Method for inducing haploids and use of said haploids in plant breeding

Abstract

The present invention belongs to the field of biotechnology and plant breeding, specifically, to a method for inducing haploidy using genes that control plant cell proliferation and its application in plant breeding. The present invention analyzes the expression profiles of rice unfertilized egg cells and embryos 5 days after fertilization, discovers two strongly expressed genes, OsCPRO1 and OsCPRO2, and reveals that these two genes can be used to induce haploid offspring. Specifically, a positive transgenic haploid inducer is produced by controlling the above transgenic plants with a promoter specifically expressed in egg cells. Furthermore, the haploid inducer can also be combined with the MiMe system to produce apomixis-free reproductive species, thereby producing cloned seeds.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology and plant breeding, and specifically relates to a method for controlling genetically induced haploids using plant cell proliferation and its application in plant breeding. [Background technology]

[0002] Compared with traditional breeding methods, haploid induction technology can obtain homozygous varieties in two generations, not only shortening the breeding period but also saving unnecessary labor costs. There are mainly two sources of haploids: one is from spontaneous formation in nature, and the other is obtained by artificial induction. Among these, the routes of spontaneous haploid formation in nature are mainly female parthenogenesis, male parthenogenesis and asexual reproduction, but the occurrence frequency of these three routes is very low (Zhang Zheng, 2007. Overview of research on haploid breeding of agricultural crops. Shandong Agricultural Science, 5: 122-125. DOI: 10.14083 / j.issn.1001-4942. 2007.05.025; Cui Ting, Li Yali, Qiao Linyi, Guo Huijuan, Dong Yanhui, Ren Yongkang and Tang Chaohui, 2016. Methods for haploid breeding of wheat and its research progress. Shanxi Agricultural Science, 44(1): 106-109. DOI: 10.3969 / j.issn.1002-2481. 2016.01.28.), which cannot meet the demand for breeding. Breeders usually incorporate artificially induced haploid methods to aid in breeding.

[0003] There are many methods to induce haploidy using artificial techniques, and the induction rate has been significantly improved compared to the frequency of natural occurrence. Among them, the methods to induce female parthenogenesis include radiation induction, chemical induction, heterologous pollen stimulation, and induction using male parthenogenesis induction lines (Ai Zhiguo, Haiyan, Kang Minghui, and Zhao Yongying, 2011. The pathway of haploid production and its application in crop genetic breeding. Henan Agricultural Science, 40(11):17-21. DOI:10.15933 / j.cnki.1004-3268.2011.11.009). Methods for inducing female parthenogenesis include pollen exosporium culture, anthium exosporium culture and hymenium induction (Li Ying, Wang Jia, Ji Lexiang, Li Hao, Ye Meixia, Guo Bin, Chen Zhong, An Xinmin, 2011. Advances in research on plant haploid technology and applications. Chinese Journal of Cell Biology, 33(9):1008-1014.). The method for inducing sporophyte reproduction is called female in vitro culture, which involves culturing non-pollinating organs such as ovaries and ovules outside the body to form new diploid plants. Haploid induction technology has been applied to the breeding of many crops, including rice, wheat, corn, cotton, barley, tobacco, and rapeseed, and its development in corn is the most mature, with the creation of a series of high-frequency induction systems such as WS14, EMK, NTU Gao Yi No. 1, and Ji Gao Yi No. 3. The induction rate of No. 1 of the National University of Agriculture and Technology's High Induction Line is 4%-6%, and that of No. 3 of the Yoshitaka Induction Line is 10%. The application of these induction systems has greatly accelerated the maize breeding process. (Cai Zhuo, Xu Guoliang, Liu Xianghui, Dong Yalin, Dai Yuxian, Li Shuhua, 2007. Selection and breeding of the high-frequency haploid reproductive induction system of maize, the Yoshitaka Induction Line No. 3. Maize Science, 15(1):1-4. DOI:10.13597 / j.cnki.maize.science.2007.01.001.) In 2015, CONNER et al. (CONNER JA, MOOKKAN M, HUO H, CHAE K, OZIAS-AKINS P, 2015. A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant. PNAS, 112(36): 11205-11210.DOI: 10.1073 / pnas.1505856112.) found that the PsASGR-BBML gene of Timothy grass is expressed in the ovaries before fertilization, developing seeds, roots, and anthers, and that the PsASGR-BBML transgene can be easily used to induce haploid embryos in Timothy grass, a sexually reproducing species. Subsequent work by CONNER et al. in 2017 (CONNER JA, PODIO M, OZIAS-AKINS P, 2017. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes. Plant Reprod, 30(1): 41-52.DOI: 10.1007 / s00497-017-0298-x.) found that the PsASGR-BBML transgene induced the production of monocotyledonous haploid embryos in two major monocotyledonous crops, maize and rice.In 2019, KHANDAY et al. (KHANDAY I, SKINNER D, YANG B, MERCIER R, SUNDARESAN V, 2019. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature, 565(7737): 91-95.DOI: 10.1038 / s41586-018-0785-8.) found that expression of BBM1 in rice starts in male gametes and activates expression of the female genome, which acts throughout embryo development, and that ectopic expression of OsBBM1 in oocytes using the AtDD45 promoter can induce solitary females, with induction rates of 5% to 10% and up to 29% in T2 generation transgenic materials. By combining ectopic expression of BBM1 in egg cells with the MiMe (osd1 / rec8 / spo11-1) system, i.e., substituting mitosis for meiosis in PGCs, we obtained clonal offspring that maintained the entire genome of the parent through parthenogenesis.

