CN115927313A - Application of GhDMP8 gene as target point in improving cotton female parent haploid inductivity - Google Patents

Abstract

The invention provides application of a GhDMP8 gene as a target spot in improving the haploid inductivity of a cotton female parent, belonging to the technical field of cotton breeding. The GhDMP8 gene in cotton is knocked out by applying CRISPR/Cas9 technology, and the obtained transgenic homozygous mutant plant or the offspring thereof is used as a male parent to be hybridized with other cotton materials to generate a cotton female parent haploid. The invention obtains the serial function deletion mutation of the gene for the first time, and the female parent haploid induction function is proved through hybridization. Experiments prove that mutation of the cotton DMP8 can cause the generation of cotton female parent haploid, and influence of different planting environments on the induction rate is definite. Meanwhile, the mutant individual obtained by the method has haploid inductivity of a cotton female parent, and has important significance in breeding a novel induction line with high inductivity and improving haploid breeding efficiency of cotton.

Description

Application of GhDMP8 gene as target point in improving cotton female parent haploid inductivity

Technical Field

The invention belongs to the technical field of cotton breeding, and particularly relates to application of a GhDMP8 gene as a target spot in improving the haploid inductivity of a cotton female parent.

Background

Cotton is one of the world's important commercial crops, providing natural fiber for the textile industry. The breeding is complicated and long due to the characteristic of common cross pollination, and quite long generation selection is also needed for obtaining pure lines. Compared with the conventional breeding method which is time-consuming and labor-consuming, the cotton haploid breeding method based on cross induction can quickly obtain pure lines only by two generations, greatly shortens the commercial cost in the breeding process and the production process, and is one of important modern breeding technologies. The basic procedure of conventional haploid breeding is to obtain haploid by crossing a haploid induction line with a male parent and a common female parent material and then forming a doubled haploid pure line by doubling. At present, the only haploid induction line in cotton is the DH57-4 induction line of the sea island cotton Pima S-1, the DH57-4 is used as a female parent, and after the hybridization with upland cotton and sea island cotton, the offspring can obtain a certain proportion of haploid, and all nations breeders breed a large number of excellent induction lines on the basis of the haploid induction line, and the induction rate is continuously improved. However, the method for breeding the haploid by the hybridization based on DH57-4 as the female parent is single, has absolute dependence on the breeding female parent, and is not beneficial to breeding the cotton haploid in a large scale.

With the large application of the induction line in breeding practice, the research on the genetic mechanism of haploid induction is also deepened. However, no genetic engineering means for obtaining cotton haploids is reported at present.

Disclosure of Invention

In view of this, the invention aims to provide an application of a GhDMP8 gene as a target point in improving the cotton female parent haploid inductivity, getting rid of the dependence on a female parent material DH57-4, and having a wider application value.

The invention provides application of a GhDMP8 gene as a target point in inducing female parent haploid of cotton.

Preferably, the nucleotide sequence of the GhDMP8 gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2.

The invention provides an application of a reagent for silencing or inhibiting GhDMP8 gene expression or a reagent for knocking out GhDMP8 gene in female parent haploid breeding of cotton.

Preferably, the reagent for silencing or inhibiting the expression of the GhDMP8 gene or the reagent for knocking out the GhDMP8 gene is the GhDMP8 gene in mutant cotton; the mutation is carried out by enabling a sequence before a first transmembrane region and/or a sequence before a third transmembrane region of a GhDMP8 gene in cotton to be mutated;

the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

Preferably, the GhDMP8 gene knockout agent comprises a CRISPR/Cas9 gene knockout vector;

the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown as SEQ ID NO 5 and SEQ ID NO 6;

the reagent for inhibiting the expression of the GhDMP8 gene comprises shRNA and/or siRNA

The invention provides a preparation method for improving the haploid inductivity of a cotton female parent, which comprises the following steps:

silencing or inhibiting the expression of the GhDMP8 gene in cotton or knocking out the GhDMP8 gene in cotton to obtain transgenic cotton;

and hybridizing the transgenic cotton serving as a male parent with a female parent material to obtain a filial generation which is a cotton female parent haploid.

Preferably, the male parent is the selfed progeny of the transgenic cotton.

