CN113557955A - Haploid induction line genetic purification method based on reproductive isolation traits - Google Patents
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Abstract
本发明公开了一种基于生殖隔离性状的单倍体诱导系遗传保纯方法。本发明将生殖隔离性状基因应用于诱导系的遗传保纯工作中，形成只接受自身花粉、具有生物学隔离功能的单倍体诱导系材料。该具有生物学隔离功能的单倍体诱导系材料在商业化DH育种应用上，有助于避免外来花粉对诱导系诱导性能的影响，解决诱导系繁育过程中遗传保纯难题，为解决单倍体育种过程中关键诱导系材料的保纯工作提供了一个可行的方案，可大幅度减少诱导系繁育工作量，并能保证诱导系材料纯度；为爆裂玉米及部分甜糯玉米材料的育种工作提供了新思路，促进单倍体育种技术的推广应用。The invention discloses a genetic purity preservation method of a haploid inductive line based on reproductive isolation traits. The invention applies the reproductive isolation trait gene to the genetic purity preservation work of the inductive line, and forms the haploid inductive line material that only accepts self-pollen and has biological isolation function. The haploid inducible line material with biological isolation function can help to avoid the influence of foreign pollen on the induction performance of the inducible line in commercial DH breeding applications, and solve the problem of genetic purity preservation during the breeding process of the inducible line. The purity preservation of key inducible line materials in the process of breeding provides a feasible solution, which can greatly reduce the workload of inducible line breeding and ensure the purity of inducible line materials. New ideas were developed to promote the popularization and application of haploid breeding technology.
Description
Technical Field
The invention belongs to the field of plant genetic breeding, and particularly relates to a haploid induction line genetic purification method based on reproductive isolation traits.
Background
Maize is a typical heterosis and cross-pollination crop, and the core of breeding is to obtain an excellent inbred line. The traditional method adopts hybridization and multi-generation backcross to obtain pure lines, the period is long, and the consumed manpower, financial resources and material resources are large. The haploid breeding technology can obtain a pure line within 1-2 years through the processes of induction, identification, doubling and the like, greatly shortens the breeding period, improves the breeding efficiency, and becomes one of the mainstream technologies of corn breeding. As one of the key materials in the technical process of haploid breeding, the work of purity preservation of an induction line is important. The key inducible gene zmpla1 on maize qhir1 was the first key site found to affect haploid inductivity (Liu et al, 2017; Kelliher et al, 2017; Gilles et al, 2017); another key inducible gene, zmdmp, on qhir8 was shown to achieve a haploid inducer doubling effect based on the presence of zmpla1 (Zhong et al, 2019). The reproductive isolation trait is present in a variety of plants, and this trait is genetically controlled and exhibits cross-incompatibility between different materials. On the corn, the burst corn has the characteristic that the Ga gene for controlling reproductive isolation has one-way hybridization incompatibility, the material with the gene can be used as a male parent to pollinate with other common materials to bear fruits, and the reverse cross is generally weak.
The development and application of the haploid breeding technology can greatly improve the breeding efficiency, and the technical application of the haploid breeding technology depends on the induction of a male parent and a female parent to produce the haploid by an induction line. The haploid inductivity of the induction line depends on the homozygosity of the induction gene, and although the purity preservation in the propagation process can be realized through strict selfing, the genetic purity preservation process in the propagation process can be simplified through the aggregation of the reproductive isolation character and the induction character, the inductivity of the induction line is maintained, and a new means is provided for the efficient utilization of the induction line.
Disclosure of Invention
The invention aims to solve the technical problem of how to keep pure the haploid induction line of the corn or how to carry out pure breeding on the haploid induction line of the corn.
In order to solve the technical problems, the invention firstly provides a method for the pure reproduction of a plant haploid induction line. The method comprises the following steps:
a1, hybridizing an induction line material carrying haploid induction related genes as a female parent and a plant carrying reproductive isolation genes as a male parent to obtain a hybrid of an F1 generation;
a2 planting said F1Generation of hybrid to obtain the F1Generation of hybrid plants, said F1And (3) selfing the generation hybrid plants or backcrossing by taking the induction line material as a recurrent parent, detecting the genotype of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by selfing or backcrossing, and finally obtaining the selfed or backcrossed plants carrying the homozygous genotypes of the haploid induction related gene and the reproductive isolation gene, wherein the plants are plant haploid induction lines obtained by pure breeding.
