阅读说明：本技术 一种快速定点突变创制纯合糯性玉米种质的方法及其应用 (Method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof ) 是由 谢传晓 朱金洁 李丽娜 刘昌林 祁显涛 徐孝洁 黄晶 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种快速定点突变创制纯合糯性玉米种质的方法及其应用。属于玉米改良技术领域。本发明提供了单倍体诱导版的糯性玉米改良单倍体诱导系Hi-Edit3与Hi-Edit8；对目标自交系进行单倍体诱导；通过双分子荧光技术在幼胚、成熟籽粒及萌发籽粒的下胚轴水平高效筛选单倍体；单倍体鉴定及靶位点基因型鉴定；单倍体诱导加倍,最终实现两个世代内快速改良获得糯性玉米,具有很高的育种应用价值。(The invention discloses a method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof. Belongs to the technical field of corn improvement. The invention provides haploid induction version waxy corn improved haploid induction lines Hi-Edit3 and Hi-Edit 8; carrying out haploid induction on the target inbred line; efficiently screening haploids on hypocotyl levels of young embryos, mature grains and germinated grains by a bimolecular fluorescence technology; haploid identification and target site genotype identification; haploid is induced to double, finally, the waxy corn is obtained by quickly improving in two generations, and the breeding application value is very high.)

1. A method for creating waxy corn germplasm by rapid site-specific mutagenesis is characterized by comprising the following steps: and (3) hybridizing the corn ZmWaxy1 gene editing haploid induction line which is homozygous for the mtl mutant homozygote and the ZmWaxy1 gene knockout element and the screening element, or the corn ZmWaxy1 gene editing haploid induction line which is homozygous for the mtl and dmp mutant homozygote and the ZmWaxy1 gene knockout element and the screening element with the corn selfing line, screening and identifying the haploid of the hybridized offspring, and doubling the chromosome of the obtained haploid induction line to obtain the double haploid corn germplasm with improved glutinosity.

2. The method for creating waxy maize germplasm by rapid site-directed mutagenesis according to claim 1, wherein the preparation of maize zmwax 1 gene editing haploid inducer line homozygous for mtl mutation homozygote and for the zmwax 1 gene knockout element and the screening element comprises the following steps:

(1) screening out a transgenic negative material from an mtl mutant homozygous strain;

(2) pCPB-ZmESP: eGFP-HvASP: introducing the DsRED dual-fluorescence expression vector into a receptor material, and screening to obtain a DFP positive material;

(3) introducing ZmWaxy1-CRISPR/Cas9 editing vector into a receptor material, and screening positive materials of ZmWaxy1-CRISPR/Cas 9;

(4) hybridizing the materials screened in the steps (1) and (2) to obtain an F1 generation material;

(5) and (3) carrying out fluorescence screening on the F1 generation material, selecting a material with embryo expressing green fluorescence and endosperm expressing red fluorescence, and carrying out hybridization screening on the positive material carrying the ZmWaxy1-CRISPR/Cas9 transgenic component in the step (3) to obtain an induction line pure line Hi-Edit 3.

3. The method of claim 2, wherein the preparation of a maize ZmWaxy1 gene editing haploid inducer line homozygous for mtl and dmp mutation homozygote and for the ZmWaxy1 gene knockout element and the screening element comprises the steps of:

step A: hybridizing the DFP positive material with a ZmMTL/ZmDMP double-gene mutant material to obtain an F1 generation material;

and B: f1 generation materials are subjected to fluorescence screening, materials with embryo portions expressing green fluorescence and endosperm expressing red fluorescence are selected to be hybridized and screened with positive materials carrying ZmWaxy1-CRISPR/Cas9 transgenic components, and then the induction line pure line Hi-Edit8 is obtained.

4. The method for creating waxy maize germplasm by using rapid site-specific mutagenesis according to claim 3, wherein the screening in the step (1) is performed by a BAR test strip.

5. The method for creating waxy maize germplasm of claim 4 where in step (2) the recipient material is maize X249.

6. The method of claim 5, wherein said inbred lines of maize are B73, 159F, 159M, 4CV or 6 WC.

7. The method for creating waxy maize germplasm by using rapid site-specific mutagenesis according to claim 2, wherein the method for screening to obtain the induction line pure line Hi-Edit3 comprises the following steps:

1) sequencing ZmMTL locus by PCR amplification, sequencing and screening a mutation homozygous strain;

2) then screening ZmWaxy1-CRISPR/Cas9 transgenic element pure line selfing by ddPCR;

3) selecting a material which is not separated from the fluorescence on the selfed clusters, namely Hi-Edit 3.

