CN115843674A - Breeding method of corn haploid induction line and application thereof - Google Patents

Abstract

The invention belongs to the field of crops, and particularly relates to a breeding method of a corn haploid induction line and application thereof. The method for breeding corn haploid inducer is characterized by that it utilizes selection of haploid inducer in corn resource pool and material whose early young embryo is purple to make hybridization, then makes multiple generation backcross and self-cross to obtain the corn haploid inducer. The method disclosed by the invention can be used for quickly breeding the corn haploid induction line which has good comprehensive properties and high induction rate and is suitable for selecting early haploid embryo.

Description

Breeding method of corn haploid induction line and application thereof

Technical Field

The invention belongs to the field of crops, and particularly relates to a breeding method of a corn haploid induction line and application thereof.

Background

The creation and screening of the inbred line are the core of corn crossbreeding, and the conventional hybridization and multi-round backcross method needs eight generations of six-generation backcross and two-generation inbred to obtain the stable inbred line with the homozygosity higher than 99 percent, and has the advantages of time consumption of years, high cost and long period. In the haploid doubling technology developed in recent years, partial offspring generated by hybridizing pollen of a haploid induction line with high-quality genetic resource materials is induced to be haploid, then cell division inhibitors such as colchicine and the like are used for treating the haploid embryo at a specific stage of sprouting and seedling formation to induce chromosome set doubling and grow into a diploid plant, and selfing, pollination and fructification are performed, so that a Doubled Haploid (DH) inbred line with the homozygosity rate of 100% can be obtained only by two generations, the creation time of the maize inbred line is accelerated to the maximum extent, the cost is greatly reduced, and the method is gradually and widely applied to maize hybrid breeding. If Haploid embryos are picked for chromosome doubling at the stage of immature embryo development, and selfed to set after in vitro Culture into seedlings, then only one half-generation is required to obtain homozygous doubled haploids (Prasanna et al, 2012, double haploided Technology in Maize breeding.

The commonly used maize haploid inducer materials are mostly derived from Stock6 mutants and carry anthocyanin synthesis genes R1-nj, and haploid embryos are identified by virtue of differential expression of anthocyanin in the embryo and endosperm (Dellaporta et al, 1988, in. Because of the gene defect of the induction line, the chromosome from the parent of the induction line is completely lost in the meiosis process of partial zygotes, and the generated offspring seed contains haploid embryos, only carries one set of chromosomes of the parent of the induced material, and does not carry anthocyanin synthesis genes R1-nj, so that anthocyanin can not be expressed and accumulated and is colorless embryos; the endosperm as the triploid carries the R1-nj gene from the induction line, and can express and accumulate anthocyanin to show purple, and the scutellum (namely the endosperm) is purple, and the embryo is colorless. Because the synthesis of the zeaxanthin is complexly regulated by a plurality of genes, the expression conditions of the R1-nj genes in materials with different dent and hard-stalk genetic backgrounds are different, if the expression of the R1-nj gene of an induction line in an embryo or endosperm is easily inhibited by a parent material, the accurate identification of a haploid embryo can be directly influenced, so that the induction line with high induction rate and good anthocyanin expression in the embryo is selected to be crucial to the successful application of the double haploid technology in corn breeding.

Recent studies revealed that the genetic defect of the maize haploid inducer line is the insertion of four bases into the 3' coding sequence of a phosphoesterase gene ZmPLA specifically expressed by pollen grains, resulting in frame shift mutation and premature termination, with reduced phosphatase function after mutation (Gilles et al, 2017, embo 36, liu et al, 2017, mol Plant 10 520-522), resulting in chromosomal loss of the inducer line during meiosis, yielding partial haploid embryos. According to the principle, the gene editing technology can be used for knocking and mutating the phosphatase gene, so that corn haploid induction lines with different genetic backgrounds or haploid induction lines of other crops such as rice and the like can be created, and the haploid doubling technology is popularized and applied in breeding of other crops (Yao et al, 2018, nat Plant 4.

Since haploids are caused by the gradual loss of sperm chromosomes from the inducible line that has entered the zygote after fertilization of the ovum, and the paternal genes are expressed and function for some time, the gene editing vector carried by the paternal of the inducible line can be expressed before the loss, and the target gene of the maternal line is edited, and subjected to chromosome doubling treatment, to finally produce a gene-edited double haploid system without any inducible line genetic material (Kelliher et al, 2019, nat Biotechnol 37, 287-292 wang et al, 2019, mol Plant 12 597-602. Since the outbreeding of wheat pollinated with maize pollen can induce wheat to produce haploids, transgenic maize pollen can be used to induce wheat haploids and site-directed gene mutation editing based on the same principle (Budhagatallli et al, 2020, plant Biotechnol J18.

The gene editing technology based on the CRISPR of the bacterially acquired immune system utilizes SpCas9 endonuclease and guide gRNA to form a nucleoprotease complex, and recognizes the target by base pairing of 20bp long RNA sequence in gRNA and edited DNA sequence, and has high specificity, strong enzyme cutting activity, simple and easy experimental design, so that the CRISPR/Cas9 gene editing system is rapidly and successfully applied in different animal and Plant systems (Chen et al, 2019, annu Rev Plant biol70 667-697. However, the application of gene editing in many important crops which are difficult to genetically transform, such as corn and soybean, is limited, and the establishment of a general efficient genetic transformation system for the crops is a necessary prerequisite for the application of gene editing. Currently, efficient transformation of maize is limited to a few experimental materials such as HiII or inbred lines B104 etc (Frame et al, 2006, plant Cell Rep 25, 1024-1034. Because the genetic traits of the experimental inbred lines are poor, the experimental inbred lines cannot be directly used for modern breeding, transformed offspring of the experimental inbred lines can be used for breeding after being introduced into inbred lines with excellent traits through hybridization and multi-generation backcross transformation, and the breeding application of gene editing is severely restricted by the process. Moreover, the corn crossbreeding is a process of continuously creating and screening excellent inbred lines, and even if a genotype-independent corn transformation technology is successfully developed, a transformation system needs to be established and completed aiming at each newly created excellent inbred line in a time-consuming and labor-consuming manner, so that the gene editing and breeding are difficult to realize effectively in time.

The haploid induction system is transformed by the gene editing vector, any corn material is induced to generate the haploid by the descendant of the transgenic induction system containing the gene editing vector, part of the target gene of the haploid is possibly edited in the process, an edited double-haploid system is formed after the chromosome is doubled, the bottleneck problem that different corn self-bred lines are difficult to transform is thoroughly solved, the gene editing of any corn material can be realized only by establishing a genetic transformation system using the induction system as a receptor, and the obtained descendant is a homozygous edited double-haploid system which can be directly used for breeding and screening. If the used material is a mature excellent inbred line, the edited double haploid system is an improved inbred line; if the material is hybrid material, the edited double haploid system is newly created selfing line.

Although the currently used haploid induction system has good induction rate and large powder scattering amount, the development of the immature embryos is late, the immature embryos with the length less than 3 mm are difficult to develop, the development is obvious after 24 hours of in vitro illumination, and the haploid induction system is not suitable for early identification and selection of the haploid immature embryos; the color development of the endosperm of the mature grains is lighter, the area is smaller, and the selection of the haploid grains is not facilitated.

Disclosure of Invention

Anthocyanin biosynthesis in corn is regulated by complex genes, and a plurality of resource materials can synthesize and accumulate enough anthocyanin in early young embryos for more than ten days after pollination to show large-area purple. The method introduces excellent characters such as early rapid color development and the like into a haploid inducing line through hybridization, and combines the molecular marker screening with the multi-generation backcross breeding, so that the breeding of the inducing line with early embryo color development, synchronous flowering phase, large powder scattering amount, high pollen activity, good inductivity and excellent comprehensive characters can improve the haploid embryo identification accuracy, improve the preparation efficiency of a double haploid system and reduce the corn hybridization breeding cost, and can also be used as a universal genetic transformation receptor material to combine a double haploid technology to carry out the corn gene editing breeding which is not limited by the genotype.

The invention mainly aims to breed a new corn haploid induction line. Hybridizing a corn haploid induction line and a resource material with an early young embryo being purple as parents, sowing the obtained F1 generation seeds with the induction line at proper time, stripping the young embryo after backcross and pollination for 13 days, illuminating, selecting the young embryo with early and obvious color development, culturing into seedlings, culturing in a greenhouse, carrying out molecular marker analysis during the period, screening and retaining ZmPLA genes carrying 4bp insertion homozygous mutation, carrying out 5 single plants with high genetic background recovery rate, selecting the single plants with good comprehensive properties when the seedlings are cultured to be mature and loose powder, then backcrossing with the induction line, repeating the process, and carrying out self-copulation and solid ear harvesting after 5-6 times of backcrossing. And (3) sowing in the field, selecting a line with orderly offspring and good comprehensive characters to test the single-time induction rate, selfing, reserving seeds, and finally breeding an improved induction line for later use according to the induction rate, the development conditions of embryo and endosperm and other comprehensive character expressions. The main experimental steps comprise: 1) Hybridizing, backcrossing and selfing for breeding; 2) Culturing and screening immature embryo tissues and forming seedlings; 3) Screening ZmPLA induced mutation molecular markers; 4) Screening the genetic background recovery molecular marker of the induction line; 5) Testing the haploid inductivity; 6) Chromosome doubling by haploid tissue culture method.

