CN113317197A - Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid - Google Patents
Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid Download PDFInfo
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Abstract
The invention discloses a rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploids. Construction of PZmBD1 or PZmBD1, 2RS5GPA promoter for driving and controlling anthocyanin synthesis regulation geneZmC1，ZmR2The expression vector creates the transgenic corn with embryo/kernel rich anthocyanin, and utilizes the two-major gene containing the induction of the parthenogenesis haploid of the cornZmpla1/ZmmtlAndZmdmpthe haploid inducer line andand (3) hybridizing the anthocyanin enrichment materials, obtaining separated offspring by using a self-crossing or backcross mode, and finally detecting the single plant simultaneously containing the homozygous major gene by using a molecular marker to obtain the parthenogenesis haploid induction line. The parthenogenesis haploid induction line can realize the early identification and the direct identification of haploid embryos, obviously improve the identification efficiency of the haploid embryos, realize the identification of the haploids in different growth periods, and provide a feasible scheme for improving the haploid breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rapid chromogenic parthenogenesis haploid inducer and application thereof in identification of a corn haploid.
Background
A corn haploid breeding technology is one of important technologies for improving breeding efficiency, and is widely applied to commercial breeding of corn (Andorf C, et al, 2019.). The corn haploid breeding technology mainly comprises four parts of haploid induction, identification, doubling and DH evaluation (Chenshaojiang et al 2012). The identification of the haploid is widely concerned as a direct method for obtaining the haploid, and the technical problem of efficiently selecting the haploid young embryo in the early development stage (about 15 days after pollination) of the young embryo is the tissue culture doubled haploid. Currently, the most practical haploid identification method mainly comprises color marker identification and oil content identification. Oil identification haploids have been automated, but the development of high oil induction systems and nuclear magnetic resonance technology have been limited. The method for identifying the haploid by utilizing the color marker becomes one of the most widely applied methods for identifying the haploid at present by the advantages of intuition and convenience. Chase and Nanda (1965) will be the first timeR1-nj(Navajo) color marker introduction into maize parthenogenesis haploid inducer. The haploid can be directly identified according to the existence of the color marks of the scutellum and the aleurone layer, and the development of the haploid technology is greatly promoted. But do notR1-njIs easily influenced by material background and environment. Simultaneously, with the use of a large number of European durum and tropical germplasm, the germplasm contains a pigment synthesis inhibiting geneC1-I,C2-IdfAnd the seed is chromatically differentiated after induction, so that the haploid identification efficiency is low, and the application of haploid breeding in the germplasm is greatly limited (Chaikam, et al. 2015). Therefore, there is an urgent need for a stable and rapid corn haploid parthenogenesis haploid inducer line with color markers for such germplasm.
In addition, haploid tissue culture identification and doubling technology is continuously developed. The identification efficiency of haploid immature embryos is greatly limited due to unstable color marker expression of a conventional parthenogenesis haploid induction system, the identification of the haploid immature embryos can be realized by in vitro culture of the immature embryos, but the stripping work of the immature embryos greatly reduces the tissue culture identification efficiency of the haploid immature embryos, if heterozygosis immature embryos can develop color when the immature embryos are stripped, the in vitro culture of all the immature embryos can be avoided, the direct identification of the haploid immature embryos is realized, and the tissue culture identification efficiency of the haploid is effectively improved. The parthenogenesis haploid induction line color marker requires to drive a plurality of anthocyanin synthesis related genes together, and can be specifically expressed in grains, if the parthenogenesis haploid induction line color marker is used for haploid embryo identification, certain requirements are also provided for anthocyanin expression periods, and at present, no related report which has practical application value and directly identifies haploid embryos by utilizing the anthocyanin marker without additional culture exists.
Disclosure of Invention
The invention aims to provide a rapid chromogenic parthenogenesis haploid induction line and application thereof in identification of a corn haploid.