8. The method for creating waxy maize germplasm by using rapid site-specific mutagenesis as claimed in claim 3, wherein the method for screening to obtain the induction line pure line Hi-Edit8 comprises the following steps:

1) PCR amplification sequencing ZmMTL and ZmDMP site screening double site mutation homozygous strain;

2) then screening ZmWaxy1-CRISPR/Cas9 transgenic element pure line selfing by ddPCR;

3) selecting a material which is not separated from the fluorescence on the selfed clusters, namely Hi-Edit 8.

9. The method for creating waxy maize germplasm by rapid site-directed mutagenesis according to any of claims 1 to 8, wherein the identification comprises a method of haploid identification including immature embryo level, mature grain and hypocotyl observation of germinating seedlings;

the haploid identification method for young embryo level observation comprises the following steps: observing fluorescence of seeds by using exciting light with the wavelength of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, stripping immature embryos, and observing green fluorescence of embryo parts by using exciting light with the wavelength of 488nm, wherein diploid is used for expressing green fluorescence, and haploid is used for expressing the green fluorescence;

the haploid identification method for observing mature grains comprises the following steps: observing red fluorescence of seeds by using exciting light with the wavelength of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and then observing green fluorescence of embryo parts by using exciting light with the wavelength of 488nm, wherein diploid expresses the green fluorescence, and haploid expresses the green fluorescence;

a haploid identification method for observing the hypocotyl of a germinated seedling comprises the following steps: threshing the harvested fruit ears, soaking overnight, placing the fruit ears in quartz sand for germination, taking germinated 7d seeds, observing red fluorescence of the seeds by using exciting light of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and observing green fluorescence of a hypocotyl by using exciting light of 488nm, wherein diploid is used for expressing the green fluorescence, and haploid is used for expressing the green fluorescence.

10. Use of the method of any one of claims 1 to 9 for breeding new varieties of maize.

Technical Field

The invention relates to the technical field of corn improvement, in particular to a method for creating homozygous waxy corn germplasm by rapid site-specific mutagenesis and application thereof.

Background

Corn is an important grain crop and has wide application in the fields of feed, industrial raw materials and the like. Corn starch is an important processed product of corn, is used as a green basic raw material, has high application value and economic value, and even plays an irreplaceable role in various national economic fields such as food, pharmacy and the like, so that the cultivation and improvement of waxy corn have extremely important significance.

The CRISPR/Cas9 gene editing technology is derived from a regularly clustered interspaced short palindromic repeats CRISPR (clustered regulated interstitial short palindromic repeats) mediated acquired immune system of bacteria or archaea. The system comprises a section of artificially designed sgRNA, the target DNA sequence is recognized by the RNA through base complementary pairing, the Cas9 nuclease is guided to cut the recognized double-stranded DNA, an in-vivo double-stranded break repair mechanism is induced, and the target gene locus is edited. At present, the CRISPR/Cas9 gene editing technology is widely applied to various fields of animals, plants and the like, and particularly has great application value on crop genetic improvement.

A haploid breeding technology provides a new method for crop genetic improvement, haploid materials of corn ZmMTL and ZmDMP genes can be induced to form a haploid at present, and early-stage research shows that the haploid inductivity obtained by using ZmMTL gene mutation is between 4.7% and 11%, and the haploid inductivity of simultaneous mutation of the ZmMTL and the ZmDMP genes is between 6.9% and 11.5%.

