CN106701803A - Maize Maternal Haploid Main Inducible Gene and Its Application - Google Patents

Abstract

The invention discloses a major induction gene of a corn female parent haploid and application thereof, and also discloses a major gene for inducing and producing the corn female parent haploid and application thereof in Double Haploid (DH) breeding. The gene codes Phospholipase (PLA), and the nucleotide sequence of the gene is sequence 1. The gene has female parent haploid inducting capacity in the process of selfing or crossing with other corn materials as a male parent after mutation in a coding region. The invention obtains the serial allelic mutation of the gene for the first time, and proves the female parent haploid induction function through self-crossing and hybridization. The obtaining of the maternal haploidy has important theoretical and practical significance for haploid breeding (DH) and related researches thereof.

Description

Maize Maternal Haploid Main Inducible Gene and Its Application

technical field

The invention relates to the field of biotechnology, in particular to a maize maternal haploid main effect induction gene and its application.

Background technique

Corn is the largest crop in the world and has many uses such as food, feed and industrial processing. The increase in corn production is of great significance for supplying the current needs of food, feed and industrial processing. In the current situation where the arable land area is gradually decreasing, it is the key to cultivate high-yielding, multi-resistant and widely suitable maize hybrids. The breeding of maize hybrids depends on the selection of superior inbred lines. The traditional method of breeding inbred lines is time-consuming and laborious, and it usually takes more than 7 generations to breed a stable inbred line. In recent years, haploid breeding technology has the advantages of short breeding cycle, high efficiency, and easy combination with molecular marker-assisted breeding methods, and has gradually become the main technology for breeding maize inbred lines. At present, the haploids in maize are mainly derived from the induction of maize parthenogenetic inducible lines, that is, Stock6 or its derived inducible lines are used as male parents and produced by crossing with other materials. Embryo and endosperm color markers can be used to identify maize haploids since most inducible lines incorporate the R1-nj marker. Therefore, the efficiency of maize haploid breeding is greatly improved.

Since the method of inducing maternal haploids by producing parthenogenesis has broad application prospects and value, many scientific research institutions around the world have studied the genetic basis and biological basis of maternal haploids induced by Stock6 and its derivatives. A lot of research has been done on the basis of science. The results showed that the maize haploid trait induced by parthenogenesis in maize is heritable and controlled by multiple genetic loci. (1999) detected two genetic loci controlling induction rate traits, located on chromosome 1 and chromosome 2, respectively. Able to explain about 17% of the phenotypic variation. Barrant et al. (2008) also detected a genetic locus located on chromosome 1, which verified the results of previous studies. Prigge et al. (2012) used multiple populations to scan the whole genome and found 8 genetic loci controlling the induction rate, including the main genetic locus located at 1.04 bin of chromosome 1, which was named qhir1. Therefore, qhir1 is the QTL with the largest effect and the most important function among multiple QTLs controlling the haplotype induction rate. Dong Xin et al. (2014) fine-tuned the location of qhir1 and successfully narrowed the location range to 243Kb. The study of candidate genes of qhir1 is particularly important for the selection of new induction lines and the genetics and biological mechanism of haploid induced by parthenogenetic induction lines. In view of the widespread use of haploid breeding technology in the breeding industry, the invention It has a very wide application space and market prospect.

Contents of the invention

One object of the present invention is to provide a maize maternal haploid main effect induced gene, ZmPLA mutant gene.

The ZmPLA mutant gene provided by the present invention has a nucleotide sequence obtained by performing insertion or/and deletion or/and substitution mutations on the wild ZmPLA gene nucleotide sequence;

The nucleotide sequence of the wild ZmPLA gene is sequence 1.

Among the above-mentioned genes, the nucleotide sequence of the ZmPLA mutant gene is any one of the following 1)-4) (the following corresponds to the ZmPLA mutant genes ZmHIR1-1, ZmHIR1-2, ZmHIR1-3, ZmHIR1-Stock6 in the embodiments ):

1) A sequence obtained by inserting a T base between the 280th and 281st positions of the wild ZmPLA gene nucleotide sequence, and keeping the other bases unchanged;

2) The 271st-281th base of the wild ZmPLA gene nucleotide sequence is deleted, and the other bases are unchanged, and the obtained sequence is obtained;

3) The 281st base G of the wild ZmPLA gene nucleotide sequence is deleted, and the other bases are unchanged, and the sequence obtained;

4) CGAG is inserted after the 1569th position of the wild ZmPLA gene nucleotide sequence, and the C at the 409th position is mutated to T, the C at the 421st position is mutated to G, the T at the 441st position is mutated to C, and the T at the 887th position Mutation to G, G at position 1210 to C, T at position 1306 to C, G at position 1435 to A, C at position 1471 to A, A at position 1541 to C, The T at position 1588 was mutated to C, and the C at position 1591 was mutated to A to obtain the DNA molecule shown in the sequence. The 1687th base A is mutated to C, the 1691st base G is mutated to A, the 1706th base T is mutated to C, the 1708th base G is mutated to C, and two bases 45-46 are missing Base TA, bases 65-67 are replaced by TCG to CAA, two bases TC are inserted between bases 67-68, bases 80-81 are replaced by TT to CG, and bases 499-503 Base GTAC loss, 524th base C mutation to G, 530th base G mutation to T, 553-560 base GCATGCAT deletion, 806-809th base GTAC deletion, 1741st base G mutation to A, the 1781st base C is mutated to T, the 1787th base A is mutated to T, and the other bases remain unchanged, the obtained sequence.

The application of the above-mentioned mutant gene or the nucleotide sequence of the wild ZmPLA gene in inducing maize or other plant haploids or in double haploid (DH) breeding is also within the protection scope of the present invention.