The anthocyanin-enriched corn with anthocyanin accumulated only in the embryo and aleurone layers of corn grains is obtained by transforming corn by using an embryo or endosperm promoter to drive anthocyanin to synthesize related genes. Transferring the target gene for controlling the anthocyanin enrichment of the corn kernels into a corn parthenogenesis haploid induction line to obtain the rapid chromogenic marker parthenogenesis haploid induction line.
Fast color-developing parthenogenesisThe reproductive haploid inducing line is one inducing line containing two major genes for controlling the parthenogenesis haploid of cornZmpla1/ZmmtlAndZmdmpthe induction line of (A) and the promoter containing PZmBD1 carry a regulatory gene for controlling anthocyanin synthesisZmC1，ZmR2The individual strain pBD-C1R2 and promoter containing PZmBD1 and 2RS5GPA carry a regulatory gene for controlling anthocyanin synthesisZmC1，ZmR2Hybridizing the single plant p2Seed-C1R2, obtaining separated offspring by using a self-crossing or backcross mode, and finally detecting whether the single plant contains homozygous by using molecular markersZmpla1/ZmmtlAndZmdmpand the parthenogenesis haploid induction line which has high haploid induction rate and is marked by rapid color development is obtained by carrying a single plant of pBD-C1R2 and p2Seed-C1R 2.
The application of the rapid chromogenic parthenogenesis haploid induction line in identifying the corn haploid is that under the condition of not carrying out isolated culture, the haploid is identified directly according to the anthocyanin expression condition after pollination for more than 12 days, and the aleurone layer and the scutellum of the hybrid diploid are colored; haploid grains are represented by a colored aleurone layer and colorless scutellum, and hybrid diploids are represented by a colored aleurone layer and scutellum; the haploid bud seedling is represented by green or light red coleoptile and white root, and the hybrid diploid is represented by dark purple coleoptile and root; haploid seedlings and plant leaves are green, and hybrid diploids are purple leaf tips; haploid embryos, seeds, seedlings and plants can be directly obtained by identification without any equipment.
The application of the rapid chromogenic parthenogenesis haploid induction line in identifying corn haploid is used for identifying conventional germplasm and carrying color suppressor geneZmC1-IOf europea duroplast or thermobanding material germplasm.
The application of the rapid chromogenic parthenogenesis haploid induction line in identifying corn haploid is used for defining haploid induction gene and smoothly expressing corn MYB family transcription factors, such asZmC1And homologous genes and bHLH family genes thereof, e.g.ZmR2The plant of (1) mainly comprises wheat, rice, Arabidopsis thaliana and the like.
A corn material for specific accumulation of anthocyanin in embryo is prepared from the anthocyaninMYB family transcription factor ZmC1Or homologous gene and bHLH family gene thereofZmR2Or the homologous gene of the single plant is hybridized with the single plant containing embryo or grain specific expression promoter PZmBD1 or 2RS5GPA to prepare the recombinant plasmid.
A corn parthenogenesis haploid inducer for fast development of young embryo is prepared from the gene containing dual-effect gene for controlling the corn parthenogenesis haploid to induceZmpla1/ZmmtlAndZmdmpthe induction line is hybridized with a maize strain with embryo or seed accumulating anthocyanin, and molecular marker assisted breeding is carried out to obtain the maize parthenogenesis haploid induction line with rapid chromogenic marker.
