CN112154910B

CN112154910B - Rapid positioning and cloning method of neutral mutant bridged plant spontaneous mutant gene

Info

Publication number: CN112154910B
Application number: CN201910518813.2A
Authority: CN
Inventors: 邢永忠; 胡伟; 周天浩
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-06-16
Filing date: 2019-06-16
Publication date: 2021-10-22
Anticipated expiration: 2039-06-16
Also published as: CN112154910A

Abstract

The present invention belongs to the field of plant gene engineering technology. In particular to a method for quickly positioning and cloning spontaneous mutant genes of plants bridged by neutral mutants. The present invention aims to overcome the drawbacks of the prior art by rapidly mapping spontaneous mutant genes using neutral mutants generated by mutagenesis with a composition of EMS mutagens. In the same genetic background, neutral mutants generated by mutagenesis using EMS mutagen compositions (i.e., no macroscopic phenotypic variation) were crossed with spontaneous mutants to give F₁Continue selfing to obtain F₂And (4) generation. By screening F₂And carrying out mixed sequencing on the single plants with the extreme recessive phenotype in the population, combining sequencing data of a neutral mutant subjected to EMS mutagenesis, and quickly positioning a mutant gene by using a bioinformatics method. Compared with the known method, the method has the outstanding advantages of high screening efficiency, short time consumption, low cost and the like. The invention can be used for the rapid cloning of spontaneous mutant genes.

Description

Rapid positioning and cloning method of neutral mutant bridged plant spontaneous mutant gene

Technical Field

The present invention belongs to the field of plant gene engineering technology. In particular to a method for quickly positioning and cloning spontaneous mutant genes of plants bridged by neutral mutants. Namely a method for rapidly positioning spontaneous mutant genes by using neutral mutants generated by EMS mutagenesis.

Background

Spontaneous mutation (spontaneous mutation) refers to a mutation that occurs naturally in an organism without human intervention. Such mutations are mainly due to base mismatches, spontaneous lesions or transposon transposition during replication of the organism's DNA (Griffiths et al, An expression to genetic analysis. Macmillan, 2005). A mutant of an organism produced by spontaneous mutation is referred to as a spontaneous mutant, and such a mutant includes not only a mutant produced during normal propagation of the organism but also a mutant produced during tissue culture of the organism. Gene mapping of spontaneous mutants currently only allows for mapping by cloning (Map-based cloning), i.e.crossing a spontaneous mutant with another parent with a different genetic background to obtain F₂After population isolation, the nucleic acid differences (molecular markers) between the two parents are used for gene mapping. This generation of F by two parent hybridization₂Gene mapping method of segregating population is suitable for mapping major mutant gene causing serious variation of plant development and growth characteristicsFor example, the location and cloning of rice few tillering mutant gene MOC1 and semi-dwarf gene SD1(Li et al. Control of tillering in rice nature 2003,422: 618) -621, Monna et al. Positional cloning of rice mid-dwarfing gene, SD-1: race "green re-volume gene" encoding a mutant enzyme in nucleic acid synthesis. DNA research 2002,9:11-17) and tomato maturation inhibitory gene Rin (EBalov et al. A MADS-box gene research for fruit growing the tomato growing-in bit (science 2002,296: 343-. However, this approach is not suitable for the mapping of slightly mutated genes that cause quantitative trait variation, because F is generated in two parents that are derived from different genetic backgrounds₂Segregation of multiple other genes controlling the mutant phenotype in the population will mask the effects of the slightly mutated genes. This masking effect will result in a non-co-segregation of phenotype and genotype, which in turn will not allow the target gene to be located.

For the minor gene mapping of spontaneous mutation, gene mapping can be performed only by crossing a spontaneous mutant with another parent and homozygous background by backcrossing so that the population has only wild type and mutant phenotypes: for example, the rice LEAF type gene NAL7 is located by backcrossing (Fujino et al, NARROW LEAF 7control LEAF style medium by itself in rice 507). However, the method of gene mapping by backcross population has the following disadvantages: firstly, because the physical position of the target gene is not clear, the selection of a donor single plant in the backcrossing process is blind, and the target gene segment can be backcrossed in the backcrossing process by a little carelessness, so that the mutant gene positioning has a large risk; and secondly, even if the backcross single plant of each generation carries a mutant gene, in order to ensure that the gene effect is not influenced by other genes, the higher background purity of backcross progeny is ensured, and more backcross generations are needed. The above two factors greatly increase the time, labor and material costs of spontaneous mutant gene localization.

