Application of GhDMP8 gene as target in improving cotton maternal haploid induction rate

Abstract

The application provides application of GhDMP8 gene as a target in improving cotton maternal haploid induction rate, and belongs to the field of cotton breeding technology. A transgenic homozygous mutant plant or offspring thereof is obtained by knocking out GhDMP8 gene in cotton by using CRISPR / Cas9 technology, and the transgenic homozygous mutant plant or offspring thereof is crossed with other cotton materials to generate cotton maternal haploid. A series of functional loss mutations of the gene are obtained for the first time, and the maternal haploid induction function of the gene is proved by crossing. The experiment proves that mutation of cotton DMP8 can lead to generation of cotton maternal haploid, and it is clear that different planting environments affect the induction rate. Meanwhile, the mutant single plant obtained by the application has the ability of inducing cotton maternal haploid, and has important significance for breeding a new type of high-induction-rate induction line and improving the efficiency of cotton haploid breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of cotton breeding, and particularly relates to an application of a GhDMP8 gene as a target in improving the haploid induction rate of cotton maternal plants. Background Art

[0002] Cotton is one of the world's most important economic crops, providing natural fibers for the textile industry. However, its frequent cross-pollination makes breeding cumbersome and time-consuming, and obtaining pure lines requires considerable generations of selection. Compared to conventional breeding methods, which are time-consuming and labor-intensive, cotton haploid breeding methods based on hybrid induction can rapidly obtain pure lines in just two generations, significantly reducing the breeding process and commercial costs during production, making it a key modern breeding technology. The basic procedure of conventional haploid breeding involves hybridizing a haploid induction line with a standard female parent to obtain haploids, followed by doubling to form doubled haploid pure lines. Currently, the only haploid induction line in cotton is the DH57-4 induction line of the island cotton variety Pima S-1. Using DH57-4 as the female parent, hybridization with upland and island cotton produces a consistent proportion of haploids in the offspring. Breeders worldwide have developed numerous superior induction lines based on this approach, and the induction rate has continued to improve. However, the method of breeding haploids based on DH57-4 as the female parent is relatively simple and absolutely dependent on the breeding female parent, which is not conducive to large-scale breeding of cotton haploids.

[0003] With the extensive application of induced lines in breeding practice, the genetic mechanism of haploid induction has been studied in depth. However, there is no report on the use of genetic engineering to obtain cotton haploids. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of the GhDMP8 gene as a target in improving the haploid induction rate of cotton maternal materials, which gets rid of the dependence on the maternal material DH57-4 and has a wider application value.

[0005] The present invention provides an application of a GhDMP8 gene as a target in inducing haploids in cotton maternal plants.

[0006] Preferably, the nucleotide sequence of the GhDMP8 gene is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] The present invention provides an application of a reagent for silencing or inhibiting the expression of a GhDMP8 gene or a reagent for knocking out the GhDMP8 gene in haploid breeding of cotton maternal plants.

[0008] Preferably, the reagent for silencing or inhibiting the expression of the GhDMP8 gene or the reagent for knocking out the GhDMP8 gene is a mutated GhDMP8 gene in cotton; the mutation is by mutating the sequence before the first transmembrane region and / or the sequence before the third transmembrane region of the GhDMP8 gene in cotton;

[0009] The mutation is a deletion mutation and / or an insertion mutation and / or other mutations that can lead to loss of gene function.

[0010] Preferably, the reagent for knocking out the GhDMP8 gene includes a CRISPR / Cas9 gene knockout vector;

[0011] The CRISPR / Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0012] The reagents for inhibiting GhDMP8 gene expression include shRNA and / or siRNA

[0013] The present invention provides a preparation method for improving the haploid induction rate of cotton maternal plants, comprising the following steps:

[0014] Silencing or inhibiting the expression of GhDMP8 gene in cotton or knocking out GhDMP8 gene in cotton to obtain transgenic cotton;

[0015] The transgenic cotton is hybridized with the male and female materials, and the obtained hybrid offspring is the cotton female haploid.

[0016] Preferably, the male parent is the self-pollinated offspring of the transgenic cotton.

