Patent Application
20220403400
The sequence listing provided in the file entitled Sequence_List_v2.txt, which is an ASCII text file that was created on Jul. 12, 2022, and which comprises 52,091 bytes, is hereby incorporated by reference in its entirety.
The present invention relates to the field of agricultural biotechnology and crop genetic breeding based on genome editing technology, in particular to a preparation method of a plant maternal haploid inducer line and application thereof, in particular to an application of a parthenogenetic haploid induction gene DMP mutant obtained by using a gene-editing technology as a plant haploid inducer line in inducing a plant to generate a maternal haploid.
The breeding of elite selfed lines is the basis and key for breeding the elite hybrid by utilizing the heterosis of the crops. However, the acquisition of a more stable selfed line requires 7-8 generations by traditional breeding methods, and only 2 generations by haploid breeding technique (Weber D F, 2014), which greatly shortens the breeding cycle. At present, the technique of in vitro anther culture is the most favorite to incite the production of haploid on dicotyledonous crops, which has the disadvantages of low efficiency, high dependence on the genotype of materials, and difficulty in large-scale application. Although the introduction of the genetically modified centromere-specific histone CENH3 variant into the Arabidopsis thaliana cenh3 mutant induces haploid production, this approach produces a large number of euploids during induction (Ravi, M, et al, 2010).
At present, the gene-editing technology system has been very mature and stable, but it cannot further exert its maximum effect due to the limitation of genetic transformation on the dependence of material and genotype. Although editing can be achieved by introducing the gene-editing vector into some readily transformable recipient material and further hybridizing it to the target to be edited (Li C et al., 2017), it takes a long time to backcross to restore the background and cannot reach 100%. Haploid induction and gene-editing techniques are combined to achieve editing of target material haploid genes to obtain homozygous editing lines, which greatly shorten and widen the application range of haploid and gene-editing techniques (Kelliher, T et al., 2019; Wang, B et al., 2019; Hu, N et al., 2019). However, in dicotyledonous plants, haploid induction cannot be combined with gene-editing techniques due to the lack of the way of generation of a haploid by biological induction.
An objective of the present invention is to provide a method for preparing a plant haploid inducer line.
The preparation method of the plant haploid inducer line provided by the invention is as follows A1) or A2):
A1) silencing or inhibiting the expression and/or activity of the gene DMP in the plant genome or knocking out the gene DMP to obtain a plant haploid inducer line;
A2) inhibiting the activity of protein DMP in the plant to obtain a plant haploid inducer line; the plant is dicotyledonous;
the protein DMP is a protein represented by the following B1) or B2) or B3) or B4):
B1) a protein with the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6;
B2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6;
B3) a protein having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6;
B4) a protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 and having the same function;
the DMP protein has the following functions: the dicotyledonous plant becomes a plant haploid inducer line when the protein DMP activity is inhibited therein. The protein DMP activity is inhibited such that the protein DMP is not expressed or is inactive.
The tag in the above-mentioned B2) refers to a polypeptide or a protein expressed by fusion with a protein of interest using DNA in vitro recombination technology to facilitate expression, detection, tracking, and/or purification of the protein of interest. The protein tag may be a Flag tag, a His-tag, an MBP tag, an HA tag, a Myc tag, a GST tag, and/or a SUMO tag.
The substitution and/or deletion and/or addition of one or several amino acid residues in the above-mentioned B3) is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
The homology of 75% or more in the above-mentioned B4) maybe 80%, 85%, 90%, or 95% or more.
According to the preparation method of the plant haploid inducer line, the gene DMP is the gene shown in the following C1) or C2) or C3) or C4):
C1) a cDNA molecule or a genomic DNA molecule shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5;
C2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined by C1);
C3) a cDNA molecule or a genomic DNA molecule derived from a dicotyledonous plant and having 75% or more identity to the nucleotide sequence defined in C1);
C4) a cDNA molecule or a genomic DNA molecule that hybridizes with a nucleotide sequence defined by C1) or C2) or C3) under stringent conditions;
The DMP gene has the following functions: the dicotyledonous plant becomes a plant haploid inducer line when the DMP gene is silenced or inhibited or knocked out therein.
The term “identity” as used herein refers to sequence similarity to a native nucleic acid sequence. “Identity” includes nucleotide sequences having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.4 or SEQ ID No.6 of the present invention. Identity can be assessed with the naked eye or by computer software. With computer software, identity between two or more sequences can be expressed as a percentage (%) that can be used to assess identity between related sequences.
In the preparation method of the plant haploid inducer line, the method for silencing or inhibiting the expression and/or activity of gene DMP or knocking out gene DMP in the plant genome comprises reducing the expression amount of gene DMP in the plant genome or subjecting gene DMP in the plant genome to deletion mutation or insertion mutation or base substitution.
Furthermore, when the dicotyledonous plant is Arabidopsis thaliana, gene DMP is gene AtDMP8 and/or gene AtDMP9; protein DMP is protein AtDMP8 and/or protein AtDMP9; the method comprises silencing or inhibiting the expression and/or activity of gene AtDMP8 and/or gene AtDMP9 in the Arabidopsis thaliana genome or knocking out gene AtDMP8 and/or gene AtDMP9 or inhibiting the activity of protein AtDMP8 and/or protein AtDMP8 to obtain the Arabidopsis thaliana haploid inducer line.
When the dicotyledonous plant is tomato, the gene DMP is gene SlDMP; the DMP protein is protein SlDMP; the method comprises silencing or inhibiting gene SlDMP or knocking out gene SlDMP or inhibiting the activity of protein SlDMP in the tomato genome to obtain a tomato haploid inducer line.
Still furthermore, the silencing or inhibiting the expression and/or activity of gene AtDMP8 and/or gene AtDMP9 in the Arabidopsis thaliana genome or knocking out gene AtDMP8 and/or gene AtDMP9 comprises reducing the expression of gene AtDMP8 and/or gene AtDMP9 in the Arabidopsis thaliana genome or subjecting the gene AtDMP8 and/or gene AtDMP9 in the Arabidopsis thaliana genome to deletion mutation or insertion mutation or base substitution.