The gene positioning is based on the molecular marker, and a target site is searched through the linkage relation between the phenotype and the molecular marker. Molecular markers include more than just traditionsThe RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA) or SSR (Simple Sequence Repeats) markers, and further includes SNP (Single Nucleotide Polymorphism) and InDel (Insertion and Deletion) markers generated by sequencing. The mutation by using a chemical mutagen EMS (Ethyl methane sulfonate) can generate abundant SNP variation, and the main principle of the mutation is that the base undergoes alkylation reaction, so that the base undergoes mismatch reaction in the replication process. For example, 99% of the alkylation in EMS mutagenesis occurs in the O of G (Guanine )⁶At a position to become O^6-Ethyl guanine, results in the change of C (cytosine) to T (Thymine) which is originally complementary to G. During subsequent DNA replication, the original G/C base pairs become A/T (Ashburn M. Drosophila. A. laboratory hand book. Cold spring harborage press, 1989; Greene et al. Spectrum of chemical induced mechanisms from a large-scale-reverse-genetic-scale in Arabidopsis. genetics,2003,164: 731-740); however, there is also a very small proportion of alkylation occurring at other positions, for example, a mutation of G to 7-ethylguanine will result in a G/C to C/G or G/C to T/A mutation, whereas A results in an A/T to G/C mutation due to the 3-ethyladenine formed by alkylation (Krieg DR. Ethyl methyl methylated-induced conversion of bacterial T4rII mutations. genetics,1963,48: 561). EMS mutagenesis has been widely used in recent years for the artificial induction and gene localization of mutants, for example TILLING (Till et al. Large-scale discovery of induced points with high-throughput TILLING. Genome Res,2003,13: 524. sub.530) and MutMap technology (Abe et al. Genome sequencing reports on molecular injection in using MutMap. Nat Biotechnology, 2012,30: 174. sub.178) are based on EMS mutagenesis.

With the development of second generation sequencing technologies, gene mapping by means of sequencing has become more and more mature, such as the MutMap method (see above) and the QTL-seq method (Takagi et al. QTL-seq: rapid mapping of quantitative trap location in by floor genome sequencing of DNA from two distributed locations. plant J,2013,74: 174) 183. SNP and InDel information are identified in a sequencing mode and used for gene positioning, so that time and labor cost are greatly saved, and the method is a main direction of future gene positioning.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and rapidly locate spontaneous mutant genes by using neutral mutants generated by EMS mutagenesis. Hybridization of neutral mutants (no phenotypic variation compared to wild-type) generated using EMS mutagen compositions with spontaneous mutants in the same genetic background to yield F₁Selfing to obtain F₂And (4) generation. By screening F₂And (3) carrying out mixed sequencing on the single plants with the extreme recessive phenotype in the population, combining sequencing data of a neutral mutant subjected to EMS mutagenesis, and quickly positioning a mutant gene by means of a bioinformatics method.

The technical problem to be solved by the invention is as follows: at present, for the gene localization and cloning of spontaneous mutants, whether major genes or minor genes, F can be obtained only by crossing the spontaneous mutants with parents in other backgrounds₂The population is isolated and then subjected to map-based cloning or QTL-seq. However, the progeny phenotype of this segregating population is co-regulated by multiple genes (Quantitative Trait loci) between the two parents, at F₂In segregating populations, these quantitative trait loci are randomly combined, resulting in no co-segregation relationship between phenotype and mutant gene. To mask interference from genetic background, for example, the wild type can be hybridized with spontaneous mutants to obtain F by the method described with reference to MutMap₂Segregating the population, progeny, although producing a phenotypic segregation ratio of a single gene effect (3:1 or 1:2:1), are difficult to locate spontaneous mutant genes because of highly uniform genomes without nucleic acid variation covering the entire genome. In order to reduce the interference of genetic background on gene localization and to rapidly locate genes by sequencing, it is necessary to have a certain base separation in the whole genome of the isolated population. Thus, artificially mutagenizing a neutral mutant material to make it have nucleotide differences between the whole genome and spontaneous mutants, while the phenotype of the material is not the same as that of the wild typeA difference. F produced by hybridization of this neutral mutant material with spontaneous mutants₂The population can be used for rapid location of the mutant gene. The method can rapidly and efficiently locate the spontaneous mutant gene.

The technical scheme of the invention is as follows:

(1) the wild type variety X mutagenized by EMS was first determined based on the genetic background of spontaneous mutants, such as in the present example spontaneous mutants derived from flower 11 (hereinafter referred to as ZH11) in japonica rice variety, so the rice variety X mutagenized by the EMS mutagen composition had to be wild type ZH11 without any treatment to ensure consistent genetic background. After the EMS mutagen composition is subjected to mutagenesis treatment and selfing for 3 generations, phenotypes are observed, and families with no difference between the phenotypes and wild types are selected for subsequent invention. For example, applicants selected one of the strains and designated EMS-X, and named this material with both SNP and phenotypic variation as neutral mutants.

(2) Hybridizing the obtained EMS-X material and spontaneous mutant material to obtain F₁Inbred to give F₂Planting 400F strains of 300-₂A single plant is a population, for example, 300 plants are planted in this example.

(3) The phenotype was investigated and the segregation ratio was counted. If the character segregation ratio is in accordance with the single-gene segregation ratio, gene localization can be carried out. At F₂25-30 individuals with extreme recessive phenotype are selected from the population and subjected to mixed sequencing. If the EMS-X selfing generation is low, the step can be also at F₂Two extreme phenotype mixed pools were selected from the population for sequencing separately: the recessive mixed pool is a mixed single plant with mutant phenotype, and the dominant phenotype mixed pool is a mixed single plant with non-mutant phenotype.

(4) Analyzing and extracting EMS-X and SNP/InDel sequenced by an extreme recessive single plant mixed pool by a bioinformatics method, and then screening candidate target sites; if the last step of sequencing uses two extreme phenotype individual mixed pools, this step requires extracting the SNP/InDel between the two extreme phenotype mixed pools and then screening candidate target sites.