Preferably, the reagent for silencing or inhibiting the expression of the GhDMP8 gene or the reagent for knocking out the GhDMP8 gene is a GhDMP8 gene in mutant cotton; the mutation is realized by mutating the sequence before the first transmembrane region and/or the sequence before the third transmembrane region of the GhDMP8 gene in cotton;

the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

Preferably, the GhDMP8 gene knockout agent comprises a CRISPR/Cas9 gene knockout vector;

the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown as SEQ ID NO 5 and SEQ ID NO 6;

the reagent for inhibiting the GhDMP8 gene expression comprises shRNA and/or siRNA.

Preferably, the cotton is upland cotton or sea island cotton.

The invention provides application of a GhDMP8 gene as a target point in inducing female parent haploid of cotton. The invention obtains transgenic cotton for hybridization by serial functional deletion mutation of GhDMP8 gene to obtain maternal haploid induction function. Experiments prove that mutation of the DMP8 of the cotton can cause the generation of cotton female parent haploid, and the induction rate of the cotton female parent haploid can be improved in different planting environments. Provides a new idea for revealing the biological role of DMP in the process of generating cotton maternal haploids. Meanwhile, the mutant individual obtained by the invention has haploid induction capability of a cotton female parent, and has important significance in breeding a novel induction line with high induction rate and improving the haploid breeding efficiency of cotton.

Drawings

FIG. 1 is a schematic diagram of cotton GhDMP8 gene structure and CRISPR/Cas9 system knockout target site.

FIG. 2 shows the sequencing comparison result of the Ghdmp8-1 mutant cotton Ghdmp8 and the Ghdmp8 wild cotton.

FIG. 3 shows the ploidy and phenotype identification results of T1 generation mutant strain ghdmp8-1 and its selfed progeny haploid plant and flow cytometer.

FIG. 4 shows the T1 generation mutant line ghdmp8-1, its haploid plant in the progeny after crossing with T586 and T586 (upper panel), the result of blade ploidy identification by flow cytometry (middle panel) and the result of blade phenotype identification (lower panel).

FIG. 5 shows the identification results of the polymorphic molecular markers.

Detailed Description

The invention provides application of a GhDMP8 gene as a target spot in improving the female parent haploid inductivity of cotton.

In the present invention, the cotton is described. The cotton preferably comprises upland cotton or sea island cotton. The cotton contains double copies of GhDMP8 gene. The nucleotide sequence of the GhDMP8 gene is preferably shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the corresponding amino acid sequence is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

In the invention, ghDMP8 gene is used as a target spot, and the mutant cotton obtained by silencing or inhibiting the expression of the GhDMP8 gene or knocking out the GhDMP8 gene is used as a male parent material for cotton haploid breeding. Experiments prove that the mutant single plant obtained by the invention has haploid inductivity of cotton female parent, and the inductivity of the mutant single plant can be improved under different planting environments. Has important significance for breeding novel induction lines with high inductivity and improving the haploid breeding efficiency of cotton.

The invention provides an application of a reagent for silencing or inhibiting GhDMP8 gene expression or a reagent for knocking out GhDMP8 gene in female parent haploid breeding of cotton.

In the invention, the reagent for silencing or inhibiting the expression of the GhDMP8 gene or the reagent for knocking out the GhDMP8 gene is preferably a GhDMP8 gene in mutant cotton; the mutation is realized by mutating the sequence before the first transmembrane region and/or the sequence before the third transmembrane region of the GhDMP8 gene in cotton. The mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function. The reagent for inhibiting the expression of the GhDMP8 gene preferably comprises shRNA and/or siRNA. The reagent for knocking out the GhDMP8 gene preferably comprises a CRISPR/Cas9 gene knock-out vector; the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6. The method for mutating the pre-sequence of the first transmembrane region and/or the pre-sequence of the third transmembrane region of the GhDMP8 gene in the cotton genome by using CRISPR/Cas9 comprises the following steps: and introducing the CRISPR/Cas9 vector expressing the sgRNA into the target cotton to obtain the transgenic cotton. The CRISPR/Cas9 vector is preferably a recombinant vector obtained by inserting DNA molecules shown in SEQ ID NO 5 and SEQ ID NO 6 into BsaI sites of a sgRNA-Cas9 double-expression vector. The resulting recombinant vector is introduced into cotton, preferably by Agrobacterium transformation.

