摘要
为了对水稻低镉积累基因进行精准检测和世代跟踪,提高低镉积累水稻品种的分子标记辅助育种(MAS)选育效率,基于籼稻品种9311及其低镉积累突变体lcd1材料7号染色体上OsNRAMP5基因第7外显子单核苷酸的突变,参照四引物扩增受阻突变PCR(Tetra-primer amplification refractory mutation system-PCR,Tetra-primer ARMS-PCR)原理设计了共显性功能标记nra5fun,且采用PCR方法分别对18份籼稻、18份粳稻及金23B/lcd1杂交F_(1)代材料进行分子标记特异性验证。电泳结果表明,含OsNRAMP5基因SNP突变材料lcd1可扩增出大小为723 bp、210 bp的条带,含野生型OsNRAMP5基因的材料9311可扩增出大小为723 bp、557 bp的条带,金23B/lcd1 F_(1)代杂合型材料可扩增出大小为723 bp、557 bp、210 bp的条带,共显性标记nra5fun得到的电泳条带与引物设计时预测的目标片段完全吻合;Sanger测序结果表明,电泳扩增条带片段的序列与目的基因的DNA序列和SNP突变位点的序列一致;18份籼稻和18份粳稻种质资源均能扩出大小为723 bp、557 bp 2个目的条带,说明这些材料均不含有OsNRAMP5基因的单个SNP突变,表明该标记特异性较高。因此,开发的功能标记nra5fun能够准确、高效地识别水稻低镉积累基因的纯合突变型、纯合野生型和杂合型,可在水稻低镉积累分子育种中推广应用。
The purpose of this study is to achieve accurate detection and generation tracking of low cadmium accumulation genes in rice,and improve the efficiency of molecular marker assisted selection(MAS).There was a single nucleotide mutation in exon 7 of OsNRAMP5 gene on chromosome 7 between the indica rice 9311 and the mutation lcd1,and the co-dominance functional marker nra5fun was developed according to tetra-primer amplification refractory mutation system-PCR.PCR was used to verify the molecular marker specificity of 18 indica rice,18 japonica rice and Jin23B/lcd1 hybrid F_(1) materials.Electrophoretic detection results showed that the mutant material lcd1 containing OsNRAMP5 gene could be amplified 723 bp and 210 bp bands,the 9311 containing wild-type OsNRAMP5 gene could be amplified 723 bp and 557 bp bands,and the heterozygote Jin23B/lcd1 could be amplified 723 bp,557 bp and 210 bp bands.The electrophoresis bands obtained by co-dominant marker nra5fun were consistent with those of the predicted target fragments.Sanger sequencing results showed that the sequence of amplified fragment was consistent with the DNA sequence of the target gene and the sequence of single nucleotide polymorphism(SNP)sites.Two target bands with lengths of 723 bp and 557 bp were amplified from 18 indica and 18 japonica germplasm resources,indicating that these cultivars did not contain single SNP mutation of OsNRAMP5 gene.So,the marker had high specificity.In conclusion,the functional marker nra5fun can accurately and efficiently identify homozygous mutant,homozygous wild type and heterozygous type of low cadmium accumulation genes in rice,and it can be widely used in the breeding of low cadmium rice cultivars.
作者
于江辉
李焱瑶
秦冠男
翁绿水
蒋显斌
邓力华
YU Jiang-hui;LI Yan-yao;QIN Guan-nan;WENG Lü-shui;JIANG Xian-bin;DENG Li-hua(Institute of Subtropical Agriculture,Chinese Academy of Sciences/Key Laboratory of Agro-ecological Processes in Subtropical Region,Changsha 410125,China;Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding,Nanning 530007,China)
出处
《江苏农业学报》
CSCD
北大核心
2022年第2期289-295,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金联合基金项目(U19A2025)。
