一种新的水稻香味基因功能标记的开发与应用

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...

Development and Application of a New Functional Marker of Fragrant Gene in Rice (Oryza sativa L.)

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.