Silencing or suppressing the expression of the ZmPLA gene in the target plant genome or knocking out the application of the ZmPLA gene in the production of plant haploids is also within the protection scope of the present invention;

Alternatively, the application of silencing or inhibiting the expression of the ZmPLA gene in the target plant genome or knocking out the ZmPLA gene in the production of plant maternal haploids is also within the protection scope of the present invention.

The above application is to silence or suppress or knock out the expression of the ZmPLA gene in the genome of the target plant to obtain a transgenic plant, and then use the transgenic plant for hybridization or selfing to obtain a maternal haploid.

In the above application, the silencing or inhibiting the expression of the ZmPLA gene in the genome of the target plant or knocking out the ZmPLA gene is to reduce the expression of the ZmPLA gene in the genome of the target plant or to cause deletion or insertion mutation;

In the above application, the deletion or insertion mutation of the ZmPLA gene in the target plant genome is the first exon and/or the second exon and/or the third exon and/or the third exon of the ZmPLA gene in the target plant genome. / or deletion or insertion mutation of the fourth exon;

Or the way to cause deletion or insertion mutation of the ZmPLA gene in the target plant genome is CRISPER/Cas9 and/or TELLEN technology and/or T-DNA insertion and/or EMS mutagenesis.

In the above application, the method for causing deletion or insertion mutation of the first exon of the ZmPLA gene in the genome of the target plant is CRISPER/Cas9;

Or the material for silencing or inhibiting the expression of the ZmPLA gene in the target plant genome or knocking out the ZmPLA gene is a material that causes deletion or insertion mutation of the first exon of the ZmPLA gene in the target plant genome;

The substance that causes deletion or insertion mutation of the first exon of the ZmPLA gene in the genome of the target plant is a CRISPER/Cas9 system;

The target sequence of the CRISPER/Cas9 system is base 264-286 in the first exon shown in sequence 3;

The sgRNA sequence of the CRISPER/Cas9 system is sequence 4.

The application of silencing or inhibiting the expression of ZmPLA gene in the target plant genome or knocking out ZmPLA gene in Double Haploid (DH) breeding or hybrid breeding based on DH lines is also within the protection scope of the present invention.

Or silencing or suppressing the expression of the ZmPLA gene in the genome of the target plant or knocking out the ZmPLA gene in the double haploid system (Double Haploid, DH) breeding or the application of the hybrid breeding based on the DH line is also the protection scope of the present invention .

The above-mentioned target plants are corn or other plants.

Another object of the present invention is to provide a material for silencing or inhibiting the expression of the ZmPLA gene in the target plant genome or for knocking out the ZmPLA gene.

The substance provided by the present invention includes the CRISPER/Cas9 system, and the target sequence of the CRISPER/Cas9 system is base 264-286 in the first exon shown in sequence 3.

Among the above substances, the sgRNA sequence of the CRISPER/Cas9 system is sequence 4.

The technical scheme of the present invention is as follows: through candidate gene prediction, a gene encoding phospholipase (PLA) named ZmPLA has been obtained in the qhir1 interval, and the gene has successfully obtained the mutant of the target gene through CRISPER/Cas9 site-directed mutation technology and transgenic tests Materials, use heterozygous genotype mutants and homozygous genotype mutants to cross other maize materials, and verify that the ZmPLA mutant material can induce the function of maternal haploid as the male parent, and the sequence mutation has no function The ZmPLA gene was named ZmHIR1. The artificial site-directed mutation of the gene ZmPLA adopts the CRISPER/Cas9 site-directed mutation technology to modify the first exon of the ZmPLA gene, so that the bases of the first exon are replaced, deleted and/or inserted. When CRISPER/Cas9 is modified, the modified target site is designed to be 20 bp in length, located at bases 264-286 in exon 1 of ZmPLA, and the target site sequence is: GCTGCAGGAGCTGGACGGACCGG.

The ZmPLA artificial site-directed mutant produced by the CRISPER/Cas9 site-directed mutagenesis technology at the target site body is characterized in that the CRISPER/Cas9 gene modification technology causes a 1bpT base between 280-281 base positions at the modified target site Base insertion to obtain the ZmPLA gene mutant, the first exon sequence after the base insertion, the gene after the base insertion is named ZmHIR1-1, the mutant offspring can produce about 1% to 2% of the maize female parent Ploidy

The ZmPLA artificial site-directed mutant produced by the CRISPER/Cas9 site-directed mutagenesis technology at the target site body is characterized in that the CRISPER/Cas9 gene modification technology causes deletion of GAGCTGGACGG between 271-281 base positions at the modified target site , to obtain the ZmPLA gene mutant, the first exon sequence after the deletion of the base, and the gene after the deletion of the base is named ZmHIR1-2, and about 1% to 2% of maize maternal haploids can be produced in the offspring of the mutant

The artificial site-directed mutant of ZmPLA produced by the CRISPER/Cas9 site-directed mutagenesis technology at the target site body is characterized in that the CRISPER/Cas9 gene modification technology causes the deletion of the 281st base G at the modified target site to obtain the ZmPLA gene Mutant, the first exon sequence after the base deletion, the gene after the base deletion is named ZmHIR1-3, and about 1% to 2% of maize maternal haploids can be produced in the offspring of the mutant

The present invention also provides a mutant gene sequence of a known maize maternal haploid induction line Stock6, and named it ZmHIR1-Stock6, and its characteristic ZmHIR1-Stock6 leads to self-crossing or hybridization with other materials as a male parent Haploid appears. The sequence is obtained by the present invention through candidate gene prediction and sequencing, and a transgenic test proves that the loss of function of the gene leads to the generation of maize female parent haploid.

The present invention also provides the application of the artificial site-directed mutant of the gene ZmPLA in maize haploid breeding.