In the embodiment of the invention, the deletion mutation of 39 bases from the 12 th base of the 5' end of the sequence before the first transmembrane region in the sequence shown in SEQ ID NO. 1 of the GhDMP8 gene in the target cotton genome and/or the deletion CCC mutation of the 372 nd base of the sequence before the third transmembrane region are realized by a gene editing technology; 2, the sequence before the first transmembrane region of the GhDMP8 gene in the cotton genome is subjected to deletion mutation of 7 bases from the 15 th base of the 5' end and/or deletion CCC mutation of the 372 nd base of the sequence before the third transmembrane region by using a gene editing technology; 2, a sequence before a first membrane spanning region of a GhDMP8 gene in a cotton genome generates deletion mutation of 7 bases from a 15 th base of a 5' end and/or generates deletion mutation of 47 bases from a 372 nd base of a sequence before a third membrane spanning region by a gene editing technology; 2, a sequence before a first membrane spanning region of a GhDMP8 gene in a cotton genome generates deletion mutation of 40 bases from the 12 th base of a 5' end and/or inserts a base C between the 373 th base and the 374 th base of a sequence before a third membrane spanning region by a gene editing technology; 2, a sequence before a first membrane spanning region of 2 in a cotton genome generates deletion mutation of 7 bases from 15 th base of a 5' end and/or inserts a base C between 373 th base and 374 th base of a sequence before a third membrane spanning region; the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO 2 in the cotton genome is subjected to deletion mutation of 3 bases ATG from the 17 th base of the 5' end and/or deletion of a base C between the 373 th base and the 375 th base of the sequence before the third transmembrane region by using a gene editing technology.

In the invention, the mutant cotton is used as a male parent to be hybridized with other cotton of the same species to obtain a filial generation. And identifying the filial generation. Preferably, the identification method carries out haploid character identification, leaf ploidy identification and molecular marker identification on filial generations, and if all identification results are haploids according to the 3 methods, the plant is a cotton female parent haploid; if the result of identification by any one of the methods is not a haploid, the plant is not a cotton female parent haploid plant.

The invention provides a preparation method for improving the induction rate of cotton female parent haploid, which comprises the following steps:

silencing or inhibiting the expression of the GhDMP8 gene in cotton or knocking out the GhDMP8 gene in cotton to obtain transgenic cotton;

and hybridizing the transgenic cotton serving as a male parent with a female parent material to obtain a filial generation serving as a cotton female parent haploid.

The invention silences or inhibits the expression of the GhDMP8 gene in cotton or knocks out the GhDMP8 gene in cotton to obtain transgenic cotton.

In the invention, the preferred choice of silencing or inhibiting the expression of the GhDMP8 gene in cotton or knocking out the GhDMP8 gene in cotton is the GhDMP8 gene in mutant cotton; the mutation is realized by mutating the sequence before the first transmembrane region and/or the sequence before the third transmembrane region of the GhDMP8 gene in cotton. The mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function. The reagent for inhibiting the expression of the GhDMP8 gene preferably comprises shRNA and/or siRNA. The reagent for knocking out the GhDMP8 gene preferably comprises a CRISPR/Cas9 gene knock-out vector; the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6. The method for mutating the pre-sequence of the first transmembrane region and/or the pre-sequence of the third transmembrane region of the GhDMP8 gene in the cotton genome by using CRISPR/Cas9 comprises the following steps: and (3) introducing the CRISPR/Cas9 vector for expressing the sgRNA into cotton to obtain transgenic cotton. The CRISPR/Cas9 vector is preferably a recombinant vector obtained by inserting DNA molecules shown in SEQ ID NO 5 and SEQ ID NO 6 into BsaI sites of a sgRNA-Cas9 double-expression vector. The obtained recombinant vector is introduced into cotton, preferably by agrobacterium transformation. The transgenic cotton preferably adopts a primer pair consisting of a primer DMP8-JF shown in SEQ ID NO. 7 and a primer DMP8-JR shown in SEQ ID NO. 8 to carry out PCR amplification, an amplification product is obtained to carry out sequencing, and whether the gene in different strains of the transgenic cotton is mutated or not is identified. The plants with the mutated genes are marked as positive T0 generation transgenic cotton. Wherein DMP8-JF:5'-CACCCCTTAGGCGAGTTTTT-3' (SEQ ID NO: 7); DMP8-JR:5'-TCATGTCCTGGGAAAACACA-3' (SEQ ID NO: 8).

After the positive transgenic cotton is obtained, the transgenic cotton is used as a male parent to be hybridized with a female parent material, and the obtained filial generation is identified as the cotton female parent haploid.

