RAPID DETECTIOM OF SINGLE NUCLEOTIDE POLYMORPHISM (SNP) IN RICE USING HIGH RESOLUTION MELTING ANALYSIS ON MICROFLUIDIC CHIP

Molecular breeding of rice is crucial for feeding the world’s rapidly growing population, for limited land resources, superior grain quality, and severe environmental situation[1]. Single nucleotide polymorphism(SNP) is regarded as the third genetic marker, having the potential to increase the speed and cost-effectiveness of genotyping and breeding applications, as well as routine genetic diversity analysis, linkage mapping, and marker-assisted selection in rice[2]. High resolution melting (HRM) analysis is a new methodology for mutation scanning that has gained considerable popularity in recent years[3]. The advantages of HRM for variant scanning include rapid turn-around times, closed-system that greatly reduces contamination risk, directly analysis after PCR, and unconsidering novel variants or genotyping known type[4]. However, due to HRM requiring high temperature resolution during heating process, traditional tube method is difficult to meet the demands for SNP detection in rice molecular breeding. Here we reported a microfluidic chip and a real-time PCR system integrated high precise thermal cycler and online fluorescence detection module (Fig.1/2) [5]. This platform ingeniously utilized the advantage of microfluidic chip, such as heat fast transfered and well-distributed in the chamber. We used Oryza sativa L. and 9311 as the experimental material, and selected a few important SNPs for breeding as our research objects by comparing genomes database of two species. The result demonstrated that this platform can rapidly detected the SNPs through PCR and HRM analysis on the chip (Fig.3/4). It also indicated that the device can be applied widely in applications screening of mutants in molecular breeding.