In the method described above, the plant may be any one of:
1) a dicotyledonous plant;
2) (ii) a monocotyledonous plant which is,
3) a plant of the order of the gramineae,
4) a plant belonging to the family of the Gramineae,
5) a plant of the genus Zea, which plant is selected from the group consisting of Zea mays,
6) corn.
The dicot may include, but is not limited to, oilseed rape, sunflower, peanut, tomato, and the like. The monocot may include, but is not limited to, wheat, rice, and the like.
In the methods described above, the haploid induction related genes can be the maize zmpla1 and zmdmp genes. The reproductive isolation gene can be a maize Ga1-S gene. The haploid induction related gene can also be other haploid induction genes, such as a CENH3 gene, an ig gene or a PLD3 gene. The reproductive isolation gene (cross-incompatible gene) may also be other reproductive isolation genes such as SPRI1, AtLURE1, UI3a, and the like.
In the method described above, the plant haploid inducer line can be the maize haploid inducer line CAU 5. The plant haploid inducer can also be other corn haploid inducer materials. The plant haploid induction line can also be used as induction line material of other plant haploids.
In the methods described above, the plant carrying the reproductive isolation gene can be corn material 401D. The plant material carrying the reproductive isolation gene can also be other maize materials containing the reproductive isolation gene. The plant material carrying the reproductive isolation gene can also be other plant materials carrying the reproductive isolation gene. The reproductive isolation gene can also be other reproductive isolation genes, such as SPRI1, AtLURE1, UI3a, and the like.
In the method described above, the method for detecting the genotypes of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by selfing or backcrossing in A2 can comprise performing PCR amplification detection on a plant to be detected by using primers. The plant containing the segment of the reproductive isolation gene in the PCR amplification product can be a plant carrying the reproductive isolation gene A1.
The primers described above may be the primer pairs described in P1 and/or P2:
p1, and DNA molecules shown in sequence 1 and sequence 2 in the sequence table;
p2, and DNA molecules shown in sequence 3 and sequence 4 in the sequence table.
In the method, the primer can be a primer pair described in P1, and the segment of the reproductive isolation gene can be a DNA molecule shown in a sequence 5 in a sequence table. The primer can also be a primer pair of P2, and the segment of the reproductive isolation gene can be a DNA molecule shown as a sequence 6 in a sequence table.
Any of the following uses of the above described method also fall within the scope of the invention:
d1, application of the method in plant induction line purification breeding;
d2, use of the method described above in a plant haploid production process;
d3, and the application of the method in the popularization of plant haploid breeding technology.
The application of the said reproductive isolation gene in the above-mentioned method in the construction of plant purity-maintaining haploid inducer line also belongs to the protection scope of the present invention.
The application of the said reproductive isolation gene in the above-mentioned method in plant induction line purification breeding also belongs to the protection scope of the present invention. The invention aims to provide a method for carrying out purity preservation work on a special corn haploid induction line by utilizing a reproductive isolation gene.
Experiments prove that: the invention utilizes a high-frequency haploid induction line CAU5 bred by Chinese agricultural breeding as a female parent, uses a cracked corn material 401D with unidirectional reproductive isolation performance as a male parent, and introduces reproductive isolation gametophyte genes in the cracked corn into the haploid induction line by a backcross method on the basis of molecular marker assisted selection to construct a haploid induction line pCAU5 which is incompatible with unidirectional hybridization. Through field verification, the haploid induction line has good fruit when selfing or pollinating other materials (including dent type, hard grain type and partial cracked corn) as a male parent, but has no fruit when pollinating the materials by common corn (without reproductive isolation gene). In addition, ZD958 test shows that the induction rate of the induction system can reach 10%.
The invention applies the reproductive isolation character to the genetic purification work of an induction line to form an induction line material which only receives self pollen and has a biological isolation function. The material is applied to commercialized DH breeding, is beneficial to avoiding the influence of foreign pollen on the induction performance of an induction line, and solves the problem of genetic purity preservation in the breeding process of the induction line.
The method provided by the invention provides a feasible scheme for solving the problem of purity preservation of key inducing line materials in the haploid breeding process, can effectively reduce the probability of field impurity bearing in breeding work of key materials such as inducing lines and the like, can reduce the breeding work task of the inducing lines, and improves the corn breeding efficiency.