[0004] In 2017, KELLIHER et al. (KELLIHER T, STARR D, RICHBOURG L, CHINTAMANANI S, DELZER B, NUCCIO ML, GREEN J, CHEN ZY, MCCUISTON J, WANG WL,LIEBLER T, BULLOCK P, MARTIN B, 2017. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature, 542(7639): 105-109.DOI: 10.1038 / nature20827.) found that the MTL gene in maize is expressed in the sperm cytoplasm and that a transcoding mutation induces haploid production with an induction rate of 6.7%. In 2018, Yao et al. (YAO L, ZHANG Y, LIU CX, LIU YB, WANG YL, LIANG DW, LIU JT, SAHOO G, KELLIHER T, 2018. OsMATL mutation induces haploid seed formation in indica rice. Nature Plants,4(8): 530-533.DOI: 10.1038 / s41477-018-0193-y.) discovered OsMTL, which is homologous to ZmMTL, in order to develop a haploid induction gene in rice. They then discovered that knocking out this gene induced the production of haploids in rice. The induction rate was 2% to 6%, proving that MTL is also involved in the formation of mediated haploids in rice and can be used to construct a haploid induction system in rice.In 2019, WANG et al. (WANG C, LIU Q, SHEN Y, HUA YF, WANG JJ, LIN JR, WU MG, SUN TT, CHENG ZK, MERCIER R,WANG KJ, 2019. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nature Biotechnology, 37(3): 283-286.DOI: 10.1038 / s41587-018-0003-0.) knocked out the OsMTL gene in hybrid rice CY84 and obtained mtl haploid inducer material in the background of hybrid rice, with an induction rate of about 5% and a maturity rate of about 10%. Furthermore, by simultaneously knocking out four endogenous rice genes, REC8, PAIR1, OSD1, and MTL, we will obtain Fix (Fixation of hybrid) material that can induce apomixis, thereby completing the fixation of hybrid vigor.

[0005] Although there are many methods to induce haploidy, there are very few genes that can achieve haploid induction by single gene control, and even fewer control genes that can be used to produce clonal seeds through rice apomixis. Currently, only two genes, OsBBM1 and OsMTL, are used. Summary of the Invention

[0006] By analyzing the expression profiles of unfertilized rice egg cells and 5-day-old embryos, two genes with high expression were found, and it was finally determined that two new genes with haploid induction ability were found in rice, which were named OsCPRO1 (CELLPROLIFERATION1) and OsCPRO2 (CELLPROLIFERATION2). Here, the gene symbol of OsCPRO1 is LOC_Os03g47740, and the gene symbol of OsCPRO2 is LOC_Os12g43950. These two haploid induction genes can be applied to rice apomixis to fix the hybrid advantage and realize the formation of clonal seeds.

[0007] We discovered two new genes, OsCPRO1 (LOC_Os03g47740) and OsCPRO2 (LOC_Os12g43950), that have the ability to induce haploid cells in rice. The haploid cells induced by these genes have a high maturity rate and can be applied to rice apomixis.