The bred haploid inducing line can be directly used for preparing doubled haploid, and can also be used as a transformation receptor material for corn genetic transformation research and development, the haploid inducing line carrying a gene editing carrier tool after transformation can also induce haploid, the gene editing carrier is introduced into egg cells through the natural insemination process, the editing and haploid induction of target genes are synchronously realized, and a doubled haploid system of gene editing is directly obtained after chromosome doubling. Since haploid induction is essentially genotype independent, inbred line production and synchronized gene editing of any genetic background maize material can be achieved by the double haploid generation process described above, provided that a haploid inducer line is transformed with the gene editing vector.

Specifically, the technical scheme of the invention is as follows:

the first aspect of the invention discloses a method for breeding a corn haploid inducer, which comprises the steps of selecting a haploid inducer in a corn resource pool to hybridize with a material developing early of a immature embryo, then backcrossing for multiple generations, and selfing to obtain the corn haploid inducer.

Preferably, the method comprises the following steps:

s1: hybridizing, backcrossing and selfing for breeding;

s2: culturing and screening immature embryo tissues and forming seedlings;

s3: and testing the haploid induction rate to obtain the corn haploid induction line.

It should be understood that the present invention is not limited to the above steps, and may also include other steps, such as before step S1, between steps S1 and S2, between steps S2 and S3, and after step S3, and other additional steps, without departing from the scope of the present invention.

As used herein, "material that develops color early in immature embryo" refers to a maize variety that can express anthocyanin biosynthesis genes in large amounts early in immature embryo development. Maize contains anthocyanin biosynthesis genes, which are regulated by various factors to have different levels of expression in different tissues. Some corn varieties can express a large amount of anthocyanin biosynthesis genes in early development stage of the young embryo, and the generated anthocyanin enables the young embryo to be purple after the young embryo is exposed to light; some corn varieties can not express anthocyanin biosynthesis genes in the development period of the immature embryos, the immature embryos are milk white, and the milk white can be kept even after illumination. Accordingly, it is possible to distinguish a diploid embryo containing an anthocyanin biosynthesis gene from a haploid embryo lacking the anthocyanin biosynthesis gene.

More preferably, in S1, a corn haploid induction line KY8556 is selected to be hybridized with purple immature embryo resource material S8360 to obtain F1 hybrid seeds.

It should be understood that in S1, the haploid inducer line is not limited to KY8556, the purple immature embryo resource material is not limited to S8360, and those skilled in the art can select any suitable haploid inducer line and purple immature embryo resource material as required to implement the technical solution of the present invention, and all of them are within the protection scope of the present invention.

In some preferred embodiments of the present invention, KY8556 is backcrossed with F1, and the single plant with better characteristics is selected for the next round of backcross until 5 or 6 rounds of backcross are completed, and then 1 to 3 rounds of selfing are carried out to obtain stable candidate induction lines.

Preferably, in S3, the candidate induction line is used for hybridizing with other corn materials to seed, tissue culture identification and/or haploid grain identification of haploid embryos are carried out, and the candidate induction line with high haploid incidence becomes a new corn haploid induction line.

Specifically, any maize breeding intermediate hybrid material expected to be used for preparing an inbred line, an old inbred line, a hybrid and the like can be used as a female parent, a haploid inducer line is used as male parent pollen for pollination, due to the genetic defect of the haploid inducer line, about 10% of the embryos of seeds in offspring generated by the hybridization of the two lose the whole set of chromosomes from the male parent, only one set of chromosomes (1 n) from the female parent is reserved, and the embryos become haploid embryos. The chromosome can be restored into a homozygous 2n double haploid state after doubling, so that an inbred line can be quickly obtained.

Preferably, the single plant with better properties is screened for the next round of backcross by adopting a method of inducing mutation molecular markers and/or recovering molecular markers from genetic background by using gene ZmPLA.

Preferably, in S2, selecting young embryos with early color development, dark color and large area, transferring the young embryos to a culture bottle containing a growth culture medium, germinating to form seedlings, transplanting the seedlings into a large flowerpot for culture when the seedlings grow to 6-7 cm, and applying a proper amount of fertilizer until the young embryos bloom and fruit.

In some embodiments of the present invention, the method for breeding the corn haploid inducer line comprises:

1. conventional breeding of early induction system materials: the corn haploid induction line Stock6 is discovered at the end of the 50 th century earlier, so that the corn haploid breeding is possible. Stock6 has serious defects in the aspects of inductivity, reproductive performance and the like, and male flowers have poor temperature sensitivity, poor pollen dispersing property, poor self-fruiting property and the like. In order to improve the unfavorable traits and adapt to different ecological environments, different breeders make a great deal of effort, and the genes are widely applied through various genetic improvements, have obvious purple marker traits, namely anthocyanin synthesis genes R1-nj, rely on anthocyanin to differentially express and identify haploid embryos in embryos and endosperms, and have been introduced from the United states before (Dazhuo et al, breeding of a maize high-frequency haploid reproductive induction line Jigao induction line No. 3, maize science 2007 (15): 1-4). In order to breed a corn haploid induction line suitable for the Harbin region, the characteristics of Stock6 materials are improved by using precocity and disease-resistant materials in the region through a conventional pedigree breeding method.

(1) And (3) breeding of a haploid induction line: disease-resistant early-maturing variety Longnam No. 4 of the self-breeding of the Zaofeng breed is taken as a female parent (Black cultivation Auyu 2009004; xue Jisheng and the like, the key points of breeding and cultivation of the Longnam No. 4 of a new corn variety, heilongjiang agricultural science 2010 (6): 168), a Stock6 material is taken as a male parent to obtain an F1 generation by hybridization, and then the F1 generation is taken as the female parent, and the Stock6 material is taken as the male parent to obtain a backcrossed BC1F generation, and then 6 generations of selfing and pedigree breeding are carried out. Selecting 3-5 single plants in each generation, sowing seeds of each single plant in a single row with the length of 5 meters and 20 holes, sowing Longnan No. 4 female parents and Stock6 material male parents at the same period as a control, observing and comparing the conditions of field natural infection of large leaf spot and head smut of different single plants and comparing the conditions with the control plants, and selecting disease-resistant single plants; observing and comparing the powder scattering and silking time of different single plants and comparing with a control plant, and selecting an early-maturing single plant; observing and comparing the grain colors of different single plants and comparing with a control plant, and selecting an embryo purple single plant; finally, each generation selects 3-5 single plants with good performance of each character, and the next generation selects 3-5 single plants with good disease resistance, prematurity and embryo purple according to the same process and method to reserve, and eliminates strains with non-ideal performance of characters. The haploid inductivity of the selected single plant is tested from each generation after BC1F3 generation, namely, pollen of the selected single plant of the inducting line is hybridized with a female parent test variety, after seeds are harvested, the haploid is judged according to purple performance of endosperm and embryo in the seeds, the seeds with purple endosperm and embryo are hybrid diploids, the seeds with purple endosperm and no purple embryo are haploid from the female parent, the highest single plant with the haploid inductivity of more than 6 percent is preferably selected, and the haploid inductivity of the single plant line KY8556 is tested for subsequent haploid induction.

(2) Breeding purple grain resource materials: in order to enhance and induce the character of single-plant-line KY8556 purple grains, a disease-resistant, early-maturing and deep-purple grain waxy corn hybrid Jinnuo No. 10 is taken as a material (Jinju jade 2014020; dong Ligong and the like, the breeding and utilization of a new black waxy corn variety Jinnuo No. 10, agricultural science and technology communication 2014 (12): 155-157), selfing is carried out for 4 generations, 3-5 single plants of the disease-resistant, early-maturing and deep-purple grains are selected for each generation, each single plant is single-row with the length of 5 meters and 20 holes, the Jinnuo No. 10 is sown at the same period as a control, the natural field infection conditions of different single plants are observed and compared with the control plants, and the disease-resistant single plants are selected; observing and comparing the powder scattering and spinning time of different single plants, comparing with a control plant, and selecting an early-maturing single plant with large powder scattering amount; observing and comparing the grain colors of different single plants and comparing the grain colors with the control plants, and selecting the single plants with deep purple grains; finally, each generation selects 3-5 individual plants with good performance of various properties, the next generation selects 3-5 individual plants with disease resistance, precocity and dark purple grains according to the same process and method to reserve, eliminates strains with unsatisfactory performance of properties, and initially selects a single strain S8360 as a purple grain resource material for further improving and inducing the single strain KY8556.