In the present invention, the hybridization is preferably a pollen hybridization. The method for crossing pollen in the present invention is not particularly limited, and any pollen crossing method known in the art may be used. The female parent material is preferably wild type cotton HM-1. In the embodiment of the invention, T586 is taken as an example of a maternal material, and the preparation method of the cotton maternal haploid is concretely explained.

In the present invention, the male parent is preferably an inbred progeny of the transgenic cotton. The selfing progeny of the transgenic cotton is preferably T1 generation plant homozygous mutant obtained by selfing the transgenic cotton, and selfing is carried out again to obtain selfing progeny.

Obtaining filial generation, and preferably carrying out the following 3 methods identification on the single plant of the filial generation: haploid character identification, leaf ploidy identification and molecular marker identification, wherein identification results are all haploids, and the plant is a cotton female parent haploid; if the result of the identification of any method is not haploid, the plant is not the cotton female parent haploid plant. The plant character identification is carried out, preferably, the plant fertility phenotype is observed, and the haploid has the characteristics of short plant, narrow leaf, compact plant type, male sterility and the like; tetraploids are characterized by high plants, wide leaves, scattering and normal fertility. The invention utilizes 1 pair of molecular marker primers to identify the filial generation individual plant. 1 pair of molecular marker primers is preferably LS-F:5'-TACAAAGCCTACCCCATCGT-3' (SEQ ID NO: 9); LS-R:5'-TGGAGAGAGGGTGGACTTGT-3' (SEQ ID NO: 10). The genome DNA of inbred lines HM-1 and T586 is used as a template for amplification detection, the PCR product is 1048bp in T586, the amplification band size of HM-1 is 915bp, agarose gel electrophoresis can be used for resolution, the PCR product of T586 is larger, the electrophoresis speed is slow, and the amplification product fragment in HM-1 is smaller, the electrophoresis speed is fast. Thus, the band of the amplification product detected by T586 is above the band detected by HM-1 (in FIG. 5, lane 1 is the T586 band type, and lane 2 is the HM-1 band type). If the plant to be tested only has a T586 band (in FIG. 5, lane 3), the plant is considered to have no paternal material banding pattern and is therefore a maternal haploid plant. If the bands of T586 and HM-1 are present in the individual plants in the progeny of the cross (FIG. 5, lane 4), the plants are considered to be the progeny of the normal cross and tetraploid. And (3) identifying the ploidy of the leaves by using a flow cytometry technology, and if the nucleus signal peak of the plant to be detected appears near 100, determining that the nucleus signal peak is enriched in the nucleus signal intensity of the tetraploid and has the same position as that of the tetraploid, wherein the plant to be detected is tetraploid. And if the nuclear signal peak of the plant to be detected appears near 50, determining that the plant to be detected is a haploid plant. The embodiment of the invention proves that haploid can be generated by selfing after GhDMP8 gene mutation, and the haploid can be hybridized with other materials, cotton female parent haploid can be obtained in progeny, and the inductivity of the haploid can be improved in different planting environments.

The application of the GhDMP8 gene provided by the invention as a target in improving the preparation of cotton maternal haploids is described in detail below with reference to the examples, but the invention is not to be construed as limiting the scope of the invention.

sgRNA-Cas9 dual expression vector and HM-1: are described in the literature: wang P, zhang J, sun L, ma YZ, xu J, liang SJ, ding JW, tan JF, zhang QH, tu LL, danielh, jin SX, zhang xl. High efficiency multiplex gene editing in using CRISPR/Cas9 system. Plant biotechnology journal,2018;16 (1): 137-150; the public is available from the cotton institute of the Chinese academy of agricultural sciences.

T586: are described in the literature: ni Xiyuan, wang Xuede, sun Zhidong SSR marker localization of cotton phenotypic trait genes, journal of Cotton gossypii 2003;15 (6) 357-360; the public is available from the cotton institute of the Chinese academy of agricultural sciences.

Polymorphism marking primer: are described in the literature: cai CP, zhang XY, niu EL, zhao L, li NN, wang LM, ding LY, guo wz.ghpsy, a phytoene synthases gene, is related to the red plant phenotype in upland cotton (Gossypium hirsutum L.). Molecular biology reports.2014;41 (8) 4941-4952; andres R J, coneva V, frank M H, et al.modifications to a LATE MERISTEM IDENTITY gene area response for the major leaf maps of upper cotton (Gossypium hirsutum L.). Proceedings of National Academy of sciences.2017;114 (1) e57-e66. The public is available from the Cotton research institute of Chinese academy of agricultural sciences.