The basic principle of the present invention is as follows: for the candidate gene ZmPLA, the target site sequence is designed on the first exon of the gene, and the first exon of the ZmPLA gene is subjected to mutation screening by the method of CRISPER/Cas9 site-directed mutation , to obtain a transgenic mutant with loss of ZmPLA gene function. After selfing of the successfully mutated individual plants, the T1 generation seeds obtained were planted again, and the pollen of the homozygous mutants and heterozygous mutants of the T1 generation plants was used to cross two maize hybrids Zhengdan 958 and Jingke 968 , to obtain offspring. The offspring of the hybrid was planted in the field, and whether the maternal haploid appeared in it was verified according to the growth of the offspring in the field, molecular markers, and flow cytometry ploidy identification.

The experiment of the present invention proves that the mutation of ZmPLA can lead to the generation of maize maternal haploid, which lays an important foundation for revealing the genetic and biological mechanism of maize maternal haploid generation. At the same time, the mutant single plant obtained by this experiment or this method has the haploid induction ability of the female parent of maize, which is of great importance for breeding new induced lines, further improving the induction rate, and improving the haploid breeding efficiency of maize. meaning.

Description of drawings

Figure 1 is a schematic diagram of the ZmPLA gene structure and the setting of target sites using Crisper/Cas9 technology.

Figure 2 shows the detection and sequencing results of CRISPER-mediated site-directed mutation of ZmPLA gene by PCR and Sanger sequencing.

Figure 3 is a photograph of the haploids that appeared after ZmPLA was crossed with hybrids Zhengdan 958 and Jingke 968.

Figure 4 shows the results of ploidy identification of haploid leaves in the field.

Figure 5 shows the results of field haploid molecular marker identification.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the method for inducing maize maternal haploid

1. Mapping of genes related to maize maternal haploid phenotype

By mapping the induction rate-related QTL in the maize maternal haploid Stock6-derived inducible line, the main QTL-qhir1 controlling haploid induction was obtained, and by functional annotation and candidate gene prediction of the genes in the mapping interval, A candidate gene ZmPLA was finally identified.

2. Obtaining maize maternal haploid induction ability after knocking out the maize ZmPLA gene

1. CRISPER/Cas9 system to knock out ZmPLA gene in maize

1) Selection of sgRNA sequence

Figure 1 is a schematic diagram of gene structure and target sites.

The genome sequence of the maize ZmPLA gene is shown in Sequence Table 1. The sequence of the first exon of the maize ZmPLA gene is shown in Sequence Table 2 (Seq. 2 is at position 91-450 of Sequence 1).

The target site sequence is designed on the first exon sequence of the maize ZmPLA gene, the length is 21bp, and it is located at the 264th-286th base position of the first exon.

The target site sequence is GCTGCAGGAGCTGGACGGACCGG (SEQ ID NO: 3).

The target site design sgRNA sequence is GCUGCAGGAGCUGGACGGACCGG (sequence 4), and the coding DNA molecule of the sgRNA is sequence 3.

2), construction of CRISPER/Cas9 vector

The CRISPER/Cas9 vector is to insert the coding DNA molecule of the sgRNA shown in sequence 3 in the sequence table into the pBUN411 vector (recorded in the following documents: Xing H L, Dong L, Wang Z P, et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants [J]. BMC plant biology, 2014, 14(1): 1.) The vector obtained.

3), the acquisition of genetically modified corn

The CRISPER/Cas9 vector was transformed into Agrobacterium competent cell EHA105 through heat shock transformation to obtain the recombinant strain EHA105/CRISPER/Cas9 vector.

Competent cells of Agrobacterium EHA105 were purchased from Huayueyang Biotechnology Co., Ltd., and the public can obtain them by purchasing

Then, the recombinant bacteria EHA105/CRISPER/Cas9 vector was transformed into maize Xu178 (recorded in the following literature) using the Agrobacterium infection method (recombinant Agrobacterium was propagated at 28°C, and the amplified bacterial solution was used to infect corn immature embryos). : Xiang Yan, Wu Daqiang, Jiang Haiyang, et al. Establishment of mature embryo regeneration system for excellent maize inbred lines[J]. Journal of Laser Biology, 2007,16(5):649-654. (obtained from the improvement center) immature embryos were screened, differentiated and rooted to obtain T0 generation transgenic maize plants.

4), identification of mutated ZmPLA gene transgenic maize

The leaves of T0 transgenic maize plants were collected, and genomic DNA was extracted as a template, and PCR amplification was performed with the following primers to obtain PCR amplification products of different lines.

ZmPLA mutant sequence detection primers:

1240F:CCCUCGACGAGUAUCUAUAGC

1240R: GAAGAUGAUAGGCUGCAGC.

The PCR amplification products of different lines were subjected to Sanger sequencing, and the sequencing results were compared with the first exon (sequence 2) of the wild-type maize ZmPLA gene to identify whether the ZmPLA gene was mutated in different lines of T0 transgenic maize .

The results are as follows: among the 21 T0 generation transgenic maize plants, the ZmPLA gene in 8 plants was mutated, the specific mutation form is as follows, partly as shown in Figure 2:

The ZmPLA mutant gene ZmHIR1-1 is a DNA molecule shown in the sequence obtained by inserting a T base between the 280th and 281st positions of the ZmPLA gene nucleotide sequence 1;

The ZmPLA mutant gene ZmHIR1-2 is a DNA molecule shown in the sequence obtained by deleting 11 bases from the 271st to 281st positions of the ZmPLA gene nucleotide sequence 1.

The ZmPLA mutant gene ZmHIR1-3 is the DNA molecule shown in the sequence obtained by the deletion of the 281st base G in the nucleotide sequence 1 of the ZmPLA gene;

The plants with mutations in the ZmPLA gene were recorded as positive T0 transgenic maize.

5) Genotype identification of transgenic maize with mutation of ZmPLA gene in T1 generation

Harvest the seeds of the positive T0 generation transgenic corn obtained in the above 1, and then sow them to obtain the T1 generation transgenic corn.