Drawings
FIG. 1 is agarose electrophoresis picture of key gene locus for molecular marker detection. A is ZmPLA1 genotype identification, B is ZmDMP genotype identification, and C is reproductive isolation gene identification.
FIG. 2 is agarose electrophoresis picture of key gene locus of new target induction system containing reproductive isolation gene. A is ZmPLA1 genotype identification, B is ZmDMP genotype identification, and C is reproductive isolation gene identification.
FIG. 3 is a diagram of the plant and ear of the new target induction line pCAU 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The haploid inducer CAU5 used in the following examples was provided by chenshaojiang task group, the chinese agricultural university, the average induction rate of which was about 10%, and which had the R1-nj color marker, disclosed in the document "chenshaojiang, liliang, lihaochuan corn haploid breeding technique [ M ]. china agricultural university press 2009", which was publicly available from chinese agricultural university to repeat the present invention.
The popcorn maize line 401D was maintained by chenshaojiang topic group of chinese agricultural university (related documents: muluke, gumingxin, chenshaojiang. "selective hybridization affinity phenomenon of maize cross-incompatibility gene Ga1-s observation". 2012 national maize genetics and breeding institute and new variety display spectacular treatises and abstract collections.ed. {4}, 2012, 47. the public can obtain from the applicant, only used for experiments of repeating the present invention, and can not be used for other purposes), the existence of the reproductive isolation gene locus was ensured according to molecular marker detection, and the field verification shows that the one-way hybridization incompatibility.
Tris saturated phenol, chloroform, alcohol, isopropanol and agarose in the following examples are all products of Beijing GJIPROTENG Biotechnology Co.
The Mix reagents for PCR amplification in the following examples are available from Erdela biotech, Beijing.
Example 1 construction of haploid inducer lines with unidirectional reproductive isolation Properties
Breeding process of haploid inducer line with unidirectional reproductive isolation performance
In order to polymerize the unidirectional reproductive isolation character and the haploid induction character, the reproductive isolation character gene is introduced into the haploid induction line, and finally the corn haploid induction line is kept pure and bred, firstly, CAU5 (molecular marker detects that key induction site gene ZmPLA1 and ZmDMP contained in the haploid induction line are recessive homozygous) is taken as female parent, and cracked corn line 401D (molecular marker detects that reproductive isolation gene contained in the corn line, namely unidirectional hybridization incompatibility gene Ga1-S dominant homozygous) is taken as male parent to carry out hybridization, so that F with heterozygous key induction site and hybridization incompatibility gene site is obtained1Generation hybrid seeds; planting F1Generation of hybrid seed to obtain F1Generation hybrid maize plants, using CAU5 as recurrent parent (female parent), and F1Backcrossing the generation hybrid corn (male parent) for 3 generations, detecting the genotype of the reproductive isolation gene locus in each generation, selecting the corn with the Ga1-S genotype homozygous or heterozygous for backcrossing, observing and recording the field phenotype and the seed character to obtain BC3F1Generation; selection of BC homozygous or heterozygous for the Ga1-S genotype and recessive homozygous for the ZmPLA1 and ZmDMP genes3F1Selfing the single plant for 2 generations, wherein each generation is paired with Ga1-S, ZmPLAnd (3) carrying out molecular marker detection on the genotypes of the A1 and ZmDMP genes, and finally selecting a plant in which the genotype of the Ga1-S locus is dominant homozygous and the genotypes of the ZmPLA1 and the ZmDMP genes are recessive homozygous as a one-way cross incompatible haploid induction line which is named as pCAU 5.
Wherein, the method for detecting the genotypes of the key induction site genes ZmPLA1 and ZmDMP and the reproductive isolation site gene Ga1-S by molecular markers is shown in the fourth step.