[0017] Preferably, the reagent for silencing or inhibiting the expression of the GhDMP8 gene or the reagent for knocking out the GhDMP8 gene is a mutated GhDMP8 gene in cotton; the mutation is by mutating the sequence before the first transmembrane region and / or the sequence before the third transmembrane region of the GhDMP8 gene in cotton;

[0018] The mutation is a deletion mutation and / or an insertion mutation and / or other mutations that can lead to loss of gene function.

[0019] Preferably, the reagent for knocking out the GhDMP8 gene includes a CRISPR / Cas9 gene knockout vector;

[0020] The CRISPR / Cas9 gene knockout vector comprises sgRNAs with nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0021] The reagent for inhibiting the expression of the GhDMP8 gene includes shRNA and / or siRNA.

[0022] Preferably, the cotton is upland cotton or sea island cotton.

[0023] The present invention provides a method for using the GhDMP8 gene as a target in inducing haploid maternal cotton. The present invention obtains transgenic cotton through a series of loss-of-function mutations in the GhDMP8 gene, and then hybridizes and obtains its maternal haploid induction function. Experiments of the present invention have shown that mutations in cotton DMP8 can lead to the production of maternal haploid cotton, and different planting environments can increase its induction rate. This provides a new approach to revealing the biological role of DMP in the production of maternal haploid cotton. At the same time, the mutant individual plants obtained by the present invention have the haploid induction ability of the maternal cotton, which is of great significance for breeding new induced lines with high induction rates and improving the efficiency of cotton haploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the cotton GhDMP8 gene structure and CRISPR / Cas9 system knockout target sites.

[0025] Figure 2 This is the sequencing comparison result of cotton GhDMP8 mutant plant ghdmp8-1 and wild-type cotton GhDMP8 gene.

[0026] Figure 3 The haploid plants in the T1 generation mutant line ghdmp8-1 and its self-pollinated progeny, as well as the leaf ploidy and phenotypic identification results were identified by flow cytometry.

[0027] Figure 4 The T1 generation mutant line ghdmp8-1, the haploid plants in its hybrid offspring with T586, and T586 (upper panel), the leaf ploidy results identified by flow cytometry (middle panel), and the leaf phenotype identification results (lower panel).

[0028] Figure 5 The results of polymorphic molecular marker identification. DETAILED DESCRIPTION

[0029] The present invention provides an application of a GhDMP8 gene as a target in improving the haploid induction rate of cotton maternal plants.

[0030] In the present invention, the cotton preferably includes upland cotton or sea island cotton. Cotton contains two copies of the GhDMP8 gene. The nucleotide sequence of the GhDMP8 gene is preferably as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the corresponding amino acid sequence is as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0031] In the present invention, the GhDMP8 gene is targeted and, by silencing or inhibiting its expression or knocking out the GhDMP8 gene, the resulting mutant cotton is used as the male parent material for cotton haploid breeding. Experiments have shown that the mutant individual plants obtained using the present invention have the haploid induction ability of the cotton maternal parent, and their induction rate can be improved in different planting environments. This is of great significance for breeding new induced lines with high induction rates and improving the efficiency of cotton haploid breeding.

[0032] The present invention provides an application of a reagent for silencing or inhibiting the expression of a GhDMP8 gene or a reagent for knocking out the GhDMP8 gene in haploid breeding of cotton maternal plants.

[0033] In the present invention, the reagent for silencing or inhibiting GhDMP8 gene expression or knocking out GhDMP8 gene preferably mutates the GhDMP8 gene in cotton; the mutation is achieved by mutating the sequence preceding the first transmembrane region and / or the sequence preceding the third transmembrane region of the GhDMP8 gene in cotton. The mutation is a deletion mutation and / or an insertion mutation and / or other mutation that can lead to loss of gene function. The reagent for inhibiting GhDMP8 gene expression preferably comprises shRNA and / or siRNA. The reagent for knocking out GhDMP8 gene preferably comprises a CRISPR / Cas9 gene knockout vector; the CRISPR / Cas9 gene knockout vector comprises an sgRNA with nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6. Using CRISPR / Cas9 to mutate the sequence preceding the first transmembrane region and / or the sequence preceding the third transmembrane region of the GhDMP8 gene in the cotton genome comprises the following steps: introducing a CRISPR / Cas9 vector expressing the sgRNA into the target cotton to obtain transgenic cotton. The CRISPR / Cas9 vector is preferably a recombinant vector obtained by inserting the DNA molecules shown in SEQ ID NO: 5 and SEQ ID NO: 6 into the BsaI site of the sgRNA-Cas9 dual expression vector. The obtained recombinant vector is introduced into cotton, preferably by Agrobacterium transformation.