(5) Calculating the SNP index value through the extracted SNP/InDel, drawing, combining the variation types (such as SNP causing frame shift mutation, missense mutation and early termination mutation) of the target sites extracted in the last step, and selecting the final candidate gene.

(6) And (3) performing candidate gene sequencing on the spontaneous mutant and the EMS-X by using a first-generation sequencing technology, and verifying whether second-generation sequencing data and an analysis result are correct. Simultaneously designing molecular markers (SNP, InDel) according to the nucleotide difference of the two candidate genes or directly sequencing, and performing sequencing on the F₂Cosegregation assays were performed in the population.

(7) If the co-segregation test shows that the candidate gene and the phenotype are co-segregated, the transgene verification of the candidate gene can be carried out.

Biological material deposit information (examples):

the applicant names the rice materials obtained by mutagenesis by using the EMS mutagen composition as rice EMS-ZH11 and Oryza sativa L.EMS-ZH11, and delivers the rice materials to China, Wuhan university China center for type culture Collection in 2019, 5 months and 27 days, wherein the preservation number is CCTCC NO: P201908.

The invention has the following beneficial effects:

neutral mutants generated by mutagenesis with the EMS mutagen composition have abundant SNP variations compared to spontaneous mutants. In gene mapping, these SNPs can be used as molecular markers for mapping. While phenotypically, this F₂Apart from the separation on the target character phenotype, the group does not have the separation of other phenotypes theoretically, so that the group has abundant nucleotide polymorphism, the phenotype is not interfered by other genes, and the group is very suitable for the rapid positioning of spontaneous mutant genes.

Although the applicants 'neutral mutant bridging method employs the same data analysis method as MutMap, the application targets of the applicants' method and the MutMap method are significantly different: the applicants' method is directed primarily to spontaneous mutations in which the background is the same as the wild type, except for the target gene. The MutMap method aims at inducing mutation, and the induced mutant has huge variation in background besides the target gene. The basic process of the two methods is also different: the method of MutMap is to use mutant and homologous generated by EMS mutagenesisA background wild-type rice variety was crossed, then at F₂And selecting single plants with extreme phenotypes from the population, and performing mixed sequencing. Applicants' method is to first induce wild-type rice material using an EMS mutagen composition to produce neutral mutants, and to hybridize with spontaneous mutants, followed by mutation at F₂And selecting single plants with extreme phenotypes from the population, and performing mixed sequencing. The MutMap method is to hybridize the mutant with the wild type rice, while the present invention utilizes spontaneous mutants to hybridize with neutral mutants.

Drawings

FIG. 1: the invention is a flow chart. Description of reference numerals: wild type X is a species with the same background as the spontaneous mutant. Obtaining M after mutagenesis by EMS reagent₁Generation, selfing for 3 generations to obtain M₄And selecting a single plant which has no difference with the wild type phenotype, namely EMS-X, and performing second-generation sequencing. Subsequent hybridization of EMS-X and spontaneous mutants to F₁And selfing to obtain F₂Selecting F₂And mixing medium-extreme recessive phenotype single plants and then performing second-generation sequencing. Extracting SNP and InDel by comparing sequencing data of an extreme invisible single plant mixing pool and EMS-X, calculating the numerical value of the SNP index, selecting a target site and drawing a picture of the SNP index; in this step, the dominant phenotype individual can also be subjected to mixed sequencing, and data analysis is performed by comparing the sequencing data of the extreme recessive individual mixed pool and the dominant phenotype mixed pool, as shown in the dotted line part in fig. 1. And (4) combining the target sites (possibly more than one target site) screened in the last step and the SNP index information, and selecting the most possible candidate genes. And then sequencing candidate genes of the EMS-X and the spontaneous mutant to verify whether the bioinformatics analysis result is accurate. Then designing molecular markers based on nucleotide differences between candidate genes, at F₂Cosegregation assays were performed in the population. If the coseparation detection shows that the candidate gene prediction is correct, the next candidate gene transgene verification can be carried out.

FIG. 2: EMS-ZH11 and 3t mutant phenotype. Description of reference numerals: EMS-ZH11 in panel A of FIG. 2 is flower 11 (ZH 11 for short) of the EMS-treated rice variety, EMS-X without phenotypic variation, and 3t mutant is a spontaneous mutant with few tillered leaves with yellowish color; FIG. 2B is a graph showing the difference in leaf color phenotype between EMS-ZH11 and 3t mutant; panel C in figure 2 is an assay for chlorophyll and carotenoids of the two parents, where there is a difference in chlorophyll a (P <0.05), marked with an "+" in figure 2.

FIG. 3: SNP index scattergram. Description of reference numerals: the abscissa represents 12 different chromosomes of rice, and the ordinate represents the value of abs. The larger the number and the greater the likelihood of linkage or co-segregation of the candidate gene genes.

FIG. 4: candidate gene sequencing and F of EMS-ZH11 and 3t mutant₂And (4) carrying out population coseparation detection. Description of reference numerals: panel A of FIG. 4 shows a comparison of the sequencing of candidate genes. The mutation occurs in adjacent positions, with a 2bp deletion occurring in the intron region and a 1bp deletion occurring in the exon region, resulting in a frame shift mutation in the amino acid. These two mutations also resulted in a mutation in the restriction site of the restriction enzyme SalI, which allows the design of a Clean Amplified Polymorphic Sequence (CAPS) marker for F₂Carrying out co-segregation detection on the population; FIG. 4B is a diagram of using CAPS tag pairs F₂The population was identified as co-segregating, where "W" represents the Wild type genotype (Wild type), "M" represents the 3t Mutant genotype (Mutant), and "H" represents the heterozygous genotype (Heterozygate).