Design of molecular marker primer
Detection primers for ZmPLA1 on QTL site qhir1 and ZmDMP induction site on qhir8 of CAU5 induction line are detected by using self-developed primer sequences of Chenshaojiang topic groups of China university of agriculture respectively:
PLA1-F：5'-ACGTGGAGACAGGGAGGTAC-3'；
PLA1-R：5'-GCTTCTGGGGTTGATGGCAG-3'。
pla1-F：5'-ACGTGGAGACAGGGAGCGAG-3'；
pla1-R：5'-GCTTCTGGGGTTGATGGCAG-3'。
chr9-76s1F：5'-CAAAACCACTTCAATCCAGGT-3'；
chr9-76s1R：5'-GTACGTCAGGAGTCCGGAGA-3'。
the sequence of the one-way hybridization incompatibility gene Ga1-S (Zm00001d048936) was downloaded through NCBI website. Autonomously designed primers CG17-F/R are used for carrying out PCR amplification and bidirectional sequencing on DNA of parent materials CAU5 and 401D to obtain gene sequences of the two, and then Primer pairs CG19-F/R and CG13-F/R are designed by using a Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) tool on an NCBI website according to a sequencing result, and are used for detecting Ga1-S genes, wherein the specific sequences are as follows:
CG17-F：5'-ACACGACCATTAGCGAGTCC-3'；
CG17-R：5'-ATCCCGAGAAAGGGTATTGCC-3'。
CG 19-F: 5'-ACACGACCATTAGCGAGTCC-3' (sequence 1 in the sequence table);
CG 19-R: 5'-ACCTTTCGACTGTCTGGACG-3' (SEQ ID NO: 2 in the sequence Listing).
CG 13-F: 5'-ACACGACCATTAGCGAGTCC-3' (SEQ ID NO: 3 in the sequence Listing);
CG 13-R: 5'-CGGCGTATACAACGTCGTAACC-3' (sequence 4 in the sequence table), wherein the CGGC of the 4 basic groups at the CG13-R5' end is supplemented according to the content requirement of the CG of the primer sequence).
Third, DNA extraction
Maize material was sampled from individual leaves at the five-leaf stage and strictly numbered, cryopreserved, and genomic DNA extracted as described by Murray and Thompson (1980).
Fourth, genotype identification
And (3) performing PCR amplification by using the extracted DNA as a template and adopting each F/R primer pair in the second step.
PCR amplification reaction System: DNA 1. mu.L, F/R primers 0.5. mu.L each, Mix 5. mu.L, ddH2O3. mu.L, 10. mu.L of paraffin oil was added.
The PCR amplification reaction conditions are shown in Table 1.
TABLE 1 PCR amplification reaction conditions for different primer pairs
Agarose gel electrophoresis: preparing 2% agarose gel, detecting the PCR amplification product by electrophoresis, and carrying out sequencing verification at 160V for 12 minutes.
PLA 1-F/R: wild type ZmPLA1/ZmPLA1 (i.e. dominant homozygous genotype) and heterozygous genotype ZmPLA1/ZmPLA1 materials have bands (155bp), and recessive homozygous genotype ZmPLA1/ZmPLA1 materials have no bands. See specifically a1 in fig. 1 and a in fig. 2;
pla 1-F/R: bands (141bp) exist in recessive homozygous genotype Zmpla1/Zmpla1 and heterozygous genotype ZmPLA1/Zmpla1 materials, and no band exists in wild type, i.e. dominant homozygous genotype ZmPLA1/ZmPLA1 materials. See specifically a2 in fig. 1 and a in fig. 2;
chr9-76s 1F/R: the wild type, namely the dominant homozygous genotype ZmDMP/ZmDMP material exists in an upper band (491bp), the heterozygous genotype ZmDMP/ZmDMP material shows upper and lower double bands (491bp and 333bp), and the recessive homozygous genotype is the ZmDMP/ZmDMP material only has a lower band (333 bp). See in particular B in fig. 1 and B in fig. 2.
CG 17-F/R: the size of the dominant homozygous genotype Ga1-S/Ga1-S is about 965bp band, and the size of the recessive homozygous genotype Ga1/Ga1 material is about 860bp band. See in particular C3 in fig. 1.
CG 19-F/R: the dominant homozygous genotype Ga1-S/Ga1-S and heterozygous genotype Ga1-S/Ga1 materials have a 747bp band (sequence 5 in a sequence table), and the genotype Ga1/Ga1 materials have no band. See in particular C1 in fig. 1 and C in fig. 2.
CG 13-F/R: recessive homozygous genotype Ga1/Ga1 and heterozygous genotype Ga1-S/Ga1 materials have 741bp bands (sequence 6 in a sequence table), and dominant homozygous genotype Ga1-S/Ga1-S materials have no bands. See in particular C2 in fig. 1 and C in fig. 2.
