通量检测方案的建立. 作物学报, 2021, 47: 770–779.
Wang R, Shi L J, Tian H L, Yi H M, Yang Y, Ge J R, Fan Y M, Ren J, Wang L, Lu D L, et al. Identification of SNP core primer and establishment of high throughput detection scheme for purity identification in maize hybrids. Acta Agron Sin, 2021, 47: 770–779 (in Chinese with English abstract).

[25] 易红梅, 王凤格, 赵久然, 王璐, 郭景伦, 原亚萍. 玉米品种SSR标记毛细管电泳荧光检测法与变性PAGE银染检测法的比较研究. 华北农学报, 2006, 21(5): 64–67.
Yi H M, Wang F G, Zhao J R, Wang L, Guo J L, Yuan Y P. Comparison of two maize SSR detection methods: capillary electrophoresis with fluorescence detection method and denaturing PAGE silver-staining detection method. Acta Agric Boreali-Sin, 2006, 21(5): 64–67 (in Chinese with English abstract).

[26] 仇律雯, 杨扬, 范亚明, 田红丽, 易红梅, 王璐, 任洁, 葛建镕, 王凤格, 陆大雷. 国家东南区鲜食糯玉米品质及农艺性状与SSR标记遗传多样性分析. 江苏农业科学, 2022, 50(18): 130–135.
Qiu LW, Yang Y, Fan Y M, Tian H L, Yi H M, Wang L, Ren J, Ge J R, Wang F G, Lu D L. Genetic diversity analysis of quality, agronomic traits and SSR markers of fresh waxy maize in Southeastern China. Jiangsu Agric Sci, 2022, 50(18): 130–135 (in Chinese with English abstract).

[27] 雷开荣. SSR标记与玉米籽粒赖氨酸含量的关系及优质蛋白玉米(QPM)的分子标记辅助选择. 重庆大学硕士学位论文, 重庆, 2005.
Lei K R. The Relation between SSR Marker and the Lysine Content of Maize and Molecule Marker Selection for QPM. MS Thesis of Chongqing University, Chongqing, China, 2005 (in Chinese with English abstract).