The invention has the beneficial effects that: by utilizing the parthenogenesis haploid induction line disclosed by the invention to be hybridized with conventional corn, anthocyanin can be biosynthesized and accumulated in a large amount for more than 12 days after pollination, so that haploid can be effectively identified. Compared with the conventional parthenogenesis haploid induction line, the parthenogenesis haploid induction line created by the invention can realize the direct identification of haploid embryos, greatly improve the efficiency of haploid embryo identification and reduce the workload of embryo taking and the consumption of culture materials. In addition, haploids in different growth periods, including immature embryos, seeds, seedlings and plants, can be identified, so that the comprehensive multiple identification of the haploids is realized, and the accuracy of the haploids identification can be greatly improved. In addition, the color marker of the parthenogenesis haploid inducer can be stably expressed in different maternal material backgrounds, and can be used for conventional parthenogenesis haploid inducerZmR1-njThe germplasm difficult in marker identification comprises haploid identification of germplasm such as European scleroid and tropical blood margin, and the germplasm materials contain color marker suppressor genesZmC1-I。ZmC1-IFor theZmC1Is dominant and affects competitivelyZmC1AndZmB/ZmRthe combination of family transcription factors and the interference of MBW complex on anthocyanin synthesis functional genesZmA2，ZmBz1Etc., thereby affecting the accumulation of anthocyanins. The invention uses embryo-specific or seed-specific expression strong promoter driverZmC1Expression, breakingZmC1-ITo pairZmC1The inhibition effect and the interference on the transcriptional activation of functional genes enable anthocyanin to be synthesized and accumulated under the conditions. For increasing jadeAn effective method is provided for the identification accuracy of the parthenogenesis haploids; meanwhile, the problem of difficulty in identifying the haploid of the hard-grain type and the hot strip material is solved, and in addition, a feasible scheme is provided for improving the efficiency of identifying the haploid embryo, so that the development of a haploid breeding technology is promoted.
Drawings
FIG. 1 is a diagram showing the structure of the pBD-C1R2 vector.
FIG. 2 is a breeding process of a rapid chromogenic parthenogenesis haploid inducer.
FIG. 3 shows induction of rapid color development parthenogenesis haploid inducible line.
FIG. 4 shows the identification of haploid by a rapid chromogenic parthenogenesis haploid inducer.
FIG. 5 shows molecular marker identification haploids (haploids contain only female parent chromosome set and are represented by 1 band, and hybrid diploids contain both male parent chromosome set and female parent chromosome set and are represented by 2 bands).
FIG. 6 shows haploid detection by flow cytometry.
FIG. 7 shows the identification of K22 haploid embryos.
FIG. 8 shows the identification of Yu 87-1 haploid embryos.
FIG. 9 shows the structure of the p2Seed-C1R2 vector.
FIG. 10 shows that Lx103-1 background induces Zhengdan 958 haploid differentiation effect.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Zea mays L hybrid Zhengdan 958 is the female parent in the present example (publicly available from Beijing German agro-farming Co., Ltd.); corn parthenogenesis haploid inducer: conventional haploid parthenogenesis-inducing lines CAU5, and CAU6 in the literature "Dongxi, 2014, maize haploid inducer genesqhir1Published in the research on the fine positioning and the breeding of the novel parthenogenesis haploid inducerAvailable from the university of Chinese agriculture. Contains embryo specific expression bidirectional promoter PZmBD and seed specific expression bidirectional promoter 2RS5GPA driveZmC1、ZmR2The germplasm Lx59(pBD-C1R2) and Lx103-1(p2Seed-C1R2) were created by the Chenomei researchers group, institute of Biotechnology, Chinese academy of agricultural sciences, and publicly available from the institute of Biotechnology, Chinese academy of agricultural sciences.
Example 1 creation of maize parthenogenesis haploid inducer lines
First, corn material preparation
Haploid parthenogenesis induction lines CAU5 and CAU6 are planted in the experimental base of China agricultural university, Tokyo, Beijing in 7 months in 2018, Lx59 is planted in the transgenic base of China agricultural academy of sciences, a carrier pBD-C1R2 is carried, the carrier structure is shown in figure 1, the carrier is matched with F1 in the same year, and then the parthenogenesis haploid induction line with haploid induction capability and containing the target expression carrier is created. The parthenogenesis haploid induction line is hybridized with a female parent material Zhengdan 958 to carry out haploid identification accuracy and haploid induction rate statistics, and haploid identification can be carried out after anthocyanin color marker expression.