The breeding of excellent materials needs a fast and efficient technology, the genetic homozygosis of the traditional breeding technology needs 8 generations, and the Double Haploid (DH) breeding technology can obtain pure lines only by two generations, so that the method has important technical advantages and breeding practical value. Although the double haploid breeding technology can shorten the breeding period, a certain agronomic trait cannot be directionally and accurately changed; the CRISPR/Cas9 gene editing technology achieves the purpose of accurately improving target traits, the improvement of the target traits of a receptor material is completed by hybridizing a transgenic material carrying a target gene mutation CRISPR-Cas9 transgenic element with the receptor material to be improved and editing the target gene of the receptor material by using the introduced CRISPR-Cas9 transgenic element in the earlier stage, however, the process needs to obtain high background reversion in the BC2 generation through self-crossing, screening, and converting a receptor into waxy germplasm and carrying no transgenic element

Therefore, how to provide a method for creating waxy maize germplasm by rapid site-directed mutagenesis is an urgent problem to be solved by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof. Any corn variety improvement to waxy quality can be achieved in two generations. According to the invention, based on early-stage mutation of the ZmMTL gene and superposition of a haploid induction system of a double-fluorescence screening system, a CRISPR-Cas9 transgenic element capable of performing site-specific mutation of the ZmWaxy1 gene is further superposed, so that breeding for rapidly improving the waxy character of the corn through haploid induction mediation is realized. Meanwhile, a corn ZmMTL and ZmDMP double-gene mutation homozygote superposition double-fluorescence screening system and a haploid induced editing ZmWaxy1 gene strain of a CRISPR-Cas9 element edited by ZmWaxy1 gene are created. And (2) re-crossing the receptor material and a haploid induction gene editing line, obtaining parthenogenesis haploid by using ZmMTL or ZmMTL and ZmDMP mutation homozygote, screening the haploid by matching with a double fluorescence system, finally obtaining waxy haploid of the improved receptor material, and obtaining the target improved material by doubling by using a chromosome doubling technology to obtain monoploid and diploid for selfing.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for creating waxy corn germplasm by rapid site-specific mutagenesis comprises the following steps: and (3) hybridizing the corn ZmWaxy1 gene editing haploid induction line which is homozygous for the mtl mutant homozygote and the ZmWaxy1 gene knockout element and the screening element, or the corn ZmWaxy1 gene editing haploid induction line which is homozygous for the mtl and dmp mutant homozygote and the ZmWaxy1 gene knockout element and the screening element with the corn selfing line, screening and identifying the haploid of the hybridized offspring, and doubling the chromosome of the obtained haploid induction line to obtain the double haploid corn germplasm with improved glutinosity.

Preferably: the preparation of the corn ZmWaxy1 gene editing haploid induction line which is homozygous for mtl mutation homozygote and ZmWaxy1 gene knockout element and screening element comprises the following steps:

(1) screening out a transgenic negative material from an mtl mutant homozygous strain;

(2) pCPB-ZmESP: eGFP-HvASP: introducing the DsRED dual-fluorescence expression vector into a receptor material, and screening to obtain a DFP positive material;

(3) ZmWaxy1-CRISPR/Cas9 editing vector is introduced into a receptor material, and positive materials of ZmWaxy1-C RISPR/Cas9 are screened;

(4) hybridizing the materials screened in the steps (1) and (2) to obtain an F1 generation material;

(5) and (3) carrying out fluorescence screening on the F1 generation material, selecting a material with embryo expressing green fluorescence and endosperm expressing red fluorescence, and carrying out hybridization screening on the positive material carrying the ZmWaxy1-CRISPR/Cas9 transgenic component in the step (3) to obtain an induction line pure line Hi-Edit 3.

Further: the doubling is carried out by adopting colchicine and adopting a immature embryo tissue culture method or a seedling root soaking method.

Preferably: the preparation of maize zmwax 1 gene editing haploid inducer lines homozygous for mtl and dmp mutant homozygotes and for the zmwax 1 gene knockout element and the screening element comprises the following steps:

step A: hybridizing the DFP positive material with a ZmMTL/ZmDMP double-gene mutant material to obtain an F1 generation material;

and B: f1 generation materials are subjected to fluorescence screening, materials with embryo portions expressing green fluorescence and endosperm expressing red fluorescence are selected to be hybridized and screened with positive materials carrying ZmWaxy1-CRISPR/Cas9 transgenic components, and then the induction line pure line Hi-Edit8 is obtained.

Preferably: screening in the step (1) is carried out by a BAR test strip.

Preferably: the receptor material in the step (2) is corn X249.

Preferably: the maize inbred line is B73, 159F, 159M, 4CV or 6 WC.