Example 1

Method for inducing and generating cotton female parent haploid by using gene GhDMP8

2 copies exist in the genome sequence of the wild cotton GhDMP8 gene (GhDMP 8 represents two genes of GhDMP8-A and GhDMP8-D, and only two homologous genes of GhDMP8-A and GhDMP8-D exist in upland cotton), as shown in SEQ ID NO 1 and SEQ ID NO 2 of the sequence table, the first transmembrane region sequence is shown as SEQ ID NO 3 and SEQ ID NO 4 of the sequence table from the 56 th to 78 th amino acids of the 5 'end, the second transmembrane region sequence is shown as SEQ ID NO 3 and SEQ ID NO 4 of the sequence table from the 88 th to 107 th amino acids of the 5' end, the third transmembrane region sequence is shown as SEQ ID NO 3 and SEQ ID NO 4 of the sequence table from the 145 th to 167 th amino acids of the 5 'end, and the fourth transmembrane region sequence is shown as SEQ ID NO 3 and SEQ ID NO 4 of the sequence table from the 187 th to 206 of the 5' end.

1. Cotton GhDMP8 gene knockout by CRISPR/Cas9 system

The GhDMP8 gene structure and CRISPR/Cas9 system knockout target site are schematically shown in figure 1.

1. 2 common target sites of SEQ ID NO 5 and SEQ ID NO 6 are designed on the upstream sequences of the first transmembrane region and the third transmembrane region of two homologous cotton GhDMP8 genes, and the lengths of the two target sites are 23bp.

Target site SEQ ID NO 5:5'-TTTGATGCCAATTCCATGGTGGG-3'.

Target site SEQ ID NO 6:5'-TACGGTTTCGTCACCCCCAACGG-3'.

2. The DNA molecules shown in SEQ ID NO 5 and SEQ ID NO 6 are inserted into the BsaI site of the sgRNA-Cas9 double expression vector to obtain a CRISPR/Cas9 knockout vector (sequencing verification is carried out).

3. And (3) introducing the CRISPR/Cas9 knockout vector prepared in the step (2) into an agrobacterium competent cell LB4404 to obtain a recombinant bacterium LB4404/CRISPR/Cas9. And then transforming the recombinant strain LB4404/CRISPR/Cas9 into cotton HM-1 hypocotyls by adopting an agrobacterium-mediated cotton hypocotyl genetic transformation method (carrying out 28 ℃ propagation on recombinant agrobacterium and infecting the cotton hypocotyls by using the bacterial solution after propagation), and obtaining T0-generation transgenic cotton plants after selection, screening, differentiation and rooting.

4. And (3) collecting the T0 generation transgenic cotton plant leaves obtained in the step (3), extracting genome DNA as a template, and carrying out PCR amplification by using a primer pair consisting of a sequence 7 primer DMP8-JF and a sequence 8 primer DMP8-JR to obtain amplification products of different strains.

DMP8-JF:5’-CACCCCTTAGGCGAGTTTTT-3’(SEQ ID NO:7)；

DMP8-JR:5’-TCATGTCCTGGGAAAACACA-3’(SEQ ID NO:8)。

Connecting PCR amplification products of different strains to a pGEM-T-easy vector, selecting a monoclonal for sequencing, comparing the sequencing result with a corresponding sequence of a target site of a wild cotton GhDMP8 gene, and identifying whether the gene in different strains of the T0 generation transgenic cotton is mutated.

The results are as follows: the genes in 1T 0 transgenic cotton plant are mutated, and the specific mutation forms are shown in figure 2, namely: the difference between the Ghdmp8-1 gene of the mutant plant and the Ghdmp8 gene of the wild-type cotton HM-1 is that the deletion mutation of 39 bases is generated from the 12 th base of the 5' end of the sequence before the first transmembrane region of SEQ ID NO. 1 of the sequence table and/or the deletion CCC mutation is generated at the 372 nd base of the sequence before the third transmembrane region; 2, the sequence before the first transmembrane region of the sequence table is subjected to 7-base deletion mutation from the 15 th base of the 5' end and/or the sequence before the third transmembrane region is subjected to deletion CCC mutation from the 372 nd base of the sequence; 2, the sequence before the first transmembrane region of the sequence table is subjected to 7 base deletion mutation from the 15 th base of the 5' end and/or the sequence before the third transmembrane region is subjected to 47 base deletion mutation from the 372 nd base of the sequence; 2, the sequence before the first transmembrane region of the sequence table is subjected to deletion mutation of 40 bases from the 12 th base at the 5' end and/or a base C is inserted between the 373 th base and the 374 th base of the sequence before the third transmembrane region; 2, the sequence before the first transmembrane region of the sequence table is subjected to deletion mutation of 7 bases from the 15 th base of the 5' end and/or a base C is inserted between the 373 rd base and the 374 th base of the sequence before the third transmembrane region; 2 of the sequence table, the sequence before the first transmembrane region of the sequence table is subjected to deletion mutation of 3 bases ATG from the 17 th base of the 5' end and/or the base C is deleted between the 373 th base and the 375 th base of the sequence before the third transmembrane region.