To identify whether the ZmPLA gene of the T1 generation transgenic maize is a mutant genotype, the details are as follows: Genomic DNA of the T1 generation transgenic maize is used as a template, and the ZmPLA mutation sequence is used to detect primers: 1240F: CCCTCGACGAGTATCTATAGC and 1240R: GAAGATGATAGGCTGCAGC for amplification, and the PCR product is amplified. Sanger sequencing was used to classify the genotypes of T1 generation transgenic maize according to the sequencing results.

In the sequencing results, the sequence with bimodal characteristics starting from the target site sequence is a heterozygous genotype, and it is a heterozygous ZmPLA gene mutation in T1 generation transgenic maize (ZmPLA gene mutation in one homologous chromosome, homologous ZmPLA gene is not mutated in the other chromosome);

The sequence with specific unimodal characteristics from the target site sequence is compared with the first exon (sequence 2) of the maize ZmPLA gene. If they are the same, they are wild type and no mutation occurs. The following analysis will not consider it; if there is a mutation , then it is the homozygous mutation obtained after selfing of the T0 generation plants, and it is the homozygous mutation of the ZmPLA gene mutation in the T1 generation transgenic maize (the ZmPLA gene is mutated in both homologous chromosomes). The T1 transgenic maize heterozygous ZmPLA gene mutant lines include ZmHIR1-1 and ZmHIR1-2, and the mutation types of each line are as follows:

One of the homologous chromosomes in the ZmHIR1-1 homologous chromosome of the T1 transgenic maize ZmPLA gene mutation heterozygous line contains a ZmPLA mutant gene, which is a T base inserted between the 280th and 281st positions of the ZmPLA gene nucleotide sequence 1 Base, and the DNA molecule shown in the sequence obtained by keeping other bases unchanged, the other contains the wild-type ZmPLA gene;

One of the homologous chromosomes in the T1 transgenic maize ZmPLA gene mutation heterozygous line ZmHIR1-2 contains a ZmPLA mutant gene, and the mutant gene is the deletion of GAGCTGGACGG bases at positions 271-281 of the nucleotide sequence 1 of the ZmPLA gene, The DNA molecule shown in the sequence obtained by keeping other bases unchanged, and the other one contains the wild-type ZmPLA gene;

The two homologous chromosomes of the T1 transgenic maize ZmPLA gene mutation homozygous line ZmHIR1-3 both contain the ZmPLA mutant gene. The DNA molecule shown in the sequence obtained with the base unchanged.

2. Identification of the haploid induction ability of mutants obtained by knocking out the ZmPLA gene in maize by CRISPER/Cas9 system

1) Identification of haploid induction ability of ZmPLA gene mutant single plant of T1 heterozygous genotype transgenic maize

(1) Field phenotyping

The pollen of ZmHIR1-1 and ZmHIR1-2 heterozygous mutant lines of ZmPLA gene in T1 generation transgenic maize were respectively awarded to hybrid Zhengdan 958 (Du Chunxin, Cao Chunjing, Cao Qing, et al. Breeding of maize hybrid Zhengdan 958 and application[J]. Maize Science, 2006,14(6):43-45 or obtained from ORG Seed Industry Co., Ltd.) and hybrid Jingke 968 (hybrid Jingke 968 was purchased from Beijing Tunyu Seed Industry Co., Ltd. Responsible company, the product number is Tunyu Jingke 968, the public can purchase it through Beijing Tunyu Seed Industry Co., Ltd.) to obtain hybrid offspring;

The T1 generation transgenic maize ZmPLA gene heterozygous mutant line ZmHIR1-2 was selfed to obtain selfed offspring.

The offspring obtained above were sown in the field, and the phenotype of the offspring was observed. The haploid had the characteristics of short plant, narrow leaves, overshoot, compact plant type, and male sterility, while the diploid showed tall plants and large leaves. Large, loose, normal fertility.

The offspring of wild-type maize (without ZmPLA gene mutation) and hybrids were used as controls. The number of tests for each strain is shown in Table 1.

The statistical results are shown in Table 1 and Figure 3:

Among the 54 offspring of the ZmPLA heterozygous mutant line ZmHIR1-1 of the T1 generation transgenic maize crossed with the hybrid Zhengdan 958, one plant exhibited haploid traits, and was proposed to be a haploid plant;

Among the 50 offspring of the T1 transgenic maize ZmPLA heterozygous mutant line ZmHIR1-1 crossed with the hybrid Jingke 968, one plant exhibited haploid traits and was proposed to be a haploid plant;

Among the 93 offspring of the ZmHIR1-2 hybrid ZmPLA heterozygous gene mutant line of the T1 generation transgenic maize crossed with the hybrid Zhengdan 958, 2 of the 93 offspring showed haploid traits, and were proposed to be haploid plants;

Among the 57 offspring of the T1 transgenic maize ZmPLA heterozygous mutant line ZmHIR1-2 crossed with the hybrid Jingke 968, 2 of the 57 offspring showed haploid traits, and were proposed to be haploid plants;

Among the 27 offspring of the ZmHIR1-2 self-crossed transgenic maize ZmPLA heterozygous mutant line of the T1 generation, one plant exhibited haploid traits was obtained, which was proposed to be a haploid plant.

(2) Leaf ploidy detected by flow cytometry

The above (1) ZmHIR1-1 and hybrid progeny identified 2 plants showing haploid traits, ZmHIR1-2 and hybrid progeny identified 4 plants showing haploid traits, ZmHIR1 One of the plants identified and obtained from the -2 selfed progeny exhibited haploid traits was tested by flow cytometry, and the method was as follows:

Extract the nucleus of the young leaves of the plant to be tested, and use diploid corn leaves as a contrast; then use a flow cytometer to detect the signal, first detect the diploid nucleus signal, and set the diploid nucleus signal peak position to 100 (due to The genetic material in the diploid cell is double that of the genetic material in the haploid cell, therefore, the signal peak of the haploid cell nucleus appears near 50); if the signal peak of the plant to be tested appears near 100, it is considered to be It is the same as the enrichment position of diploid nucleus signal intensity, and the plant to be tested is diploid. If the nuclear signal peak of the tested plant appears near 50, the tested plant is considered to be a haploid plant.