FIG. 5: tillering number frequency distribution in the F2 population. Description of reference numerals: the different colors in the figure are classified according to the genotype identified by the CAPS marker, and the arrows indicate the number of tillers corresponding to the two materials.

FIG. 6: candidate gene transformation vectors. Panel A in FIG. 6 is pU2301-3 XFLAG vector backbone, vector Picture origin (Sun Q. and Zhou D. Proc Natl Acad Sci U S A,2008,105: 13679-; panel B of FIG. 6 shows the vector backbone of candidate gene OS07G0141400-pU2301-3 XFLAG.

FIG. 7: 3t mutant transgene verification. Description of reference numerals: panel A in FIG. 7 is a control of candidate gene over-expression negative and positive individuals, wherein #3 and #8 are over-expression negative individuals and #1, #2 and #4 are over-expression positive individuals; panel B in FIG. 7 is a positive test for the DNA level of the transgenic individual; panel C in FIG. 7 is a comparison of leaf color phenotype; the D plot in fig. 7 is an assay of chlorophyll and carotenoids of transgenic negative and positive individuals, where the over-expressed positive individual has a significant difference in chlorophyll a compared to the negative (P <0.01), which has been marked with an "x".

Detailed Description

By referring to the flow chart of fig. 1, this example rapidly located and cloned the tiller-less leaf color yellowish mutant gene.

The following examples further define the invention and describe the detailed procedures for the novel method to locate spontaneous mutant genes. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1: obtaining wild rice material EMS-ZH11 by using 0.4% EMS mutagenesis

The applicant obtains a spontaneous mutant gene with less tillering and yellow leaf color, and the gene is derived from a medium flower 11 of a japonica rice variety. According to the process of the present invention, EMS-ZH11 is obtained by first treating wild type mesoflower 11 (specifically, the term "wild type" as used in the technical documents of the present invention means the original material which is substantially not genetically modified by any human or non-human in the art with respect to the mutant. for the sake of brevity, "wild type" as used in the present invention is a term of art in the plant field and does not mean any other meaning or any derivative thereof, and mesoflower 11 is a rice variety commonly used in the art and is a material commonly used in rice research and creation). In the present invention, 0.4% ethyl methanesulfonate (EMS for short, an abbreviation of the chemical mutagen; for the sake of descriptive convenience, the present invention will be directly written as EMS) mutagen composition (preparation method: 61mL of 0.2mol/L Na₂HPO₄A solution; 39mL of 0.2mol/L NaH₂PO₄A solution; supplemental ddH₂O to 200mL 0.1M phosphate buffer formulated to pH 7.0Liquid; 800. mu.L of EMS stock solution was added to the mixture in a volume ratio of 0.4% EMS stock solution to 0.4% PBS buffer to obtain a 0.4% EMS mutagen composition. )

(1) Preparation of reagents:

phosphate buffer (0.2mol/L Na)₂HPO₄): weighing Na₂HPO₄(Sigma _ FW 142)28.4g in 1000ml ddH₂O。

0.2mol/L NaH₂PO₄: weighing NaH₂PO₄·2H₂O31.2 g in 1000mL ddH₂And (4) in O.

Inactivating agent (Inactivation solution): 0.1mol/L NaOH, 20% w/v Na₂S₂O₃(Sodium Thiosulfate), 4g NaOH was weighed and dissolved in 1000mL ddH₂In O, 200g of Na is weighed₂S₂O₃Dissolving in the solution, and mixing.

(2) Seed cleaning: bagging seeds of flower 11 of wild rice variety with mesh bag, cleaning with tap water, removing dust, and finally dH₂And O cleaning for 2-3 times.

(3) Seed soaking: washing the seeds at room temperature (24-28 deg.C) with dH₂Soaking in O for 24 h. Preparation of EMS phosphate buffer: in an amount of 61mL Na₂HPO₄Solution +39mL NaH₂PO₄Solution +100mL ddH₂O ratio 0.1M phosphate buffer (pH 7.0) was prepared, mixed well and added with 800 μ L EMS (Sigma USA) stock (about 0.8/200 ═ 0.4% by volume), the amount of phosphate buffer needed to cover 1-2cm of seeds.

(4) EMS processing: the whole mesh bag with the seeds was put into phosphate buffer of EMS reagent composition at room temperature on a shaker at 140rpm for 18 h.

(5) Cleaning: firstly pouring EMS solution into a container, and pouring the EMS solution to the greatest extent; then washing with tap water, collecting the waste liquid of the previous two times of washing as EMS solution, and then washing for 24 hours under the tap water; EMS solution treatment: mixing the inactivated Solution with EMS Solution in equal amount, standing for 24h, delivering to a hazardous waste recovery unit for uniform treatment, and soaking other EMS pollution appliances in the inactivated Solution for 24h and then discarding.

(6) Accelerating germination: the whole mesh bag is wrapped by absorbent paper and then placed in a 37-degree thermostat and washed once every 12 h.

