| 一种新的水稻香味基因功能标记的开发与应用 |
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| 引用本文： | 刘文静,胡文彬,周政,刘烨,赵正洪,徐庆国.一种新的水稻香味基因功能标记的开发与应用[J].热带作物学报,2022,43(4):675-683. |
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| 作者姓名： | 刘文静 胡文彬 周政 刘烨 赵正洪 徐庆国 |
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| 作者单位： | 1.湖南农业大学,湖南长沙 4101282.湖南省水稻研究所,湖南长沙 4101253.杂交水稻国家重点实验室,湖南长沙 410125 |
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| 基金项目： | 湖南省农业科技创新资金项目(2017LM0409);湖南省农业科技创新资金项目(2021CX18) |
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| 摘 要： | 香味是影响稻米品质的一个重要性状,Badh2基因突变是水稻产生香味的主要原因,水稻香味产生主要有Badh2基因第2、4、5、7、8外显子突变类型,其中研究最多的是第7外显子突变类型。为了提高水稻香味性状检测的准确性、安全性和效率,本研究根据水稻Badh2基因突变（第7外显子8 bp缺失、3 bp突变）的序列设计开发了3条分子标记引物,分别命名为Fgr-FF、Fgr-NF和Fgr-R,利用该三引物分子标记法可准确扩增出Badh2第7外显子突变基因,并鉴定其纯杂合情况。本标记利用PCR扩增中引物5°端保守性差的特点,将31 bp外源DNA序列添加至Fgr-NF引物的5°端,将Badh2基因第7外显子突变类型的非香型和香型特征条带差异由8 bp扩大至41 bp,通过琼脂糖凝胶就能准确、清晰、快速地检测出水稻植株中香味基因的纯合、杂合和无3种基因型。利用该三引物分子标记的PCR扩增结果表明：该标记在香型水稻中扩增出一条115 bp的特征条带,在非香型水稻中扩增出一条156 bp的特征条带,而在杂合型水稻中能同时扩增出115 bp和156 bp两条特征条带。本研究利用该三引物功能标记对30个水稻品种香味基因型进行检测,结果检测出香味纯合植株9株,非香纯合植株20株,该检测结果与2种表型鉴定结果相符;杂合植株1株,这是由于水稻香味基因是隐性基因,杂合型水稻叶片表现为非香,部分籽粒表现为香;使用传统的香味鉴定方法容易对香味杂合型水稻产生误判,而通过基因检测结果和表型验证表明新开发香味三引物功能标记准确可靠;利用三引物功能标记对94个F2代单株的香味基因进行检测,结果检测出香味纯合植株23株,非香纯合植株44株,杂合植株27株,符合1∶2∶1孟德尔遗传的分离比例。该研究既避免了水稻香味鉴定与检测中KOH法和咀嚼法的主观误差,也解决了聚丙烯酰胺凝胶电泳的毒性强、耗时长等问题,大幅度地提高了香型水稻品种的选择效率。
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| 关 键 词： | 水稻 香味基因 分子标记 |
| 收稿时间： | 2021-12-13 |
Development and Application of a New Functional Marker of Fragrant Gene in Rice (Oryza sativa L.) |
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| LIU Wenjing,HU Wenbin,ZHOU Zheng,LIU Ye,ZHAO Zhenghong,XU Qingguo.Development and Application of a New Functional Marker of Fragrant Gene in Rice (Oryza sativa L.)[J].Chinese Journal of Tropical Crops,2022,43(4):675-683. |
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| Authors: | LIU Wenjing HU Wenbin ZHOU Zheng LIU Ye ZHAO Zhenghong XU Qingguo |
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| Institution: | 1. Hunan Agricultural University, Changsha, Hunan 410128, China2. Hunan Rice Research Institute, Changsha, Hunan 410125, China3. State Key Laboratory of Hybrid Rice, Changsha, Hunan 410125, China |
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| Abstract: | Fragrance is an important trait which affects the quality of rice. The mutation of Badh2 is the main contribution of fragrance in rice, and there are mainly several types of exon mutation, such as the second exon, the fourth exon, the fifth exon, the seventh exon and the eighth exon of Badh2, and the mutation of the seventh exon is the most studied one. In order to improve the accuracy, safety and efficiency of rice fragrant detection, the molecular marker with three primers (3-primer marker)was designed based on the mutation of the seventh exon of Badh2 (eight bp deletion and three bp mutation), and three primers were named Fgr-FF, Fgr-NF and Fgr-R respectively. The mutation of the seventh exon of Badh2 could be detected accurately by the three3-primers molecular marker, and the homozygous and heterozygous of fragrant rice were identified. This marker took advantage of the poor conservation of the 5' end of primers in polymerase chain reaction (PCR), added the 31 bp exogenous deoxyribonucleic acid (DNA) to the 5' end of the primer Fgr-NF. This marker enlarged the differences of length between the non-fragrant and fragrant bands of PCR from 8 bp to 41 bp, and the homozygous, heterozygous and non-fragrant genotypes of rice could be accurately, clearly and quickly detected by agarose gel electrophoresis. The results of PCR amplification with the three-primer marker indicated that: 115 bp DNA fragment in the fragrant rice, 156 bp DNA fragment in the non-fragrant rice, both 115 bp and 156 bp DNA fragments in the heterozygous rice. The 30 rice varieties were detected by the 3-primer marker. And the results showed that: there were nine fragrant homozygous genotype varieties and twenty non-fragrant homozygous genotype varieties, which were consistent with the two phenotypic identification methods, KOH method and chewing method. There was one heterozygous genotype variety, and its genotype identification results was inconsistent with the phenotypic identification results, because the fragrant gene of rice was recessive gene, the leaves of heterozygous rice were non-fragrant, while some grains were fragrant. The traditional fragrant identification methods were easy to misjudge the fragrance of heterozygous rice. However, These results of gene detection and phenotypic verification showed that the 3-primer marker designed by this study was accurate and reliable. The 94 individual plants from F2 generation were detected by the 3-primer marker, and the results also showed that there were 23 fragrant homozygous plants, 44 non-fragrant homozygous plants and 27 heterozygous plants, which accorded with the 1∶2∶1 ratio of Mendelian genetic law. This study not only excluded the subjective error of KOH method and chewing method, but also avoided the toxicity and time consuming of polyacrylamide gel electrophoresis, and greatly improved the breeding efficiency of fragrant rice varieties. |
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| Keywords: | rice fragrant gene molecular marker |
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