The number of tests for each strain is shown in Table 1.

The results are shown in Figure 4, the upper figure is the result of flow cytometry detection of wild-type maize, and the lower figure is the result of flow cytometry detection of T1 generation transgenic corn ZmPLA gene mutation heterozygous strain;

The result is as follows:

Two phenotype-identified pseudo-haploids in the hybrid offspring of ZmHIR1-1 were detected by flow cytometry, and their ploidy was all haploid, which were recorded as the ZmPLA heterozygous gene mutation in the T1 transgenic maize Line ZmHIR1-1 is a pseudo-haploid plant.

Four phenotype-identified pseudo-haploids in the hybrid offspring of ZmHIR1-2 were detected by flow cytometry, and their ploidy was all haploid, which were recorded as ZmPLA heterozygous gene mutation in T1 transgenic maize Line ZmHIR1-2 is a pseudo-haploid plant.

One phenotype of the pseudo-haploid identified in the ZmHIR1-2 selfed offspring was detected by flow cytometry, and its ploidy was all haploid, which was recorded as the ZmPLA heterozygous mutant line of T1 transgenic maize ZmHIR1 -2 pseudo-haploid plants.

(3) Molecular marker identification

Randomly design 30 pairs of molecular markers on the genome, and use the transgenic material Xu178 (Xiang Yan, Wu Daqiang, Jiang Haiyang, et al. Establishment of a mature embryo regeneration system for an elite maize inbred line[J]. Acta Laser Biology, 2007, 16(5) :649-654., which the public can obtain from the National Maize Improvement Center of China Agricultural University) and the genomic DNA of the hybrids Zhengdan 958 and Jingke 968 were used as templates for amplification and polymorphic molecular marker screening, and finally a pair of molecular Marker, the PCR product is 500bp in Xu178, and the length of the product in hybrid Zhengdan 958 and hybrid Jingke 968 is 300bp, which has a large difference and can be distinguished by agarose gel electrophoresis. The PCR product of Xu178 is larger, The electrophoresis speed is slow, but the PCR product fragments of the hybrid Zhengdan 958 and the hybrid Jingke 968 are small and the electrophoresis speed is fast. Therefore, the band of Xu178 is located above the bands of the hybrid Zhengdan 958 and the hybrid Jingke 968. (In Figure 5, lanes 3 and 4 are the band patterns of the hybrid Zhengdan 958 and hybrid Jingke 968, and lane 5 is the band pattern of Xu178)

For the 2 pseudo-haploid plants that appeared in the hybrid progeny of the above-mentioned T1 generation heterozygous mutant line ZmHIR1-1 and the hybrid progeny and the T1 heterozygous gene mutant strain ZmHIR1-2 that appeared in the hybrid progeny Genomic DNA extraction, PCR and agarose band detection were performed on the 4 pseudo-haploid plants. If the individual plant to be tested only had the band of Zhengdan 958 (Figure 5, lane 1), it was considered that the individual plant did not have the male parent material The banding pattern is therefore maternal haploid. If there are bands of Xu178 and Zhengdan 958/Jingke 968 in the offspring of the hybrid (Figure 5, lane 2), it is considered that the offspring of the hybrid is a normal hybrid and is diploid.

The results are shown in Figure 5, M: Marker, 5 is the Xu178 band of the male parent, 4 is the Zhengdan 958 band of the female parent, 3 is the Jingke 968 band of the female parent, 1 is the haploid band of the offspring, 2 It is the heterozygous diploid pattern in the offspring.

The molecular marker identification results are as follows:

The molecular marker identification results of the phenotype-identified pseudo-haploids in the offspring of the two ZmHIR1-1 hybrids showed that they were all haploid plants of the female parent.

The molecular marker identification results of the phenotype-identified pseudo-haploids in the offspring of the four ZmHIR1-2 hybrids showed that they were all maternal haploid plants.

Therefore, if the offspring of a heterozygous transgenic line and a hybrid or a single plant of a self-crossed offspring of a heterozygous transgenic line is identified as haploid according to any of the above three identification methods, the plant is considered to be haploid. Or the candidate is maize maternal haploid; if the identification results of the above three methods are not haploid, then the plant is not or the candidate is not maize maternal haploid.

Statistics of the above identification results are shown in Table 1, haploid induction rate (%)=(number of haploids/total number of tested plants)*100, it can be seen that after ZmPLA gene mutation is hybridized with other materials, it can be obtained in offspring maize female parent haploid.

Table 1 The frequency of occurrence of haploid plants in the test offspring of heterozygous mutant lines

Note: The control is the offspring obtained after pollination with wild-type Xu178 material and hybrids Zhengdan 958 and Jingke 968.

2) Identification of haploid induction ability in single plant of T1 homozygous genotype transgenic maize ZmPLA gene mutation

(1) Field phenotyping

The pollen of ZmHIR1-3, a homozygous mutant line of ZmPLA gene in T1 generation transgenic maize, was given to hybrid Zhengdan 958 to obtain hybrid offspring;

The T1 generation transgenic maize ZmPLA gene heterozygous mutant line ZmHIR1-3 was selfed to obtain selfed offspring.

The offspring obtained above were sown in the field, and the phenotype of the offspring was observed. The haploid had the characteristics of short plant, narrow leaves, overshoot, compact plant type, and male sterility, while the diploid showed tall plants and large leaves. Large, loose, normal fertility.