2. And (3) rapid improvement and breeding of induction system materials: the haploid induction line strain KY8556 has the defects of late immature embryo development, small and weak purple range of grains, asynchronism between male and female flowering phases and the like, and needs to be further improved. A biotechnology research and development center is newly established in the breed reclamation industry, all-weather greenhouse cultivation, immature embryo tissue culture regeneration, genetic background molecular marker screening, target gene molecular marker screening and other biotechnology means are utilized, purple immature embryo phenotype screening and haploid inductivity measurement are combined, 4 generations of hybridization or backcross can be carried out every year, 8 generations of breeding can be completed within more than two years, and further improvement of a haploid induction line can be rapidly completed.

(1) Screening hybrid single plants: selecting a haploid induction line strain KY8556 and a purple kernel strain S8360 bred by the conventional breeding method, sowing in the field at proper time, selecting 5 single plants with normal development, bagging female and male ears before silk drawing and ear drawing, performing artificial pollination and hybridization, and harvesting ears after normal fruiting and hybridization to obtain F1 hybrid seeds.

(2) Backcrossing for multiple generations: sowing an induction line KY8556 and the F1 hybrid seeds in a greenhouse, selecting 10 single plants with normal development, bagging female and male ears before silking and heading, and manually carrying out KY8556 XF 1 backcross pollination. Harvesting young ears after about 13 days, after surface disinfection and sterilization, stripping young embryos in an aseptic operation workbench, developing color by illumination, selecting the young embryos with deep color development, large area and fast color development, carrying out tissue culture to form seedlings, selecting about 50 robust seedlings, and transplanting the robust seedlings as BC1F1 plants to a greenhouse for cultivation.

Timely sowing an induction line KY8556 in a greenhouse to ensure the flower-season meeting with the BC1F1 plants, bagging male and female ears before silk and ear sprouting, and manually carrying out KY8556 xBC 1F1 backcross pollination. And (3) collecting young ears after about 13 days, repeating the young ear tissue culture screening and seedling formation, if necessary, carrying out target gene ZmPLA mutation marker screening and molecular marker screening for induction system KY8556 genetic background recovery, and selecting 5 single plants with the highest recovery rate and better comprehensive phenotypic characters for next round of backcross until 5 or 6 rounds of backcross are completed to obtain BC5F1 and BC6F1 plants.

(3) Selfing: and (3) cultivating BC5F1 and BC6F1 plants which are subjected to 5-round and 6-round backcross breeding in a greenhouse, bagging female and male ears before silking and heading, manually performing selfing and pollination on 5 plants, continuously cultivating until the ears are naturally mature, and harvesting BC5F2 and BC6F2 seeds from single ears. And (2) sowing BC5F2 and BC6F2 seeds in the field in a corn growth season, reserving about 20 plants in each ear, performing phenotype evaluation at each growth stage, selecting plants with regular and consistent progeny, bagging female and male ears before ear emergence and silk emergence, performing artificial selfing and pollination on 5 plants, continuously cultivating until the ears are naturally mature, harvesting BC5F3 and BC6F3 seeds from a single ear, sowing the seeds as a candidate induction system and an induced material in a proper period, and testing the haploid induction effect by hybridization.

3. And (3) young embryo tissue culture screening and seedling establishment: the young maize embryos about 13 days after pollination can be directly grown into seedlings through tissue culture, the young maize embryos are cultured and developed in a greenhouse and normally bloom, loose powder and fruit, a long seed maturation stage is not needed, the time from the seedlings to the next generation seedlings is not more than 3 months, and the breeding of 4 generations can be carried out every year.

(1) And (3) young embryo tissue culture screening: taking young ears of which the young embryos are about 3 mm long about 13 days after pollination, disinfecting and sterilizing the ears by using 75% alcohol, placing the young ears in a culture dish with the diameter of 150 mm, picking the young embryos by using a scalpel, upwards placing a shield piece on an induction culture medium, placing the culture dish in a culture room with the temperature of 26-28 ℃, and continuously illuminating for 24 hours to enable the young embryos to develop color by light.

(2) And (3) selecting young embryo seedlings: selecting about 50 young embryos with early color development, dark color and large area, transferring the young embryos to a glass culture bottle containing a growth culture medium, and germinating to form seedlings. When the seedlings grow to about 6-7 cm, transplanting the seedlings into a larger flowerpot for culturing, and applying a proper amount of fertilizer until the seedlings bloom and fruit. During this period, leaves from these T0 plants can be sampled and DNA extracted for genotyping.

4. Screening of target gene ZmPLA mutant molecular markers: the corn haploid induction line is caused by insertion mutation of phosphatase gene ZmPLA, and homozygous single plants of the induction line can be quickly and accurately screened by using ZmPLA mutation specific molecular markers.

(1) ZmPLA gene sequencing of haploid induction line KY 8556: functional genes of maize haploid inducer lines have been identified as calcium ion independent phosphatase GRMZM2G471240 gene ZmPLA (Ca 2+ -independent phospholipase A2), with an insertion frame shift mutation of 4 base CGAG at the 3' end of its coding sequence leading to its reduced function, resulting in chromosomal loss of the inducer line during meiosis (Gilles et al, 2017, embo 36 707-717 liu et al, 2017, mol Plant 10. According to the sequenced ZmPLA gene GRMZM2G471240 sequence (https:// phytozome.jgi.doe.gov/pz/port.html) of inbred line B73, a plurality of pairs of PCR primers are designed, KY8556 genomic DNA is used as a template, the ZmPLA gene sequence of KY8556 induction line is amplified in a segmented mode and sequenced, and whether the 3' end of the coding sequence contains the same CGAG insertion frame shift mutation with 4 bases or not is detected.

(2) Mutation ZmPLA gene specific PCR markers: the ZmPLA-KY gene of the induction line KY8556 has 4 base CGAG insertion mutations at the 3' end of the coding sequence, and has a plurality of point mutations at the downstream which are different from the B73 wild ZmPLA gene sequence, the primers with the ZmPLA-KY sequence specificity are designed according to the differences, the genomic DNA of KY8556 and S8360 is respectively used as a template for PCR amplification, only the KY8556 template can successfully amplify a specific fragment with the expected length, and S8360 does not have any PCR product, so the ZmPLA-KY gene specificity PCR marker can be used for identifying and screening induction line plants.

(3) Quantitative qPCR detection of the copy number of the inducible mutant gene ZmPLA-KY: the induction line KY8556 as a diploid inbred line contains 2 copies of ZmPLA-KY mutant genes, S8360 contains 2 copies of ZmPLA wild type genes, the hybrid F1 of KY8556 and S8360 contains one copy of ZmPLA-KY and one copy of ZmPLA respectively, 50% of backcross progeny BC1F1 of F1 and KY8556 contains 2 copies of ZmPLA-KY, and the other 50% of plants contain one copy of ZmPLA-KY and one copy of ZmPLA respectively. The fluorescent quantitative PCR can estimate the copy number of ZmPLA-KY contained in the BC1F1 plant, the plant containing 2 copies of ZmPLA-KY is selected for continuous backcross with KY8556, the subsequent backcross progeny are not separated, and all the 2 copies of ZmPLA-KY mutant genes are stably contained.

5. Genetic background recovery molecular marker screening: because KY8556 and S8360 strains have different sources and have genetic background difference, parents and backcross progeny can be analyzed by a molecular marker genotyping method, and progeny single strains with high genetic background recovery rate are rapidly screened.

(1) Induction line KY8556 genetic background screen for BC1F 1: in order to improve the recovery screening efficiency of KY8556 and S8360 filial generations, parent and multi-generation backcross progeny are analyzed by a KASP (Kompetitive Allelele Specific PCR) genotyping method (Semagn et al, 2013, mol Breeding 33, 1-14), and 5 plants with the highest KY8556 genotype recovery rate are selected for each generation to carry out next generation backcross. A total of 384 SNP (Single Nucleotide Polymorphism) markers were used to analyze KY8556 and S8360 parents, and effective SNP markers with Polymorphism between the two were identified. The markers are used for analyzing BC1F1 transplanted plants which have good early embryo color development and are tissue-cultured into seedlings, statistical analysis is carried out to select plants which have the highest background recovery rate and are homozygous for ZmPLA-KY mutant genes, and then the plants and KY8556 are subjected to next generation backcross.

(2) Induction line KY8556 genetic background screen for BC2F 1: after backcrossing BC1F1 and KY8556, selecting early-stage immature embryos by tissue culture, developing color, transplanting the tissue culture seedling plants to survive, sampling leaves, extracting genome DNA, and analyzing the single plant recovery rate by using the markers still showing polymorphism. And (4) selecting the single plant with the highest background recovery rate through statistical analysis, and then carrying out next generation backcross with KY8556.

(3) Induction line KY8556 genetic background screen for BC3F 1: after backcrossing BC2F1 and KY8556, selecting early-stage immature embryos by tissue culture, developing color, transplanting the tissue culture seedling plants to survive, sampling leaves, extracting genome DNA, and analyzing the single plant recovery rate by using the markers still showing polymorphism. And (4) selecting the single plant with completely recovered background through statistical analysis, and then carrying out next generation backcross with KY8556.

6. And (3) testing the haploid inductivity: the single plant of the induction line primarily bred needs to be subjected to haploid induction rate test, and finally, single plant mooring species with good comprehensive properties and high induction rate are selected for later use.