[0008] Thus, the present invention provides a haploid-inducing protein having a POX superfamily domain and a homeobox domain. Specifically, the POX superfamily domain includes a TELL domain and a SKY domain, and is SKYLKAAQELLDEVVSV. Preferred is OsCPRO1 or OsCPRO2 protein, the amino acid sequences of which are set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. No. 6 or derived from the homologous genes of the following species, preferably 90%, 95% or more, more preferably 98%, 99% or more of the homologous genes being as follows: rice (Oryza sativa), Oryza meyeriana var, maize (Zeamays), wheat (Triticuma estivum), hard wheat (Triticum turgidum subsp.), wild wheat (Triticum dicoccoides), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), Aegilopstauschii, rye (Secalecereale), oat (Avenasativa), buckwheat (Fagopyrum esculentum), naked barley (Hordeum vulgare), Job's tears (Coixlac ryma-jobi), foxtail millet (Setaria italica), rice (Digitaria exilis), sorghum (Sorghum bicolor), millet (Panicum miliaceum), marshmallow (Zizania palustris), eleusine (Eleusine coracana), yam (Zizania palustris), cassava (Manihotesculenta), potato (Solanum tuberosum), Chinese yam (Dioscorea polystachya), peanut (Arachis hypogaea), groundnut (Arachis duranensis), soybean (Glycinemax), wild soybean (Glycinesoja), cosmic grass (Vigna angularis), rice bean (Vignaumbellata),Lentil (Lensculinaris), Green bean (Vignaradiata), Cowpea (Vignaunguiculata), Common bean (Phaseolusvulgaris), Broad bean (Viciafaba), Pea (Pisumsativum), Sword bean (Canavaliagladiata), Pigeon pea (Cajanuscajan), Lupinusalbus, Mucunapruriens, Lupinusangustifolius, Kalopogoni Calopogonium mucunoides, Cicerarietinum, Sesame, Linumusitatissimum, Brassicanapus, Sunflower, Ricinus communis, Carthamus tinctorius, Betavulgaris, Sugarcane, Gossypium officinarum, Cotton pp), Cannabis sativa, Selaginella moellendorffii, Lemon eucalyptus, Tetracentronsinense, Prosopis alba, Taxus chinensis, Olive, Abrusprecatorius, Panicum willigatum mvirgatum, Rhamnella rubrinervis, Caryaillinoinensis, Camellia sinensis, Nicotianatabacum, Pennisetum purpureum, Lolium rigidum, Miscanthus lutarioriparius, Setariaviridis,Pennisetum alopecuroides, Sorghum sudanense, Thalictrum thalictroides, Eragrostis curvula, Brachypodium distachyon, Lotus corniculatus L, Arabidopsis thaliana, Physco Physcomitriumpatens, Ceratodonpurpureus, Brassicaoleracea, Brassicajuncea, Brassicarapa, Brassicaoleracea, Lactucasativa, Spinaciaoleracea, Raphanussatius ivus, Chinese cabbage (Brassicarapa), Cucurbita pepo, Apium graveolens, Allium tuberosum, Daucus carota, Solanum melongena, Lycopersicone sculentum, Cucumissativus, Benincasa hisspida, Cucurbita Cucurbitamoschata, Luffacylindrica, Cucumismelo, Potentilla anserina, Hemerocalliscitrina, Lactucasativa, Asparagus officinalis, Capsicumannuum, Allium sativum,Allium fistulosum, Helianthus tuberosus, Coriandrum sativum, Watermelon (Citrullus lanatus), Pear (Pyrus spp), Peach (Prunus persica), Apricot (Armeniacavulgaris), Plum (Prunus salicina), Jujube (Ziziphus jujuba), Vaticamangachapoi, Apple (Maluspumila), Coral apple (Malus asiatica), Cherry (Cerasus spp), Juglansregia, strawberry (Fragaria×ananassa), grape (Vitisvinifera), pineapple (Ananascomosus), Coptischinensis, Astragalussinicus, ginseng (Panaxginseng), angelica (Angelicasinensis), honeysuckle (Lonicerajaponica), bloodroot (Artemisiaargyi), mint (Menthacanadensis), orchid (Cymbidiumssp), lily (Liliumbrownii), tulip (Tulipagesneriana).

[0009] Similarly, the present invention provides a gene encoding a protein which is OsCPRO1 or OsCPRO2, the gene symbol of OsCPRO1 is LOC_Os03g47740, and the gene symbol of OsCPRO2 is LOC_Os12g43950; or BEL1-like homeodomain protein 6 from maize, Sequence ID: PWZ21223.1; or BEL1-like homeodomain protein 7 from wheat, Sequence ID: XP_044365192.1; or BEL1-like homeodomain protein 1 from soybean, Sequence ID: XP_003543416.1; or BEL1-like homeodomain protein 2 from peanut, Sequence ID: XP_003543416.1. 1, Sequence ID: XP_016170518.1, or a gene having at least 90%, 95% or more, more preferably 98% or 99% or more of the same species-derived homologue as the above gene, and still having the ability to induce haploidy.

[0010] More preferably, the nucleotide sequence of OsCPRO1 gene is as set forth in SEQ ID NO:1, or is more than 90%, 95%, more preferably more than 98%, 99% or more of the homologous gene derived from rice and has haploid inducibility. The nucleotide sequence of OsCPRO2 gene is as set forth in SEQ ID NO:4, or is more preferably more than 98%, 99% or more of the homologous gene derived from rice and has haploid inducibility.

[0011] Also preferred is a CDS nucleotide sequence of OsCPRO1 as set forth in SEQ ID NO:2, or a nucleotide sequence derived from rice that has haploid inducibility and has greater than 98% or 99% identity thereto and still has haploid inducibility. The CDS nucleotide sequence of OsCPRO2 as set forth in SEQ ID NO:5, or a nucleotide sequence derived from rice that still has haploid inducibility and has greater than 98% or 99% identity thereto.

[0012] The present invention provides gene cassettes, recombinant proteins, recombinants or host cells comprising the genes.

[0013] The present invention provides for the use of said proteins or said genes in derived plant haploids.

[0014] The present invention further provides a method for utilizing a transgenic plant haploid, the method comprising the steps of: introducing a recombinant expression vector containing a gene into a plant to obtain a positive transgenic line, the gene being controlled by an egg cell-specific expression promoter.Preferably, the plant includes monocotyledonous and dicotyledonous plants.

[0015] Preferably, the egg-specific expression promoter is the AtDD45 egg-specific expression promoter, more preferably, its nucleotide sequence is as shown in SEQ ID NO: 7. The introduction is performed by Agrobacterium-mediated or gene shot or gene editing.

[0016] More specifically, the method includes the steps of investigating the field phenotypic traits of wild-type plants and T0 generation transgenic positive plants; harvesting seeds of the T0 generation transgenic plants and performing a germination process to obtain T1 generation transgenic lines; performing haploid testing on the T1 generation transgenic plants using Indel mark or flow cytometry ploidy testing techniques to obtain haploid materials; and combining the haploid-derived materials with the MiMe system to obtain apomixis materials.