[0034] In an embodiment of the present invention, a gene editing technology is used to cause a 39-base deletion mutation starting from the 12th base at the 5' end in the sequence before the first transmembrane region of the GhDMP8 gene shown in SEQ ID NO: 1 in the target cotton genome and / or a CCC deletion mutation occurs at the 372nd base of the sequence before the third transmembrane region; the gene editing technology is used to cause a 7-base deletion mutation starting from the 15th base at the 5' end in the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO: 2 in the cotton genome and / or a CCC deletion mutation occurs at the 372nd base of the sequence before the third transmembrane region; the gene editing technology is used to cause a 7-base deletion mutation starting from the 15th base at the 5' end in the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO: 2 in the cotton genome and / or a 47-base deletion mutation occurs at the 372nd base of the sequence before the third transmembrane region; the gene editing technology is used to cause a 7-base deletion mutation starting from the 15th base at the 5' end in the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO: 2 in the cotton genome and / or a 47-base deletion mutation occurs at the 372nd base of the sequence before the third transmembrane region; the gene editing technology is used to cause a 7-base deletion mutation starting from the 15th base at the 5' end in the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO: NO:2 The sequence before the first transmembrane region undergoes a deletion mutation of 40 bases starting from the 12th base at the 5' end and / or a base C is inserted between the 373rd and 374th bases of the sequence before the third transmembrane region; the gene editing technology is used to cause the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO:2 in the cotton genome to undergo a deletion mutation of 7 bases starting from the 15th base at the 5' end and / or a base C is inserted between the 373rd and 374th bases of the sequence before the third transmembrane region; the gene editing technology is used to cause the sequence before the first transmembrane region of the GhDMP8 gene SEQ ID NO:2 in the cotton genome to undergo a deletion mutation of 3 bases ATG starting from the 17th base at the 5' end and / or a base C is deleted between the 373rd and 375th bases of the sequence before the third transmembrane region.

[0035] In the present invention, the mutant cotton plant is used as the male parent and hybridized with another cotton plant of the same species to obtain hybrid offspring. The hybrid offspring are then identified. The identification method preferably includes performing haploid trait identification, leaf ploidy identification, and molecular marker identification on the hybrid offspring. If the identification results of all three methods are haploid, the plant is a haploid plant of the cotton maternal parent; if the identification results of any of the above methods are not haploid, the plant is not a haploid plant of the cotton maternal parent.

[0036] The present invention provides a preparation method for improving the haploid induction rate of cotton maternal plants, comprising the following steps:

[0037] Silencing or inhibiting the expression of GhDMP8 gene in cotton or knocking out GhDMP8 gene in cotton to obtain transgenic cotton;

[0038] The transgenic cotton is hybridized with the male and female materials, and the obtained hybrid offspring is the cotton female haploid.

[0039] The invention silences or inhibits the expression of GhDMP8 gene in cotton or knocks out GhDMP8 gene in cotton to obtain transgenic cotton.

[0040] In the present invention, silencing or inhibiting the expression of the GhDMP8 gene in cotton or knocking out the GhDMP8 gene in cotton is preferably performed by mutating the GhDMP8 gene in cotton; the mutation is performed 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 mutation that can cause loss of gene function. The reagent for inhibiting the expression of the GhDMP8 gene preferably includes shRNA and / or siRNA. The reagent for knocking out the GhDMP8 gene preferably includes a CRISPR / Cas9 gene knockout vector; the CRISPR / Cas9 gene knockout vector contains an sgRNA with a nucleotide sequence as shown in SEQ ID NO: 5 and SEQ ID NO: 6. The method of using CRISPR / Cas9 to mutate the sequence before the first transmembrane region and / or the sequence before the third transmembrane region of the GhDMP8 gene in the cotton genome comprises the following steps: introducing a CRISPR / Cas9 vector expressing the sgRNA into cotton to obtain transgenic cotton. The CRISPR / Cas9 vector is preferably a recombinant vector obtained by inserting the DNA molecules shown in SEQ ID NO: 5 and SEQ ID NO: 6 into the BsaI site of the sgRNA-Cas9 dual expression vector. The obtained recombinant vector is introduced into cotton, preferably by Agrobacterium transformation. The transgenic cotton is preferably amplified by PCR using a primer pair consisting of primers DMP8-JF shown in SEQ ID NO: 7 and primers DMP8-JR shown in SEQ ID NO: 8. The amplified products are sequenced to identify whether the genes in different strains of transgenic cotton have mutated. Plants with gene mutations are recorded 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).