The result is as follows:

Among the 256 offspring of the T1 generation transgenic maize ZmPLA homozygous mutant line ZmHIR1-3 and the hybrid Zhengdan 958, 4 individual plants with haploid traits were obtained, which were proposed to be haploid plants;

Among the 30 selfed offspring of the ZmHIR1-3 transgenic maize ZmPLA homozygous mutant line of the T1 generation, 2 individual plants showing haploid traits were obtained, which were proposed to be haploid plants.

(2) Leaf ploidy detected by flow cytometry

Four pseudo-haploids in the offspring of the T1 generation transgenic maize ZmPLA homozygous mutant line ZmHIR1-3 crossed with the hybrid Zhengdan 958, and two pseudo-haploids in the offspring of ZmHIR1-3 homozygous mutation Individual plants were tested by flow cytometry, the method is as follows:

Extract the nuclei of the young leaves of the plants to be tested, and use wild-type maize (ZmPLA gene unmutated, diploid) leaves as a control; then use flow cytometry to detect signals, first detect diploid nuclei signals, and diploid The nuclear signal peak position is set to 100 (because the genetic material in the diploid cell is twice the genetic material in the haploid cell, therefore, the haploid cell nuclear signal peak appears around 50); if the signal of the plant to be tested If the peak appears near 100, it is considered to be the same as the position where the signal intensity of diploid nuclei is enriched, and the plant to be tested is diploid. If the plants to be tested are small, the detection quantity of each line is shown in Table 2.

The results are shown in Figure 4, the upper figure is the result of flow cytometry detection of wild-type maize, and the lower figure is the result of pseudo-haploid flow cytometry detection of the progeny of the T1 transgenic corn ZmHIR1-3 homozygous line;

The result is as follows:

Four pseudo-haploids in the offspring of ZmHIR1-3 and Zhengdan 958 were detected by flow cytometry, and their ploidy was all haploid.

The ploidy of the 2 pseudo-haploid plants produced by selfing offspring of the ZmHIR1-3 homozygous mutant material were all haploid after flow cytometry detection.

(3) Molecular marker identification

30 pairs of agarose molecular markers were randomly designed on the genome, and the genomic DNA of the transgenic material Xu178 and the hybrids Zhengdan 958 and Jingke 968 were used as templates for amplification and polymorphic molecular marker screening to obtain a pair of molecular markers. The PCR product is 500bp in Xu178, but the product length of the hybrid Zhengdan 958 and the hybrid Jingke 968 is 300bp, which has a large difference, which can be distinguished by agarose gel electrophoresis. The Xu178 PCR product is larger and the electrophoresis speed is higher. However, the PCR product fragments of the hybrid Zhengdan 958 and the hybrid Jingke 968 are smaller and the electrophoresis speed is fast. Therefore, the band of Xu178 is located above the bands of the hybrid Zhengdan 958 and the hybrid Jingke 968. (In Figure 5, lanes 3 and 4 are the band patterns of the hybrid Zhengdan 958 and hybrid Jingke 968, and lane 5 is the band pattern of Xu178)

Genomic DNA extraction, PCR and agarose band detection were performed on the four pseudo-haploid plants of the ZmHIR1-3T1 generation transgenic maize homozygous mutant line selected from the field and Zhengdan 958 hybrid offspring. If there is only the band of Zhengdan 958 (Figure 5, lane 1), it is considered that the single plant does not have the band pattern of the male parent material, so it is a maternal haploid. If there are bands of Xu178 and Zhengdan 958 in the offspring of the hybrid (Figure 5, lane 2), it is considered that the offspring of the hybrid is a normal hybrid and is diploid.

The results are shown in Figure 5, M: Marker, 5 is the Xu178 band type, 4 is the hybrid Zhengdan 958 band type, 3 is the hybrid Jingke 968 band type, 1 is the haploid band type in the offspring, and 2 is the offspring Homozygous diploid band type;

The result is as follows:

The four pseudo-haploids obtained from the hybrid offspring of T1 generation transgenic maize ZmHIR1-3 gene homozygous mutant line and Zhengdan 958 were all maternal haploids after identification by molecular markers.

Therefore, if a homozygous transgenic line and a hybrid offspring individual plant or a homozygous transgenic line self-crossed offspring individual plant is identified as haploid according to any of the above three identification methods, the plant is Or the candidate is maize maternal haploid; if the identification results of the above three methods are not haploid, then the plant is not or the candidate is not maize maternal haploid.

The results are shown in Table 2, induction rate (%)=(number of haploid plants/total number of tested plants)*100, it can be seen that after the ZmPLA gene mutation is crossed with other materials, maize female parent haploid can be obtained in the offspring .

Table 2 The frequency of occurrence of haploid plants in the test offspring of heterozygous mutant lines

3. Genotype identification of maize maternal haploid Stock6

Stock6 is the first reported special material capable of inducing maternal haploids in maize (Coe EH (1959) Aline of maize with high haploid frequency. Am Nat 93:381–382). After fine mapping of the main QTL for induction rate and Candidate gene prediction found that compared with B73, there were multiple SNP mutations and a 4bp insertion in the gene ZmPLA of Stock6 (Table 3), which made the gene lose its normal function. After site-directed mutation of the ZmPLA gene of wild-type maize materials by using Crisper technology, it was proved that after the gene mutation was used as the male parent to pollinate other materials, a certain frequency of haploids could appear in the offspring. The ZmPLA gene in the Stock6 genome is a mutated sequence obtained by mutating the gene ZmPLA shown in Sequence 1 as follows, and is named ZmHIR-Stock6.

Table 3 Exon mutant forms of gene ZmPLA

The 5'UTR region of the gene ZmPLA is mutated as follows: two bases TA are missing at bases 45-46, bases 65-67 are replaced by TCG with CAA, and two bases are inserted between bases 67-68 Base TC, bases 80-81 are replaced by TT to CG

The mutations in the intron region of the gene ZmPLA are: 499-503 base GTAC deletion, 524 base C mutation to G, 530 base G mutation to T, 553-560 base GCATGCAT deletion, 806-809 base The base GTAC is missing.