Example 2 evaluation of the Properties of the one-way incompatible haploid inducer pCAU5
One-way incompatibility verification
In 5 months of 2020, the target inducible line pCAU5 obtained in example 1 and various conventional materials (including commercial hybrid ZD958, ND678 and JK968 and conventional inbred lines C229, Chen04_065 and Chen04_125 bred by Chenshaojiang task group, in which ZD958 is purchased from Beijing Denong species Co., Ltd., ND678, C229, Chen04_065 and Chen04_125 are provided by Chenshaojiang task group of Chenshaojiang university, and JK968 is purchased from Beijing Shuxin agriculture GmbH, and the conventional materials do not contain Ga1-S gene and R1-nj color marker gene) are planted in different plots at Shanzhuang test station of China agricultural university.
A land parcel I: three repeat regions were set, 100 pCAU5 plants and 50 commercial hybrids ZD958, ND678 and JK968 plants were planted in each repeat region, and staggered planting ensured that the flowering phases met. The material carrying the Ga1-S gene is not contained around the plot. Strictly castrating pCAU5 before powder scattering and bagging the female ear; pollen of three hybrids ZD958, ND678 and JK968 (as male parents) is mixed in equal proportion in the flowering period, then the pCAU5 material (as female parents) is pollinated, the fruiting condition of pCAU5 is observed 25 days after pollination, and the fruiting of pCAU5 is 0;
and (2) land parcel II: three repeat regions were provided, each repeat region was seeded with 100 pCAU5, 50 each of the commercial hybrids ZD958, ND678 and JK968, where the hybrids were distributed around pCAU5 and staggered planting ensured that the flowering phases met. The material carrying the Ga1-S gene is not contained around the plot. Strictly castrating pCAU5 before pollen scattering and not bagging female ears, and carrying out open pollination; pCAU5 set was observed about 25 days after pollen dispersal for the hybrids, and pCAU5 set was 0;
and (3) land parcel III: three repetitive areas are provided, 50 plants of 100 pCAU5, conventional inbred lines C229, Chen04-065 and Chen04-125 are planted in each repetitive area, wherein the inbred lines and the pCAU5 are planted at intervals, and staggered planting ensures that the flowering phases meet. No other inducible materials were included around the plot. All materials were open pollinated. Observing the colors of a conventional inbred line and a pCAU5 grain aleurone layer after fruit ears are harvested, wherein pCAU5 is fruited normally, the grain aleurone layer is provided with R1-nj color markers (the R1-nj color marker gene is dominant marker, and the heterozygous genotype or dominant homozygous genotype grain aleurone layer is provided with color), and the aleurone layer is purple; the seeds of the conventional inbred line not only contain the seeds with the color marks of R1-nj, but also have the color marks of R1-nj, namely normal yellow seeds.
And (4) land parcel IV: three replicate regions were set up, 100 pCAU5 plants were planted per replicate region, the pCAU5 was strictly emasculated and the ears were bagged before pollen dispersal, and all materials were strictly selfed. After the ears are harvested, the pCAU5 fructification condition and the color of the aleurone layer of the seeds are observed, all single plants are normally fructified, the aleurone layer of the seeds is marked with the color of R1-nj, and the aleurone layer is purple.
The results show that the haploid induction line pCAU5 carries reproductive isolation genes, is an induction line material with a biological isolation function, can eliminate the influence of exogenous pollen when mixed with conventional materials for open pollination, and realizes the purity preservation of the induction line.
Second, induction rate test
In 5 months of 2020, three repeat regions were set up at Shanzhuang test station of Chinese agricultural university, 100 pCAU5 and 500 ZD958 were planted in each repeat region, and staggered planting ensured that the florescence met. Before pollen scattering, carrying out genotype detection on pCAU5, selecting a material with two key induction sites of zmpla1 and zmdmp genotype as recessive homozygous genotype and reproductive isolation gene Ga1-S genotype as dominant homozygous genotype (the corresponding genotype combination of the three genes of the material is zmpla1/zmpla1+ zmdmp/zmdmp + Ga1-S/Ga1-S) to carry out induction rate test on ZD958 pollination, and pollinating 5 ZD958 strains by each pCAU5, wherein the total number of 500 ZD958 strains are obtained. And (2) identifying the haploid grain by utilizing an R1-nj color marker after the fruit cluster is harvested (when ZD958 is pollinated by utilizing pCAU5, the obtained diploid embryo and aleurone layer have purple color markers, while the haploid aleurone layer has color but the haploid embryo has no color, and the haploid grain can be identified from the diploid grain according to the color difference on the embryo). Fruit ears with less than 100 single ear fruits and containing a plurality of seeds without R1-nj color markers are discarded, and the average haploid inductivity of pCAU5 to ZD958 can reach 10 +/-1.5% by statistics. Wherein, the single haploid inductivity calculation formula is as follows: the induction rate is haploid number/total fructification number.