(7) Homozygous background: m₁Generations (first generation of EMS processing) were directly harvested and planted M without observation of phenotype₂And (4) generation. Then selfing the first generation to obtain M₃This step was to eliminate heterozygous SNPs by selfing, making the background more homozygous.

(8) Selecting EMS-ZH 11: 30 plants were planted in each family of M3 generation. The pedigree-growing material was selected as the follow-up material in the M3 generation (M4) and sequenced.

Example 2: mixed sequencing of gene-localized populations and extremely recessive individuals

(1) And (3) parent phenotype investigation: the spontaneous mutant used in this example was a few tillers and a yellowish leaf color, and the two parents were found to have significant differences in tiller number, plant height and chlorophyll a by examining major agronomic traits (table 1 and fig. 2).

TABLE 1 EMS-ZH11 and 3t mutants major phenotypic differences

(2) Hybridizing the 3t mutant serving as a female parent and the EMS-ZH11 serving as a male parent to obtain F₁Seed (since this cross cannot identify true and false hybrids by molecular markers, the female parent must be a spontaneous mutant when crossed, so that at F₁Selection of correct hybrids by phenotype: f for restoring phenotype of all hybrid species to wild type phenotype₁Single plant, i.e. the correct hybrid).

(3) Planting F₁Generation, identifying true and false hybrid through phenotype, and selecting correct single plant to harvest F₂And (4) seeds. Planting 300 Strain F₂And (4) carrying out phenotype investigation 30 days after the seedlings are transplanted, and investigating tillering number and leaf color phenotype. The applicant finds that the tillering number and the leaf color are co-separated, the leaf color of a single plant with the tillering number less than 4 is yellowish, and the general leaf color of a single plant with the tillering number more than 5 is normal. 300F plants were removed based on leaf color₂Protective rows of the population and head and tail two of each row, applicant for the remaining 224F₂The groups are classified: wherein 163 plants with normal leaf color and 61 plants with yellow leaf color are subjected to chi-square detection according to the separation ratio of 3:1 (normal leaf color: yellow leaf color) of the two phenotypes, and chi-square detection is carried out²＝0.60<χ² _0.05,13.84, P is 0.44, as desired. The frequency distribution of tillering numbers conformed to a bimodal distribution (see FIG. 5). Indicating that the mutant is a single-gene recessive mutant.

(4) 30 individual plants with tillering number less than 3 and yellow leaf color are selected, and the same amount of leaf blades are mixed and sent to a company for sequencing.

In this example, the tillering number and plant height in step (1) can be statistically investigated by naked eyes, and the chlorophyll content is measured mainly by extracting total chlorophyll from leaves and measuring absorbance value, as follows:

the rice seeds are disinfected and then placed in a rooting culture medium, the rooting culture medium is placed in an incubator at 28 ℃ (light/dark, each 12 hours), after 10 days of culture, rice seedlings with consistent growth vigor in each pot are poured into the middle of two leaves and are cut into pieces, the mixture is stirred and mixed evenly, about 50mg of leaves are weighed by an analytical balance, 8mL of 95% ethanol is added, the leaves are extracted for 24 hours in a dark place at 4 ℃, and the leaves are turned upside down and mixed evenly from time to time.

The absorbance values at 665nm, 649nm and 470nm were measured using UV spectrophotometer DU640 as a blank control with 95% ethanol of the leach liquor. Chlorophyll content was calculated using a modified Lichtensalter Method (Lichtensalter HK. chlorophenyls and carotenoids: pigments of photosynthetic biomemers, Method enzyme. Elsevier,1987.350-382.), as follows:

chlorophyll a: c_a＝13.95*A665-6.88*A649

Chlorophyll b: c_b＝24.96*A649-7.32*A665

Carpesium bicolor (Royle) JosepalumB, carotene: c_X·C＝(1000*A470-2.05*C_a-114.8*C_b)/245

Chlorophyll pigment content (mg/g) ═ C V N/W

In the formula: c is pigment content (mg/g); v is the volume (L) of the extract; n is the dilution multiple; w is the fresh mass (g) of the sample. Note: a665, A649, A470 refer to the absorbance at 665, 649, 470nm wavelength, respectively.

Example 3: sequencing data analysis

(1) The compressed data is checked for correctness by the MD5 command.

(2) The quality of the sequencing was assessed using FastQC software (http:// www.bioinformatics.babraham.ac.uk/projects/FastQC /).

(3) Data analysis was performed by reference to the method of Wachsman (Wachsman et al. A SIMPLE pipeline for mapping point statistics. plant Physiol,2017,174: 1307; 1313), with the specific codes https:// github. com/wacguy/SIMPLE-1.8.1. There are two improvements, improvement part 1: if a site is defined as being dominant as "+" and recessive as "-", the criteria for Wachsman to screen candidate genes are to screen all the sequencing reads at a site of the mutant mixed pool (mut) as-/-, and the sequencing reads at the site of the corresponding wild-type mixed pool (wt) as +/+ and +/-sites. Whereas applicants have used extreme mutant phenotype mixed pools (muts) and EMS-mutagenized "wild-type" (wt), applicants 'wt theoretically is homozygous (only +/+) after selfing, and thus, applicants' selection criteria is to screen mut mixed pools for all reads that are sequenced-/-, and for all reads that are wt +/+, when screening candidate genes. The improved part 2: the calculation formula of the SNP index of Wachsman is ratio, wt. ratio-mut. ratio, wherein wt. ratio is the proportion of reference genome reads to total reads in the wt mixing pool; ratio is the ratio of reference genomic reads to total reads in an extremely recessive phenotype mixed pool mut. The applicants modify the formula of SNP index to ABS, namely the absolute value of (wt-mut), so that the map of the SNP index is more intuitive.