Preferably: the method for screening and obtaining the induction line pure line Hi-Edit3 comprises the following steps:

1) sequencing ZmMTL locus by PCR amplification, sequencing and screening a mutation homozygous strain;

2) then screening ZmWaxy1-CRISPR/Cas9 transgenic element pure line selfing by ddPCR;

3) selecting a material which is not separated from the fluorescence on the selfed clusters, namely Hi-Edit 3.

Preferably: the method for screening and obtaining the induction line pure line Hi-Edit8 comprises the following steps:

1) PCR amplification sequencing ZmMTL and ZmDMP site screening double site mutation homozygous strain;

2) then screening ZmWaxy1-CRISPR/Cas9 transgenic element pure line selfing by ddPCR;

3) selecting a material which is not separated from the fluorescence on the selfed clusters, namely Hi-Edit 8.

Preferably: identifying a haploid identification method comprising observation of the hypocotyl of the young embryo level, mature grain and germinating seedling;

haploid identification method for young embryo level observation: observing fluorescence of seeds by using exciting light with the wavelength of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, stripping immature embryos, and observing green fluorescence of embryo parts by using exciting light with the wavelength of 488nm, wherein diploid is used for expressing green fluorescence, and haploid is used for expressing the green fluorescence;

the haploid identification method for observing mature grains comprises the following steps: observing red fluorescence of seeds by using exciting light with the wavelength of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and then observing green fluorescence of embryo parts by using exciting light with the wavelength of 488nm, wherein diploid expresses the green fluorescence, and haploid expresses the green fluorescence;

a haploid identification method for observing the hypocotyl of a germinated seedling comprises the following steps: threshing the harvested fruit ears, soaking overnight, placing the fruit ears in quartz sand for germination, taking germinated 7d seeds, observing red fluorescence of the seeds by using exciting light of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and observing green fluorescence of a hypocotyl by using exciting light of 488nm, wherein diploid is used for expressing the green fluorescence, and haploid is used for expressing the green fluorescence.

Further: and screening the red fluorescent protein and the green fluorescent protein in 18d of young embryos, mature seeds and 3d of germinated seeds after pollination.

The invention also provides application of the method in breeding new corn varieties.

Has the advantages that: the haploid induced mutant gene, the haploid assisted screened bimolecular fluorescent protein and the CRISPR-Cas9 element of the fixed-point editing ZmWaxy1 gene are polymerized together in a hybrid polymerization and molecular assisted screening mode, and each point is guaranteed to be homozygous.

According to the technical scheme, compared with the prior art, the invention discloses a method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof, and compared with waxy maize improved haploid induction lines Hi-Edit3 and Hi-Edit8 of a haploid induction version provided by the invention in the prior art; carrying out haploid induction on the target inbred line; efficiently screening haploids on hypocotyl levels of young embryos, mature grains and germinated grains by a bimolecular fluorescence technology; haploid identification and target site genotype identification; haploid is induced to double, finally, the waxy corn is obtained by quickly improving in two generations, and the breeding application value is very high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of a gene editing vector structure targeting ZmMTL gene provided by the present invention.

FIG. 2 is a schematic structural diagram of a gene editing vector targeting ZmWaxy1 gene provided by the invention.

FIG. 3 is a schematic diagram of a dual-fluorescent protein expression vector provided by the present invention.

FIG. 4 is a diagram showing the result of ZmMTL genotype analysis of Hi-Edit3 inducible line material according to the present invention.

Fig. 5 is a graph showing the copy number identification result of the CRISPR-Cas element of the Hi-Edit3 inducible material provided by the invention.

FIG. 6 is a flow chart of the creation and modification of the rapid waxy maize modified haploid inducer line Hi-Edit3 provided by the invention.

FIG. 7 is a diagram showing the result of ZmMTL genotype analysis of Hi-Edit8 inducible line material according to the present invention.

FIG. 8 is a diagram showing the result of ZmDMP genotype analysis of Hi-Edit8 inducible line material provided by the present invention.

Fig. 9 is a graph showing the copy number identification result of the CRISPR-Cas element of the Hi-Edit8 inducible material provided by the invention.

FIG. 10 is a flow chart of the creation and modification of the rapid waxy maize modified haploid inducer line Hi-Edit8 provided by the invention.

FIG. 11 is a diagram of a double-fluorescent system for haploid selection at the level of immature embryos.