[0017] The only haploid induction genes discovered with current technology are OsBBM1 and OsMTL, which are used to artificially create apomixis. The two novel genes with haploid induction ability discovered in this invention (OsCPRO1 and OsCPRO2) have been confirmed to be applicable to apomixis for producing cloned seeds, and have high application value. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the plant types of the T0 generation OsCPRO series haploid derivatives, including: CY84: control; OsCPRO-ee: haploid derivatives; Percentage: maturity rate; Proportion scale=10 cm. [Diagram 2] Figure 2 shows the spike types of T0 generation OsCPRO series haploid derivatives, including: CY84: control; OsCPRO-ee: haploid derivatives; Percentage: maturity rate; Proportion scale=5cm. [Diagram 3] Figure 3 shows the genotypes of the Indel marks used for haploid testing. [Figure 4] Figure 4. Indel-marked examination of haploid and double haploid materials in the T1 generation. 16A, C84, CY84: parental controls; numbers: numbers of haploid and diploid materials. [Diagram 5] Figure 5 shows flow cytometry ploidy testing of haploid and double haploid materials in the T1 generation, where A: haploid and diploid controls; B: haploid and double haploid materials. [Figure 6] Figure 6 shows the comparison of the properties of haploid material and wild type CY84. A: Comparison of plant types, scale=10 cm; B: Comparison of spike types, scale=5 cm. [Figure 7] Figure 7 shows the comparison of properties between double haploid materials and wild-type CY84, where A: comparison of plant types, scale=10cm; B: comparison of spike types, scale=5cm. [Figure 8] Figure 8 shows the comparison of properties of haploid material and wild type Arabidopsis of Col-0 ecotype. A: wild type, B: haploid, proportional scale = 1 cm. [Figure 9] FIG. 9 is an outline of the technical direction of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be further described with reference to a combination of examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0020] Example 1: Discovery and characterization of OsCPRO1 and OsCPRO2 By analyzing the expression profiles of unfertilized rice egg cells and 5-day-old embryos, two genes with high expression were found, and finally, it was confirmed that two new genes with haploid induction ability were found in rice, which were named OsCPRO1(CELLPROLIFERATION1) and OsCPRO2(CELLPROLIFERATION2), respectively. Here, the gene symbols of OsCPRO1 and OsCPRO2 are LOC_Os03g47740 and LOC_Os12g43950, respectively.

[0021] 1. OsCPRO1 (LOC_Os03g47740) (1) The sequence of the OsCPRO1 (LOC_Os03g47740) gene is shown in SEQ ID NO:1. (2) The CDS sequence of OsCPRO1 (LOC_Os03g47740) is shown in SEQ ID NO:2. (3) The amino acid sequence of OsCPRO1 (LOC_Os03g47740) is shown in SEQ ID NO:3. (4) Key structural domains of OsCPRO1 (LOC_Os03g47740): [1] POX superfamily domain (pfam07526): A family of protein domains associated with nuclear function. They include the TELL domain: LQNKMAKLMAMLDEVDRKYKHYYHQMQIVVSSFDMVAGSGAAKPYTAVALQTISKHFRCLKDAINDQINVIRKKLGEEESSSGKEGKLTRLRYIDQQLRQQRAFQQYGLLQ and the SKY structural domain: SKYLKAAQELLDEVVSV. [2] Homeobox domain (pfam05920): This is a homeobox transcription factor KN structural domain that is conserved from fungi to humans and plants. They were first identified in eukaryotes as TALE homeobox genes (including KNOX and MEIS genes).

[0022] 2. OsCPRO2 (LOC_Os12g43950) (1) The sequence of the OsCPRO2 (LOC_Os12g43950) gene is shown in SEQ ID NO:4. (2) The CDS sequence of OsCPRO2 (LOC_Os12g43950) is shown in SEQ ID NO:5. (3) OsCPRO2 (LOC_Os12g43950) The amino acid sequence is as shown in SEQ ID NO:6. (4) Key structural domains of OsCPRO2 (LOC_Os12g43950): [1] POX superfamily domain (pfam07526): A family of protein domains associated with nuclear function. They include the TELL structural domain: LQNKMAKLMAMLDEVDRKYKHYYHQMQTVVSSFDVVAGPGSAKPYTAVALQTISRHFRCLKDAINDQINVIRKKLGEEENSSGKEGKLTRLRYIDQQLRQQRAFQQYGMIP and the SKY structural domain: SRYLKAAQELLDEVVSV. [2] Homeobox domain (pfam05920): This is a homeobox transcription factor KN domain that is conserved from fungi to humans and plants. They were first identified in eukaryotes as TALE homeobox genes (including KNOX and MEIS genes).

[0023] 3. Species containing the cognate genes OsCPRO1 and OsCPRO2 Using the NCBI database, we compared the amino acid sequences of corn, wheat, soybean, and peanut for homology. The results of the comparison of the amino acid sequences of the homologous genes OsCPRO1 and OsCPRO2 are as follows.