Example 4: candidate gene assaySequence and F₂Population cosegregation assay

According to the results of the analysis in example 4, the target sites were obtained, see Table 2. Two mutations occur at adjacent positions of the target site, one is deleted for 2bp, and the other is deleted for 1 bp. Also, the SNP index pattern demonstrates this result (see FIG. 3).

TABLE 23 t mutant candidate site information Table

Sequencing and verifying a target gene: using the primers "OS 07G 0141400-seq" in Table 3, applicants amplified candidate gene sequences of EMS-ZH11 and the 3t mutant, respectively, using rTaq enzyme (Bao bioengineering, Dalian, Ltd.). The amplification system used was as follows: 10 × Buffer, 2 μ L; 40-60ng of DNA template; 2mM dNTP, 1.5. mu.L; 10 μ M primer (F + R), 0.8 μ L; rTaq, 0.2. mu.L; by ddH₂The amount of O was made up to 20. mu.L. The PCR reaction procedure used was: 4min at 95 ℃; (94 ℃ 30sec, 58 ℃ 1min, 72 ℃ 90sec)35 cycles; 72 ℃ for 7 min; 25 ℃ for 1 min. The amplified product was then sent to a commercial sequencing company for sequencing, again using the sequencing primer "OS 07G 0141400-seq". As shown in FIG. 4A, the 3t mutant did show two deletions in adjacent positions. Wherein a 2bp deletion occurs in the intron region and a 1bp deletion occurs in the exon region, resulting in a mutation in the encoded amino acid.

Based on the nucleotide information of the mutation sites, applicants designed CAPS markers "OS 07G 0141400-CAPS" (Table 3) vs F₂The population is subjected to coseparation detection (wherein the wild type is 870bp, the mutant type is 867bp, and the heterozygosity is 867/870bp), and the amplification method is the same as the above. The subsequent cleavage was carried out using Fast Digest Fast-cutting enzyme Sal I from Thermo Scientific: taking 10 mu L of amplification product; 10 Xfast Digest Green Buffer 1.5. mu.L; fast Digest Sal I0.2. mu.L; complement ddH₂And performing agarose gel electrophoresis detection after enzyme digestion for 4 hours at 37 ℃ when the volume of the product is between 15 and 15 mu L: wherein the products of wild type amplification are changed into 554bp and 316bp bands after enzyme digestion; the product of heterozygote amplification is changed into 870/867bp, 554bp and 316bp after enzyme digestion, and three bands are displayed; and mutant due toThe restriction site is mutated, the size of the restriction product is still 867bp, one band (FIG. 4B). The detection result shows that all the single plants of the mutant genotype show the phenotypes of few tillers and yellowish leaf color, while the heterozygous genotype and the wild genotype have basically consistent phenotypes and normal leaf color, but the tillering number is between 5 and 18, and the genes controlling the tillering number and the leaf color belong to dominant genes.

TABLE 3 candidate Gene sequencing and Co-isolation detection primers

Example 5: candidate gene transgene validation

The applicant amplified the full genomic length (1111bp) of the candidate gene and ligated into pU2301-FLAG vector driven by the maize ubiquitin promoter using the "OE-OS 07G 0141400" primer (Table 4) (FIG. 6). The vector is a plant overexpression vector modified by key laboratories in the crop genetic improvement country of the applicant, and is obtained by modifying a maize ubiquitin promoter, a 3 XFLAG tag and an NOS (synthase polymerization signal) terminator by inserting a maize Rice 706 Rice codes H3K9 methylated Rice for floral organ cultivation. PNAS, 2008). The pU2301-FLAG vector was digested with Fast Digest Kpn I, and the PCR product and the digested vector product were recovered using a recovery kit from Fermentas corporation (see the description, cat # K0513) and ligated in one step (Gibson DG. enzymatic assembly of overlapping DNA fragments, Method enzyme. Elsevier, 2011.349-361). The vector was subsequently transformed into Agrobacterium EHA105 and transformed into the 3t mutant.

TABLE 4 candidate Gene sequencing overexpression primers

Then, positive identification is carried out on the transgenic plant, the used marker is CAPS marker "OS 07G 0141400-CAPS", if the transgenic plant is a positive individual plant, the CAPS marker genotype will show heterozygosity, three bands; the negative individuals showed only one band due to the cleavage site mutation (FIG. 7B). Subsequently, chlorophyll content of the positive and negative individuals was measured, and it was found that chlorophyll a content of the positive individuals was increased and tillering number also tended to increase (fig. 7D).

The above examples illustrate that neutral mutants mutagenized using EMS mutagen compositions can rapidly localize spontaneously mutated genes.