FIG. 12 is a schematic diagram of a double fluorescence system for haploid selection at the mature grain level provided by the present invention.

FIG. 13 is a diagram of haploid selection at hypocotyl site by the double fluorescence system of the present invention.

FIG. 14 is a diagram showing the genotype analysis results of haploid plants edited at site ZmWaxy1, wherein DH-1-DH-7 are haploid plants obtained by induction screening of B73, 159F, 159M, 4CV and 6WC materials by using Hi-Edit8 haploid inducer.

FIG. 15 is a ploidy identification chart of haploid plants edited at ZmWaxy1 site provided by the invention, wherein Debris-represents cell fragments, Aggregates-represents cell Aggregates, Dip G1-represents DNA content at G1 stage, Dip G2-represents DNA content at G2 stage, and Dip S-represents DNA content at S stage.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof.

Wherein, the construction and transformation of ZmMTL-CRISPR/Cas9, ZmWaxy1-CRISPR/Cas9 gene editing vector and double-fluorescence screening vector:

FIG. 1: the target sequence gggtcaacgtggagacaggg of ZmMTL was designed in its vicinity using CRISPR-P2.0 with reference to the natural mutation site of MTL, and pCPB-Ubi was constructed using a commercial recombination kit (NEB, E2621L): cas9 carrier, the method of overlapping PCR introduces the designed target sequence into sgRNA expression box, and then the sgRNA expression box is recombined and constructed to pCPB-Ubi: and (3) obtaining a ZmMTL-CRISPR/Cas9 editing vector on a Cas9 vector (see a Chinese academy of agricultural sciences academic paper, construction of a corn maternal parthenogenesis haploid induction system and a high-efficiency bifluorescent protein haploid identification system created by gene editing, 2018).

FIG. 2: the CRISPR-P2.0 is used for designing and screening a target sequence gaggttcagctccgggtagtcgg of ZmWaxy1, and a commercial recombinant kit is used for constructing pCPB-Ubi: cas9 vector, a target sequence is introduced into a sgRNA expression cassette through overlapping PCR, and the sgRNA expression cassette is recombined and constructed into pCPB-Ubi: and (3) obtaining a ZmWaxy1-CRISPR/Cas9 editing vector on a Cas9 vector (see a university of Anhui agriculture university academic paper & ltcorn RNA polymerase III recognized promoter activity identification & Waxy1 gene editing & gt 2016).

FIG. 3: constructing a double-fluorescence screening system, including expressing green fluorescent protein by an embryo-specific promoter and expressing red fluorescent protein by an endosperm-specific promoter, and constructing pCPB-ZmESP by using a commercial recombinant kit (NEB, E2621L): eGFP-HvASP: DsRED Double-fluorescent expression vector (Double-fluorescent Proteins, DFP, see the academic papers of Chinese academy of agricultural sciences: "construction of haploid induction system of maize maternal parthenogenesis and haploid identification system of efficient Double-fluorescent protein", 2018) by gene editing.

And (3) sending the three constructed vectors to a medium-variety genetic transformation platform for transformation, wherein the genetic transformation is completed by a conventional agrobacterium-mediated method, and the transformed acceptor materials are all corn X249.

Example 1

A maize ZmWaxy1 gene editing haploid induction line based on mtl mutation homozygote and homozygous for ZmWaxy1-CRISPR/Cas9 and DFP transgenic elements is created and named as Hi-Edit3

Maize haploid inducer line with ZmMTL gene edited, carrying zmwax 1-CRISPR/Cas9 and DFP vector, but not ZmMTL-CRISPR/Cas9 vector:

the positive material of ZmMTL-CRISPR/Cas9 is screened and transformed by glufosinate, a T1 material is obtained by selfing, ZmMTL gene screening mutant homozygous strains (MTL-F: cggtggctccgcaacaac and MTL-R: ttgggttgatggcagagacg) are sequenced in a T1 generation material, the genotype of the obtained MTL homozygous mutant is shown in figure 4, and then the material with negative deletion elements is screened by a BAR test strip. And respectively screening the positive material of the transformed DFP and the positive material of ZmWaxy1-CRISPR/Cas9 by using a BAR test strip, and hybridizing the positive strain of the DFP transgene with an mtl mutation homozygous strain without a ZmMTL-CRISPR/Cas9 transgenic element to obtain an F1 material. Fluorescence screening was performed on the F1 generation material, and a material in which the embryo portion expressed green fluorescence and the endosperm expressed red fluorescence was selected. The fluorescence screening system needs to use a handheld excitation light source, can generate two light sources of 488nm and 520nm, and respectively observes two kinds of fluorescence of eGFP and DsRED. The screened material is then hybridized with a positive material carrying a ZmWaxy1-CRISPR/Cas9 transgenic component, and finally, a gene editing induction line of improved ZmWaxy1 which can induce and form haploid and is provided with a double fluorescence screening system is obtained.

