基于HRM体系的稻瘟病抗性基因Pi2特异性分子标记的开发及应用

摘要

Pi2 is a broad-spectrum rice blast resistance gene to the physiological races of rice blast fungus and has great application value in rice blast resistance breeding.But the wide application of the gene in rice marker-assisted selection (MAS) breeding process is seriously hindered because the operation of the developed molecular markers in the marker assisted selection of the gene is complex and difficult to achieve high throughput and accurate detection.High resolution melting analysis (HRM) is a recently developed PCR-based DNA polymorphism detection technology that is easy to operate and with high resolution,low cost,and accurate results.In this study,we took sequence alignment analysis of the genomic sequence of Pi2 gene with that of Pi9,Piz-t,PigmR and PigmS,and found 6 specific single nucleotide polymorphism (SNP) loci in Pi2 gene.Then we sequenced the DNA flanking the 6 specific SNPs of Pi2 gene in 12 rice varieties.Sequence alignment showed that they were divided into 6 categories.Based on these differences,6 pairs of primers were designed for genotyping the Pi2 gene in different rice varieties based on the HRM analysis.Primer pair Pi2-HRMF1/R3 was found to be able to classify 12 rice accessions into 6 types with high stability and reliability,which were in good agreement with the gene sequence.Through the HRM-detection of Pi2 in 23 rice sterile line and maintainer line materials,we found that none of them contained the gene.Therefore,the Pi2 gene can be introduced into these sterile lines and maintainer lines via MAS breeding by using the Pi2-HRMF1/R3 primer pair combined with the HRM technology,in order to enhance the blast disease resistance of hybrid varieties which are derived from these sterile lines and maintainer lines.When the Pi2-HRMF 1/R3 primer pair was used to analyze the segregating populations from the crosses between Huazhan,which carries the Pi2 gene,and four other recurrent parents which do not have the Pi2 gene,and found that the marker could well distinguish the Pi2 donor parent from the recurrent parents as well as the heterozygous genotype in the Pi2 gene locus in the offspring.Compared with the previously reported Pi2 gene specific molecular markers,the molecular marker developed in this study is more efficient in detection and easier in operation.Therefore,the Pi2 gene specific molecular marker combined with the HRM technology in this study can be used for efficient and accurate identification of the Pi2 gene in genetic resources and for high throughput screen of the descendants for improved resistance to blast disease based on Pi2.The method will serve as an efficient and accurate technical support to molecular breeding in a large scale in the future.%Pi2是一个对稻瘟病生理小种具有广谱抗性的基因,对于水稻(Oryza sativa)的稻瘟病抗性育种具有非常重要的应用价值,但目前针对该基因的分子标记辅助选择(marker-assisted selection,MAS)育种所开发的分子标记操作繁杂,难以实现批量以及准确检测,严重阻碍了该基因在水稻MAS育种过程中的大量应用.本研究通过对Pi2基因及其复等位基因进行序列比对,找出其特异性变异,开发成基于高分辨率熔解曲线分析(high resolution melting analysis,HRM)技术检测的与Pi2基因完全共分离的分子标记.通过对23份两系不育系和三系保持系材料进行Pi2基因鉴定,发现它们都不含有该基因.对4个Pi2基因改良的后代分离群体进行分子标记跟踪检测,发现该标记能够很好地区分供体亲本华占与其他受体亲本以及华占与各受体在Pi2基因处的杂合基因型.与已经报道的Pi2特异性分子标记相比,本研究所开发的分子标记的检测效果更好,操作更加简便.因此,本研究中的Pi2基因特异性分子标记结合HRM的检测方法能够高效准确地进行Pi2基因的资源鉴定和对抗病性改良后代群体的高通量检测筛选,为今后的水稻高效、准确、大规模分子育种提供技术支持.