Development Of Rice SNP Marker Based On HRM Technology And Its Application In Gene Location

SNP(Single Nucleotide Polymorphism)is a kind of variation in a single nucleotide that occurs at a specific position in the genome. SNP is widely distributed across the genome, and it has been applied in a lot of studies related to genetics, functional genomics and molecular breeding. In the recent years, hundreds of thousands of SNPs have been identified in rice(Oryza sativa L.)through high-throughput genome sequencing. Development of low-cost and high-efficient SNP genotyping methods would help to use these massive SNPs high efficiently and economically. The HRM(High-Resolution Melting)analysis is such a low-cost and medium- to high-throughput genotyping approach, which achieved SNP genotyping through real-time monitoring of the melting curves of double-strand DNA fragments. In previous studies in our lab, several HRM-based DNA markers of cloned key genes have been developed and used in rice breeding programs. Herein, a set of HRM-based rice whole-genome SNP markers was firstly developed, and then it was used in analyzing genetic diversity and mapping of a mutated gene in rice. The main results are as following:(1)According to rice SNP data from the Hap Rice and RICE6 K, 1871 SNPs randomly distributed throughout the genome were selected as candidates for developing HRM-based markers, and 1069 of them were successfully developed primer pairs to amplify short fragments suitable for HRM genotyping.(2) Amplification of the 1069 primer pairs was conducted by using DNA of 6 diverse rice cultivars including Nipponbare(Temperate japonica rice), Francis(Tropical japonica rice),Yuefengxinzhan(conventional indica rice from Southern China), Wufeng B(indica rice, B-line from Southern China), R173(indica rice, R-line from Southern China) and Kasalath(indica, rice from South Asia). And then, amplicons of each pair was subjected to HRM analysis process. The amplicons from 865 pairs showed single melting peaks, and totally 599 of them displayed polymorphism between at least two rice cultivars. Therefore, the 599 primer pairs were successfully transferred into HRM-based SNP markers, and their average density across rice genome was 1.6 Mb per SNP. The constructed HRM-SNP genetic map provides a total of 48 SNP markers which can use on a location of both sides of the 24 important traits genes that has been cloned in rice, It has a higher value in the important gene marker-assisted selection breeding.(3) Taken genotyping results at the 599 SNP markers as input, genetic diversity among the 6 rice accessions was calculated. The Nipponbare and Wufeng B showed the maximum polymorphism with a polymorphic rate of 75.63%(453 of 599 SNPs); while the Yuefengxinzhan and R173 showed lowest polymorphism with a polymorphic rate of 34.06%(204 of 599 SNPs). Within the 4 indica rice cultivars, R173 and Yuefengxinzhan have the most similar genetic background, the followed is Wufeng B, and the last is Kasalath; the genetic background between Nipponbare and indica varieties is the furthest, and Francis followed.(4) According to the development of genomic HRM-SNP markers, we using an F2 population to preliminary locate the spotted leaf mutant genome of spl32. With the method of BSA(Bulked Segregation Analysis)and recessive-class analysis to finish the SNP genotyping and association studies respectively, and then located the mutant gene on chromosome 11 of 22.35 Mb-24.07 Mb.