Second, creation of fast developing parthenogenesis haploid induction line
Hybridizing parthenogenesis haploid inducing line with Lx59 plant containing target expression vector pBD-C1R2, backcrossing, and screening generation by combining molecular markerZmpla1/ZmmtlAndZmdmpthe individual of (1).Zmpla1/ZmmtlAndZmdmpdetection primers are described in the literature "Zhong Y, Liu C, Qi X, et al, Mutation of ZmDMP enghances halogenated introduction in mail [ J]Nature Plants, 2019, 5(6):575 & 580, "publicly available from the university of Chinese agriculture. The specific breeding process is shown in figure 2. To pair ofZmpla1/ZmmtlAndZmdmpthe homozygous single plant with the expression vector is used for detecting the induction rate and is used for evaluating the haploid identification accuracy rate and subsequently parthenogenesis haploid induction line single plantAnd (4) strain screening.
Identification of triple or haploid
The conventional parthenogenesis Haploid induction line can be used for inducing and identifying Haploid Immature Embryos in the literature "Chen C, Xiao Z, Zhang J, et al]Plants, 2020, 9(6):739 ", after crossing of a conventional parthenogenetic haploid inducer line,R1-njthe normal expression of the marker occurs about 20 days after pollination, the young embryos are more than 12 days after pollination, and after the whole young embryos are completely stripped, the isolated culture is needed, the culture condition is 24 hours of illumination, the temperature is 26-28 ℃, the humidity is 60%, the continuous culture is more than 24 hours, so that the heterozygous diploid can accumulate a large amount of anthocyanin and show purple, and the haploid young embryos are identified. In the haploid embryo identification, the embryo taking method refers to the document, but the heterozygous embryo 12 days after pollination has a large amount of anthocyanin accumulated on the ear, namely the heterozygous embryo presents a clear and identifiable purple color, the haploid embryo can be directly identified without additional culture, and the haploid embryo can be directly stripped for subsequent doubling or seedling culture. The induction rate of the parthenogenesis haploid induction line is generally below 15%, namely 75% of seeds on the hybrid ears are heterozygous diploids, so that the method has no obvious utilization value, the method for obtaining all the embryos in vitro to culture and identify the haploids greatly limits the efficiency of identifying the haploid embryos, and a large amount of manpower, material resources and financial resources are wasted. The parthenogenesis haploid induction line created by the method can improve the haploid embryo identification efficiency by 100%/15% =6.6 times, saves the time of in vitro culture, and greatly improves the haploid embryo identification efficiency. In addition, in vitro immature embryo culture is used for identifying haploid, heterozygous diploid immature embryoR1-njThe color marker expression is greatly influenced by the background of the parent material, the culture condition and the components of the culture medium, and the haploid identification accuracy is about 80 percent. The technology of the invention does not need the step of re-culturing the heterozygous immature embryo in vitro, and the pigment expression is stable, thus, the stable and high-efficiency haploid identification accuracy can be kept.
In European hard grains and partial temperate zone germplasm, shield slices or paste with different degrees appear in hybrid ears in the haploid induction process due to the fact that the germplasm contains pigment synthesis inhibiting genesThe problem of chromatism of the powder layer greatly limits the application of haploid breeding technology in the germplasms. The color marker of the parthenogenesis haploid inducer created by the invention is not influenced by material background, and can be directly used for carrying ZmC1-ILeading to haploid induction and identification of heterochromatic germplasm of heterozygous diploid embryo.
Example 2 method for identifying haploid embryos of maize
Production of mono-and haploid
1. Hybridization of
(1) Hybrid parents
Taking Zhengdan 958 as a female parent and a conventional parthenogenesis haploid induction line CAU5 as a male parent, and hybridizing to obtain a hybrid progeny 1;
taking Zhengdan 958 as a female parent and a conventional parthenogenesis haploid induction line CAU6 as a male parent, and hybridizing to obtain a hybrid progeny 2;
taking Zhengdan 958 as a female parent, and quickly developing a parthenogenesis haploid induction line as a male parent, and hybridizing to obtain a hybrid progeny 3;
(2) specific method of hybridization
Before the female parent filament is discharged, castration and female ear bagging are carried out on the female parent filament; and (3) cutting the filaments at the same time after the filaments are spitted out, uniformly pollinating for about 15 days in the next day, and obtaining hybrid progeny with the embryo length of 2.0-4.0 mm.
