Development Of SNP Markers Based On Transcriptome Sequencing Of Xu 781 And Xushu 18 In Sweetpotato, Ipomoea Batatas(L.) Lam.

Single Nucleotide Polymorphism(SNP) had been thought of the most potential third-generation molecular markers in recent years. We had developed 1,386 SNPs candidate loci from the RNA-seq data of Xushu 18 and Xushu781 with significant difference in starch content, dry matter yield and resistant to stem nematode. Three methods were selected to detect SNP loci between Xu 781 and Xushu 18 respectively, including two specific amplification methods, Allele-specific PCR(AS-PCR) and Tetra-primer Amplification Refractory Mutation System PCR(Tetra-primer ARMS-PCR), and one enzyme digestion method, Cleaved Amplified Polymorphic Sequence(CAPS) in this study. One suitable method was confirmed by comparing their availability and validity in sweetpotato SNP marker detection. The reaction system of the suitable method was optimized further, including the annealing temperature, the concentration ratios of inner and outer primers, and the dosage of Taq DNA polymerase. According to the sequencing of SNP candidate loci, the SNP markers were detection and developed using the suitable method. The main results are as follows:1. Three methods,AS-PCR, Tetra-primer ARMS-PCR and CAPS, were compared for their availability and validity of SNP marker detection, and the reaction system of the suitable method was optimized further. Agarose gel electrophoresis results showed that Tetra-primer ARMS-PCR was a rapid, accurate, efficient and cost-effective SNPs detection method in sweetpotato, and efficient results could be gotten when the concentration ratios of inner and outer primers was 0.4 μmol/L: 0.2 μmol/L, the dosage of Taq DNA polymerase was 1.25 U in 25 μL PCR reaction system under optimum annealing temperatures.2. Total 153 sets primers were designed in Tetra-primer ARMS-PCR based on 1,386 SNP loci between Xu 781 and Xushu 18. PCR products of 103 sets primers were different between Xu 781 and Xushu 18 by detection of agarose gel electrophoresis, the rate of difference was 67.97%. The results showed that Tetra-primer ARMS-PCR was suitable for detectiing SNP loci and developing SNP markers in sweetpotato.Tetra-primer ARMS-PCR was simple, accurate, and cheap method to detect SNP loci and to develop SNP markers without special equipments in sweetpotato effectively. It can be applied to high density genetic maps construction and functional molecular marker development in sweetpotato.