[0041] After obtaining the positive transgenic cotton, the present invention hybridizes the transgenic cotton as a male parent with a female parent material, and the obtained hybrid offspring is identified as the cotton female parent haploid.

[0042] In the present invention, the hybridization is preferably pollen hybridization. The present invention does not particularly limit the pollen hybridization method; pollen hybridization methods well known in the art may be employed. The maternal material is preferably wild-type cotton HM-1. In the present embodiment, the preparation method of a cotton maternal haploid is specifically described using T586 as the maternal material.

[0043] In the present invention, the male parent is preferably the self-pollinated offspring of the transgenic cotton. The self-pollinated offspring of the transgenic cotton is preferably the T1 generation plant homozygous mutant obtained by self-pollination of the transgenic cotton and then self-pollinated again to obtain the self-pollinated offspring.

[0044] To obtain hybrid offspring, the present invention preferably identifies the hybrid offspring using the following three methods: haploid trait identification, leaf ploidy identification, and molecular marker identification. If all identification results are haploid, the plant is a cotton maternal haploid plant; if any of the above identification methods do not identify the plant as haploid, the plant is not a cotton maternal haploid plant. The plant trait identification preferably observes the plant fertility phenotype. Haploids are characterized by short stature, narrow leaves, compact plant shape, and male sterility; while tetraploids are characterized by tall plants, broad, spreading leaves, and normal fertility. The present invention utilizes a pair of molecular marker primers to identify the hybrid offspring. The preferred pair of molecular marker primers is as follows: LS-F: 5'-TACAAAGCCTACCCCATCGT-3' (SEQ ID NO: 9); LS-R: 5'-TGGAGAGAGGGTGGACTTGT-3' (SEQ ID NO: 10). Using genomic DNA from inbred lines HM-1 and T586 as templates, amplification detection was performed. The PCR product in T586 was 1048 bp, while the amplified band in HM-1 was 915 bp. This could be resolved using agarose gel electrophoresis. The PCR product in T586 was larger and the electrophoresis was slow, while the amplified product in HM-1 was smaller and the electrophoresis was fast. Therefore, the amplified product band detected in T586 was above the band detected in HM-1 ( Figure 5 In the test, the first lane is T586 and the second lane is HM-1. If the plant to be tested has only T586 band ( Figure 5 If the hybrid offspring has both T586 and HM-1 bands ( Figure 5, the 4th lane), then the plant is considered to be the offspring of normal hybridization and is a tetraploid. Flow cytometry is used to identify leaf ploidy. If the nuclear signal peak of the plant to be tested appears near 100, it is considered to be enriched with the tetraploid nuclear signal intensity and in the same position, and the plant to be tested is a tetraploid. If the nuclear signal peak of the plant to be tested appears near 50, then the plant to be tested is considered to be a haploid plant. The embodiments of the present invention prove that after the GhDMP8 gene mutates, not only can self-pollination produce haploids, but also hybridization with other materials can obtain cotton maternal haploids in the offspring, and different planting environments can improve its induction rate.

[0045] The following describes in detail the application of the GhDMP8 gene provided by the present invention as a target in improving the preparation of cotton maternal haploids in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] sgRNA-Cas9 dual expression vector and HM-1: described in the literature: Wang P, Zhang J, Sun L, Ma YZ, Xu J, Liang SJ, Deng JW, Tan JF, Zhang QH, Tu LL, Daniell H, Jin SX, Zhang XL. Highefficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system. Plant biotechnology journal, 2018; 16(1): 137-150.; publicly available from the Cotton Research Institute, Chinese Academy of Agricultural Sciences.