The main steps of genetic transformation, culture medium and methods for their formulation used in this example are as follows:

(1) reagent and solution abbreviations

The abbreviations for the phytohormones used in the medium of the present invention are as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin ); KT (Kinetin ); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indoleacetic acid); 2,4-D (2, 4-dichlorphenoxyacetic acid, 2,4-Dichlorophenoxyacetic acid); AS (acetosyringone); CH (Casein enzymic Hydrolysate, hydrolyzed Casein); g418(Geneticin ); DMSO (Dimethyl Sulfoxide); n6max (N6 macronutrient solution); n6mix (N6 trace element composition solution); MSmax (MS macronutrient component solution); MSmix (MS microelement component solution)

(2) Main solution formulation

1) N6 medium macroelement mother liquor (prepared as 10-fold concentrated solution (10 ×)):

the reagents are dissolved one by one, and then distilled water is used for fixing the volume to 1000mL at room temperature.

2) Preparing N6 culture medium microelement mother liquor (according to 100 times of concentrated solution (100X))

The above reagents were dissolved at room temperature and made up to 1000mL with distilled water.

3) Iron salt (Fe)₂EDTA) stock solution (prepared as 100X concentrate)

3.73g of disodium ethylene diamine tetraacetate (Na)₂EDTA·2H₂O) and 2.78g FeSO₄·7H₂Dissolving O respectively, mixing, fixing the volume to 1000mL by using distilled water, carrying out warm bath at 70 ℃ for 2 hours, and storing at 4 ℃ for later use.

4) Vitamin stock solution (prepared according to 100X concentrated solution)

Adding distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use.

5) MS culture medium macroelement mother liquor (MSmax mother liquor) (prepared according to 10X concentrated solution)

6) MS culture medium microelement mother liquor (MSmin mother liquor) (prepared according to 100X concentrated solution)

7) Preparation of 2,4-D stock solution (1 mg/mL):

weighing 2, 4-D100 mg, dissolving with 1mL of 1N potassium hydroxide for 5 minutes, adding 10mL of distilled water to dissolve completely, diluting to 100mL, and storing at room temperature.

8) Preparation of 6-BA stock solution (1 mg/mL):

weighing 6-BA 100mg, dissolving with 1mL of 1N potassium hydroxide for 5 minutes, adding 10mL of distilled water to dissolve completely, then fixing the volume to 100mL, and storing at room temperature.

9) Preparation of stock solution of Naphthylacetic acid (NAA) (1 mg/mL):

weighing NAA 100mg, dissolving with 1mL of 1N potassium hydroxide for 5 minutes, adding 10mL of distilled water to dissolve completely, diluting to 100mL, and storing at 4 ℃ for later use.

10) Formulation of Indoleacetic acid (IAA) stock solution (1 mg/mL):

weighing 100mg of IAA, dissolving with 1mL of 1N potassium hydroxide for 5 minutes, adding 10mL of distilled water to dissolve completely, then fixing the volume to 100mL, and storing at 4 ℃ for later use.

11) Preparation of glucose stock solution (0.5 g/mL):

weighing 125g of glucose, dissolving with distilled water to a constant volume of 250mL, sterilizing, and storing at 4 ℃ for later use.

12) Preparation of AS stock solution:

0.392g of AS was weighed, dissolved in 10mL of DMSO, and dispensed into 1.5mL centrifuge tubes and stored at 4 ℃ until use.

13)1N potassium hydroxide stock solution:

5.6g of potassium hydroxide is weighed, dissolved by distilled water to be 100mL, and stored at room temperature for later use.

(3) Culture medium formula for rice genetic transformation

1) Induction medium

Adding distilled water to 900mL, adjusting pH to 5.9 with 1N potassium hydroxide, boiling to 1000mL, packaging into 50mL triangular flask (25 mL/bottle), sealing, and sterilizing by conventional method (for example, sterilizing at 121 deg.C for 25 min, the following method for sterilizing culture medium is the same as that for the present culture medium).

2) Subculture medium

Adding distilled water to 900mL, adjusting pH to 5.9 with 1N potassium hydroxide, diluting to 1000mL, boiling, packaging into 50mL triangular flask (25 mL/bottle), sealing, and sterilizing.

3) Co-culture medium

Adding distilled water to 250mL, adjusting pH to 5.6 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved by heating and 5mL of glucose stock solution and 250. mu.L of AS stock solution were added before use and dispensed into petri dishes (25mL per dish).

4) Suspension culture medium

Adding distilled water to 100mL, adjusting pH to 5.4, packaging into two 100mL triangular bottles, sealing, and sterilizing according to the above method.

1mL sterile glucose stock solution and 100. mu.L AS stock solution were added prior to use.

5) Selection medium

Adding distilled water to 250mL, adjusting pH to 6.0, sealing, and sterilizing as above.

The medium was dissolved before use and 250. mu. L G418(50mg/mL) and 400. mu.L CN (250mg/mL) were added and dispensed into petri dishes (25 mL/dish). (Note: the concentration of carbenicillin in the first selection medium was 400mg/L, and the concentration of carbenicillin in the second and subsequent selection media was 250 mg/L).

6) Differentiation medium

Distilled water was added to 900mL and the pH was adjusted to 6.0 with 1N potassium hydroxide.

Adding distilled water to 1000mL, boiling, packaging into 50mL triangular flask (50 mL/bottle), sealing, and sterilizing.

7) Rooting culture medium

Distilled water was added to 900mL and the pH was adjusted to 5.8 with 1N potassium hydroxide.

Adding distilled water to 1000mL, boiling, packaging into rooting tube (25 mL/tube), sealing, and sterilizing.