The mutations in the 3'UTR region of the gene ZmPLA were as follows: the 1741st base G was mutated to A, the 1781st base C was mutated to T, and the 1787th base A was mutated to T.

Compared with the ZmPLA wild-type gene 4 in the above-mentioned inducible line Stock6, the SNP and Insertion mutations occurred, the specific mutation forms are as follows:

The ZmHIR-Stock6 mutation sequence is the insertion of CGAG after the 1569th position of the ZmPLA gene nucleotide sequence 1, and the C at the 409th position is mutated to T, the C at the 421st position is mutated to G, the T at the 441st position is mutated to C, and the 887th position is mutated The T at position is mutated to G, the G at position 1210 is mutated to C, the T at position 1306 is mutated to C, the G at position 1435 is mutated to A, the C at position 1471 is mutated to A, and the A at position 1541 is mutated to C, the T at position 1588 is mutated to C, the C at position 1591 is mutated to A, and the DNA molecule shown in the obtained sequence is obtained. The 1687th base A is mutated to C, the 1691st base G is mutated to A, the 1706th base T is mutated to C, the 1708th base G is mutated to C, and two bases 45-46 are missing Base TA, bases 65-67 are replaced by TCG to CAA, two bases TC are inserted between bases 67-68, bases 80-81 are replaced by TT to CG, and bases 499-503 Base GTAC loss, 524th base C mutation to G, 530th base G mutation to T, 553-560 base GCATGCAT deletion, 806-809th base GTAC deletion, 1741st base G mutation to A, the 1781st base C is mutated to T, and the 1787th base A is mutated to T.

The above-mentioned CGAG insertion after base 1482 resulted in a frameshift mutation in this gene. SNP variants located at bases 319, 331, and 1120 resulted in amino acid changes that also affected protein function.

The haploid traits, leaf ploidy detected by flow cytometry and molecular marker identification in maize maternal haploid Stock6 offspring all proved to have haploid.

Therefore, no matter which mutation of the maize ZmPLA gene leads to loss of function, it can form maize maternal haploid.

sequence listing

<110> China Agricultural University

<120> Maize Maternal Haploid Major Inducible Gene and Its Application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1795