(1) Haploid induction: KY8556 induction line is used as a control, the same corn material is used for testing the haploid induction effect of the bred improved induction line, and because the induction rates of different materials are generally different, multiple materials are used for testing the same induction line simultaneously. And properly adjusting staggered-period sowing according to the growth period difference of different induced corn materials and induction lines to ensure that the flowering periods of the male parent and the female parent meet each other. The plants are bagged before heading and spinning, and the induced materials are artificially pollinated by the inducing line pollen.

(2) Tissue culture identification of haploid embryos: and (3) after pollination for about 13 days, taking clusters for sterilization and disinfection, picking young embryos with the length of about 3 mm, placing the young embryos on a culture dish containing an induction culture medium with the scutellum facing upwards, after 24 hours of illumination, displaying most of the embryos as purple, and after all diploid embryos are developed after 48 hours, counting and counting the haploid induction rate.

(3) Identification of haploid grains: and (4) continuously cultivating the material which finishes the induced series pollination, waiting for the fruit clusters to naturally mature, threshing after harvesting, and identifying the haploids according to the color development condition of the grains. The endosperm is purple, the embryo is white and is a haploid, the endosperm and the embryo which are both purple are normal hybrid seeds of an induction line and an induced material, the individual endosperm is white and is a seed of pollen pollution pollination fructification from other sources, and the percentage of the haploid in the total seed number is the haploid inductivity.

In a second aspect, the invention discloses a corn haploid inducer line obtained by the method.

The third aspect of the invention discloses a transgenic haploid induction line, which is used as a receptor material for gene editing vector transformation to obtain a transgenic haploid induction line carrying a gene editing vector.

In a fourth aspect of the present invention, there is disclosed a method for doubling haploid chromosomes induced from the above-mentioned corn haploid inducer line or the above-mentioned transgenic haploid inducer line, comprising: and hybridizing the corn haploid induction line with any corn material, transferring the haploid young embryo generated by induction to a doubling culture medium containing a cell mitosis inhibitor, culturing for 12-48 hours to induce chromosome doubling, and performing selfing and fructification after tissue culture and seedling establishment.

Preferably, the cell mitosis inhibitor is colchicine.

In some embodiments of the invention, the method of haploid tissue culture method chromosome doubling comprises:

(1) Doubling chromosome of haploid embryo: selecting the above tissue culture colorless haploid embryo, transferring to doubling medium containing colchicine or other mitosis inhibitor at appropriate concentration, and culturing for 12-48 hr to induce chromosome doubling. The concentration and treatment time of colchicine or other doubling agents are adjusted according to different materials, and the low concentration or the too short treatment time can cause low doubling rate, and the too high or too long treatment can damage cell growth and inhibit the development of young embryos into seedlings.

(2) Tissue culture seedling transplantation: the young embryo is cultured on a growth culture medium for about 1 week to germinate into seedlings, the seedlings grow to be more than 5 cm after 2-3 weeks, the roots are developed, when the main roots of the seedlings grow to be 5 cm and are robust, the seedlings are transplanted into a seedling tray containing vermiculite nutrient soil, the seedlings are adaptively cultured in a 16/8 hour illumination artificial climate chamber or a greenhouse at 28 ℃/24 ℃ for about 1 week until new leaves grow out, the seedlings are transplanted into a field or a larger flowerpot for cultivation, and watering and fertilizing are carried out at proper time until the seedlings blossom and fruit.

(3) Selfing, pollination and fructification of doubled plants: successfully doubled haploid plants are fertile, mostly capable of producing fertile pollen and capable of pollen dispersal, but some plants may be difficult to fruit successfully due to male-female asynchronism and the like. Bagging, artificial selfing and pollination are carried out in time before the double haploid plants are subjected to heading and spinning, the double haploid plants are naturally mature and fructified, double haploid system seeds are harvested for sowing in the next year and are used as new selfing lines for evaluation and screening.

In a fifth aspect, the invention discloses a maize double haploid system obtained according to the method described above.

In a sixth aspect, the invention discloses the method, the corn haploid inducer line, the transgenic haploid inducer line and the application of the corn doubled haploid line in corn breeding.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

Compared with the prior art, the invention has the following remarkable advantages and effects:

(1) The corn haploid induction line with good comprehensive character and high induction rate is bred. The improved haploid induction line with early and obvious immature embryo color development, good fruit set, high induction rate and high comprehensive characters is obtained by hybridizing the existing haploid induction line with purple immature embryo resource materials, backcrossing for multiple generations and selfing breeding.

(2) A tissue culture chromosome doubling treatment method of corn haploid is developed and published, haploid embryos can be identified and selected at the early development stage of immature embryos, and a cell division inhibitor is used for treatment in the tissue culture process, so that the doubling efficiency is improved. The regeneration seedlings normally develop, loose powder and seed, and double haploid seeds can be obtained in the same growing season, so that the time for preparing the double haploid inbred line is shortened.

(3) The ZmPLA gene sequence of the improved corn induction line is sequenced and published, has a plurality of characteristic differences with the corresponding sequence of B73, and a coding region at the 3' end contains 4 bases of CGAG insertion mutation, so that the ZmPLA gene coding sequence is terminated in advance, and the ZmPLA gene sequence is a gene basis with haploid induction capability.

(4) The bred haploid induction line has large pollen scattering amount, good pollen activity, high maturing rate and strong purple gene expression. The embryo and scutellum endosperm of mature seeds are completely dark purple, the color is uniform, and accurate selection of haploid seeds in the mature seed period is facilitated after the haploid is induced and generated.

(5) The bred haploid induction line has better comprehensive characters and good tissue culture differentiation and regeneration capability, can be used as a universal receptor material for genetic transformation, the obtained transgenic haploid induction line is hybridized and backcrossed with other inbred lines for multiple times, and the transformed gene can be introduced into other inbred lines for transgenic breeding.

(6) The gene editing carrier is used to transform the bred haploid inducing line, the obtained transgenic inducing line carries the gene editing carrier, and is hybridized with other hybrid materials or inbred lines to induce and generate haploid, in the process, a part of haploid corresponding target gene can be edited, and the chromosome is doubled to obtain the gene-edited double haploid inbred line, so that the homozygous editing improvement of the target gene is directly realized.

Drawings

FIG. 1 is a conventional route diagram for breeding haploid inducer early-stage corn material.

FIG. 2 is a molecular marker-assisted breeding route diagram of a corn haploid inducer line.

FIG. 3 is a schematic diagram showing the DNA sequence alignment of the maize phosphatase gene ZmPLA.

FIG. 4 is a schematic amino acid sequence alignment of the maize phosphatase gene ZmPLA.

FIG. 5 is a schematic diagram of the bred haploid inducer ears and grains.

FIG. 6 is a schematic diagram showing the comparison of the coloration of the young embryo of the hybrid progeny of the haploid inducer line and the induced material.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

The embodiment mainly relates to a method for breeding a corn haploid induction line and doubling a haploid tissue culture chromosome. Hybridizing a corn haploid induction line KY8556 and a resource material S8360 with purple early immature embryos as parents, backcrossing F1 offspring and KY8556, stripping the immature embryos after pollination for 13 days, illuminating, selecting the immature embryos with early and obvious color development, culturing into seedlings, culturing in a greenhouse, carrying out molecular marker analysis during the period, screening 5 single plants which carry 4bp insertion homozygous mutation ZmPLA genes and have high genetic background recovery rate, selecting the single plants with good comprehensive properties when the plants are mature and loose powder, backcrossing with KY8556, repeating the process, and selfing to obtain solid ears after 5-6 times of backcrossing (figure 2). And (3) sowing in the field, selecting a strain with orderly offspring and good comprehensive characters to test the single-time induction rate, selfing, reserving seeds, and finally breeding an excellent induction line according to the induction efficiency and other comprehensive character performances. The technical scheme mainly comprises the following steps: 1. conventional breeding of early induction system materials; 2. rapidly improving and breeding the induction system material; 3. culturing, screening and seedling formation of immature embryo tissues; 4. screening ZmPLA mutant molecular markers of a target gene; 5. screening the genetic background recovery molecular markers of the induction line; 6. testing the haploid inductivity; 7. chromosome doubling by haploid tissue culture method. The method comprises the following specific steps:

1. conventional breeding of early induction system materials:

(1) And (3) breeding of a haploid induction line: derivative progeny of haploid inducer line Stock6 (Eder and Chalyk2002, the or Appl Genet 104. Longzhong No. 4 is a hybrid corn product of the northern wasteland reclaiming Feng variety industry, inc. (Hei Zan Ju Shen Yu 2009004; xue Jisheng, etc., the breeding and cultivation key points of the Longzhong No. 4 new corn variety, hei Longjiang agricultural science 2010 (6): 168), has good comprehensive properties of prematurity, disease resistance, etc., and can be obtained by the public from the northern wasteland reclaiming Feng variety industry.