The silencing or inhibiting the expression and/or activity of gene SlDMP in the tomato genome or knocking out gene SlDMP comprises reducing the expression amount of gene SlDMP in the tomato genome or subjecting gene SlDMP in the tomato genome to deletion mutation or insertion mutation or base substitution.
The method of reducing gene DMP expression in the plant genome comprises RNAi interference. The RNAi interference involves single stranded RNA interference, such as miRNA, or double-stranded RNA interference, such as siRNA, dsRNA, shRNA, etc.
The method of subjecting the gene DMP in the plant genome to deletion mutation or insertion mutation or base substitution comprises CRISPR/Cas9 or TELLEN or T-DNA insertion or EMS mutagenesis.
Furthermore, the method of subjecting the gene DMP in the plant genome to deletion mutation or insertion mutation, or base substitution comprises CRISPR/Cas9. The method of subjecting the gene DMP in the plant genome to deletion mutation or insertion mutation or base substitution comprises the following steps: introducing a CRISPR/Cas9 vector containing the target sequence into a plant to obtain a plant haploid inducer line.
In a specific example of the present invention, when the dicotyledonous plant is Arabidopsis thaliana, the target sequence of CRISPR/Cas9 is at positions 98-117 of SEQ ID No. 1, positions 290-309 of SEQ ID No. 3, positions 368-387 of SEQ ID No. 3, and positions 509-528 of SEQ ID No. 1. The CRISPR/Cas9 vector containing the target sequence is a recombinant vector obtained by connecting a DNA molecule represented by SEQ ID No. 7 (sgRNA expression element), a DNA molecule represented by SEQ ID No. 9 (Cas9 expression element), and a DNA molecule represented by SEQ ID No. 10 (fluorescent protein expression element) to a pICSL4723 vector through a golden gate method.
In a specific example of the present invention, when the dicotyledonous plant is tomato, the target sequences of CRISPR/Cas9 are at positions 76-95 of SEQ ID No. 5 and positions 247-266 of SEQ ID No. 5. The CRISPR/Cas9 vector containing the target sequence is a recombinant vector obtained by connecting a DNA molecule represented by SEQ ID No. 8 (sgRNA expression element), a DNA molecule represented by SEQ ID No. 11 (Cas9 expression element), a DNA molecule represented by SEQ ID No. 10 (fluorescent protein expression element), and a DNA molecule represented by SEQ ID No. 12 (NptII expression element) to a pICSL4723 vector through a golden gate method.
Another object of the present invention is to provide a method for preparing a plant haploid inducer line, comprising the step of selfing the plant haploid inducer line for at least one time, particularly for one time.
The preparation method of the plant haploid inducer line further comprises the step of screening gene DMP mutants. The gene DMP mutant is a plant with gene DMP mutation, and the gene DMP mutant is a gene DMP heterozygous mutant or a gene DMP homozygous mutant.
According to the preparation method of the plant haploid inducer line, the plant haploid inducer line specifically comprises an Arabidopsis thaliana mutant line T1-34, an Arabidopsis thaliana mutant line T1-6, an Arabidopsis thaliana mutant line T1-11, an Arabidopsis thaliana mutant line T1-19, an Arabidopsis thaliana mutant line T1-24, an Arabidopsis thaliana mutant line T1-25, an Arabidopsis thaliana mutant line T1-28, an Arabidopsis thaliana mutant line T1-32, an Arabidopsis thaliana mutant line T2-33, an Arabidopsis thaliana mutant line T2-38, a tomato mutant line sldmp-1 or a tomato mutant line sldmp-2; the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-34 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID No. 1;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-6 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID No. 1, and a fragment deletion occurs in the other chromosome at positions 113-114 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a fragment insertion occurs in both chromosomes at positions 160-161 of SEQ ID No. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID No. 13;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-11 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) insertion occurs at positions 114-115 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a base (T) insertion occurs in one chromosome at positions 160-161 of SEQ ID No. 3, and a fragment insertion occurs in the other chromosome at positions 160-161 of SEQ ID No. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID No. 13;
the difference between the genome of the Arabidopsis thaliana mutant line T1-19 and the genome of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurs in one chromosome at position 114 of SEQ ID No. 1, and a fragment deletion occurs in the other chromosome at positions 115-511 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-560 of SEQ ID No. 3, and a fragment deletion occurs in the other chromosome at positions 161-564 of SEQ ID No. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-24 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-560 of SEQ ID No. 3, and a fragment deletion occurs in the other chromosome at positions 159-160 of SEQ ID No. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-25 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurs in one chromosome at position 114 of SEQ ID No. 1, and a fragment CGT insertion occurs in the other chromosome at positions 114-115 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-162 of SEQ ID No. 3, and a base (A) insertion occurs in the other chromosome at positions 160-161 of SEQ ID No. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-28 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) deletion occurs in the other chromosome at position 114 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, and a base (A) insertion occurs in both chromosomes at positions 160-161 of SEQ ID No. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-32 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-511 of SEQ ID No. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, and a base (C) deletion occurs in both chromosomes at position 161 of SEQ ID No. 3;
the difference of the genomic DNA of the Arabidopsis thaliana mutant line T2-33 and the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP9, a base (T) insertion occurs in both chromosomes at positions 160-161 of SEQ ID No. 3, and a fragment insertion occurs in both chromosomes at positions 561-562 of SEQ ID No. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID No. 14;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T2-38 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in both chromosomes at positions 115-127 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a base (T) and a base (G) insertion occurs in both chromosomes at positions 160-161 of SEQ ID No. 3 and positions 562-563 of SEQ ID No. 3, respectively;
the difference between the genome DNA of the tomato mutant line sldmp-1 and the genome DNA of wild-type tomato AC only lies in that in a gene encoding SlDMP protein, and a base (C) insertion occurs in both chromosomes at positions 92-93 of SEQ ID No. 5;
the difference between the genome DNA of the tomato mutant line sldmp-2 and the genome DNA of wild-type tomato AC only lies in that in a gene encoding protein SlDMP, a fragment deletion occurs in both chromosomes at positions 93-249 of SEQ ID No. 5.
