Rapid mapping and cloning method for plant spontaneous mutation genes bridged by neutral mutant

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to rapid mapping and cloning method for plant spontaneous mutation genes bridged by a neutral mutant. The invention aims to overcome the defects in the prior art, and the neutral mutant produced by mutagenesis of an ethylmethane sulfonate (EMS) mutagenic agent composition is utilized to rapidly mapthe spontaneous mutation genes. Under the same genetic background, the neutral mutant (namely, no macroscopic phenotypic variation) produced by mutagenesis of the EMS mutagenic agent composition is hybridized with a spontaneous mutant to obtain F1, and selfing is continued to obtain an F2 generation. By performing screening and hybrid sequencing of extremely recessive phenotypic single plants in an F2 population, combining with EMS mutated neutral mutant sequencing data, and utilizing a bioinformatics method, the mutation genes are rapidly mapped. Compared with known methods, the rapid mappingand cloning method has the outstanding advantages of high screening efficiency, short consumed time, low cost and the like. The rapid mapping and cloning method can be used for rapid cloning of the spontaneous mutation genes.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. In particular, it relates to a rapid positioning and cloning method of a plant spontaneously mutated gene bridged by a neutral mutant. That is, the method of quickly locating the spontaneous mutant gene by using the neutral mutant produced by EMS mutagenesis. Background technique [0002] Spontaneous mutation refers to the mutation that occurs naturally in organisms without human interference. This mutation is mainly derived from base mismatches, spontaneous lesions, or transposon transpositions during the DNA replication of organisms (Griffiths et al. An introduction to genetic analysis. Macmillan, 2005). The mutants produced by organisms due to spontaneous mutation are called spontaneous mutants, which include not only mutants produced during normal reproduction of organisms, but also mutants produced by organisms during tissue culture. The gene localization of spontaneous ...

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Problems solved by technology

However, the method of gene mapping through backcross populations has the following disadvantages: First, because the physical location of the target gene is not clear, the selection of donor individual plants in the backcross process is blind, and a little carelessness may result in The backcross process backcrosses the target gene segment, which makes the location of the mutant gene more risky; second, even if each generation of backcross single plant carries the mutant gene, in order to prevent the gene effect from being affected by other genes, it is necessary to ensure that the backcross offspring Higher background homozygosity requires more backcross generations

During subsequent DNA replication, the original G / C base pair becomes A / T (Ashburner M. Drosophila. A laboratory handbook. Cold spring harbor laboratory press, 1989; Greene et al. Spectrum of chemically induced mutations from a large -scale reverse-geneticscreen in Arabidopsis.Genetics,2003,164:731-740); but there is also a small part of the alkylation occurs in other positions, such as G mutation to 7-ethylguanine will lead to G / C to The mutation of C / G or G / C to T / A, and the 3-ethyladenine formed by alkylation of A leads to the mutation of A / T to G / C (Krieg DR. Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants. Genetics, 1963, 48:561)

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