Abstract：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-HRMF1/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.