2. Obtaining young maize embryos
Taking hybrid ears about 15 days after pollination, disinfecting the ears and stripping young Embryos, wherein the specific steps are described in the reference of "Chen C, Xiao Z, Zhang J, et al, Development of in Vivo Haploid inductor Lines for Screening Haploid embryo organisms in Maize [ J ]. Plants, 2020, 9(6): 739"; near the axis side, flat in surface, and has a smaller embryo body composed of embryo bud, embryo axis, and radicle.
Identification of dihaploid
When the parthenogenesis haploid induction system is CAU5 or CAU6, the cluster grains are not synthesized with anthocyanin 15 days after pollination and are all represented as yellow grains, the young embryos are not colored, after culture is needed, haploid identification can be carried out according to the fact that the young embryo scutellum is accumulated with pigment, the hybrid diploid is colored, and the haploid young embryos are colorless.
When the parthenogenesis haploid induction line is a rapid chromogenic induction line, a visible anthocyanin marker exists 12 days after pollination, and about 15 days after pollination, namely when the haploid embryo is in a conventional identification period, a large amount of anthocyanin is accumulated in a hybrid diploid embryo and an aleurone layer, so that a colorless embryo, namely a pseudohaploid can be directly stripped and cultured in the practical application process (figures 3 and 4).
Statistics of triple and haploid inductivity
And (3) taking the hybrid ears about 15 days after pollination, stripping immature embryos, and respectively counting the haploid number (non-chromogenic immature embryos), the number of aborted embryo seeds (no embryos but normal endosperm development) and the heterozygous seed number (chromogenic immature embryos), so as to calculate the haploid inductivity of the single ear = haploid number/(haploid number + embryo aborted embryo seeds + heterozygous seeds) × 100%.
TABLE 1 haploid embryo induction statistics
Quadruple and haploid accuracy detection
192 and 194 single plants are randomly selected to induce Zheng 958 haploids 61 and 51, and two pairs of polymorphic molecular markers are used for detecting haploids, one haploid band and two heterozygous diploid bands. The results (figure 5) show that the wrong selection numbers of the haploids obtained by the induction of 192 and 194 are respectively 1 and 2, the haploidy accuracy rate = (pseudohaploid number-wrong haploid number)/pseudohaploid number is 100%, and the haploidy identification accuracy rates are both more than 95% and respectively reach 96.08% and 98.36%. 155 colored embryos are randomly selected to be identified as a pseudodiploid, and molecular marker detection finds that two strips exist, so that the diploid identification accuracy is 100%, and the haploid selection missing rate is 0.
Detecting molecular markers:
Chr5-214.6-F(5’-3’): AGCAGCACATCTTGTGGATGAGA；
Chr5-214.6-R(5’-3’): CATCATGTTGAAGGGGCATTTGT；
Chr6-163.9-F(5’-3’): GGATGGGAGAACTGGGTGAGG；
Chr6-163.9-R(5’-3’): GCTCGGAAGGCAATGAAATCG；
TABLE 2 identification of haploids by molecular marker detection
Cell flow ploidy detection:
haploid immature embryos are selected for seedling formation after molecular marker screening, and then 20 strains are randomly selected for flow cytometry detection to find that the haploid immature embryos are all haploid (figure 6). A detection instrument: partec CyFlow Space, kit: partec CyStain UV precision P. And a result judgment method comprises the following steps: the fluorescence intensity of the sample peak X-Mean (abscissa) is proportional to the cellular DNA content, so the ploidy is judged from the proportional relationship between the sample X-means.