[0047] T586: recorded in the literature: Ni Xiyuan, Wang Xuede, Sun Zhidong. Localization of cotton phenotypic trait genes using SSR markers. Cotton Science. 2003; 15(6): 357-360. The public can obtain it from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0048] Polymorphic marker primers: recorded in the literature: Cai CP, Zhang XY, Niu EL, Zhao L, Li NN, Wang LM, Ding LY, Guo WZ. GhPSY, a phytoene synthase gene, is related to the red plant phenotype in upland cotton (Gossypium hirsutum L.). Molecular biology reports. 2014; 41(8): 4941-4952.; Andres RJ, Coneva V, Frank MH, et al. Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for themajor leaf shapes of upland cotton (Gossypium hirsutum L.). Proceedings of the National Academy of Sciences. 2017; 114(1): e57-e66. The public can obtain them from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0049] Example 1

[0050] Method for inducing cotton maternal haploid using gene GhDMP8

[0051] There are two copies of the GhDMP8 gene in the genomic sequence of wild-type cotton (GhDMP8 represents two genes, GhDMP8-A and GhDMP8-D, since only two homologous genes, GhDMP8-A and GhDMP8-D, exist in upland cotton), as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. The sequence of its first transmembrane region is shown in SEQ ID NO: 3 and SEQ ID NO: 4 from the 5' end amino acids 56 to 78 in the sequence listing, the sequence of the second transmembrane region is shown in SEQ ID NO: 3 and SEQ ID NO: 4 from the 5' end amino acids 88 to 107 in the sequence listing, the sequence of the third transmembrane region is shown in SEQ ID NO: 3 and SEQ ID NO: 4 from the 5' end amino acids 145 to 167 in the sequence listing, and the sequence of the fourth transmembrane region is shown in SEQ ID NO: 3 and SEQ ID NO: 4 from the 5' end amino acids 187 to 206 in the sequence listing.

[0052] 1. CRISPR / Cas9 system knockout of cotton GhDMP8 gene

[0053] Schematic diagram of the GhDMP8 gene structure and CRISPR / Cas9 system knockout target sites is shown in Figure 1 .

[0054] 1. Two common target sites, SEQ ID NO: 5 and SEQ ID NO: 6, were designed on the upstream sequences of the first transmembrane region and the third transmembrane region of two homologous cotton GhDMP8 genes, both with a length of 23 bp.

[0055] Target site SEQ ID NO: 5: 5'-TTTGATGCCAATTCCATGGTGGG-3'.

[0056] Target site SEQ ID NO: 6: 5'-TACGGTTTCGTCACCCCCAACGG-3'.

[0057] 2. Insert the DNA molecules shown in SEQ ID NO:5 and SEQ ID NO:6 into the BsaI site of the sgRNA-Cas9 dual expression vector to obtain a CRISPR / Cas9 knockout vector (sequencing verified).

[0058] 3. Introduce the CRISPR / Cas9 knockout vector prepared in step 2 into competent Agrobacterium cells LB4404 to obtain recombinant bacteria LB4404 / CRISPR / Cas9. The recombinant bacteria LB4404 / CRISPR / Cas9 are then transformed into cotton HM-1 hypocotyls using the Agrobacterium-mediated genetic transformation method (recombinant Agrobacterium is propagated at 28°C and the propagated bacterial solution is used to infect cotton hypocotyls). After selection, screening, differentiation, and rooting, T0-generation transgenic cotton plants are obtained.

[0059] 4. Collect the leaves of the T0 transgenic cotton plants obtained in step 3, and extract genomic DNA as a template. Perform PCR amplification using a primer pair consisting of sequence 7 primer DMP8-JF and sequence 8 primer DMP8-JR to obtain amplified products of different strains.

[0060] DMP8-JF:5'-CACCCCTTAGGCGAGTTTTT-3' (SEQ ID NO:7);

[0061] DMP8-JR: 5'-TCATGTCCTGGGAAAACACA-3' (SEQ ID NO: 8).

[0062] The PCR amplification products of different strains were connected to the pGEM-T-easy vector, and single clones were picked for sequencing. The sequencing results were compared with the corresponding sequences of the target site of the wild-type cotton GhDMP8 gene to identify whether the gene in different strains of T0 generation transgenic cotton had mutated.

