Molecular marker detection method based on next-generation sequencing technology

Abstract

The invention belongs to the technical field of molecular marker-assisted breeding, and discloses a molecular marker detection method based on a next-generation sequencing technology. Amplicons of allsingle plants at important agronomic trait-related loci are obtained at a time through multiple PCR by using designed specific primers of the multiple loci and a PCR reaction system and an analysis process according to the principle of amplicon sequencing, primers containing different tag sequences are used to carry out one-time amplification on the amplicons, the amplicons of all the plants areexpanded. With the different tag sequences, the amplicons undergo next-generation sequencing, and sequencing results are analyzed to obtain the genotypes of all the single plants at a time. A high-throughput detection system constructed in the invention is used to quickly and accurately detect the disease-resistant quality-related genes which are widely used in existing breeding practices.

Description

technical field [0001] The invention belongs to the technical field of molecular marker-assisted breeding, and in particular relates to a molecular marker detection method based on next-generation sequencing technology. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] Molecular markers currently used in breeding work are generated by converting functional loci in genes related to agronomic traits or closely linked variant loci into AFLP, CAPS, SSR, indel, SCAR and other markers. For example, the double SNP marker NL2-3 for the detection of Ty-1 (Gennaipeng et al.2014), the SCAR marker ToMV for the detection of Tm-2a (Foolad et al.2012), etc. This type of technology mainly relies on the specificity of PCR amplification and restriction endonuclease, and finally distinguishes genotypes by detecting the presence and size of target bands by agarose gel electrophoresis and polyacrylamide gel electrophoresis...

AI Technical Summary

Problems solved by technology

[0008] The molecular markers currently used in breeding practice depend on whether the mutation site is a restriction site (CAPS marker), whether it is an insertion-deletion (indel marker), whether it is sequence-specific (SCAR marker), microsatellite variation (SSR marker ), limited in the selection of variable sites

[0009] The genotype is judged by displaying band differences on agarose or polyacrylamide gel electrophoresis, and the labor cost is high

[0010] Low throughput, usually only 1 to 10 marker sites can be detected per PCR

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