<212>DNA

<213> Artificial sequence

<220>

<223>

<400> 1

agttcatcac taatcacact tattgtgccc tcgacgagta tctatagcta gctcattaat 60

cgattcgggg gtgtgttgtc gaaggcggca atggcgagct actcgtcgcg gcgtccatgc 120

aatacctgta gcacgaaggc gatggccggg agcgtggtcg gcgagcccgt cgtgctgggg 180

cagagggtga cggtgctgac ggtggacggc ggcggcgtcc ggggtctcat cccgggaacc 240

atcctcgcct tcctggaggc caggctgcag gagctggacg gaccggaggc gaggctggcg 300

gactacttcg actacatcgc cggaaccagc accggcggtc tcatcaccgc catgctcacc 360

gcgcccggca aggacaagcg gcctctctac gctgccaagg acatcaacca cttttacatg 420

cagaactgcc cgcgcatctt tcctcagaag tgagtccgat gctgccgcca ttgttcttgc 480

atccatccag catcgtacgt acgtcctcta tacatctgcg gatcatcatg tgcgcatgtt 540

tgtggcatgc atgcatgcat gtgagcagga gcaggcttgc ggccgccatg tccgcgctga 600

ggaagccaaa gtacaacggc aagtgcatgc gcagcctgat taggagcatc ctcggcgaga 660

cgagggtaag cgagacgctg accaacgtca tcatccctgc cttcgacatc aggctgctgc 720

agcctatcat cttctctacc tacgacgtac gtacgtcgtc acgaatgatt catctgtacg 780

tcgtcgcatg cgaatggctg cctacgtacg ccgtgcgcta acatactcag ctctttccta 840

tctgctgcgc caatttgcag gccaagagca cgcctctgaa gaacgctctg ctctcggacg 900

tgtgcattgg cacgtccgcc gcgccgacct acctcccggc gcactacttc cagactgaag 960

acgccaacgg caaggagcgc gaatacaacc tcatcgacgg cggtgtggcg gccaacaacc 1020

cggtaactga ctagctaact ggaaaacgga cgcacagact ccatgtccat ggcggcccac 1080

aaggtcgatg ctaattgttg cttatgtatg tcgcccgatt gcacatgcgt agacgatggt 1140

tgcgatgacg cagatcacca aaaagatgct tgccagcaag gacaaggccg aggagctgta 1200

cccagtgaag ccgtcgaact gccgcaggtt cctggtgctg tccatcggga cggggtcgac 1260

gtccgagcag ggcctctaca cggcgcggca gtgctcccgg tggggtatct gccggtggct 1320

ccgcaacaac ggcatggccc ccatcatcga catcttcatg gcggccagct cggacctggt 1380

ggacatccac gtcgccgcga tgttccagtc gctccacagc gacggcgact acctgcgcat 1440

ccaggacaac tcgctccgtg gcgccgcggc caccgtggac gcggcgacgc cggagaacat 1500

gcggacgctc gtcgggatcggggagcggat gctggcacag agggtgtcca gggtcaacgt 1560

ggagacagggg aggtacgaac cggtgactgg cgaaggaagc aatgccgatg ccctcggtgg 1620

gctcgctagg cagctctccg aggagaggag aacaaggctc gcgcgccgcg tctctgccat 1680

caacccaaga ggctctagat gtgcgtcgta cgatatctaa gacaagtggc tttactgtca 1740

gtcacatgct tgtaaataag tagactttat tttaataaaa cataaaaata tatat 1795

<210> 2

<211> 360

<212>DNA

<213> Artificial sequence

<220>

<223>

<400> 2

atggcgagct actcgtcgcg gcgtccatgc aatacctgta gcacgaaggc gatggccggg 60

agcgtggtcg gcgagcccgt cgtgctgggg cagagggtga cggtgctgac ggtggacggc 120

ggcggcgtcc ggggtctcat cccgggaacc atcctcgcct tcctggaggc caggctgcag 180

gagctggacg gaccggaggc gaggctggcg gactacttcg actacatcgc cggaaccagc 240

accggcggtc tcatcaccgc catgctcacc gcgcccggca aggacaagcg gcctctctac 300

gctgccaagg acatcaacca cttttacatg cagaactgcc cgcgcatctt tcctcagaag 360

<210> 3

<211> 23

<212>DNA

<213> Artificial sequence

<220>

<223>

<400> 3

gctgcaggag ctggacggac cgg 23

<210> 4

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223>

<400> 4

gcugcaggag cuggacggac cgg 23

Claims (10)

1.ZmPLA mutators, its nucleotides sequence is classified as will be inserted or/and lacked on wild ZmPLA gene nucleotide series Lose or/and Substitution, obtain sequence；

The wild ZmPLA gene nucleotide series are sequence 1.

2. ZmPLA mutators according to claim 1, it is characterised in that：

The nucleotides sequence of the ZmPLA mutators is classified as following 1) -4) in any one：

1) to insert T bases between wild ZmPLA gene nucleotide series the 280th and 281, other bases are constant, obtain Sequence；

2) it is that the 271st -281 bit bases of wild ZmPLA gene nucleotide series are lacked, other bases are constant, the sequence for obtaining；

3) for the wild bit base G of ZmPLA gene nucleotide series the 281st is lacked, other bases are constant, the sequence for obtaining；

4) to insert CGAG after wild ZmPLA gene nucleotide series the 1569th, and the C of the 409th sport T, the 421st C sport G, the T of the 441st sports C, and the T of the 887th sports G, and the G of the 1210th sports C, the T of the 1306th C is sported, the G of the 1435th sports A, and the C of the 1471st sports A, and the A of the 1541st sports C, the T of the 1588th C is sported, the C of the 1591st sports A, the DNA molecular shown in sequence for obtaining.1687th bit base A sports C, the 1691 bit base G sport A, and the 1706th bit base T sports C, and the 1708th bit base G sports C, and 45-46 bases position lacks It is that base replaces with CAA by TCG to lose two bases TA, 65-67, and two base TC are inserted between 67-68 bases position, the 80-81 bit bases replace with CG by TT, and 499-503 bit bases GTAC missings, 524 bit base C sport G, and 530 bit base G dash forward It is changed into T, 553-560 bit bases GCATGCAT missings, 806-809 bit bases GTAC missings, the 1741st bit base G sports A, 1781st bit base C sports T, and the 1787th bit base A sports T, and other bases are constant, the sequence for obtaining.

3. mutator or the wild ZmPLA gene nucleotide series described in claim 1 or 2 produces plant list in induction Times body or the application in Doubled haploid line breeding.

4. silence or suppress the expression of ZmPLA genes in purpose Plant Genome or knock out ZmPLA genes in single times of production plant Application in body；

Or, silence or the material of the expression or knockout ZmPLA genes that suppress ZmPLA genes in purpose Plant Genome are planted in production Application in thing female parent monoploid.

5. application according to claim 4, it is characterised in that：

The silence suppresses the expression of ZmPLA genes in purpose Plant Genome or knocks out ZmPLA genes to make purpose plant The reduction of ZmPLA gene expression amounts or generation missing or insertion mutation in genome.

6. application according to claim 5, it is characterised in that：

It is described to make ZmPLA genes in purpose Plant Genome that missing or insertion mutation to occur to make purpose Plant Genome for described ZmPLA genes First Exon and/or Second Exon and/or the 3rd extron and/or the 4th extron generation missing are inserted Enter mutation；

Or described to make ZmPLA genes in purpose Plant Genome that missing or the mode of insertion mutation to occur be CRISPER/Cas9 And/or TELLEN technologies and/or T-DNA insertions and/or EMS mutagenesis.

7. application according to claim 6, it is characterised in that：

It is described to make that ZmPLA genes First Exon in purpose Plant Genome occurs missing or the mode of insertion mutation is CRISPER/Cas9；

Or the expression of ZmPLA genes or the material of knockout ZmPLA genes are to make in the silence or suppression purpose Plant Genome In purpose Plant Genome there is the material of missing or insertion mutation in ZmPLA genes First Exon；

It is described to make that ZmPLA genes First Exon in purpose Plant Genome occurs missing or the material of insertion mutation is CRISPER/Cas9 systems；

The target sequence of the CRISPER/Cas9 systems is 264-276 bit bases in the 1st extron shown in sequence 3；

The sgRNA sequences of the CRISPER/Cas9 systems are sequence 4.

8. silence or suppress purpose Plant Genome in ZmPLA genes expression or knock out ZmPLA genes Doubled haploid line select Educate or breeding hybridized based on DH systems in application；

Or silence or suppress the expression of ZmPLA genes in purpose Plant Genome or knock out the material of ZmPLA genes in double lists times Application in system seed selection or breeding hybridized based on DH systems.

9. silence or suppress purpose Plant Genome in ZmPLA genes expression or knock out ZmPLA genes material, including CRISPER/Cas9 systems, the target sequence of the CRISPER/Cas9 systems is 264- in the 1st extron shown in sequence 3 286 bit bases；The sgRNA sequences of the CRISPER/Cas9 systems are sequence 4.

10. the material according to any described applications of claim 3-8 or claim 9, it is characterised in that：The plant It is corn or other plant.