The plants with the mutated genes are marked as positive T0 generation transgenic cotton.

5. And (4) harvesting the positive T0 generation transgenic cotton obtained in the step (4), sowing and selfing to obtain T1 generation transgenic cotton. Identifying whether the gene of T1 generation transgenic cotton is a mutant genotype, the specific method comprises the following steps: taking the genome DNA of the T1 generation transgenic cotton as a template, carrying out PCR amplification by using a primer pair consisting of a sequence 7 primer DMP8-JF and a sequence 8 primer DMP8-JR, sequencing a PCR product, and classifying the genotype of the T1 generation transgenic cotton according to a sequencing result.

In the sequencing result, (1) the sequence with the bimodal characteristics from the target site sequence is a heterozygous genotype, and is a T1 generation transgenic cotton heterozygous genotype mutation (the gene in one homologous chromosome is mutated, and the gene in the other homologous chromosome is not mutated); (2) Comparing a sequence with a specific single peak characteristic from a target site sequence with a target site sequence of a GhDMP8 gene of a cotton wild type HM-1, if the sequence is the same, the sequence is the wild type and has no mutation, and the following analysis does not consider the sequence; if the mutation exists, the mutation is homozygous mutation obtained after the T0 generation plant is selfed, and the mutation is homozygous for the T1 generation transgenic cotton gene (two GhDMP8 genes of homologous chromosomes are mutated).

Through analysis, the T1 generation transgenic cotton homozygous gene mutant line has ghdmp8-1, and the mutation types are as follows: the T1 generation transgenic cotton gene mutation homozygous line GhDMP8-1 contains mutant gene GhDMP8 in both At and Dt genomes (the difference with the GhDMP8 gene of wild type cotton HM-1 is that the sequence before the first transmembrane region of SEQ ID NO:1 of the sequence table is subjected to deletion mutation of 39 bases from the 12 th base of the 5 'end and/or the sequence before the third transmembrane region is subjected to deletion CCC mutation At the 372 nd base; the sequence before the first transmembrane region of SEQ ID NO. 2 of the sequence table is subjected to deletion mutation of 7 bases from the 15 th base At the 5' end and/or deletion CCC mutation of the 372 nd base of the sequence before the third transmembrane region, the sequence before the first transmembrane region of SEQ ID NO. 2 of the sequence table is subjected to deletion mutation of 7 bases from the 15 th base At the 5 'end and/or deletion mutation of 47 bases from the 372 nd base of the sequence before the third transmembrane region, the sequence before the first transmembrane region of SEQ ID NO. 2 of the sequence table is subjected to deletion mutation of 40 bases from the 12 th base At the 5' end and/or insertion of a base C between the 373 th base and the 374 th base of the sequence before the third transmembrane region, the sequence before the first transmembrane region of SEQ ID NO. 2 of the sequence table is subjected to deletion mutation of 7 bases from the 15 th base At the 5 'end and/or insertion of a base C between the 373 th base and the 374 th base of the sequence before the third transmembrane region, and the sequence after the 375 th base deletion mutation of the first transmembrane region and/or deletion of the 374 th base of the 375 g sequence before the 5' end.

2. Identification of haploid induction capability of mutant obtained by knocking cotton GhDMP8 gene out through CRISPR/Cas9 system

(1) Phenotypic identification

Selfing the T1 generation homozygous gene mutant strain GhDMP8-1, continuously observing phenotypes of 2 pseudohaploid plants appearing in filial generations of the T586, and comparing HM-1 wild type cotton (the GhDMP8 gene is not mutated) with T586. The haploid has the characteristics of short plant, narrow and small leaf, compact plant type, male sterility and the like; tetraploids are shown as tall plants, wide leaves, cloggy, normal fertility (fig. 3 and 4).