[0063] The results are as follows: A gene mutation occurred in one T0 transgenic cotton plant. The specific mutation was as follows: Figure 2 As shown, that is, the difference between the GhDMP8 gene of the mutant plant ghdmp8-1 and the wild-type cotton HM-1 is that the sequence before the first transmembrane region of SEQ ID NO: 1 in the sequence listing has a deletion mutation of 39 bases starting from the 12th base at the 5' end and / or a deletion CCC mutation at the 372nd base of the sequence before the third transmembrane region; the sequence before the first transmembrane region of SEQ ID NO: 2 in the sequence listing has a deletion mutation of 7 bases starting from the 15th base at the 5' end and / or a deletion CCC mutation at the 372nd base of the sequence before the third transmembrane region; the sequence before the first transmembrane region of SEQ ID NO: 2 in the sequence listing has a deletion mutation of 7 bases starting from the 15th base at the 5' end and / or a deletion mutation of 47 bases at the 372nd base of the sequence before the third transmembrane region; NO:2 The sequence before the first transmembrane region undergoes a deletion mutation of 40 bases starting from the 12th base at the 5' end and / or base C is inserted between the 373rd and 374th bases of the sequence before the third transmembrane region; SEQ ID NO:2 in the sequence listing has a deletion mutation of 7 bases starting from the 15th base at the 5' end and / or base C is inserted between the 373rd and 374th bases of the sequence before the third transmembrane region; SEQ ID NO:2 in the sequence listing has a deletion mutation of 3 bases ATG starting from the 17th base at the 5' end and / or base C is deleted between the 373rd and 375th bases of the sequence before the third transmembrane region.

[0064] Plants with gene mutations were recorded as positive T0 generation transgenic cotton.

[0065] 5. Harvest seeds from the positive T0 transgenic cotton obtained in step 4 and then sow and self-pollinate to obtain T1 transgenic cotton. Identify whether the genes of the T1 transgenic cotton have a mutant genotype by the following method: Using genomic DNA from the T1 transgenic cotton as a template, perform PCR amplification using a primer pair consisting of sequence 7 primer DMP8-JF and sequence 8 primer DMP8-JR. Sequence the PCR product, and classify the genotype of the T1 transgenic cotton based on the sequencing results.

[0066] In the sequencing results, (1) the sequence with a double peak characteristic from the target site sequence is a heterozygous genotype, which is a heterozygous gene mutation of the T1 generation transgenic cotton (the gene in one homologous chromosome is mutated, and the gene in the other homologous chromosome is not mutated); (2) the sequence with a specific single peak characteristic from the target site sequence is compared with the target site sequence of the GhDMP8 gene of the wild type cotton HM-1. If they are the same, it is a wild type and no mutation has occurred, and it will not be considered in the following analysis; if there is a mutation, it is a homozygous mutation obtained after self-pollination of the T0 generation plant, which is a homozygous gene mutation of the T1 generation transgenic cotton (both GhDMP8 genes on the homologous chromosomes have mutated).

[0067] After analysis, the T1 generation transgenic cotton homozygous gene mutation strain ghdmp8-1 has the following mutation types: the T1 generation transgenic cotton gene mutation homozygous strain ghdmp8-1 contains the mutant gene ghdmp8 in both At and Dt genomes (the difference from the GhDMP8 gene of the wild-type cotton HM-1 is that the sequence before the first transmembrane region in SEQ ID NO:1 in the sequence listing has a deletion mutation of 39 bases starting from the 12th base at the 5' end and / or a deletion CCC mutation at the 372nd base of the sequence before the third transmembrane region; the sequence before the first transmembrane region in SEQ ID NO:2 in the sequence listing has a deletion mutation of 7 bases starting from the 15th base at the 5' end and / or a deletion CCC mutation at the 372nd base of the sequence before the third transmembrane region; the sequence before the first transmembrane region in SEQ ID NO: NO:2 has a deletion mutation of 7 bases starting from the 15th base at the 5' end of the sequence before the first transmembrane region and / or a deletion mutation of 47 bases starting from the 372nd base at the sequence before the third transmembrane region; SEQ ID NO:2 in the sequence listing has a deletion mutation of 40 bases starting from the 12th base at the 5' end of the sequence before the first transmembrane region and / or a base C is inserted between the 373rd and 374th bases at the sequence before the third transmembrane region; SEQ ID NO:2 in the sequence listing has a deletion mutation of 7 bases starting from the 15th base at the 5' end of the sequence before the first transmembrane region and / or a base C is inserted between the 373rd and 374th bases at the sequence before the third transmembrane region; SEQ ID NO:2 in the sequence listing has a deletion mutation of 3 bases ATG starting from the 17th base at the 5' end of the sequence before the first transmembrane region and / or a base C is deleted between the 373rd and 375th bases at the sequence before the third transmembrane region.).