Third, survey of agronomic characters
In 5 months of 2020, three repeat regions were established at the Shanzhuang test station at the university of agriculture in China, and 100 strains each of pCAU5, CAU5 and 401D were grown in each repeat region. Agronomic traits such as plant height, ear position, flowering phase, pollen dispersing duration, tassel branch, tassel main branch length and the like of pCAU5, CAU5 and 401D were investigated (FIG. 3), and the data are as follows:
TABLE 2 comparison of agronomic traits for novel inducible lines containing the reproductive isolation genes pCAU5, CAU5, 401D (mean. + -. standard deviation)
The result shows that the haploid inducer pCAU5 with reproductive isolation performance obtained by the method also has excellent agronomic characters such as short growth period, low tassel position, high tassel branch number and the like. Therefore, the induction system material has a biological isolation function, has excellent agronomic characters and has a wide application prospect in production.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
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Claims (10)
1. The method for the purity preservation and propagation of the plant haploid induction line is characterized by comprising the following steps: the method comprises the following steps:
a1, hybridizing an induction line material carrying haploid induction related genes as a female parent and a plant carrying reproductive isolation genes as a male parent to obtain a hybrid of an F1 generation;
a2 planting said F1Generation of hybrid to obtain the F1Generation of hybrid plants, said F1Hybrid plant of generationsSelfing the plants or backcrossing the plants by taking the induction line material as recurrent parents, detecting the genotypes of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by selfing or backcrossing, and finally obtaining the selfing or backcrossed plants carrying the genotypes of the haploid induction related gene and the reproductive isolation gene which are homozygous genotypes, wherein the plants are the plant haploid induction lines obtained by the pure breeding.
2. The method of claim 1, wherein: the plant is any one of the following plants:
1) a dicotyledonous plant;
2) (ii) a monocotyledonous plant which is,
3) a plant of the order of the gramineae,
4) a plant belonging to the family of the Gramineae,
5) a plant of the genus Zea, which plant is selected from the group consisting of Zea mays,
6) corn.
3. The method according to claim 1 or 2, characterized in that: the haploid induction related genes are maize zmmpla 1 and zmdmp genes; the reproductive isolation gene is a corn Ga1-S gene.
4. A method according to claim 2 or 3, characterized in that: the plant haploid inducer is a corn haploid inducer CAU 5.
5. The method according to any one of claims 2-4, wherein: the plant carrying the reproductive isolation gene is corn material 401D.
6. The method according to any one of claims 1-5, wherein: the method for detecting the genotypes of the haploid induction related gene and the reproductive isolation gene in each generation of plants obtained by self-crossing or back-crossing in A2 comprises the step of carrying out PCR amplification detection on a plant to be detected by using a primer, wherein a plant containing a segment of the reproductive isolation gene in a PCR amplification product is a plant carrying the reproductive isolation gene A1;
the primer is the primer pair of P1 and/or P2:
p1, and DNA molecules shown in sequence 1 and sequence 2 in the sequence table;
p2, and DNA molecules shown in sequence 3 and sequence 4 in the sequence table.
7. The method of claim 6, wherein: the primer is the primer pair P1, and the segment of the reproductive isolation gene is a DNA molecule shown as a sequence 5 in a sequence table; and/or the primer is the primer pair P2, and the segment of the reproductive isolation gene is a DNA molecule shown as a sequence 6 in a sequence table.
8. Use of the method of any one of claims 1 to 7 for any one of the following:
d1, use of the method of any one of claims 1 to 7 for the maintenance of purity in a plant induction line;
d2, use of the method of any one of claims 1-7 in a plant haploid production process;
d3, the method of any one of claims 1-7, in the popularization of plant haploid breeding technology.
9. Use of the reproductive isolation gene of any one of claims 1-7 in the construction of a plant-sparing haploid inducer line.
10. Use of the reproductive isolation gene in a method according to any one of claims 1 to 7 in the maintenance of purity in a plant induction line.
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