Another object of the present invention is to provide a method for preparing a plant haploid.
The preparation method of the plant haploid provided by the invention comprises the following steps: selfing the plant haploid inducer line or the offspring thereof prepared by the method or crossing the plant haploid inducer line or the offspring thereof with other plant materials as a male parent to obtain the selfed offspring or the cross-species offspring, namely the plant haploid; the plant is a dicotyledonous plant.
Furthermore, the preparation method of the plant haploid further comprises the following steps: performing fluorescent labeling identification and/or haploid traits identification and/or leaf ploidy identification and/or molecular marker identification on the selfed offspring or the cross-species offspring single plant, and selecting the offspring single plants identified as haploids by at least one method as plant haploids.
Furthermore, the fluorescent labeling identification method may be performed as follows: crossing the plant haploid inducer line carrying the fluorescent protein expression element as a male parent with a female parent to obtain a cross-species offspring, and judging whether the seed to be tested is a haploid or a diploid by detecting whether the seed of the cross-species offspring has a fluorescent signal; if the seed to be tested shows no fluorescence or weak fluorescence, the seed is a haploid or is a candidate; if the seed to be tested shows strong fluorescence, the seed is a diploid or a candidate. Furthermore, whether the seeds to be tested are fluorescent or not is detected by fluorescent light. Furthermore, the male parent carries a TagRFP fluorescent protein expression element driven by the promoter AtOLEO1, so that it can be judged whether it is a haploid or a diploid depending on whether the hybrid offspring seed has red fluorescence.
The haploid trait identification method can be performed as follows: if the plant to be tested has the traits of dwarf, narrow leaves growing upward, compact plant type, and male sterility, the plant is a haploid or a candidate; if the plant to be tested has the traits of high plant, broad and scattered leaves, and normal fertility, the plant is a diploid or a candidate.
The leaf ploidy identification method can be performed as follows: extracting cell nuclei of young leaves of a plant to be tested, and taking diploid plant leaves as a control; detecting the signal by a flow cytometry instrument, firstly detecting the diploid nuclear signal and setting the diploid nuclear signal peak at 50 (since the genetic materials in diploid cells are twice that in haploid cells, the haploid nuclear signal peak appears around 25). If the nuclear signal peak of the plant to be tested appears around 25, the plant is haploid or a candidate; and if the signal peak of the plant to be tested appears around 50, and the signal intensity enrichment position of the plant to be tested is the same as that of the diploid nucleus, the plant is diploid or a candidate.
The molecular marker identification may be performed as follows: carrying out PCR amplification by adopting a polymorphic primer between a male parent (a female parent haploid inducer line) and a female parent, and judging whether a plant to be tested is haploid or diploid according to a PCR amplification product; if the amplification product of the plant to be tested only has the banding pattern of the female parent without the banding pattern of the male parent, the plant is haploid or a candidate; if the amplification product of the plant to be tested has a heterozygous banding pattern of a male parent and a female parent, the plant is diploid or a candidate.
The plant haploid inducer line and the plant haploid prepared by the method also fall within the scope of the invention.
The plant haploid inducer line and the plant haploid include not only cells, tissues, and organs derived from the plant haploid inducer line and the plant haploid, such as seeds, leaves, fruits, stems, flowers, roots, but also propagation materials derived from the plant haploid inducer line and the plant haploid, such as a group consisting of pollen, ovaries, ovules, germs, endosperms, egg cells, cleavage, roots, root tips, hypocotyls, cotyledons, stems, leaves, flowers, anthers, seeds, meristematic cells, protoplasts, and tissue cultures.
Gene AtDMP8 in the Arabidopsis thaliana mutant line or gene AtDMP9 in the Arabidopsis thaliana mutant line or gene SlDMP in the tomato mutant line in the above method also falls within the scope of the present invention.
Any of the following uses D1) to D5) also falls within the scope of the present invention:
D1) application of the plant haploid inducer line prepared by the method in cultivating plant haploids;
D2) application of a substance for silencing or inhibiting the expression and/or activity of gene AtDMP8 and/or gene AtDMP9 in the plant genome or knocking out gene AtDMP8 and/or gene AtDMP9 or a substance for inhibiting the activity of protein AtDMP8 and/or protein AtDMP8 in cultivating plant haploid inducer lines or plant haploids;
D3) application of a substance for silencing or inhibiting the expression and/or activity of gene SlDMP in the plant genome or knocking out gene SlDMP or a substance for inhibiting the activity of protein SlDMP in cultivating plant haploid inducer lines or plant haploids;
D4) application of protein AtDMP8 or related biological material thereof and/or protein AtDMP9 or related biological material thereof in regulating induction rate of plant haploid inducer lines or improving induction rate of plant haploid inducer lines or cultivating plant haploid inducer lines or plant haploids;
D5) application of SlDMP protein or related biological materials thereof in regulating induction rate of plant haploid inducer lines or improving induction rate of plant haploid inducer lines or cultivating plant haploid inducer lines or plant haploids.
In any of above-mentioned uses or methods, protein AtDMP8 is a protein represented by a1) or a2) or a3) or a4):
al) a protein with the amino acid sequence shown in SEQ ID No. 2;
a2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID No. 2;
a3) a protein having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 2;
a4) a protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 2 and having the same function; protein AtDMP9 is a protein represented by the following b 1) or b2) or b3) or b4):
b1) a protein with the amino acid sequence shown in SEQ ID No. 4;
b2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID No. 4;
b3) a protein having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 4;
b4) a protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 4 and having the same function.
The protein SlDMP is a protein represented by the following c1) or c2) or c3) or c4):
c1) a protein with the amino acid sequence shown in SEQ ID No. 6;
c2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID No. 6;
c3) a protein having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 6;
c4) a protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 6 and having the same function.