Hybridizing to obtain an F1 generation by taking Longnan reclaimed No. 4 as a female parent and a Stock6 material as a male parent in Harbin in 2012 in spring, backcrossing to obtain a first generation BC1F1 by taking the F1 generation of Longnan reclaimed No. 4/Stock 6 as the female parent and the Stock6 as the male parent in Hainan in 2012, and then carrying out 6-generation selfing and pedigree breeding. Sowing BC1F1 purple seeds in Harbin in 2013 spring, selecting 3-5 single plants in each generation, sowing seeds of each single plant in a single row with length of 5 meters and 20 holes, sowing Longnan No. 4 female parent and Stock6 material male parent in the same period as a control, observing and comparing the conditions of field natural infection of different single plants with large leaf spot and head smut, comparing the conditions with the control plants, and selecting disease-resistant single plants; observing and comparing the powder scattering and silking time of different single plants and comparing with a control plant, and selecting an early-maturing single plant; observing and comparing the grain colors of different single plants with those of control plants, and selecting purple single plants; finally, each generation selects 3-5 single plants with good performance of each character, and the next generation selects 3-5 single plants with good disease resistance, prematurity and embryo purple according to the same process and method to reserve, and eliminates strains with non-ideal performance of characters. The haploid inductivity of the selected single plant is tested from each generation after BC1F3 generation, namely, pollen of the selected single plant of the inducting line is hybridized with a female parent test variety, each plant is subjected to test crossing by more than 5 materials, each combination test crossing has at least 3 ears, after seeds are harvested, the haploid is judged according to purple performance of endosperm and embryo in the seeds, the seeds with purple endosperm and embryo are hybrid diploid, the seeds with purple endosperm and embryo without purple are haploid from the female parent, the haploid inductivity reaches more than 6 percent, the 3-5 single plants with the highest preference are subjected to next generation selection, 6 generations of selfing breeding are performed in total as shown in figure 1, the plant with the highest inductivity is named as KY8556, and the plant is used as a haploid inducting line for trial.

During the trial use period of a KY8556 strain, the material is found to have the good properties of precocity, disease resistance, large powder scattering amount, high induction rate and the like. In a haploidy induction experiment in a Harbin region in 2016, different breeding materials are selected and planted in 55 cells, according to the needs of different materials, each cell consists of 1-3 lines with the length of 6 meters and the length of 25 holes, tassels and female ears are respectively sleeved before ear emergence and silk laying, pollen of KY8556 is used for pollination manually, after fruit ears are mature, threshing is carried out, the haploidy induction condition is counted, the endosperm is purple, the embryo is haploid grains without purple, the endosperm and the embryo are hybrid diploid grains with purple color, the haploidy induction rate is up to 12.83 percent at most, 3.95 percent at least and 9.43 percent on average (Table 1). However, the KY8556 strain still has a plurality of defects to influence the use of the strain, including smaller purple area of grains, lighter color, later and light purple color development of immature embryos, shorter and small plants, dissimilarity in male and female flowering phases and the like, and further genetic improvement needs to be carried out by using purple grain resource materials with better comprehensive properties to remove or improve the property defects.

TABLE 1 Harbin 2016 Induction of haploids with KY8556 for different breeding materials

(2) And (3) breeding the purple seed resource material: jinnuo No. 10 is a hybrid bred by the institute of corn of agricultural sciences of Shanxi province (Jinju Yu 2014020; dong Ligong, etc., a new black waxy corn variety, jinnuo No. 10, and agricultural science and technology communication 2014 (12): 155-157), and has the characteristics of dark purple grains and purple cob, consistent female and male panicle flowering stages, 13-15 tassel branches, large pollen amount, strong stress resistance, large leaf spot resistance, small leaf spot, rough dwarf and dwarf mosaic disease. The public is available from the academy of agricultural sciences of shanxi province. The filial generation of Jinnuo No. 10 hybrid is continuously selfed for 4 generations to obtain a purple waxy corn material, 3 ears (namely F2 generation) 2016 self-bearing Jinnuo No. 10 are selected for Haerbin sowing in spring, each ear is single row with the length of 5 meters and 20 holes, the Jinnuo No. 10 is sown as a contrast, the tassels and the female ears are respectively sleeved before silking, and artificial selfing pollination is carried out. Observing and comparing the natural field infection conditions of different single plants and comparing with the control plants, and selecting disease-resistant single plants; observing and comparing the powder scattering and spinning time of different single plants, comparing with a control plant, and selecting an early-maturing single plant with large powder scattering amount; observing and comparing the seed colors of different single plants with those of control plants, and selecting the single plants with deep purple seeds; finally, 3-5 strains with good performance of each character are selected to eliminate the strains with non-ideal performance of the character. Planting 3-5 single plants with disease resistance, prematurity and dark purple seeds in the Hainan in the third Hainan in the winter of 2016, the Harbin in the spring of 2017 and the Hainan in the winter of 2017 according to the same method and the same process and method. Finally, selecting a single plant with good resistance and deep purple grain color as a purple grain resource material, and naming the S8360 strain (figure 1).

2. And (3) rapid improvement and breeding of induction system materials:

(1) Screening hybrid single plants: and (3) selecting a haploid inducing line material KY8556 strain and a purple young embryo resource material S8360 strain, sowing in the field at proper time, selecting 5 normally developed single plants, respectively bagging male and female ears before silk drawing and ear drawing, carrying out artificial pollination hybridization, and normally fruiting and harvesting ears to obtain F1 hybrid seeds.

(2) And (3) backcross screening for multiple generations: sowing an induction line KY8556 and the F1 hybrid seeds in a greenhouse, selecting 10 single plants with normal development, bagging female and male ears before silking and heading, and manually carrying out KY8556 XF 1 backcross pollination. And (3) taking young ears about 13 days later, after surface disinfection and sterilization, stripping young embryos in an aseptic operation workbench, developing color by illumination, selecting young embryo tissues with deep color development, large color development area and quick color development to culture seedlings, selecting about 50 robust seedlings, and transplanting the robust seedlings as BC1F1 plants into a greenhouse for cultivation. Sampling young plant leaves, extracting genome DNA, respectively carrying out ZmPLA mutation marker screening on a target gene and molecular marker screening for genetic background recovery of an induction line KY8556, and reserving 5 BC1F1 single plants which contain ZmPLA homozygous mutation, have the highest recovery rate and have better comprehensive phenotypic characters for next generation backcross.

Timely sowing KY8556 in a greenhouse to ensure the plants to meet the flowering phase of the BC1F1 plants, bagging male and female ears before silking and heading, and manually carrying out KY8556 xBC 1F1 backcross pollination. And (3) collecting young ears after about 13 days, repeating the young ear tissue culture screening and seedling formation, carrying out target gene ZmPLA mutation marker screening and molecular marker screening for inducing system KY8556 genetic background recovery, and selecting 5 single plants with highest recovery rate and better comprehensive phenotypic characters for next round of backcross until 5 or 6 rounds of backcross are completed to obtain plants BC5F1 and BC6F 1. Because the young embryo tissue culture seedling is not subjected to the normal seed maturation process to directly obtain the next generation of plants, the time of the generation of hybridization and backcross is greatly shortened, the planting in a greenhouse is not limited by seasons, one generation can be completed about every three months, and 4 rounds of backcross can be performed every year.

(3) Selfing and screening: and (3) cultivating the BC5F1 and BC6F1 plants which are subjected to 5-round and 6-round backcross breeding in a greenhouse, bagging female and male ears before silking and heading, artificially selfing and pollinating 5 plants, continuously cultivating until the ears are naturally mature, and harvesting BC5F2 and BC6F2 seeds from a single ear. The method comprises the steps of sowing BC5F2 and BC6F2 seeds in a field in a corn growing season, reserving about 20 plants in each ear, performing phenotype evaluation at each growing stage, selecting plants with regular and consistent progeny, bagging female and male ears before silk drawing and ear drawing, performing artificial selfing and pollination on 10 plants, continuously cultivating until the ears are naturally mature, harvesting BC5F3 and BC6F3 seeds from a single ear, and observing and screening the ears with good embryo and scutellum color development as candidate induction lines (figure 5).

Multiple induced materials are sown in proper staggered period in the same season, pollen of the BC5F2 and BC6F2 plants is pollinated, and the haploid induction effect of each candidate induction line is tested. The BC5F3 and BC6F3 seeds can be sowed in the field in the next year, and the evaluation such as character performance, induction rate test and the like is continuously carried out until an ideal improved induction line is bred.

3. And (3) young embryo tissue culture screening and seedling establishment:

(1) And (3) young embryo tissue culture screening: taking young ears with young embryos about 3 mm long about 13 days after pollination, spraying 75% alcohol on the surfaces of the ears for disinfection, spraying a layer of bract, and finally cutting off the excessive parts of the top and the base of the corn by using a debranning knife. Placing the young corn ears without the bracts into a beaker, placing the beaker in a super clean workbench, firstly soaking and cleaning for 5 minutes by using 75% alcohol, replacing fresh 75% alcohol, continuously soaking and sterilizing for 15 minutes (the alcohol in the step can be used once again for soaking and sterilizing in the first step), and finally cleaning for 3 times by using sterile distilled water, wherein each time lasts for 2 minutes.