In order to ensure the application efficiency of the system, a pure line needs to be obtained, namely, the target material is a ZmMTL-CRISPR/Cas 9-free transgenic element, and the ZmMTL locus is subjected to homozygous mutation; homozygous for the DFP transgene element; ZmWaxy 1-material homozygous for CRISPR/Cas9 transgenic element.

The specific experimental procedures included:

1. using a gene editing induction system of improved ZmWaxy1 which can induce to form haploid and is provided with a double fluorescence screening system as a material, carrying out PCR amplification sequencing on a ZmMTL locus, and sequencing and screening a mutation homozygous strain;

detection primers: MTL-F: cggtggctccgcaacaac, MTL-R: ttgggttgatggcagagacg, respectively;

amplification system, using high fidelity enzyme (TOYOBO, KOD-201): buffer 5. mu.L, dNTP 5. mu.L, MgSO₄2. mu.L of F primer, 1.5. mu.L; 1.5 mu L of R primer and 1 mu L of DNA, and adding water to supplement to 50 mu L; the PCR product is sent to Liuhe Huada company for sequencing;

2. then screening a pure line of the ZmWaxy1-CRISPR/Cas9 transgenic element by ddPCR, wherein the result is shown in figure 5;

designing a Cas9 amplification primer on the vector, and designing an amplification primer on a corn reference gene ADH 1;

cas 9-F: TATCGAAGTTGGTCATCCGC, respectively; cas 9-R: ACCAGAAAGAGCGAGGAAAC, respectively; ADH-F: GAATGTGTGTTGGGTTTGCAT, respectively; ADH-R: TCCAGCAATCCTTGCACCTT, respectively; cas9 probe: FAM-ACCAGAAAGAGCGAGGAAAC-BHQ 1; ADH probe: HEX-TGCAGCCTAACCATGCGCAGGGTA-BHQ 1.

Probe method mixture configuration (BIO-RAD, L002053B): ddPCRMix 11. mu.L; cas9-F2 μ L, Cas 9-R2 μ L, ADH-F2 μ L, ADH-R2 μ L; cas9 probe 0.55 μ L; ADH probe 0.55 u L, DNA 1 u L; make up to 22 μ L with water;

clamping the micro-droplets into a bracket, adding 20ul of reaction liquid into a middle row of holes, adding 70ul of micro-droplets generated by a probe method into each hole of the lowest row, and covering a silica gel pad; the carrier is placed in a droplet generator and the lid is closed to generate droplets. The resulting microdroplets were transferred to a PCR plate, sealed, placed in a PCR instrument, PCR program: 10min at 95 ℃; 30s at 94 ℃, 60s at 61 ℃ and 40 cycles; 10min at 98 ℃; after the PCR is finished, the PCR product is put into a droplet detector to read the number of droplets.

Whether the material is a homozygous line (the ratio is 1) can be judged by calculating the number ratio of positive droplets of Cas9 and ADH1, the optimally screened individuals are selfed, a material which is not separated by fluorescence on selfed ears is selected, a DFP transgenic element is homozygous, and finally an inducible line pure line is screened and named as Hi-Edit3, as shown in FIG. 6.

Example 2

A maize ZmWaxy1 gene editing haploid induction line based on mtl and dmp mutation homozygote and homozygous for ZmWaxy1-CRISPR/Cas9 and DFP transgenic elements is created and named as Hi-Edit8

Bar test strip detects positive material with DFP transgenic element, and when crossing with ZmMTL and ZmDMP double gene mutant material (commercial induction line H3), F1 offspring selects material with embryo expressing green fluorescence and endosperm expressing red fluorescence. The fluorescence screening system needs to use a handheld excitation light source, can generate two light sources of 488nm and 520nm, and respectively observes two kinds of fluorescence of eGFP and DsRED. The screened material is then hybridized with a positive material carrying a ZmWaxy1-CRISPR/Cas9 transgenic component, and finally an improved haploid induction line with improved ZmWaxy1 of a double-fluorescence screening system is obtained.