The protein AtDMP8 or AtDMP9 or SlDMP related biomaterial is any one of the following d1) to d12):
d1) a nucleic acid molecule encoding protein AtDMP8 or AtDMP9 or SlDMP;
d2) an expression cassette comprising the nucleic acid molecule of d1);
d3) a recombinant vector containing the nucleic acid molecule of d1);
d4) a recombinant vector containing the expression cassette of d2);
d5) a recombinant microorganism containing the nucleic acid molecule of d1);
d6) a recombinant microorganism containing the expression cassette of d2);
d7) a recombinant microorganism containing the recombinant vector of d3);
d8) a recombinant microorganism containing the recombinant vector of d4);
d9) a transgenic plant cell line comprising the nucleic acid molecule of d1);
d10) a transgenic plant cell line containing the expression cassette of d2);
d11) a transgenic plant cell line containing the recombinant vector of d3);
d12) a transgenic plant cell line comprising the recombinant vector of d4).
Furthermore, the nucleic acid molecule of d1) is a gene shown in the following 1) or 2) or 3) or 4):
1) a cDNA molecule or a genomic DNA molecule with the coding sequence shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5;
2) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in 1) and encoding protein AtDMP8 or AtDMP9 or SlDMP;
3) a cDNA molecule or a genomic DNA molecule derived from a dicotyledonous plant and having 75% or more identity with the nucleotide sequence defined in 1) and encoding protein AtDMP8 or AtDMP9 or SlDMP;
4) a cDNA molecule or a genomic DNA molecule hybridizing with the nucleotide sequence defined in 1) or 2) or 3) under stringent conditions and encoding protein AtDMP8 or AtDMP9 or SlDMP;
wherein SEQ ID No. 1 is a nucleic acid molecule encoding protein AtDMP8, SEQ ID No. 3 is a nucleic acid molecule encoding protein AtDMP9, and SEQ ID No. 5 is a nucleic acid molecule encoding protein SlDMP.
Furthermore, the substance knocking out gene AtDMP8 and/or gene AtDMP9 or the substance knocking out gene SlDMP is the CRISPR/Cas9 vector containing the target sequence as described above.
In any one of the above-mentioned uses or methods or plants, wherein the plants are dicotyledonous, specifically Arabidopsis thaliana or tomato; specifically, Arabidopsis thaliana may be wild-type Arabidopsis thaliana (Col-0) or ms1; specifically, the tomato may be wild-type tomato AC or Micro-Tom or F1 generation material obtained by crossing tomato AC with Micro-Tom.
The following examples facilitate a better understanding of the present invention but do not limit the present invention. The experimental methods in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. In the quantitative experiments of the following examples, triplicate experiments are set up, and the results are averaged.
The pICSL4723 vector in the following examples is described in the document “Castel, B. and L. Tomlinson, et al. (2019). Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis thaliana. PloS one, 14(1).”, publicly available from the Chinese Agricultural University, the test material is used only for repeating the relevant experiments of the present invention and cannot be used for other purposes.
Wild-type Arabidopsis thaliana Col-0 and ms1 in the following examples are described in the literature “Rosso, M. G. and Y. Li, et al. (2003). An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53 (1-2): 247-59.”, publicly available from China Agricultural University, the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
Wild-type tomato AC in the following examples is described in the literature “ Yuan, G. and C. Jia, et al. (2010). Effect of brassinosteroids on drought resistance and abscisic acid concentration in tomato under water stress. Scientia Horticulturae 126 (2): 103-108.”, publicly available from China Agricultural University, the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
The wild-type tomato Micro-Tom in the following examples is described in the literature “Sun, H. and S. Uchii, et al. (2006). A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant and Cell Physiology 47 (3): 426-431.”, publicly available from China Agricultural University, the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
The CDS sequence of gene AtDMP8 in the invention is shown in positions 95-826 of SEQ ID No. 1 in the sequence listing, and the amino acid sequence of the protein encoded by gene AtDMP8 is shown in SEQ ID No. 2.
The CDS sequence of gene AtDMP9 in the invention is shown in positions 141-875 of SEQ ID No. 3 in the sequence listing, and the amino acid sequence of the protein encoded by gene AtDMP9 is shown in SEQ ID No. 4.
The CDS sequence of gene SlDMP in the invention is shown in positions 1-678 of SEQ ID No. 5 in a sequence table, and the amino acid sequence of the protein encoded by gene SlDMP is shown in SEQ ID No. 6.
I. Genes AtDMP8 and/or AtDMP9 were knocked out using a CRISPR/Cas9 system Genes AtDMP8 and/or AtDMP9 in Arabidopsis thaliana were knocked out by a CRISPR/Cas9 system to obtain gene AtDMP8 and/or gene AtDMP9 knockout Arabidopsis thaliana mutants. The specific steps were as follows:
1. Selection of sgRNA Sequences
Target site sequences were designed on gene AtDMP8 and/or gene AtDMP9, respectively, and were 20 bp in length.
The target site 1 was located at positions 98-117 of SEQ ID No. 1 and 144-163 of SEQ ID No. 3, and the sequence of sgRNA target site 1 was GAGAAAACAGAGGAAAGCGT. (SEQ ID NO.15)
Target site 2 was located at positions 290-309 of SEQ ID No. 3, and the sequence of sgRNA target site 2 was AAGAGGTCGAAAACGTCGCA. (SEQ ID NO.16)
The target site 3 was located at 368-387 of SEQ ID No. 3, and the sequence of sgRNA target site 3 was TCAAGAGTGTTCCTGTCGGA. (SEQ ID NO.17)
The target site 4 was located at positions 509-528 of SEQ ID No. 1 and 558-577 of SEQ ID No. 3, and the sequence of sgRNA target site 4 was ATGACAACCGCGAGTCCACG (SEQ ID NO.18).