Placing the sterilized fruit cluster in a culture dish with the diameter of 150 mm, cutting off the surface of the fruit cluster by about 3 mm by using a scalpel, inserting the tip of the scalpel between the endosperm and the seed coat at the side of the seed particle facing the upper end of the fruit cluster and inclining by about 30 degrees from top to bottom, picking out the immature embryo, placing a shield slice upwards on an induction culture medium (shown in table 2), inverting the culture dish on a tissue culture frame, and carrying out continuous illumination for 24 hours to ensure that the immature embryo is developed by light, wherein the immature embryo is developed into purple after about 12 hours.

TABLE 2 culture medium for haploid chromosomal tissue culture doubling

(2) Culturing immature embryo tissues into seedlings: selecting about 50 immature embryos with early color development, dark color and large area, transferring the immature embryos into a glass culture bottle containing a growth medium (table 2), continuously culturing, and germinating into seedlings. When the seedlings grow to about 6-7 cm and the main root system is about 5 cm and strong, opening a culture bottle cap and adding a small amount of distilled water to keep humidity, hardening the seedlings for 24 hours, carefully clamping the plants out by using tweezers to avoid damaging stems and roots, washing culture media attached to the roots by using clear water, transplanting the plants into a seedling tray containing vermiculite nutrient soil, covering the seedling tray to keep humidity, culturing the plants in a 16/8 hour illumination artificial climate chamber or a greenhouse at 28 ℃/24 ℃ for about 1 week until new leaves grow out, transplanting the seedlings into a larger flowerpot for culturing, and applying a proper amount of fertilizer until the seedlings bloom, loose powder and fruit. During this period, the leaves of these plants can be sampled and DNA extracted for genotyping.

4. Screening of target gene ZmPLA mutant molecular markers:

(1) Sequencing ZmPLA gene of haploid induction line KY 8556: the functional gene of the maize haploid inducer line has been identified as the calcium ion independent phosphatase GRMZM2G471240 gene ZmPLA (Ca 2+ -independent phospholipase A2), whose 1452bp long expression sequence comprises a CDS sequence of 1287bp, a protein sequence encoding 428 amino acids (SEQ ID NO: 1-3). Insertion frame shift mutations of 4 base CGAG at the 3' end of its coding sequence lead to a reduction in its function, resulting in chromosomal loss of the inducible line during meiosis (Gilles et al, 2017, embo 36, liu et al, 2017, mol Plant 10. According to the sequenced ZmPLA gene GRMZM2G471240 sequence (https:// phytozome.jgi.doe.gov/pz/port.html) of inbred line B73, a plurality of pairs of PCR primers (SEQ ID NO: 8-30) are designed, KY8556 genome DNA is used as a template, the ZmPLA gene sequence of KY8556 induction line is amplified in a segmented mode and sequenced, and whether the 3' end of the coding sequence contains the insertion frame-shifting mutation of CGAG with 4 bases or not is detected.

Sampling KY8556 plant leaves, extracting genome DNA by using a DNA rapid extraction method based on SDS (Sodium dodecyl sulfate), carrying out segmented amplification by using a plurality of pairs of GRMZM2G471240 (ZmPLA) sequence specific primers of B73, and sequencing a corresponding gene ZmPLA-KY of KY8556. The PCR reaction system used Takara One Shot LA PCR kit (Takara Bio Inc.), and 20. Mu.l of the PCR reaction system contained: mu.l of 2X One Shot LA PCR mix, 0.5. Mu.l of 10. Mu.M forward primer, 0.5. Mu.l of 10. Mu.M reverse primer, 7.0. Mu.l of sterile water, and finally 2.0. Mu.l of genomic DNA from 50 ng/. Mu.l of sample. The PCR reaction conditions were 94 ℃ denaturation for 5 min, then 94 ℃ denaturation for 30 sec, 60 ℃ annealing for 1 min, and 72 ℃ extension for 1 min for 30 cycles, and finally 72 ℃ extension for 5 min and held at 4 ℃.

Mu.l of the PCR amplification product was separated by 1% agarose gel electrophoresis, and the remaining PCR product, from which the specific length fragment was successfully amplified, was directly sequenced using PCR primers, or cloned into pCR2.1 TOPO vector using vector-specific M13F or M13R (SEQ ID NO:33, 34) primers according to the method provided by TOPO TA Cloning Kit (Invitrogen). Because the ZmPLA gene sequences of B73 and KY8556 have multiple differences, some primer pairs can not amplify PCR fragments, different primers need to be designed and tried again, and finally, the primer pairs ZmPLA-F3/ZmPLA-R3 (SEQ ID NO:12, 13), zmPLA-F7/ZmPLA-R4 (SEQ ID NO:20, 15) and ZmPLA-F2/ZmPLA-R2 (SEQ ID NO:10, 11) successfully amplify 811, 901 and 605bp mutually overlapped fragments respectively, and the ZmPLA-KY sequence of a part KY8556 is assembled after sequencing. To obtain a longer 3 'end sequence, zmPLA-F5 (SEQ ID NO: 16) and a matched pair of 6 different 3' end primers ZmPLA-R5, zmPLA-R7, zmPLA-R9, zmPLA-R10, zmPLA-R11, and ZmPLA-R12 (SEQ ID NO:17, 21, 25, 26, 27, 28) were used, but none of them succeeded in amplifying a specific PCR fragment.

To obtain a longer ZmPLA end sequence of KY8556, primers ZmPLA-F9, zmPLA-R13, and ZmPLA-R14 (SEQ ID NO:24, 29, 30) were designed based on the above partially assembled ZmPLA sequence of KY8556, and Inverse PCR amplification (Green and Sambrook 2019) was used, first a KY8556 genomic DNA sample was cut into fragments with a plurality of different restriction enzymes, a portion of the fragments was self-ligated into circles by adding T4 DNA ligase, then first round PCR amplification was performed with ZmPLA-F8 and ZmPLA-R13 primers (SEQ ID NO:22, 29), then second round PCR amplification was performed with ZmPLA-F9 and ZmPLA-R14 using the first round PCR reaction as a template (SEQ ID NO:24, 30) and after obtaining a specific fragment, the specific fragment was determined by Cloning into a vector for RmPLA 13, rmPLA-M13 (SEQ ID NO:33, rmPCR vector after Cloning into the ZmPLA-R14 (SEQ ID NO:1, rmPLA 13) provided by TOPO TA Cloning Kit (Invitrogen).

Summarizing the sequencing results, comparing and analyzing with the ZmPLA gene fragment sequence of B73, finally obtaining 2495bp long ZmPLA-KY gene fragment sequence (SEQ ID NO: 5) of KY8556, and the corresponding ZmPLA gene fragment sequence of B73 is 2522bp long (SEQ ID NO: 4). Through comparison analysis, the two sequences have a plurality of differences in coding regions, upstream and downstream, and non-coding regions such as introns, and therefore, a molecular fingerprint marker specific to KY8556 and a derivative induction line thereof can be developed (FIG. 3). The 3' end of ZmPLA gene coding sequence of KY8556 has insertion frame shift mutation of CGAG with 4 bases, which causes the coding sequence to terminate early, the predicted CDS sequence length is 1200bp (SEQ ID NO: 6), the coded mutant ZmPLA-KY phosphatase is 399 amino acids (SEQ ID NO: 7) longer, 29 amino acids are deleted at the carboxyl end and 20 differential sequences are provided compared with 428 amino acid wild type ZmPLA phosphatase of B73, and in addition, three amino acid point mutations are distributed in the middle (figure 4).

(2) Mutation ZmPLA-KY gene specificity PCR marker: the ZmPLA-KY gene of the induction line KY8556 has 4 base CGAG insertion mutations at the 3' end of the coding sequence, and has a plurality of point mutations at the downstream which are different from the B73 wild ZmPLA gene sequence, primers ZmPLA-F8 and ZmPLA-R8 (SEQ ID NO:22, 23) with ZmPLA-KY sequence specificity are designed according to the differences, genomic DNA of KY8556 and S8360 are respectively used as templates for PCR amplification, only the KY8556 template successfully amplifies an expected specific fragment with the length of 150bp, and S8360 does not have any PCR product, so the primer pair ZmPLA-F8 and ZmPLA-R8 can be used as a mutation ZmPLA-KY gene specificity PCR marker for identification and screening of induction line plants.

(3) Quantitative qPCR detection of the copy number of the inducible mutant gene ZmPLA-KY: an induction line KY8556 serving as a diploid inbred line contains 2 copies of ZmPLA-KY mutant genes, S8360 contains 2 copies of ZmPLA wild type genes, the hybrids F1 of KY8556 and S8360 respectively contain one copy of ZmPLA-KY and ZmPLA, 50% of backcross progeny BC1F1 groups of F1 and KY8556 contain 2 copies of ZmPLA-KY, and the other 50% of plants respectively contain one copy of ZmPLA-KY and ZmPLA. The number of ZmPLA-KY copies contained in the BC1F1 plant can be estimated by fluorescent quantitative PCR, and a plant containing 2 ZmPLA-KY copies is selected for continuous backcross with KY8556. Backcross offspring of the gene locus are not separated, and all stably contain 2 copies of ZmPLA-KY mutant genes.