In order to ensure the application efficiency of the system, pure lines need to be obtained, namely the target materials are ZmMTL and ZmDMP, the DFP transgenic element is homozygous, and the ZmWaxy1-CRISPR/Cas9 transgenic element is homozygous.

The specific experimental procedures included: the improved haploid induction line of the improved ZmWaxy1 with a double fluorescence screening system is used as a material, the ZmMTL and ZmDMP sites (MTL-F: cggtggctccgcaacaac, MTL-R: ttgggttgatggcagagacg; DMP-F: agattagcagttggtggtagaga and DMP-R: catcacctcgtccatctcctt) are subjected to PCR amplification sequencing, the double site mutation homozygous strain is screened, and the genotype sequencing result is shown in figure 7 and figure 8. Then, a ZmWaxy1-CRISPR/Cas9 transgenic element pure line is screened by ddPCR (the specific operation is the same as that of Hi-Edit3), the result is shown in figure 9, the finally screened single plant is selfed, a material which is not separated by fluorescence on a selfed ear is selected, the DFP transgenic element is homozygous, an induction line pure line is screened finally, and the induction line pure line is named as Hi-Edit8, which is shown in figure 10.

Example 3

Haploid induction and screening of maize inbred lines B73, 159F, 159M, 4CV and 6WC by using maize ZmWaxy1 gene editing haploid induction line Hi-Edit8 or Hi-Edit3

The created maize ZmWaxy1 gene is used for editing haploid induction lines Hi-Edit3 and Hi-Edit8 respectively to carry out maize inbred lines B73, 159F, 159M, 4CV and 6WC for hybridization, hybrid progeny materials are obtained, and haploid screening and identification are carried out at the level of immature embryos and mature grains respectively.

The identification of haploids includes the level of immature embryos, the observation of the hypocotyls of mature kernels and germinating seedlings (Hi-Edit3 and Hi-Edit8 screening methods are the same, and Hi-Edit3 is taken as an example below).

Haploid identification method at the immature embryo level: taking the fruit cluster of 18d after pollination, disinfecting the fruit cluster, observing the fluorescence of the kernel by using exciting light of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, stripping immature embryos, and observing green fluorescence of embryo parts by using exciting light of 488nm, wherein diploid expresses the green fluorescence, and haploid expresses the green fluorescence, as shown in figure 11.

The haploid identification method of mature grains comprises the following steps: and (3) taking the mature ears after harvesting, observing red fluorescence of grains by using excitation light of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and observing green fluorescence of embryo parts by using excitation light of 488nm, wherein diploid expresses green fluorescence and haploid expresses no green fluorescence, as shown in figure 12.

A haploid identification method for observing the hypocotyl of a germinated seedling comprises the following steps: harvesting the fruit ears, threshing, soaking overnight, placing in quartz sand for germination, taking germinated 7d seeds, observing red fluorescence of the seeds by using excitation light of 520nm, excluding seeds without red fluorescence of endosperm caused by pollination of other pollen, and observing green fluorescence of hypocotyl by using excitation light of 488nm, wherein diploid expressing green fluorescence and haploid expressing no green fluorescence is shown in figure 13.

Example 4

ZmWaxy1 locus improved maize inbred line B73, 159F, 159M, 4CV and 6WC haplotype

And (3) haploid genotype identification: DNA was extracted and ZmWaxy1 sequencing demonstrated that the homozygous mutant material was an improved acceptor material (ZmWaxy 1-F: agcctcaacaacaacccatactt, ZmWaxy 1-R: gagatgagctcctcggcgtag) and the determination of the screened and edited haploid genotype is shown in FIG. 14.

Ploidy identification of edited haploids: the DNA content of this material was confirmed by flow cytometry to be half of that of the female parent, indicating an induced haploid, as shown in figure 15.

The obtained haploid induction system is doubled by a colchicine root soaking method, and finally a double haploid system with improved waxy character can be obtained.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.