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

A technology of spontaneous mutation and cloning method, which is applied in the field of plant genetic engineering and can solve problems such as unclear physical location of target genes

Active Publication Date: 2021-01-01
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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 generat

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Obtaining wild-type rice material EMS-ZH11 by mutagenesis with 0.4% EMS

[0031] The applicant obtained a spontaneous mutant gene with few tillers and yellowish leaf color, which was derived from the japonica rice variety Zhonghua 11. According to the process of the present invention, the wild-type Zhonghua 11 must first be treated with EMS reagents according to the background of the spontaneous mutant (Special note: the "wild-type" material described in the technical documents of the present invention essentially refers to the relative Raw materials that have not undergone any artificial or non-artificial genetic improvement for mutants. For concise expression, "wild type" involved in the present invention is a technical term in the field of plants, and does not refer to other meanings or derived meanings; Zhonghua 11 is a common rice variety in the field, and is a material often used in rice research and inventions) EMS-ZH11 was obtained. In the present in...

Embodiment 2

[0044] Example 2: Mixed sequencing of gene mapping populations and extremely recessive individual plants

[0045] (1) Parental phenotype investigation: The spontaneous mutant used in this example is a mutant with few tillers and yellowish leaf color. Through the investigation of the main agronomic traits, it was found that the two parents had the same number of tillers, plant height and chlorophyll a. Significant difference (Table 1 and figure 2 ).

[0046] Table 1 Main phenotypic differences between EMS-ZH11 and 3t mutants

[0047]

[0048] (2) F 1 Seeds (Since this hybridization cannot identify true and false hybrids through molecular markers, the female parent must be a spontaneous mutant during hybridization, so that the F 1 The generation selects the correct hybrid according to the phenotype: any hybrid whose phenotype returns to the wild-type phenotype is F 1 single plant, that is, the correct hybrid).

[0049] (3) planting F 1 generation, identify true and fal...

Embodiment 3

[0059] Example 3: Analysis of sequencing data

[0060] (1) Check whether the compressed data is correct through the MD5 command.

[0061] (2) FastQC software (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) was used to evaluate the sequencing quality.

[0062] (3) Refer to Wachsman's method for data analysis (Wachsman et al. A SIMPLE pipeline formatting point mutations. Plant Physiol, 2017, 174:1307-1313), see https: / / github.com / wacguy / Simple-1.8 for the specific code. 1. There are two improvements, improvement part 1: If a site is defined as dominant as "+" and recessive as "-", Wachsman's criteria for screening candidate genes is to screen the sequencing reads of a site in the mutant mixed pool (mut) All are - / -, and the sequencing reads of the corresponding wild-type mixed pool (wt) at this site are + / + and + / - sites. However, the applicant uses the extreme mutation phenotype mixed pool (mut) and the "wild type" (wt) induced by EMS. After selfing, the applican...

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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 ...

Claims

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Application Information

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IPC IPC(8): A01H1/02A01H1/06C12Q1/6869G16B20/50G16B20/20
CPCA01H1/02A01H1/06C12Q1/6869G16B20/20G16B20/50
Inventor 邢永忠胡伟周天浩
Owner HUAZHONG AGRI UNIV
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