Detection of whether or not parthenogenesis haploid induction line vector exists in penta-haploid and haploid
109 haploids obtained by molecular marker screening are detected by using Bar gene detection primers and are all found to be negative, so that the obtained haploids do not carry a transgenic screening marker Bar gene. The primers used were:
Bar-F (5’-3’): GGCGGTCTGCACCATCGTC；
Bar-R (5’-3’): CATGCCAGTTCCCGTGCTTGA。
example 3 for a composition comprisingZmC1Suppressor gene (A)ZmC1-I) Haploid induction experiment of selected material
Color suppression gene detection
Subjecting multiple inbred lines toZmC1-IThe gene detection shows that K22 and Yu 8701 containZmC1-IGene, and the use of the gene containing R1-njThe diploid embryos obtained by the marked conventional parthenogenesis haploid induction line are poor in coloration, and specific detection primers are as follows:
C1-IF(5’-3’): TACACTCGCCCTCATAGCAG
C1-IR(5’-3’): CAAACCTGCACCCACACAC
first, haploid embryo identification
Planting in experiment station of Beijing Chinese agriculture university in 5 months in 2020ZmC1-IThe germplasm K22 and Yu 8701 are matched with a conventional parthenogenesis haploid induction line CAU6 and a rapid chromogenic parthenogenesis haploid induction line. Taking embryos 15 days after pollination, and finding that the embryos are cultured for 0 h after CAU6 is induced (K22 in figure 7 and 8701 in figure 8), the hybridized embryos are basically not developed, and the embryos are slightly developed after 24 h of culture; after the induction line of the rapid chromogenic parthenogenesis haploid is induced, the induction line is cultured for 0 h, the hybridized immature embryo is developed, the haploid can be directly identified, and after the induction line is cultured for 24 h, the color development of the heterozygous immature embryo is more obvious and is obviously superior to that of a CAU6 induction group.
EXAMPLE 4 Omni-directional Multimarker identification of haploids
Firstly, preparation of fast color developing material
On the basis of a pBD-C1R2 vector, a 2RS5GPA promoter is added to carry a gene for controlling anthocyanin synthesis regulationZmC1，ZmR2Namely, an embryo-specific expression promoter PZmBD1 and an endosperm-specific expression promoter are simultaneously used for respectively driving and controlling anthocyanin synthesis regulation genesZmC1，ZmR2Obtaining a Seed anthocyanin specific expression vector p2Seed-C1R2, wherein the vector structure is shown in figure 9, and creating a Seed rapid color development transgenic material Lx 103-1.
Second, creation of fast developing parthenogenesis haploid induction line
Hybridizing a parthenogenesis haploid induction line CAU6 and a rapid grain color development material Lx103-1 to obtain F1Then obtaining separated offspring by means of selfing or backcrossing, finally utilizing molecular marker to detect that the gene contains homozygosisZmpla1/ZmmtlAndZmdmpmeanwhile, a parthenogenesis haploid induction line with high haploid induction rate and rapid color development is obtained from a single plant carrying a transformation event p2Seed-C1R 2.
Differential evaluation of triplex and haploid
Utilizing a rapid chromogenic parthenogenesis haploid induction system carrying a p2Seed-C1R2 carrier to induce an Zheng bill 958, and then respectively identifying haploid young embryos, mature seeds, seedlings with the length of 1-3 cm after pollination for about 15 days, seedlings and plants, which are shown in the following concrete expression (see figure 10):
identification of haploid immature embryos: after a rapid chromogenic parthenogenesis haploid induction line carrying a p2Seed-C1R2 carrier is hybridized with an Zheng 958, embryos are taken about 15 days after pollination, and the result shows that the hybridized diploid immature embryos have obvious anthocyanin accumulation, become purple and become colorless, so that the direct identification of the haploid immature embryos can be realized.
Identification of haploid mature seeds: after a rapid chromogenic parthenogenesis haploid induction line carrying a p2Seed-C1R2 carrier is hybridized with an Zheng 958, mature grains are harvested, and the result shows that the diploid and haploid aleurone layers of the hybridization show obvious color marks, while in the aspect of the embryo part of the grains, the diploid embryo of the hybridization shows purple red, the haploid embryo shows colorless, and the grain longitudinal cutting can show that the anthocyanin content difference of the embryo parts of the two parts is obvious, so that the identification of the mature grains of the haploid can be realized.
Identification of haploid sprouts: the method is characterized in that the fast chromogenic parthenogenesis haploid induction line carrying a p2Seed-C1R2 carrier and the Zheng 958 hybrid progeny mature Seed are subjected to germination accelerating and sprouting, and the result shows that heterozygous diploid shows obvious purple leaf sheath and purple root, the haploid bud sheath shows green or light purple, and the root is white, so that the identification of haploid bud seedlings can be realized according to the color marking expression of the bud sheath and the root.