[0068] II. Identification of the Haploid-Inducing Ability of Mutants Obtained by Knocking Out the Cotton GhDMP8 Gene Using the CRISPR / Cas9 System

[0069] (1) Phenotypic identification

[0070] The T1 generation homozygous gene mutation line ghdmp8-1 was self-pollinated, and the phenotypes of the two pseudohaploid plants that emerged from the hybridization with T586 were continuously observed. HM-1 wild-type cotton (GhDMP8 gene non-mutated) and T586 were used as controls. Haploids have characteristics such as short plants, narrow leaves, compact plant shape, and male sterility; tetraploids have characteristics such as tall plants, wide and spreading leaves, and normal fertility ( Figure 3 and Figure 4 ).

[0071] (2) Flow cytometry identification of leaf ploidy

[0072] A total of two pseudohaploid plants showing haploid banding patterns identified in the above-mentioned 1) self-pollination and hybrid offspring were subjected to flow cytometry testing as follows: the nuclei of young leaves of the test plant were extracted, and tetraploid cotton leaves were used as a control; the signal was then detected using a flow cytometer, first detecting the control tetraploid nuclear signal, and setting the tetraploid nuclear signal peak position to 100 (because the genetic material in tetraploid cells is twice that in haploid cells, the haploid nuclear signal peak position appears near 50). If the signal peak of the test plant appears near 100, it is considered that it is enriched with the tetraploid nuclear signal intensity and has the same position, and the test plant is tetraploid. If the nuclear signal peak of the test plant appears near 50, it is considered that the test plant is a haploid plant.

[0073] The results are as follows Figure 3 and Figure 4 As shown, Figure 3 The middle left picture shows the flow cytometric detection results of ghdmp8-1 cotton, and the right picture shows the flow cytometric detection results of haploid plants in the T1 generation of transgenic cotton self-pollinated progeny. Figure 4 The middle left picture shows the flow cytometric detection results of ghdmp8-1 cotton, and the right picture shows the flow cytometric detection results of pseudohaploid plants in the T1 generation of transgenic cotton hybrid offspring.

[0074] The results showed that two pseudohaploids identified by phenotype in the self-pollinated offspring of ghdmp8-1 and the hybrid offspring with T586 were haploid after flow cytometry detection, and were recorded as T1 generation transgenic cotton homozygous gene mutation line ghdmp8-1 pseudohaploid plants.

[0075] (3) Identification of polymorphic molecular markers

[0076] Based on the red leaf and leaf shape traits of T586, 10 pairs of molecular marker primers were designed, and finally a pair of molecular markers for leaf shape traits were screened, namely sequence 9 primer LS-F and sequence 10 primer LS-R. Using the genomic DNA of the inbred lines HM-1 and T586 as templates, amplification detection was performed. The PCR product in T586 was 1048bp, while the amplified band size in HM-1 was 915bp. They can be distinguished by agarose gel electrophoresis. The PCR product of T586 is larger and the electrophoresis speed is slow, while the amplified product fragment in HM-1 is smaller and the electrophoresis speed is fast. Therefore, the amplified product band detected in T586 is above the band detected in HM-1 ( Figure 5 In the test, the first lane is T586 and the second lane is HM-1. If the plant to be tested has only T586 band ( Figure 5 If the hybrid offspring has both T586 and HM-1 bands ( Figure 5 If the plant is not hybridized, the fourth lane is considered to be a tetraploid plant.

[0077] LS-F:5'-TACAAAGCCTACCCCATCGT-3' (SEQ ID NO:9);

[0078] LS-R: 5'-TGGAGAGAGGGTGGACTTGT-3' (SEQ ID NO: 10).