(2) Flow-based leaf ploidy identification

Performing flow cytometry detection on 2 pseudohaploid plants which are identified and obtained from the 1) selfing and hybrid progeny and show haploid banding patterns in total, wherein the method comprises the following steps: extracting cell nucleuses of young and tender leaves of a plant to be detected, and taking tetraploid cotton leaves as a reference; the signal was then detected with a flow cytometer by first detecting the control tetraploid cell nucleus signal and setting the tetraploid cell nucleus signal peak to 100 (the haploid cell nucleus signal peak occurred around 50 because the genetic material in the tetraploid cell was twice that in the haploid cell). And if the signal peak of the plant to be detected appears near 100, the nuclear signal intensity of the plant to be detected and the nuclear signal intensity of the tetraploid are enriched, the positions of the nuclear signal intensities are the same, and the plant to be detected is tetraploid. And if the nuclear signal peak of the plant to be detected appears near 50, determining that the plant to be detected is a haploid plant.

The results are shown in FIG. 3 and FIG. 4, wherein the left graph in FIG. 3 is the flow cytometry result of ghdmp8-1 cotton, and the right graph is the flow cytometry result of the haploid plant in the T1 generation transgenic cotton selfing progeny. The left graph in FIG. 4 is the flow cytometry detection result of ghdmp8-1 cotton, and the right graph is the flow cytometry detection result of the pseudohaploid plant in the filial generation of T1 generation transgenic cotton.

The results show that: after 2 phenotypically identified pseudohaploids in the self-bred progeny and the T586 hybrid progeny of the ghdmp8-1 are detected by a flow cytometer, the ploidy of the pseudohaploids is haploid and is marked as a T1 generation transgenic cotton homozygous gene mutant strain ghdmp8-1 pseudohaploid plant.

(3) Polymorphic molecular marker identification

Designing 10 pairs of molecular marker primers according to T586 red leaf and leaf shape characters, and finally screening 1 pair of molecular markers for marking the leaf shape characters, namely a sequence 9 primer LS-F and a sequence 10 primer LS-R. The genome DNA of inbred lines HM-1 and T586 is used as a template for amplification detection, the PCR product is 1048bp in T586, the amplification band size of HM-1 is 915bp, agarose gel electrophoresis can be used for resolution, the PCR product of T586 is larger, the electrophoresis speed is slow, and the amplification product fragment in HM-1 is smaller, the electrophoresis speed is fast. Thus, the band of the amplification product detected by T586 is above the band detected by HM-1 (in FIG. 5, lane 1 is the T586 band type, and lane 2 is the HM-1 band type). If the plant to be tested only has a T586 band (in FIG. 5, lane 3), the plant is considered to have no paternal material banding pattern and is therefore a maternal haploid plant. If the bands of T586 and HM-1 are present in the individual plants in the progeny of the cross (FIG. 5, lane 4), the plants are considered to be the progeny of the normal cross and tetraploid.

LS-F:5’-TACAAAGCCTACCCCATCGT-3’(SEQ ID NO:9)；

LS-R:5’-TGGAGAGAGGGTGGACTTGT-3’(SEQ ID NO:10)。

The pollen of the T1 generation transgenic cotton gene homozygous mutant strain ghdmp8-1 is awarded to T586, and filial generations are obtained; sowing the obtained filial generation in a cotton research institute field of Chinese agricultural science institute, anyang city, henan province, taking three weeks of seedling leaves for genome DNA extraction and agarose banding detection, wherein the molecular marker identification result is as follows:

obtaining 1 single plant with only T586 strip from 298 offspring obtained by crossing T1 generation transgenic cotton homozygous gene mutant strain ghdmp8-1 and T586, and drawing up to haploid plant.

The PCR gel-running result of 1 pseudohaploid plant is shown in FIG. 5, wherein M, marker, lane 1 is T586,2 is HM-1,3 is hybrid haploid, and 4 is heterozygous tetraploid banding pattern in offspring.

The results show that: the pseudohaploid offspring identified by polymorphic molecular marker and flow detection of 1 ghdmp8-1 and T586 filial generation is expressed as maternal haploid plant after phenotypic identification.

Therefore, in the progeny individual plant of the homozygous transgenic line and the T586 cross, if the individual plant is identified as the haploid according to any one of the above 3 identification methods, the plant is or is selected as the cotton female haploid; if none of the 3 methods identified above is a haploid, then the plant is not, or is not a candidate for, a cotton maternal haploid.