2. Construction of CRISPR/Cas9 Vector
The CRISPR/Cas9 vector is a recombinant vector(vector structure diagram shown in
3. Acquisition of Transgenic Plants
The CRISPR/Cas9 vector obtained in step 2 was transformed into Agrobacterium competent cells GV3101 through heat shock (Agrobacterium GV3101 competent cells were purchased from Beijing Aosen Dingxin Biotechnology Co., Ltd., and publicly available) to obtain a recombinant strain GV3101/CRISPR/Cas9.
Then the inflorescence of wild Arabidopsis thaliana Col-0 was infected with the recombinant strain GV3101/CRISPR/Cas9 by adopting a transformation method of infecting Arabidopsis thaliana inflorescences with Agrobacterium (i.e. the recombinant Agrobacterium was subjected to expanding propagation at 28° C., and Arabidopsis thaliana inflorescence was infected with the propagated Agrobacterium solution), and T1 generation transgenic Arabidopsis thaliana plant was obtained after red fluorescence screening.
4. Identification of Transgenic Plants with Mutation of Gene AtDMP8 and/or Gene AtDMP9
Leaves of the T1 generation transgenic Arabidopsis thaliana plant obtained in step 3 were collected, genomic DNA was extracted as a template, and PCR amplification was conducted by adopting the following two pairs of primers respectively, to obtain PCR amplification products of different lines.
The sequences of the primers for detecting gene AtDMP8 mutant sequence were as follows:
The sequences of the primers for detecting gene AtDMP9 mutant sequence were as follows:
The PCR amplification products of different lines were subjected to Sanger sequencing, and the sequencing results were compared with AtDMP8 and AtDMP9 of wild-type Arabidopsis thaliana Col-0, respectively. The genotypes of AtDMP8 and AtDMP9 were identified according to the following principles, respectively.
If the sequence had a doublet characteristic from the target site sequence, the genotype of the line was heterozygous (i.e. gene AtDMP8 and/or gene AtDMP9 mutated on one of the two homologous chromosomes, and unmutated on the other chromosome), and the line was a T1 generation transgenic Arabidopsis thaliana heterozygous mutant line;
For a sequence with specific singlet characteristics from the target site sequence, if it was the same as the gene sequence of AtDMP8 and AtDMP9 of wild-type Arabidopsis thaliana Col-0, the genotype of the line was wild-type, that is there was no mutation on the gene sequence of AtDMP8 and AtDMP9; if it was different from the gene sequence of AtDMP8 and/or AtDMP9 of the wild-type Arabidopsis thaliana Col-0, the genotype of the line was homozygous (the gene AtDMP8 and/or gene AtDMP9 mutated on both homologous chromosomes), the line was a T1 generation transgenic Arabidopsis thaliana homozygous mutant line.
The identification results were shown in Tables 1 and 2 (Tables 1 and 2 were mutations genes AtDMP8 and AtDMP9 of T1 generation transgenic Arabidopsis thaliana, respectively): among 41 T1 generation transgenic Arabidopsis thaliana plants, there was 33 T1 generation transgenic plants with mutation of gene AtDMP8, of which 2 plants have a homozygous mutation in gene AtDMP8 and 17 plants had a biallelic mutation in gene AtDMP8. There were 28 T1 generation transgenic plants with mutation of gene AtDMP9, of which 5 plants had a homozygous mutation in gene AtDMP9 and 7 plants had a biallelic mutation in gene AtDMP9. There were 12 plants with homozygous/biallelic mutations in AtDMP8 and AtDMP9. Individuals that resulted in a frameshift mutation (deletion not a multiple of 3) among homozygous/biallelic mutant individuals were further selected for phenotypic identification. There were three types of AtDMP8 homozygous mutation/biallelic mutation, AtDMP9 homozygous mutation/biallelic mutation, and AtDMP8 and AtDMP9 homozygous mutation/biallelic mutation.
The obtained T1 generation transgenic Arabidopsis thaliana gene AtDMP8 mutant lines comprised T1-34, and the specific mutations were as follows:
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana gene AtDMP8 mutant lines T1-34 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) insertion occurred in the other chromosome at positions 114-115 of SEQ ID No. 1.
The obtained T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines comprised T1-6, T1-11, T1-19, T1-24, T1-25, T1-28, and T 1-32, and the specific mutations were as follows:
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-6 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred in one chromosome at positions 115-512 of SEQ ID No. 1, and a fragment deletion occurred in the other chromosome at positions 113-114 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a fragment insertion occurred at positions 160-161 of SEQ ID No. 3, with the nucleotide sequence of the inserted fragment shown as GTTTACACGGCGACTC (SEQ ID No. 13).
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-11 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) insertion occurred at positions 114-115 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a base (T) insertion occurred in one chromosome at positions 160-161 of SEQ ID No. 3, and a fragment insertion occurred in the other chromosome at positions 160-161 of SEQ ID No. 3, with the nucleotide sequence of the inserted fragment shown as GTTTACACGGCGACTC (SEQ ID No. 13).
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-19 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurred in one chromosome at position 114 of SEQ ID No. 1, and a fragment deletion occurred in the other chromosome at positions 115-511 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a fragment deletion occurred in one chromosome at positions 161-560 of SEQ ID No. 3, and a fragment deletion occurred in the other chromosome at positions 161-564 of SEQ ID No. 3.
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-24 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) insertion occurred in the other chromosome at positions 114-115 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a fragment deletion occurred in one chromosome at positions 161-560 of SEQ ID No. 3, and a fragment deletion occurred in the other chromosome at positions 159-160 of SEQ ID No. 3.
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-25 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurred in one chromosome at position 114 of SEQ ID No. 1, and a fragment CGT insertion occurred in the other chromosome at positions 114-115 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a fragment deletion occurred in one chromosome at positions 161-162 of SEQ ID No. 3, and a base (A) insertion occurred in the other chromosome at positions 160-161 of SEQ ID No. 3.
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-28 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred in one chromosome at positions 115-512 of SEQ ID No. 1, and a base (T) deletion occurred in the other chromosome at position 114 of SEQ ID No. 1; and in a gene encoding protein AtDMP9, a base (A) insertion occurred at positions 160-161 of SEQ ID No. 3.