(4) Agrobacterium-mediated genetic transformation procedure

1) Callus induction

Mature 3t mutant rice seeds were dehulled and then treated sequentially with 75% ethanol for 1 minute with 0.15% mercuric chloride (HgCl)₂) Disinfecting the surface of the seeds for 15 minutes;

washing the seeds with sterilized single-steaming water for 8-10 times;

placing the seeds on an induction medium;

the inoculated culture medium is placed in a dark place for culturing for 4 weeks at the temperature of 25 +/-1 ℃.

2) Callus subculture

The bright yellow, compact and relatively dry embryogenic callus was selected and cultured on subculture medium in the dark for 2 weeks at 25 + -1 deg.C.

3) Agrobacterium culture

Agrobacterium EHA105 (a strain from an Agrobacterium strain publicly used by CAMBIA corporation) was pre-cultured for two days at 28 ℃ in LA medium with corresponding resistance selection (see J. Sambulu et al, molecular cloning guidelines, third edition, King Dong Yan et al (ed.), scientific Press, 2002, Beijing);

the Agrobacterium was transferred to suspension medium and cultured on a shaker at 28 ℃ for 30 min.

4) Infection with Agrobacterium

Transferring the pre-cultured callus into a sterilized bottle;

adjusting the suspension of Agrobacterium to OD 6000.8-1.0;

soaking the callus in the agrobacterium tumefaciens suspension for 30 minutes;

transferring the callus to sterilized filter paper and sucking to dry; then placed on a co-culture medium for 2 days at a temperature of 19-20 ℃.

5) Callus wash and selection culture

Washing the callus with sterilized single distilled water until no agrobacterium is visible;

soaking in sterilized water containing 400mg/L Carbenicillin (CN) for 30min after the last washing;

transferring the callus to sterilized filter paper and sucking to dry;

transferring the callus to selective medium for 2-3 times of 2 weeks each time.

6) Differentiation

Transferring the resistant callus to a differentiation culture medium, and culturing under the illumination until green seedlings grow at the temperature of 26 ℃.

7) Rooting

The roots produced during differentiation were cut off and then transferred to rooting medium and cultured under illumination for 2-3 weeks at 26 ℃.

8) Transplanting

Washing off residual culture medium on the roots, transferring the seedlings with good root systems to a field isolation environment, and managing the field as the common field.

Claims

1. A method for rapidly positioning and cloning plant spontaneous mutant genes is characterized in that: mutagenesis agent combination utilizing Ethyl Methanesulfonate (EMS)Treating the plant with the agent to generate nucleotide variation covering the whole genome, so as to obtain a neutral mutant with SNP variation and no phenotype difference; generation of F by hybridization of said neutral mutants with spontaneous mutants₂A population for rapid localization of mutant genes;

the method comprises the following specific steps:

(1) selecting a variety X with the same genetic background as a mutagenic material according to the genetic background of a spontaneous mutant, selfing for at least more than 3 generations after mutagenesis by an EMS mutagen composition, observing a phenotype after harmful mutation is eliminated, and selecting a mutagenic single plant with no difference between the phenotype and the variety X as a neutral mutant EMS-X;

(2) hybridizing the obtained neutral mutant EMS-X with spontaneous mutant to obtain F₁Generation, selfing and planting F₂Generation group;

(3) f of investigation step (2)₂Making group phenotype, making statistics on segregation ratio, and performing gene localization if the character segregation ratio meets the single gene segregation ratio to respectively perform EMS-X and F₂Sequencing an extreme recessive phenotype mixed pool in the population;

(4) analyzing and extracting EMS-X and SNP/InDel sequenced by an extreme recessive single plant mixed pool, and then screening candidate target sites;

(5) calculating an SNP index value through the extracted SNP/InDel, drawing, and selecting a final candidate gene by combining the target site extracted in the step (4);

(6) sequencing candidate genes for spontaneous mutants and EMS-X, verifying the results in step (5), and then at F₂Performing co-segregation detection in the population;

(7) and selecting genes at the co-segregation sites according to the annotation information to perform transgene verification on the candidate genes.

2. The method for rapidly mapping and cloning a plant spontaneous mutant gene according to claim 1, wherein: replacing the method in the step (3) by respectively matching F₂Respectively sequencing two extreme phenotype mixed pools in a population, wherein the extreme phenotype mixed pools comprise a recessive mixed pool and a dominant phenotype mixed pool, and the recessive mixed pool and the dominant phenotype mixed pool areThe sex mixed pool is a mixed single plant with mutant phenotype, and the dominant phenotype mixed pool is a mixed single plant with non-mutant phenotype; and (4) replacing the method in the step (4) by respectively extracting the sequenced SNP/InDel of the two extreme phenotype mixed pools and then screening candidate target sites.

3. The method for rapidly mapping and cloning a plant spontaneous mutant gene according to claim 1, wherein: the formula of the mutagen composition comprises the following components: 61mL of 0.2mol/L Na₂HPO₄A solution; 39mL of 0.2mol/L NaH₂PO₄A solution; supplemental ddH₂O to 200mL of 0.1M phosphate buffer at pH 7.0; adding 800 mu L EMS stock solution according to the volume ratio of 0.4% of EMS stock solution to 0.4% of phosphate buffer solution to obtain 0.4% EMS mutagen composition.