[1] YONG Rui, HU Wen-Jing, WU Di, WANG Zun-Jie, LI Dong-Sheng, ZHAO Die, YOU Jun-Chao, XIAO Yong-Gui, WANG Chun-Ping. Identification and validation of quantitative trait loci for grain number per spike showing pleiotropic effect on thousand grain weight in bread wheat (Triticum aestivum L.) [J]. Acta Agronomica Sinica, 2025, 51(2): 312-323.
[2] GAO Wei-Dong, HU Chen-Zhen, ZHANG Long, ZHANG Yan-Yan, ZHANG Pei-Pei, YANG De-Long, CHEN Tao. Cloning and functional analysis of ubiquitin-conjugating enzymes TaUBC16 gene in wheat [J]. Acta Agronomica Sinica, 2024, 50(8): 1971-1988.
[3] BI Jun-Ge, ZENG Zhan-Kui, LI Qiong, HONG Zhuang-Zhuang, YAN Qun-Xiang, ZHAO Yue, WANG Chun-Ping. QTL mapping and KASP marker development of grain quality-relating traits in two wheat RIL populations [J]. Acta Agronomica Sinica, 2024, 50(7): 1669-1683.
[4] WANG Rui, SUN Bo, ZHANG Yun-Long, ZHANG Ming-Qi, FAN Ya-Ming, TIAN Hong-Li, ZHAO Yi-Kun, YI Hong-Mei, KUANG Meng, WANG Feng-Ge. Application analysis of chloroplast markers on rapid classification in maize germplasm [J]. Acta Agronomica Sinica, 2024, 50(7): 1867-1876.
[5] HAN Jie-Nan, ZHANG Ze, LIU Xiao-Li, LI Ran, SHANG-GUAN Xiao-Chuan, ZHOU Ting-Fang, PAN Yue, HAO Zhuan-Fang, WENG Jian-Feng, YONG Hong-Jun, ZHOU Zhi-Qiang, XU Jing-Yu, LI Xin-Hai, LI Ming-Shun. Analysis of differential accumulation of starch in waxy maize grain caused by the o2 mutation gene [J]. Acta Agronomica Sinica, 2024, 50(5): 1207-1222.
[6] LOU Fei, ZUO Yi-Ping, LI Meng, DAI Xin-Meng, WANG Jian, HAN Jin-Ling, WU Shu, LI Xiang-Ling, DUAN Hui-Jun. Effects of organic fertilizer substituting chemical fertilizer nitrogen on yield, quality, and nitrogen efficiency of waxy maize [J]. Acta Agronomica Sinica, 2024, 50(4): 1053-1064.
[7] ZHANG Tian-Xing, LI Meng, WU Lin-Nan, ZHAO Hui-Xian, HU Sheng-Wu, MA Meng. Development of functional markers of wheat grain size related gene TaCYP78A17 [J]. Acta Agronomica Sinica, 2024, 50(12): 3025-3034.
[8] SONG Xu-Dong, ZHU Guang-Long, ZHANG Shu-Yu, ZHANG Hui-Min, ZHOU Guang-Fei, ZHANG Zhen-Liang, MAO Yu-Xiang, LU Hu-Hua, CHEN Guo-Qing, SHI Ming-Liang, XUE Lin, ZHOU Gui-Sheng, HAO De-Rong. Identification of heat tolerance of waxy maizes at flowering stage and screening of evaluation indexes in the middle and lower reaches of Yangtze River region [J]. Acta Agronomica Sinica, 2024, 50(1): 172-186.
[9] SONG Zhao-Jian, FENG Zi-Yi, QU Tian-Ge, LYU Pin-Cang, YANG Xiao-Lu, ZHAN Ming-Yue, ZHANG Xian-Hua, HE Yu-Chi, LIU Yu-Hua, CAI De-Tian. Indica-japonica attribute identification and heterosis utilization of diploid rice lines reverted from tetraploid rice [J]. Acta Agronomica Sinica, 2023, 49(8): 2039-2050.
[10] TAO Shun-Yu, WU Bei, LIU Nian, LUO Huai-Yong, HUANG Li, ZHOU Xiao-Jing, CHEN Wei-Gang, GUO Jian-Bin, YU Bo-Lun, LEI Yong, LIAO Bo-Shou, JIANG Hui-Fang. Development and employment of InDel marker in peanut QTL mapping of oil content [J]. Acta Agronomica Sinica, 2023, 49(5): 1222-1230.
[11] YAN Xin, XIANG Chao, LIU Rong, LI Guan, LI Meng-Wei, LI Zheng-Li, ZONG Xu-Xiao, YANG Tao. Fine mapping of flower colour gene in pea (Pisum sativum L.) based on BSA-seq technique [J]. Acta Agronomica Sinica, 2023, 49(4): 1006-1015.
[12] ZHANG Yi-Ning, ZHANG Yan-Fei, WANG Min, WANG Jing-Yi, LI Long, LI Chao-Nan, YANG De-Long, MAO Xin-Guo, JING Rui-Lian. Transcription factor gene TaPHR1 involved in regulation spikelet number per spike in common wheat [J]. Acta Agronomica Sinica, 2023, 49(12): 3176-3187.
[13] HU Wen-Jing, LI Dong-Sheng, YI Xin, ZHANG Chun-Mei, ZHANG Yong. Molecular mapping and validation of quantitative trait loci for spike-related traits and plant height in wheat [J]. Acta Agronomica Sinica, 2022, 48(6): 1346-1356.
[14] DENG Zhao, JIANG Nan, FU Chen-Jian, YAN Tian-Zhe, FU Xing-Xue, HU Xiao-Chun, QIN Peng, LIU Shan-Shan, WANG Kai, YANG Yuan-Zhu. Analysis of blast resistance genes in Longliangyou and Jingliangyou hybrid rice varieties [J]. Acta Agronomica Sinica, 2022, 48(5): 1071-1080.
[15] WANG Rui, CHEN Xue, GUO Qing-Qing, ZHOU Rong, CHEN Lei, LI Jia-Na. Development of linkage InDel markers of the white petal gene based on whole-genome re-sequencing data in Brassica napus L. [J]. Acta Agronomica Sinica, 2022, 48(3): 759-769.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd