A method for rapid mapping and cloning of plant spontaneously mutated genes bridged by neutral mutants

Abstract

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. The purpose of the present invention is to overcome the defects of the prior art, and quickly locate the spontaneous mutant gene by utilizing the neutral mutant produced by mutagenesis with the EMS mutagen composition. F 1 , continue selfing to get F 2 generation. by screening F 2 The extreme recessive phenotypes in the population were mixed and sequenced, combined with the sequencing data of the neutral mutants induced by EMS, and the bioinformatics method was used to quickly locate the mutant genes. Compared with the known method, the present invention has the outstanding advantages of high screening efficiency, short time and less cost. The invention can be used for rapid cloning of spontaneously mutated 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 ...

AI Technical Summary

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)

Images