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 double mutant lines T1-32 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred in one chromosome at positions 115-511 of SEQ ID No. 1, and a base (T) insertion occurred in the other chromosome at positions 114-115 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a base (C) deletion occurred at position 161 of SEQ ID No. 3;
5. Identification of T2 Generation Transgenic Arabidopsis thaliana Genotyping
T1 generation transgenic Arabidopsis thaliana gene AtDMP8 and/or gene AtDMP9 mutant lines T1-19, T1-33, and T1-38 obtained in step 4 were selfed, seeds were sowed after harvesting to obtain T2 generation transgenic Arabidopsis thaliana. The specific method for identifying the genotypes of genes AtDMP8 and AtDMP9 of T2 generation transgenic Arabidopsis thaliana was as follows: with the genomic DNA of T2 transgenic Arabidopsis thaliana as a template, the genotypes of genes AtDMP8 and AtDMP9 in T2 transgenic Arabidopsis thaliana were identified by using mutant sequence detection primers of AtDMP8 (DMP8F1 and DMP8R1) and AtDMP9 (DMP9F1 and DMP9R2) according to the method in step 4, respectively.
The obtained T2 generation transgenic Arabidopsis thaliana gene AtDMP8 mutant homozygous lines comprised T2-33-1, T2-33-2, and T2-33-3 which have the same mutation sequences, and the specific mutations were as follows:
Sequencing identification showed that: the difference between the genomic DNA of T2 generation transgenic Arabidopsis thaliana gene AtDMP8 homozygosis mutant lines T2-33-1, T2-33-2, and T2-33-3 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in the gene encoding protein AtDMP8, a fragment at positions 115-511 of SEQ ID No. 1 was substituted with a fragment of size 202 bp. The nucleotide sequence of the fragment with the size of 202 bp was specifically as follows:
The obtained T2 generation transgenic Arabidopsis thaliana gene AtDMP9 homozygous lines comprised T2-33-4, T2-33-5, and T2-33-6 which have the same mutation sequences, and the specific mutations were as follows:
Sequencing identification showed that: the difference between the genomic DNA of T2 generation transgenic Arabidopsis thaliana gene AtDMP9 homozygosis mutant lines T2-33-1, T2-33-2, and T2-33-3 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in the gene encoding protein AtDMP9, a base (T) insertion occurred at positions 160-161 of SEQ ID No. 3, and a fragment insertion occurred at positions 561-562 of SEQ ID NO 3, with the nucleotide sequence shown as:
The obtained T2 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 homozygous mutant lines comprised T2-19, T2-38-1, and T2-38-2, with the mutation types of each line as follows:
Sequencing identification showed that: the difference between the genomic DNA of T2 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 homozygosis mutant lines T2-19 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred at positions 115-511 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a fragment deletion occurred at positions 161-564 of SEQ ID No. 3.
Sequencing identification showed that: the difference between the genomic DNA of T2 generation transgenic Arabidopsis thaliana genes AtDMP8 and AtDMP9 homozygosis mutant lines T2-38-1 and T2-38-2 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurred at position 115-127 of SEQ ID No. 1, and in a gene encoding protein AtDMP9, a fragment insertion occurred at positions 160-161 of SEQ ID No. 3 and positions 562-563 of SEQ ID No. 3.
The above T1 and T2 generation transgenic Arabidopsis thaliana mutant lines were selected for the following haploid induction capacity analysis experiments.
II. Application of Gene AtDMP8 and/or Gene AtDMP9 Knockout Arabidopsis thaliana Mutant in Inducing the Haploid Generation
(A) Identification of Haploid Selfing Induction Ability of AtDMP8 and AtDMP9 Knockout Arabidopsis thaliana Mutants
Three types of mutants obtained from genes AtDMP8 and AtDMP9 were respectively selfed to obtain selfed offspring, and haploid identification was carried out on the selfed offspring by the following method (since wild Arabidopsis thaliana Col was a homozygous selfed line, the mutant selfed offspring obtained by knocking out genes AtDMP8 and AtDMP9 on this background cannot identify haploids through molecular markers):
1. Identification of Plant Phenotype
After the selfed seeds were planted, the phenotypes of a single plant were observed, with haploidy showed dwarf, narrow leaves growing upward, compact plant type, and male sterility, while diploid showed high plant, broad and scattered leaves, and normal fertility (
2. Leaf Identification by Flow Cytometry
Flow cytometry was conducted on the plant with the haploid character obtained in step 1, wherein the specific method was as follows: cell nuclei of young leaves of a plant to be tested was extracted, and diploid Arabidopsis thaliana leaves were used as a control; the signal was then detected by a flow cytometry instrument, the diploid nuclear signal was firstly detected, and the diploid nuclear signal peak position was set at 50 (since the genetic materials in diploid cells were twice that in haploid cells, the haploid nuclear signal peak position appeared around 25). And if the nuclear signal peak position of the plant to be tested appears around 25, the plant to be tested is considered to be a haploid plant. If the signal peak of the plant to be tested appears around 50, it was considered that the signal intensity enrichment position of the plant to be tested is the same as that of the diploid nucleus, and the plant to be tested was diploid (
The identification results were counted and the induction rate was calculated according to the following formula: induction rate (%)=(number of haploid plants/total number of plants)×100. As can be seen, after simultaneous mutation of the genes AtDMP8 and AtDMP9, haploids can be obtained in selfed offspring.