[0079] Pollen from the T1 generation transgenic cotton homozygous mutant line ghdmp8-1 was transferred to T586 to obtain hybrid progeny. The hybrid progeny obtained above were sown in the experimental field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Anyang, Henan Province. Genomic DNA was extracted from leaves of three-week-old seedlings and agarose banding was performed. The molecular marker identification results are as follows:

[0080] Among the 298 offspring obtained by crossing the T1 generation transgenic cotton homozygous gene mutant line ghdmp8-1 with T586, one individual plant with only the T586 band was obtained and was proposed to be a haploid plant.

[0081] The PCR results of a pseudohaploid plant are as follows: Figure 5 As shown, M, marker, lane 1 is T586, 2 is HM-1, 3 is the hybrid haploid, and 4 is the heterozygous tetraploid banding pattern in the offspring.

[0082] The results showed that a pseudohaploid progeny of the hybrid between ghdmp8-1 and T586 was identified by polymorphic molecular markers and flow cytometry, and the phenotypic identification showed that it was a haploid plant of the maternal parent.

[0083] Therefore, if a single offspring plant of the hybridization between the homozygous transgenic line and T586 is identified as a haploid according to any of the three identification methods mentioned above, then the plant is or is a candidate for being a cotton maternal haploid; if the identification results of none of the three methods mentioned above are haploid, then the plant is not or is not a candidate for being a cotton maternal haploid.

[0084] According to the judgment results, the self-pollination induction rate and the hybridization induction rate are calculated according to Formula I and Formula II respectively.

[0085] Self-pollination induction rate (%) = (number of haploid plants / total number of self-pollination offspring) * 100% Formula I

[0086] Hybridization induction rate (%) = (number of haploid plants / total number of hybrid offspring plants) * 100% Formula II

[0087] According to the above judgment results, the number of haploid plants was 1, the total number of plants tested was 139, and the selfing induction rate was 0.71%. According to the above judgment results, the number of haploid plants was 1, the total number of plants tested was 298, and the hybridization induction rate (the sum of the selfing induction rate and the hybridization induction rate) was 0.34%. It can be seen that after the GhDMP8 gene mutation, not only can self-pollination produce haploids, but hybridization with other materials can also produce cotton maternal haploids in the offspring.

[0088] Example 2

[0089] Identification of the haploid-inducing ability of cotton GhDMP8 gene knockout mutants using the CRISPR / Cas9 system in different planting environments

[0090] Cotton maternal haploids were prepared using the method described in Example 1, except that the planting areas were the experimental fields of Manfa Village, Menghan Town, Jinghong City, Yunnan Province and the experimental fields of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Anyang City, Henan Province, for self-pollination and hybridization.

[0091] The haploid induction rate of the induced line in different environments was identified using the above method. It was found that the haploid induction rate of its offspring after planting in Jinghong City, Yunnan Province was 0.93% to 1.02%, of which the selfing induction rate (%) was: (haploid number of plants / total number of selfing offspring)*100%=(2 / 197)*100%=1.02%; the hybridization induction rate (%) was: (haploid number of plants / total number of hybrid offspring)*100%=(1 / 108)*100%=0.93%. In contrast, the haploid induction rate of cotton maternal plants obtained by the planting group in Henan Province was 0.34% to 0.71% (Example 1).

[0092] The induction rate of haploids in cotton maternal plants grown in Jinghong, Yunnan, was 0.31% to 0.59% higher than that in Anyang, Henan. This suggests that different growth environments also affect the haploid induction rate, which needs to be optimized to achieve a high induction rate.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a reagent for knocking out the GhDMP8 gene in haploid breeding of cotton maternal plants, wherein the reagent for knocking out the GhDMP8 gene comprises a CRISPR / Cas9 gene knockout vector; The CRISPR / Cas9 gene knockout vector contains sgRNA with nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO:

6.

2. A preparation method for improving the haploid induction rate of cotton maternal plants, characterized in that: The following steps are involved: Knock out the GhDMP8 gene in cotton to obtain transgenic cotton; Crossing the transgenic cotton as the male parent with the female parent material, and obtaining the hybrid offspring as the cotton female parent haploid; The reagent for knocking out the GhDMP8 gene includes a CRISPR / Cas9 gene knockout vector; The CRISPR / Cas9 gene knockout vector contains sgRNA with nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO:

6.

3. The preparation method according to claim 2, characterized in that: The male parent is the self-pollinated offspring of the transgenic cotton.

4. The preparation method according to claim 1, characterized in that The cotton includes upland cotton or sea island cotton.

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