[0024] Maize [Zea mays], BEL1-like homeodomain protein 6, Sequence ID: PWZ21223.1 Length: 643 Number of Matches: 1, Range 1: 1 to 634。 TIFF2024545555000002.tif 117170 Wheat [Triticum aestivum], BEL1-like homeodomain protein 7, Sequence ID: XP_044365192.1 Length: 638 Number of Matches: 1, Range 1: 1 to 626。 TIFF2024545555000003.tif 112170 Soybean [Glycine max], BEL1-like homeodomain protein 1, Sequence ID: XP_003543416.1 Length: 702 Number of Matches: 1, Range 1: 166 to 489。 TIFF2024545555000004.tif 72170 Peanut [Arachis ipaensis], BEL1-like homeodomain protein 1, Sequence ID: XP_016170518.1 Length: 733 Number of Matches: 1, Range 1: 278 to 488。 TIFF2024545555000005.tif51170The results showed that the homologous genes exist in species including, but not limited to, rice (Oryza sativa), Oryza meyeriana subspecies (Oryza meyeriana var), maize (Zeamays), wheat (Triticuma estivum), hard wheat (Triticum turgidum subsp.), wild wheat (Triticum dicoccoides), Urartu wheat (Triticum urartu), barley (Hordeum vu lgare, Aegilopstauschii, Rye, Oats, Buckwheat, Hordeumvulgare, Coixlacryma-jobi, Setariaitalica, Digitariaexilis, Sorghum, Sorghum bicolor, Panicum miliaceum, Zizanial atifolia, marshmallow (Zizaniapalustris), eleusine (Eleusinecoracana), yam (Zizaniapalustris), cassava (Manihotesculenta), potato (Solanumtuberosum), Chinese yam (Dioscoreapolystachya), peanut (Arachishypogaea), groundnut (Arachisduranensis), soybean (Glycinemax), wild soybean (Glycinesoja), cosmic grass (Vigil) naangularis), Rice bean (Vignaumbellata), Lentil (Lensculinaris), Green bean (Vignaradiata), Cowpea (Vignaunguiculata), French bean (Phaseolusvulgaris), Broad bean (Viciafaba), Pea (Pisumsativum), Jack bean (Canavalia gladiata), Pigeon pea (Cajanuscajan), Lupinus lubus (Lupinusalbus), Mucunapruriens (Mucunapruriens),Lupinus angustifolius, Calopogonium mucunoides, Cicerarietinum, Sesame, Linumusitatissimum, Brassicanapus, Sunflower, Ricinus communis, Carthamus tinctorius, White beet, Sugarcane, accharum officinarum, cotton (Gossypium spp), cannabis (Cannabissativa), Selaginella moellendorffii, lemon eucalyptus (Corymbia citriodora), Tetracentronsinense, Prosopisalba, Taxus chinensis, olive (Oleaeuropaea), Abras precatorius rusprecatorius, Panicum virgatum, Rhamnella rubrinervis, Caryaillinoinensis, Camellia sinensis, Nicotianatabacum, Pennisetum purpureum, Lolium rigidum, Miscanthus lutar ioriparius, Setariaviridis, Pennisetum alopeculoides, Sorghum sudanense, Thalictrum thalictroides, Eragrostis curvula, Brachypodium distachyon, Lotus corniculatus L,Arabidopsisthaliana, Physcomitriumpatens, Ceratodonpurpureus, Brassicaoleracea, Brassicajuncea, Brassicarapa, Brassicaoleracea, Lactucasativa, Spinaciaoleracea pinaciaoleracea, Raphanus sativus, Brassicarapa, Cucurbita pepo, Apium graveolens, Allium tuberosum, Daucus carota, Solanum melongena, Lycopersicone sculentum, Cucumis ativus, Benincasa hisspida, Cucurbitamoschata, Luffacylindrica, Cucumis melo, Potentilla anserina, Hemerocalliscitrina, Lactucasativa, Asparagus officinalis, Capsicum annuum Capsicumannuum, Allium sativum, Allium fistulosum, Helianthus tuberosus, Coriander (Coriandrum sativum), Watermelon (Citrullus lanatus), Pear (Pyrus spp), Peach (Prunus persica), Apricot (Armeniacavulgaris), Plum (Prunus salicina), Jujube (Ziziphus jujuba),Vaticamangachapoi, Apple (Maluspumila), Coral Apple (Malusasiatica), Cherry (Cerasusspp), Walnut (Juglansregia), Strawberry (Fragaria×ananassa), Grape (Vitisvinifera), Pineapple (Ananascomosus), Coptischinensis, Astragalussinicus, Ginseng (Panaxginseng), Angelica (Angelicasinensis), Honeysuckle (Lonicerajaponica), Red Root (Artemisiaargyi), Mint (Menthacanadensis), Orchid (Cymbidiumssp), Lily (Liliumbrownii), Tulip (Tulipagesneriana).