A. Identification of Hybridization Induction Ability of AtDMP8 and AtDMP9 Knockout Arabidopsis thaliana Mutants
The three types of mutants obtained from genes AtDMP8 and AtDMP9 were crossed with Arabidopsis thaliana ms1 materials to obtain cross-species offspring, and the haploids in the cross-species offspring were identified by the following methods:
1. Fluorescent Labeling Identification
The CRISPR/Cas9 vector carried the expression element of TagRFP (Entacmaea quadricolor) driven by the promoter AtOLEO1. Since the promoter AtOLEO1 was specifically expressed in mature seed embryos, the fluorescent signal of TagRFP may be observed by fluorescent light. Therefore, the mutant carrying the expression element as a male parent was hybridized with other non-fluorescent female parent materials to obtain seeds, wherein embryos of diploid seeds showed strong red fluorescence due to having the genome of the male parent, while embryos of haploid seeds showed no fluorescence or weak fluorescence due to being derived from the female parent (
2. Molecular Marker Identification
The seeds with no fluorescence and weak fluorescence identified in step 1 were further planted, the genomic DNA was extracted, and PCR amplification was conducted adopting AtDMP8 and AtDMP9 knockout Arabidopsis thaliana mutant polymorphic primer 092B02-F(092B02-F: CAGCTGAGATGAACGAGTTGTCTT) (SEQ ID NO.97), 092B02-R (092B02-R: TCTTTTGAGTCACTCCGTATGTCC) (SEQ ID NO.98), and LB-o8474(LB-o8474: ATAATAACGCTGCGGACATCTACATTTT) (SEQ ID NO.99), and the amplified product was subjected to agarose banding pattern detection if the size of the amplified product of the individual plant to be tested was 500 bp, showing 1 band, it was considered the individual plant band to be the Arabidopsis thaliana ms1 banding pattern without banding pattern of the male parent material, the individual plant was female parent haploid. If the size of the amplification products of the individual plant to be tested was 500 bp and 1094 bp, showing 2 bands, the individual plant band was considered to be a heterozygous banding pattern of Arabidopsis thaliana ms1 and a transgenic Arabidopsis thaliana mutant line, the individual plant was an offspring of a normal hybrid and was diploid (
3. Identification of Mature Plants Phenotype
The phenotypes of the plants identified in steps 1 and 2 were further observed, with haploidy showed dwarf, narrow leaves growing upward, compact plant type, and male sterility, while diploid showed high plant, broad, and scattered leaves, and normal fertility.
4. Leaf Identification by Flow Cytometry
Flow cytometry was conducted on the plant with the haploid character obtained in step 3, wherein the specific method was as follows: cell nuclei of young leaves of a plant to be tested was extracted, and diploid Arabidopsis thaliana leaves were used as a control; the signal was then detected by a flow cytometry instrument, the diploid nuclear signal was firstly detected, and the diploid nuclear signal peak position was set at 50 (since the genetic materials in diploid cells were twice that in haploid cells, the haploid nuclear signal peak position appeared around 25). And if the nuclear signal peak position of the plant to be tested appears around 25, the plant to be tested is considered to be a haploid plant. If the signal peak of the plant to be tested appears around 50, it was considered that the signal intensity enrichment position of the plant to be tested is the same as that of the diploid nucleus, and the plant to be tested was diploid
The identification results were counted and the induction rate was calculated according to the following formula: induction rate (%)=(number of maternal haploid plants/total number of plants)×100. As can be seen, after gene AtDMP8 mutation, gene AtDMP9 mutation, and simultaneous mutation of genes AtDMP8 and AtDMP9, they were subjected to hybridization with other materials, and female parent haploid may be obtained in an offspring.
I. Gene SlDMP was Knocked Out Using the CRISPR/Cas9 System
Gene SlDMP in the tomato was knocked out by using a CRISPR/Cas9 system to obtain a gene SlDMP knockout tomato mutant. The specific steps were as follows:
1. Selection of sgRNA Sequences
A target site sequence was designed on gene SlDMP and was 20 bp in length.
The target site 1 was located at positions 76-95 of SEQ ID No. 5, and the sequence of sgRNA target site 1 was TATCTCACTAATTACCACA (SEQ ID NO.100).
Target site 2 was located at positions 247-266 of SEQ ID No. 5, and the sequence of sgRNA target site 2 was TCTCCTTTTACCAAATACTGA (SEQ ID NO.101).
2. Construction of CRISPR/Cas9 Vector
The CRISPR/Cas9 vector is a recombinant vector (vector structure diagram shown in
3. Acquisition of Transgenic Plants
The CRISPR/Cas9 vector obtained in step 2 was transformed into Agrobacterium competent cells GV3101 through heat shock (Agrobacterium GV3101 competent cells were purchased from Beijing Aosen Dingxin Biotechnology Co., Ltd., and publicly available) to obtain a recombinant strain GV3101/CRISPR/Cas9.
Then the cotyledon explant of wild tomato AC was infected with the recombinant strain GV3101/CRISPR/Cas9 by adopting a transformation method of infecting tomato cotyledon explant with Agrobacterium (i.e. the recombinant Agrobacterium was subjected to expanding propagation at 28° C., and tomato cotyledon explant was infected with the propagated Agrobacterium solution), and T0 generation transgenic tomato cotyledon explant plant was obtained after kanamycin resistance screening.
4. Identification of Transgenic Plants with Mutation of Gene SlDMP
Leaves of the T0 generation transgenic tomato plant obtained in step 3 were collected, genomic DNA was extracted as a template, and PCR amplification was conducted by adopting the following primers, to obtain PCR amplification products of different lines. The sequences of the primers for detecting gene SlDMP mutant sequence were as follows:
The PCR amplification products of different lines were subjected to Sanger sequencing, and the sequencing results were compared with gene SlDMP of wild-type tomato AC. The genotype of SlDMP was identified according to the following principles.
If the sequence had a doublet characteristic from the target site sequence, the genotype of the line was a heterozygous genotype (i.e. gene SlDMP mutated on one of the two homologous chromosomes, and unmutated on the other chromosome), and the line was a TO generation transgenic tomato heterozygous mutant line;
For a sequence with specific singlet characteristics from the target site sequence, if it was the same as the gene sequence of SlDMP of wild-type tomato AC, the genotype of the line was wild-type, that is there was no mutation on the gene sequence of SlDMP; if it was different from the gene sequence of SlDMP of the wild-type tomato AC, the genotype of the line is homozygous (the genes SlDMP mutated on both homologous chromosomes), the line was a T0 generation transgenic tomato homozygous mutant line.