Extracting genomic DNA of KY8556, KY8556xS 8360F 1 and S8360 plant leaves, respectively using the genomic DNA of the KY8556xS 8360F 1 plant as 2,1 of ZmPLA-KY mutant genes and 0 copy of control samples, using 5-fold serial dilution containing single copy of genomic DNA of the KY8556xS 8360F 1 plant as standard samples, using corn Alcohol dehydrogenase (AF 123535) as endogenous reference genes, designing primers specific to ZmPLA-KY and ZmADH genes, carrying out fluorescent green qPCR amplification, establishing respective standard curves, evaluating and confirming that PCR amplification efficiencies of two primer pairs are consistent, and carrying out copy number detection on unknown samples by using the same method after ZLA-KY copy numbers of three control samples KY8556, KY8556xS8360 and S8360 mPLA-KY genes are consistent.

Fluorescent quantitative PCR reaction system adopts

Green Realtime PCR Master Mix kit (TOYOBO Life Science), 20 microliter PCR reaction system containing: mu.l of 2X Master mix, 0.5. Mu.l of 10. Mu.M primer ZmPLA-F8 and 0.5. Mu.l of 10. Mu.M primer ZmPLA-R8 (SEQ ID NO:22, 23), 8.0. Mu.l of sterile water, and finally 1.0. Mu.l of 30 ng/. Mu.l of the genomic DNA of the sample. The PCR reaction conditions were 40 cycles of denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 10 sec, annealing at 66 ℃ for 10 sec, and extension at 72 ℃ for 15 sec. Corn alcohol dehydrogenase specific primers ZmAdh-F3 and ZmAdh-R3 were used as endogenous reference gene controls (SEQ ID NO:31, 32). When the real-time quantitative qPCR reaction is carried out, the PCR reaction of the standard sample and the sample to be detected is carried out on the same 96-well plate, and 3 are arranged on each sampleRepeating for the times, taking an average value to calculate the copy number, and identifying the BC1F1 plant with the ZmPLA-KY gene copy number of 2. After the second round of backcross is completed, the same quantitative qPCR is used for verifying that the copy numbers of the mutant ZmPLA-KY genes of all BC2F1 plants are homozygous diploid, so 3-5 rounds of backcross offspring are not verified in the same way.

5. Genetic background recovery molecular marker screening:

(1) KY8556 inducible line genetic background screening of BC1F1 population: in order to improve the recovery screening efficiency of KY8556 and S8360 filial generations, parent and multi-generation backcross progeny are analyzed by a KASP (Kompetitive Allelele Specific PCR) genotyping method (Semagn et al, 2013), and 5 plants with the highest KY8556 genotype recovery rate are selected for next generation backcross. A total of 139 effective SNP markers with Polymorphism between KY8556 and S8360 parents are firstly analyzed by using a fluorescence labeling primer pair (LGC Group, UK) of Single Nucleotide Polymorphism (SNP) with 384 Single base diversity sites. The 139 markers are used for analyzing 36 plants in total of BC1F1 transplanted plants which have good early embryo color development and are subjected to tissue culture seedling formation, and after statistical analysis, 44 markers are found to be completely restored to KY8556 genotype, and the other 95 markers still show polymorphism. The recovery rates of 36 BC1F1 plants were calculated according to the ratio of "KY8556 homozygous SNP" to "total detected SNP" detected in each plant sample, and finally 5 BC1F1 single plants KY2, 18, 19,8, 20 which had the highest background recovery rate and were confirmed to be homozygous by qPCR analysis were selected and then backcrossed with KY8556 in the next round (Table 3).

(2) Induction line KY8556 genetic background screen for BC2F1 population: after backcrossing BC1F1 and KY8556, selecting 65 BC2F1 plants with good color development of early young embryos and tissue culture seedlings by tissue culture, transplanting the plants to survive, sampling leaves to extract genome DNA, and analyzing the recovery rate of each plant by using the 95 markers still showing polymorphism. After statistical analysis, 38 markers are found to be completely restored to KY8556 genotype, and the other 57 markers still show polymorphism, but 5 of the markers cannot be used abnormally by PCR amplification typing, and only 52 effective markers remain. The recovery rates of 36 BC1F1 plants are calculated according to the proportion of the detected "KY8556 homozygous SNP" in the total detected SNPs "in each plant sample, and finally, 5 BC2F1 single plants KY18-5, 19-14, 18-3, 20-23 and 18-4 with the highest background recovery rate are selected and then are backcrossed with KY8556 in the next round (Table 3).

TABLE 3 recovery of genetic background of KY8556xS8360 backcross progeny KY8556 parent

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* Two BC3F1 single plants were selected for subsequent backcross breeding.

(3) Induction line KY8556 genetic background screen for BC3F1 population: after backcrossing BC2F1 and KY8556, selecting 71 BC3F1 plants with good color development of early young embryos and tissue culture seedlings by tissue culture, transplanting the plants to survive, sampling leaves to extract genome DNA, and analyzing the recovery rate of each plant by using the 52 markers still showing polymorphism. After statistical analysis, 52 markers of 6 samples are completely restored to KY8556 genotype, and 51 markers of 8 samples are completely restored to KY8556 genotype, so that the molecular marker screening for genetic background restoration is completed. The recovery rate of 36 BC1F1 plants is calculated according to the proportion of 'KY 8556 homozygous SNP' in 'total detected SNP' detected in each plant sample, and finally two single plants KY18-5-24 and 18-5-3 with robust growth state and good comprehensive properties are selected from BC3F1 single plants with high background recovery rate and then backcrossed with KY8556 for the next round (Table 3).

6. And (3) testing the haploid inductivity:

(1) Haploid induction: after the multiple rounds of backcrossing, selfing and various screening with KY8556, a plurality of candidate improved induction lines with stable and consistent performance of various characters are obtained, and three of the candidate improved induction lines are selected and named KY3 (cultivation induced 3), KY10 (cultivation induced 10) and KY11 (cultivation induced 11) again for haploid induction rate testing. Because the inductivity of different materials generally varies, multiple materials should be used to test the same induction line simultaneously. Using a KY8556 induction line as a control, testing the haploid induction effect of KY3, KY10 and KY11 improved induction lines by using the same corn material, and properly adjusting staggered sowing according to the growth period difference of different induced materials and induction lines to ensure that the flowering periods of the male parent and the female parent meet. Respectively bagging before plant heading and spinning, pollinating an induced material by an induction system, taking fruit ears for sterilization after pollinating for about 13 days, selecting young embryos with the length of about 3 mm for tissue culture and screening, and identifying haploid embryos according to the condition of the young embryos after illumination. Or waiting for the ears to naturally mature, threshing after harvesting, and identifying the haploids according to the color of embryos in the seeds.

(2) Tissue culture and identification of haploid embryos: taking back young corn ears, removing old bracts on the surfaces, cutting off filaments at the tops, peeling off the bracts layer by layer, spraying 75% alcohol on each layer for surface disinfection, soaking the ears in 75% alcohol for disinfection for 10 minutes, and cleaning the ears with sterile water for three times. Placing the disinfected fruit cluster in a culture dish with the diameter of 150 mm, cutting off about 2 mm of the upper part of the seed by using a scalpel, obliquely inserting the scalpel tip between the endosperm and the seed coat from the embryo-containing end of the developing seed, picking out the immature embryo positioned at the base part of the seed, placing the shield piece upwards on the culture dish containing an induction culture medium (table 2), after 12 hours of illumination, most of the embryo is purple, and if the embryo is not colored, the embryo is a haploid embryo (figure 6), and counting the haploid induction rate.

The color development of the immature embryos induced by the improved induction lines KY3, 10 and 11 has no obvious difference, the purple color is observed after about 6-7 hours of illumination, the immature embryos induced by KY8556 do not develop until 8 hours of illumination, and the color development time of the improved induction lines KY3, 10 and 11 is obviously earlier than about 1-2 hours of KY8556. When most embryos are developed after 12 hours, the developed area of the embryos of the improved induction system is obviously larger than that of KY8556, and the embryos are darker in color, so that the embryos are more suitable for accurately selecting the haploid embryos which are not developed (figure 6). The average haploid inductivity of each induction line judged according to the proportion of the chromogenic immature embryos is similar and is between 12 and 15 percent, and the haploid inductivity of different materials possibly has difference, but because the experimental data is small, the conclusion is difficult to draw, and the field large experimental data needs to be collected and analyzed and confirmed (table 4).