[0025] Example 2: Method for inducing haploid cells using OsCPRO1 and OsCPRO2 The method of inducing haploidy using OsCPRO1 and OsCPRO2 in the present invention is summarized as follows (Figure 9): [1] A specific expression promoter is used in AtDD45 egg cells to drive the CDS sequence or genomic sequence of OsCPRO1 and OsCPRO2 genes, respectively, as shown in SEQ ID NO:7, to construct an ectopic expression vector; [2] An Agrobacterium method is used to obtain transgenic plants; [3] Field phenotypic traits of wild-type CY84 and T0 generation transgenic positive plants are investigated; [4] Indel marks for haploidy testing are designed; [5] The maturation rate of T0 generation transgenic plants is measured and germination treatment is performed to obtain T1 generation transgenic plants; [6] Indel marks are used to perform haploid testing of T1 generation transgenic plants; [7] The haploid induction rate of T1 generation transgenic plants is statistically calculated; [8] The haploid induction material is combined with the MiMe system to obtain apomixis material. An overview of the technology pathway is shown in Figure 9.

[0026] Experimental Example 1 1. Experimental Materials In this example, the indica-japonica triple hybrid rice Shunyou 84 (CY84) was selected as the genetically modified material. CY84 was developed by crossbreeding the japonica-sterile Shun'e 16A as the female and the indica-restorer C84 as the male. This hybrid rice has a long growing period and is suitable for single-season late rice cultivation. It also has vigorous growth, large ears, and many grains. It has a thick stem and is resistant to lodging.

[0027] 2. Constructing Ectopic Expression Vectors The AtDD45 oocyte-specific expression promoter was selected to drive the CDS sequences of OsCPRO1 and OsCPRO2 genes, respectively, to complete the ectopic expression process.

[0028] 2.1 Obtain fragments required for vector construction for ectopic expression.

[0029] (1) Genomic DNA was extracted from Arabidopsis leaves and used as a template to amplify the AtDD45 promoter sequence.

[0030] DNA extraction method: [1] Take about 2 cm of rice leaves, put them into a 2 mL centrifuge tube containing steel beads, number them, and then add 500 μL of CTAB extraction solution. [2] Transfer the centrifuge tube to a tissue cell disrupter and vibrate and grind them at 60 Hz / sec for 2 minutes. [3] Place the ground sample in a 65 °C oven for 30 minutes, during which it was rotated upside down and shaken for 10 minutes / time. [4] Take out the sample, centrifuge at 12,000 rpm for 30 seconds, add 300 μL of chloroform in a fume hood, and mix by turning upside down. [5] Centrifuge again at 12,000 rpm for 8 minutes. After centrifugation, take 350 μL of the supernatant, put it into a sterilized centrifuge tube, add 700 μL of cooled absolute ethanol, shake gently to mix evenly, then transfer to a -20 °C refrigerator and leave it for 15 minutes. [6] After the sample has settled, place it in a centrifuge and centrifuge it at 12,000 rpm for 10 minutes, discard the supernatant, and dry it at room temperature. [7] Add 100 μL of ddH2O to dissolve it, shake gently, and store it in a refrigerator at 4°C for later use.

[0031] (2) RNA was extracted from CY84 rice spikelets and then reverse transcribed into cDNA sequences to provide templates for amplification of the CDS sequences of genes OsCPRO1 and OsCPRO2.

[0032] RNA extraction method: [1] Place 3-4 cm of rice ears in a sterile mortar precooled with liquid nitrogen, grind clockwise, and after it becomes a whitish-green powder, quickly transfer it to an RNase-free EP tube with 1 mL of Trizol and vortex for 1 minute. Mix thoroughly for 1 minute. [2] Incubate at room temperature for 5 minutes, add 200 μL of chloroform to the EP tube, vortex for 15 seconds, and leave on ice for 2 minutes. [3] High-speed refrigerated centrifugation at 4 °C, centrifugation at 12,000 rpm for 15 minutes. [4] Aspirate 500 μL of the supernatant, add 100 μL of chloroform, vortex for 15 seconds, and leave on ice for 2 minutes. [5] High-speed refrigerated centrifugation at 4 °C, centrifugation at 12,000 rpm for 15 minutes. [6] Pipette 400 μL of the supernatant into a 1.5 mL RNase-free EP tube, add 500 μL isopropanol, and mix by inversion. [7] Leave on ice for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the supernatant. [8] Add 1 mL of 75% ethanol, vortex to wash the precipitate, and centrifuge at 12,000 rpm for 10 minutes at 4°C. [9] Discard the supernatant and repeat the previous step.

[10] Leave the RNA precipitate at the bottom of the tube, suction off the remaining liquid, pat dry, add 100 μL of RNase-free water, flick to dissolve, check concentration and purity, and store at -80°C for later use.

[0033] How to convert RNA reverse transcription to cDNA: Use HiFiScript cDNA Synthesis Kit (CW2569M) to create cDNA. [1] Place the reagents and RNA template provided in the kit on ice and dissolve and store. [2] Mix the reagents according to the RNA reverse transcription system (Table 1) and centrifuge briefly to concentrate the liquid on the tube wall at the bottom of the tube. [3] Then run the reaction program and react at 42°C for 15 minutes, then at 85°C for 5 minutes. [4] After the reaction, cool the cDNA on ice for later use. TIFF2024545555000006.tif75170

[0034] (3) Designing amplification primers. Amplification primers for the AtDD45 promoter sequence and the CDS sequences of the OsCPRO1 and OsCPRO2 genes are shown in Table 2. Lowercase letters indicate adapter primers. TIFF2024545555000007.tif64170

[0035] (4) Amplification primers were used to amplify the promoter sequence and gene CDS sequence. The amplification scheme is shown in Table 3, and the amplification process is shown in Table 4. TIFF2024545555000008.tif90170

[0036] (5) After checking by 1% agarose gel electrophoresis, cut the gel to recover the required fragment and store at -20°C for later use.