5. Identification of T1 Generation Transgenic Tomato Genotyping
T0 transgenic tomato gene SlDMP mutant lines T0-33 and T0-34 obtained in step 4 were selfed, seeds were sowed after harvesting to obtain T1 generation transgenic tomato. The specific method for identifying the genotype of gene SlDMP of T1 generation transgenic tomato was as follows: with the genomic DNA of T1 transgenic tomato as a template, the genotype of gene SlDMP of T1 transgenic tomato was identified by using mutant sequence detection primers of SlDMP (SlDMPF2 and SlDMPR1) according to the method in step 4, respectively.
The finally obtained T1 generation transgenic tomato gene SlDMP homozygous mutant lines comprised sldmp-1 and sldmp-2, with the mutation types of each line as follows:
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic tomato genes SlDMP homozygosis mutant lines sldmp-1 and the genomic DNA of the wild-type tomato AC only lies in that in a gene encoding SlDMP protein, a base (C) insertion occurred at positions 92-93 of SEQ ID No. 5.
Sequencing identification showed that: the difference between the genomic DNA of T1 generation transgenic tomato genes SlDMP homozygosis mutant lines sldmp-2 and the genomic DNA of the wild-type tomato AC only lies in that in a gene encoding SlDMP protein, a fragment deletion occurred at positions 93-249 of SEQ ID No. 5.
II. Application of Gene SlDMP Knockout Tomato Mutant Inducing the Haploid Generation
(A) Plant performance and seed setting rate of SlDMP knockout tomato mutants Comparing the performance of the tomato mutant plants with gene SlDMP knocked out in the context of wild-type tomato AC and AC, it was found that the knockout of the gene SlDMP does not affect the growth of the plant (
(B) Identification of Hybridization Induction Ability of Gene SlDMP Knockout Tomato Mutants
The sldmp mutants were crossed with F1 generation material obtained by crossing tomato AC and Micro-Tom to obtain cross-species offspring, and the haploids in the cross-species offspring were identified by the following methods:
1. Fluorescent Labeling Identification
The CRISPR/Cas9 vector carried the expression element of TagRFP (Entacmaea quadricolor) driven by the promoter AtOLEO1. Since the promoter AtOLEO1 was specifically expressed in mature seed embryos, the fluorescent signal of TagRFP may be observed by fluorescent light. Therefore, the mutant carrying the expression element as a male parent was hybridized with other non-fluorescent female parent materials to obtain seeds, wherein embryos of diploid seeds red fluorescence due to having the genome of the male parent, while embryos of haploid seeds showed no fluorescence or weak fluorescence due to being derived from the female parent (
2. Molecular marker identification
The seeds with no fluorescence identified in step 1 were further planted, the genomic DNA was extracted, and PCR amplification was conducted adopting with polymorphic primers SlDMPF2+SlDMPR1 between the F1 generation obtained by crossing tomato AC with Micro-Tom and the gene SlDMP knockout tomato mutant, and the amplified product was subjected to agarose banding pattern detection or sequencing if the amplified product of the single plant to be tested showed 1 band or the sequencing result showed a single peak graph, it was considered the single plant band to be a female parent banding pattern without banding pattern of the male parent material, the single plant was the female parent haploid. And if the amplification product of the single plant to be tested showed two bands or the sequencing result showed a heterozygous peak graph, it was considered the single plant band to be a heterozygous banding pattern of the F1 generation obtained by crossing AC and the Micro-Tom of the tomato and the gene SlDMP knockout tomato mutant, and the single plant was a normal cross-species offspring and diploid.
3. Identification of Mature Plants Phenotype
The phenotypes of the plants identified in steps 1 and 2 were further observed, with haploidy showed dwarf, narrow leaves growing upward, compact plant type, and male sterility, while diploid showed high plant, broad, and scattered leaves, and normal fertility.
4. Leaf Identification by Flow Cytometry
Flow cytometry was conducted on the plant with the haploid character obtained in step 3, wherein the specific method was as follows: cell nuclei of young leaves of a plant to be tested was extracted, and diploid Arabidopsis thaliana leaves were used as a control; the signal was then detected by a flow cytometry instrument, the diploid nuclear signal was firstly detected, and the diploid nuclear signal peak position was set at 100 (since the genetic materials in diploid cells were twice that in haploid cells, the haploid nuclear signal peak position appeared around 50). And if the nuclear signal peak position of the plant to be tested appears around 50, the plant to be tested is considered to be a haploid plant. If the signal peak of the plant to be tested appears around 100, it was considered that the signal intensity enrichment position of the plant to be tested is the same as that of the diploid nucleus, and the plant to be tested was diploid (
The identification results were counted and the induction rate was calculated according to the following formula: induction rate (%)=(number of maternal haploid plants/total number of plants)×100. After gene SlDMP mutation, crossing with other materials was conducted, a female parent haploid may be obtained in the cross-species offspring.
The parthenogenetic haploid induction genes AtDMP8 and AtDMP9 are cloned from Arabidopsis thaliana. Experiments have shown that mutations of AtDMP8 and AtDMP9 can produce parthenogenetic haploid inducibility, to enable dicotyledonous crops to be induced to produce haploids via parthenogenetic means. The present invention was further verified in tomatoes, and it was also found in tomatoes that the mutation of SlDMP can produce parthenogenetic haploid inducibility. The invention lays an important foundation for broadening the application of haploid breeding technology on dicotyledonous plants and revealing the biological mechanism of parthenogenetic haploid production. Given the universality of the utilization of haploid breeding technology in the current breeding industry, the invention has very wide application space and market prospects.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910445082.3 | May 2019 | CN | national |
The present application is a U.S. National Phase of International Application Number PCT/CN2020/099680 filed Jul. 1, 2020, and claims priority to Chinese Application Number 201910445082.3 filed May 27, 2019.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/099680 | 7/1/2020 | WO |