Identification of haploid seedlings and plants: the hybrid diploid and haploid bud seedlings continue to grow to have two leaves and one heart, the bud sheaths and roots of the hybrid diploid seedlings still show purple, and the leaf tips show purple; the bud sheath of the haploid seedling is still green or light purple, the root is white, the leaf is green, the marker phenotype with the obvious color difference from that of the heterozygous diploid is provided, the haploid plant is green when the seedling is transplanted into the field, and the leaf sheath of the heterozygous diploid are light purple, so that the identification of the haploid seedling and the plant can be realized.
Claims (7)
1. A rapid color development parthenogenesis haploid inducer is characterized in that the inducer contains a two-effect gene for controlling maize parthenogenesis haploidZmpla1/ZmmtlAndZmdmpthe induction line of (A) and the promoter containing PZmBD1 carry a regulatory gene for controlling anthocyanin synthesisZmC1，ZmR2The individual strain pBD-C1R2 and promoter containing PZmBD1 and 2RS5GPA carry a regulatory gene for controlling anthocyanin synthesisZmC1，ZmR2Hybridizing the single plant p2Seed-C1R2, obtaining separated offspring by using a self-crossing or backcross mode, and finally detecting whether the single plant contains homozygous by using molecular markersZmpla1/ZmmtlAndZmdmpand the parthenogenesis haploid induction line which has high haploid induction rate and is marked by rapid color development is obtained by carrying a single plant of pBD-C1R2 and p2Seed-C1R 2.
2. The use of the rapid-chromogenic parthenogenetic haploid inducer system of claim 1 to identify maize haploids, wherein haploids are identified directly based on anthocyanin expression without in vitro culture for more than 12 days after pollination, and hybrid diploid aleurone layers and scutellum are colored; haploid grains are represented by a colored aleurone layer and colorless scutellum, and hybrid diploids are represented by a colored aleurone layer and scutellum; the haploid bud seedling is represented by green or light red coleoptile and white root, and the hybrid diploid is represented by dark purple coleoptile and root; haploid seedlings and plant leaves are green, and hybrid diploids are purple leaf tips; haploid embryos, seeds, seedlings and plants can be directly obtained by identification without any equipment.
3. Use of the rapid-chromogenic parthenogenetic haploid inducer line of claim 1 for identifying maize haploids for identifying conventional germplasm and carrying a color suppressor geneZmC1-IOf europea duroplast or thermobanding material germplasm.
4. The application of the rapid chromogenic parthenogenesis haploid inducer system of claim 1 in identifying corn haploid is characterized in that the rapid chromogenic parthenogenesis haploid inducer system is used for defining haploid induction genes and smoothly expressing corn MYB family transcription factors.
5. A corn material for embryo specific accumulation of anthocyanin is characterized in that MYB family transcription factor Zm involved in regulation and control of anthocyanin synthesis is utilizedC1Or homologous genes thereof and bHLH family genesZmR2Or homologous genes thereof, and a single plant containing an embryo specific expression promoter PZmBD1 or the same type of promoter thereof.
6. A corn material for embryo specific accumulation of anthocyanin is characterized in that MYB family transcription factor Zm involved in regulation and control of anthocyanin synthesis is utilizedC1Or homologous gene and bHLH family gene thereofZmR2Or the homologous gene of the single plant is hybridized with the single plant containing the seed specific expression promoter 2RS5GPA or the promoter of the same type.
7. A corn parthenogenesis haploid induction line with the rapid development of immature embryos is characterized in that a gene containing two main effects of controlling corn parthenogenesis haploid induction is utilizedZmpla1/ZmmtlAndZmdmpthe induction line is hybridized with a maize strain with embryo or seed accumulating anthocyanin, and molecular marker assisted breeding is carried out to obtain the maize parthenogenesis haploid induction line with rapid chromogenic marker.
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