[0037] 2.2 Enzyme-cleaved backbone vector The pCAMBIA1300-ACTIN backbone vector was digested using KpnI-HF and BamHI-HF endonucleases to remove the original ACTIN promoter sequence, create sticky end deletions, and retain all other sequences, including the original terminator sequence. The enzyme cleavage scheme is shown in Table 5. TIFF2024545555000009.tif45170

[0038] 2.3 Ligation of the recovered amplified fragment into the backbone vector The coupling system is shown in Table 6, and after mixing the system, it is coupled at 50° C. for 15 minutes, and after coupling, the product is kept on ice before use. TIFF2024545555000010.tif47170

[0039] 2.4 Converting the ligation product Method for transforming the ligation product: [1] Take out the competent cells (provided by our laboratory) stored in a -80°C refrigerator and place on ice to thaw. [2] When the competent cells (50 μL to 100 μL) reach a molten state, immediately add the ligation product. [3] Leave on ice for 30 minutes. [4] Heat shock in a 42°C water bath for 90 seconds. [5] Add 500 μL of liquid LB medium. [6] Incubate in a shaker at 37°C for 1 hour. [7] Centrifuge at low speed at 4000 rpm for 3 minutes and spread on Kan-resistant LB solid medium. [8] Incubate overnight at 37°C and perform colony PCR testing after plaque growth. [9] Select a single colony in the Kan-resistant liquid medium and incubate in a thermostatic shaker at 200 rpm at 37°C for 14 hours.

[10] Extract the plasmid and perform Sanger sequencing.

[0040] 3. Obtaining genetically modified strains The ectopic expression vector was introduced into Agrobacterium tumefaciens EHA105 by electroporation, and then introduced into calli of hybrid rice CY84 using the Agrobacterium method.

[0041] Specific transformation method: [1] Sterilize the embryos of hybrid rice CY84 seeds. [2] Inoculate the medium of induced callus. [3] After one week of culture, actively growing, light yellow and relatively loose embryo-shaped callus was selected as the recipient for transformation. [4] Infect the rice callus with EHA105 strain containing pC1300-AtDD45P-OsCPRO1 plasmid and pC1300-AtDD45P-OsCPRO2 plasmid, respectively. [5] Incubate in the dark at 25℃ for 3 days. [6] Use selection medium containing 50mg / L hygromycin to screen resistant callus and transgenic strains. [7] Select transgenic strains that grow normally.

[0042] 4. Cultivation of genetically modified strains The GM materials are grown in the GM laboratory in summer and in greenhouses in winter, with an average daytime temperature of 34°C and an average nighttime temperature of 25°C, a 12-hour light / 12-hour dark cycle, and 75% humidity.

[0043] 5. Testing for genetically modified strains 5.1 Design test primers.

[0044] A primer designed from the AtDD45 promoter sequence and a primer designed from the gene CDS sequence (spanning the intron region) were used simultaneously as test primers for transgenic positive plants. The specific primer information is shown in Table 7. The TIFF2024545555000011.tif501705.2 inspection system is shown in Table 8, and the inspection procedure is shown in Table 9. TIFF2024545555000012.tif78170

[0045] 6. Examine the phenotype of the field Field phenotyping included plant type, spike type and maturity rate. 6.1. To investigate the plant type (Figure 1) and panicle type (Figure 2), the mutant and wild-type CY84 were selected and comparatively observed (n=3). 6.2. Statistics on maturity rate: Three ears of rice are selected for each plant and statistics are performed. Calculation formula: maturity rate = (number of grains / total number of grains) x 100%.

[0046] 7. Select haploids using Indel marks Based on the whole genome sequences of hybrid rice CY84 and its two parents (16A and C84), one pair of Indel marks was designed on each rice chromosome, and a total of 12 pairs of primer marks were designed. The primer information is shown in Table 10, and the gene classification is shown in Figure 3, which is used to test the haploid induction rate of the genetically modified materials in the T1 generation. If all 12 pairs of Indel marks show the same single-band genotype as the parent 16A or C84, rather than the double-band genotype as the CY84 heterozygous genotype, the probability that it is haploid or double haploid reaches 99.98%. The specific calculation method is 1-(1 / 2) 12 = 99.98%. The inspection status of Indel marks in haploid and double haploid materials of T1 generation is shown in Figure 4.

[0047] TIFF2024545555000013.tif157170

[0048] 8. Flow cytometry polyploidy test For the haploid or double haploid plants selected for the indel mark, we further confirmed the cytodidity using flow cytometry techniques (Figure 5). The comparison of the properties of the haploid material and wild-type CY84 is shown in Figure 6, and the comparison of the properties of the double haploid material and wild-type CY84 is shown in Figure 7.