And calculating the self-crossing induction rate and the cross induction rate according to the judgment result and the formula I and the formula II respectively.

Inbred induction rate (%) = (haploid plant number/total plant number of inbred progeny) × 100% formula I

Cross induction rate (%) = (haploid strain/total strain of progeny) 100% formula II

According to the above judgment result, the haploid plant number is 1, the total plant number in the test is 139, and the selfing induction rate is 0.71%. According to the above judgment results, the number of haploid plants was 1, the total number of tested plants was 298, and the cross induction rate (sum of the selfing induction rate and the cross induction rate) was 0.34%. It can be seen that the GhDMP8 gene can generate haploid by selfing after mutation, and can be hybridized with other materials to obtain cotton female parent haploid in progeny.

Example 2

Identification of haploid induction capability of mutant obtained by knocking cotton GhDMP8 gene out by CRISPR/Cas9 system in different planting environments

The method described in example 1 was used to prepare a cotton female parent haploid, except that the planting areas were the test field of Menghan Zhenmann Famura, yunnan province, and the test field of the cotton research institute, china academy of agricultural sciences, anyang, henan province, respectively, for selfing and crossing.

The haploid inductivity of the induction system in different environments is identified according to the method, and the induction rate of the progeny haploid of the induction system after being planted in the Yunnan Jinghong city is 0.93-1.02 percent, wherein the inbred inductivity (%): (haploid plant number/total plant number of selfed progeny) × 100% = (2/197) × 100% =1.02%; hybridization induction rate (%): (haploid strain number/total hybrid progeny strain number) = 100% = (1/108) = 100% =0.93%. While the induction rate of the cotton female parent haploid obtained by the planting group in Henan region is 0.34-0.71% (example 1).

The induction rate of the cotton female parent haploid obtained by planting in Yunnan Jinghong city is improved by 0.31-0.59% compared with the induction rate of the haploid obtained by planting in Henan Anyang city. Different growth environments are shown to have influence on the haploid inductivity, and optimization is needed to realize high inductivity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. An application of GhDMP8 gene as a target point in improving the female parent haploid inductivity of cotton.

2. The use of claim 1, wherein the GhDMP8 gene has a nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2.

3. Application of a reagent for silencing or inhibiting GhDMP8 gene expression or a reagent for knocking out the GhDMP8 gene in female haploid breeding of cotton.

4. The use of claim 3, wherein the agent for silencing or inhibiting the expression of the GhDMP8 gene or the agent for knocking out the GhDMP8 gene is the GhDMP8 gene in mutant cotton; the mutation is realized by mutating the sequence before the first transmembrane region and/or the sequence before the third transmembrane region of the GhDMP8 gene in cotton;

the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

5. The use according to claim 3, wherein the GhDMP8 gene knockout agent comprises a CRISPR/Cas9 gene knockout vector;

the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown as SEQ ID NO 5 and SEQ ID NO 6;

the reagent for inhibiting the GhDMP8 gene expression comprises shRNA and/or siRNA.

6. A preparation method for improving the haploid inductivity of a cotton female parent is characterized by comprising the following steps:

silencing or inhibiting the expression of the GhDMP8 gene in cotton or knocking out the GhDMP8 gene in cotton to obtain transgenic cotton;

and hybridizing the transgenic cotton serving as a male parent with a female parent material to obtain a filial generation serving as a cotton female parent haploid.

7. The method according to claim 6, wherein the male parent is a self-bred progeny of the transgenic cotton.

8. The preparation method according to claim 6, wherein the agent for silencing or inhibiting the expression of the GhDMP8 gene or the agent for knocking out the GhDMP8 gene is the GhDMP8 gene in mutant cotton; the mutation is realized by mutating the sequence before the first transmembrane region and/or the sequence before the third transmembrane region of the GhDMP8 gene in cotton;

the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

9. The preparation method according to claim 8, wherein the reagent for knocking out the GhDMP8 gene comprises a CRISPR/Cas9 gene knock-out vector;

the CRISPR/Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences shown as SEQ ID NO 5 and SEQ ID NO 6;

the reagent for inhibiting the expression of the GhDMP8 gene comprises shRNA and/or siRNA.

10. The method of claim 9, wherein the cotton comprises Gossypium hirsutum or Gossypium barbadense.

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