TABLE 4 haploid inductivity of selected inducible lines

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(3) Identification of haploid mature grains: and (3) continuously cultivating the material which finishes the induced series pollination, waiting for the ears to be naturally mature, threshing after harvesting, and identifying the haploid according to the color development condition of the grains. The endosperm shows purple and the embryo shows light yellow and is a haploid, the endosperm and the embryo which are both purple are normal hybrid seeds of an induction line and an induced material, the individual endosperm which is light yellow can be pollinated and fruited seeds polluted by pollen from other sources, and the percentage of the haploid in the total seed number is the haploid inductivity.

7. Chromosome doubling by haploid tissue culture method:

(1) Doubling chromosome of haploid embryo: selecting the above tissue culture colorless haploid embryo, transferring to doubling medium containing colchicine with concentration of 0.005% to 0.05% or other cell mitosis inhibitor with appropriate concentration, and culturing for 12-48 hr to induce chromosome doubling. The concentration and treatment time of colchicine or other doubling agents are adjusted according to different materials, and the low concentration or the too short treatment time can cause low doubling rate, and the too high or too long treatment can damage cell growth and inhibit the normal development of young embryos into seedlings. After the chromosome doubling treatment is completed, the young embryos are transferred to a glass culture bottle containing a growth medium in time and are subjected to illumination culture in a tissue culture chamber at the temperature of 28 ℃ for 16/8 hours to form seedlings.

(2) Tissue culture seedling transplanting: culturing young embryos on a growth culture medium for about 1 week to germinate into seedlings, after 2-3 weeks, the young embryos grow to be more than about 5 cm in height and have developed root systems, when the main root systems of the seedlings grow to be 5 cm and are robust, opening a sealing film of a culture bottle, adding a small amount of distilled water to keep humidity, hardening the seedlings for 24 hours, carefully clamping the plants by using tweezers to avoid damaging stems and roots, washing the culture medium attached to the roots by using clear water, transplanting the seedlings into a seedling tray containing vermiculite nutrient soil, covering the seedling tray to keep humidity, adaptively culturing the seedlings in a 16/8 hour illumination artificial climate chamber or a greenhouse at 28 ℃/24 ℃ for about 1 week, after new leaves grow out, transplanting the seedlings into a field or a larger flowerpot to cultivate, watering and fertilizing at proper time until the seedlings bloom and fruit.

(3) Selfing, pollination and fructification of doubled plants: the haploid plants which are not doubled and the doubled and successfully doubled haploid plants are short, weak in growth, and far shorter than inbred line plants naturally germinated from seeds or hybrid plants which may be polluted, so that the hybrid plants can be distinguished and removed. Haploid plants are sterile, cannot be pollen-dispersed and fructified, and are automatically eliminated. The doubled haploid plants which are successfully doubled can be bred, most of the doubled haploid plants can produce fertile pollen, the pollen can be scattered, and the silks can be drawn at proper time, but some plants are difficult to fruit successfully due to male and female asynchronism and the like. Bagging, artificial selfing and pollination are carried out in time before the double haploid plants are subjected to heading and spinning, the double haploid plants are naturally mature and fructified, double haploid system seeds are harvested for sowing in the next year and are used as new selfing lines for evaluation and screening.

The nucleotide sequences involved in this example are shown below:

SEQ ID NO 1：Zea mays B73 phospholipase A2 gene ZmPLA GRMZM2G471240 1452bp；

SEQ ID NO 2：Zea mays B73 phospholipase A2 gene ZmPLA GRMZM2G471240 CDS 1287bp；

SEQ ID NO 3：Zea mays B73 phospholipase A2 ZmPLA GRMZM2G471240protein 428aa；

SEQ ID NO 4：Zea mays haploid inducer B73 phospholipase A2 gene fragment ZmPLA 2522bp；

SEQ ID NO 5：Zea mays haploid inducer KY8556 phospholipase A2 gene fragment ZmPLA-KY2495bp；

SEQ ID NO 6：Zea mays haploid inducer KY8556 phospholipase A2 gene ZmPLA-KY CDS1200bp；

SEQ ID NO 7：Zea mays haploid inducer KY8556 phospholipase A2 ZmPLA-KY protein 399aa；

SEQ ID NO 8：ZmPLA-F1 oligo 21bp；

SEQ ID NO 9：ZmPLA-R1 oligo 22bp；

SEQ ID NO 10：ZmPLA-F2 oligo 21bp；

SEQ ID NO 11：ZmPLA-R2 oligo 26bp；

SEQ ID NO 12：ZmPLA-F3 oligo 22bp；

SEQ ID NO 13：ZmPLA-R3 oligo 22bp；

SEQ ID NO 14：ZmPLA-F4 oligo 21bp；

SEQ ID NO 15：ZmPLA-R4 oligo 22bp；

SEQ ID NO 16：ZmPLA-F5 oligo 19bp；

SEQ ID NO 17：ZmPLA-R5 oligo 22bp；

SEQ ID NO 18：ZmPLA-F6 oligo 18bp；

SEQ ID NO 19：ZmPLA-R6 oligo 18bp；

SEQ ID NO 20：ZmPLA-F7 oligo 21bp；

SEQ ID NO 21：ZmPLA-R7 oligo 20bp；

SEQ ID NO 22：ZmPLA-F8 oligo 17bp；

SEQ ID NO 23：ZmPLA-R8 oligo 19bp；

SEQ ID NO 24：ZmPLA-F9 oligo 21bp；

SEQ ID NO 25：ZmPLA-R9 oligo 21bp；

SEQ ID NO 26：ZmPLA-R10 oligo 21bp；

SEQ ID NO 27：ZmPLA-R11 oligo 20bp；

SEQ ID NO 28：ZmPLA-R12 oligo 21bp；

SEQ ID NO 29：ZmPLA-R13 oligo 21bp；

SEQ ID NO 30：ZmPLA-R14 oligo 22bp；

SEQ ID NO 31：ZmAdh-F3 oligo 22bp；

SEQ ID NO 32：ZmAdh-R3 oligo 22bp；

SEQ ID NO 33：M13F oligo 20bp；

SEQ ID NO 34：M13R oligo 17bp。

the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims (10)

1. A method for breeding a corn haploid inducer line is characterized by comprising the following steps:

s1: hybridizing, backcrossing and selfing, wherein a haploid induction line KY8556 in a corn resource library is selected for hybridization with a purple immature embryo resource material S8360 to obtain F1 hybrid seeds;

s2: culturing and screening immature embryo tissues and forming seedlings;

s3: and (5) carrying out haploid inductivity test to obtain a corn haploid induction line.

2. The method as claimed in claim 1, wherein in step S1, the breeding method of the haploid inducer line KY8556 comprises the following steps: hybridizing by taking Longnan No. 4 as a female parent Stock6 as a male parent to obtain an F1 generation, then taking the F1 generation as the female parent and taking the Stock6 as the male parent to obtain a backcrossed BC1F1 generation, selecting 3-5 plants with optimal disease resistance, prematurity and embryo purple in the same generation of plants in each generation, wherein the haploid inductivity of the selected plants reaches more than 6 percent, and selfing for 6 generations to obtain a haploid induced plant KY8556 strain; the breeding method of the S8360 comprises the following steps: jinnuo No. 10 is taken as a material, 3-5 plants with the best disease resistance, precocity and deep purple grains in the same generation of plants are selected for each generation, and selfing is carried out for 4 generations to obtain a single-line purple grain material S8360.

3. The method as claimed in claim 1, wherein KY8556 is backcrossed with F1, the single plant with better characteristics is selected for the next round of backcross until 5 or 6 rounds of backcross are completed, and then 1 to 3 rounds of selfing are carried out to obtain stable candidate induction lines.

4. The method of claim 3, wherein in S3, the candidate induction line of claim 3 is used for hybridization fructification with other corn materials, tissue culture identification and/or haploid grain identification of haploid embryos are carried out, and the candidate induction line with high haploid incidence becomes a new corn haploid induction line.

5. The method as claimed in claim 1, wherein individuals with better properties than previous generation are screened for next round of backcross by using gene ZmPLA induced mutation molecular marker and/or genetic background recovery molecular marker.

6. The method as claimed in claim 5, wherein the nucleotide sequence of gene ZmPLA is shown in SEQ ID NO:5, respectively.

7. The method of claim 1, wherein in S2, selecting early-colored, dark-colored and large-area immature embryos, transferring the immature embryos into a culture flask containing a growth medium, germinating into seedlings, transplanting the seedlings into a larger flowerpot for culturing after the seedlings grow to 6-7 cm, and applying a proper amount of fertilizer until the young embryos flower and fruit.

8. A method for obtaining a transgenic haploid inducer line, characterized in that a transgenic haploid inducer line carrying a gene editing vector is obtained by transforming a gene editing vector using the corn haploid inducer line obtained by the method of any one of claims 1 to 7 as a recipient material.

9. A method of doubling the haploid chromosomes induced by the transgenic haploid inducer line of claim 8, comprising: and hybridizing the corn haploid induction line with any corn material, transferring the haploid young embryo generated by induction to a doubling culture medium containing a cell mitosis inhibitor, culturing for 12-48 hours to induce chromosome doubling, and performing selfing and fructification after tissue culture and seedling establishment.

10. Use of the method according to any one of claims 1 to 9 in maize breeding.

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