[0049] Flow cytometry polyploidy test method: [1] Cut fresh young leaves into 3 cm pieces, place them in a sterile glass dish, and place the dish on a tray containing ice. [2] Add 1 mL of plant lysis buffer (LB01) to the dish, chop the leaves with a blade, and soak the leaf tissue cells in the lysis buffer. [3] Aspirate the lysate in the dish, filter through a 50 μm nylon mesh, transfer to a 1.5 mL sterile centrifuge tube, and store on ice. [4] Centrifuge at 1200 rpm for 5 minutes at 4°C. [5] Discard the supernatant and add 450 μL of pre-cooled LB01, 25 μL of PI (1 mg / mL), and 25 μL of RNaseA (1 mg / mL). [6] Stain for 10 minutes on ice protected from light. [7] On-machine test of BDAccuriC6.

[0050] 9. Whole Genome Sequencing Analysis DNA is extracted from the leaves of hybrid rice CY84, the two parents of CY84, 16A and C84, and haploid plants, and whole genome sequencing is performed. The results of whole genome sequencing show that there are many different homozygous genotypes between 16A and C84. The genotypes at these positions in CY84 are heterozygous genotypes, including both 16A and C84 genotypes. The positions in the haploid plants are homozygous genotypes of 16A or C84.

[0051] Experimental Example 2 1. Experimental materials: See Experimental Example 1 2. Constructing Ectopic Expression Vectors The AtDD45 egg-cell-specific expulsion promoter was selected to drive the genomic sequences of OsCPRO1 and OsCPRO2 genes, respectively, to complete the ectopic expulsion process.

[0052] 2.1 Obtain fragments required for vector construction for ectopic expression. (1) Genomic DNA was extracted from Arabidopsis leaves and used as a template to amplify the AtDD45 promoter sequence. For the DNA extraction method, please refer to Example 1. (2) Genomic DNA is extracted from rice leaves and used as a template to amplify the genomic sequences of OsCPRO1 and OsCPRO2 genes. (3) Amplification primers are designed and used to amplify the AtDD45 promoter sequence and OsCPRO1 and OsCPRO2 genomic sequences. (4) After checking by 1% agarose gel electrophoresis, cut the gel to recover the required fragments and store them at -20°C for later use.

[0053] 2.2 Enzyme-cleaved backbone vector: See Example 1.

[0054] 2.3 Ligate the recovered amplified fragment into the backbone vector: See Example 1.

[0055] 2.4 Converting the ligation product: See Example 1.

[0056] 3. Obtain the genetically modified strain: See Example 1.

[0057] 4. Plant the genetically modified strain: See Example 1.

[0058] 5. Testing for genetically modified strains Design primers on the AtDD45 promoter sequence as test primers for transgenic plants. See Table 7 in Experimental Example 1 for primer information. See Table 8 in Experimental Example 1 for the test system. See Table 9 in Experimental Example 1 for the amplification program.

[0059] 6. Investigate the phenotype of the field: See Example Experiment 1.

[0060] 7. Screening for haploids using Indel marks: See Example Experiment 1.

[0061] 8. Flow cytometry polymorphism test: See Example 1.

[0062] 9. Whole genome sequencing analysis: See Example Experiment 1.

[0063] Experimental Example 3 1. Experimental materials: Wild Arabidopsis thaliana of Columbia (Col-0) ecotype was used as the experimental material.

[0064] 2. Constructing Ectopic Expression Vectors The AtDD45 egg-specific expression promoter was selected to drive the CDS sequences of AtCPRO1 and AtCPRO2 genes, respectively, to complete the ectopic expression process.

[0065] 2.1 Obtaining fragments required for construction of vectors for ectopic expression (1) Genomic DNA was extracted from Arabidopsis leaves and used as a template to amplify the AtDD45 promoter sequence. For the DNA extraction method, please refer to Example 1. (2) Extract RNA from young flowers of Arabidopsis thaliana of Columbia ecotype as material and reverse transcribe it into cDNA sequences to provide templates for amplification of CDS sequences of genes AtCPRO1 and AtCPRO2. For the method of extracting RNA and reverse transcribing RNA into cDNA, please refer to Example 1. (3) Amplification primers were designed and used to amplify the AtDD45 promoter sequence and the CDS sequences of the AtCPRO1 and AtCPRO2 genes. For the amplification scheme, see Table 3 in Example 1. For the amplification program, see Table 4 in Example 1. (4) After checking by 1% agarose gel electrophoresis, cut the gel to recover the required fragments and store them at -20°C for later use.

[0066] 2.2 Enzyme-cleaved backbone vector: See Example 1.

[0067] 2.3 Ligate the recovered amplified fragment into the backbone vector: See Example 1.

[0068] 2.4 Converting the ligation product: See Example 1.

[0069] 3. Obtaining genetically modified strains The ectopic expression vector is introduced into Agrobacterium tumefaciens GV310 strain by electroporation, and the ectopic expression vector is introduced into Col-0 ecotype wild Arabidopsis thaliana using the Agrobacterium-mediated flower dipping method.

[0070] The specific transformation method is: [1] Immerse the inflorescences of T0 generation Arabidopsis thaliana in a suspension of Agrobacterium tumefaciens GV310 strain containing a certain concentration of the target transformation plasmid. [2] Cultivate under specific conditions. [3] Collect the seeds of T0 generation Arabidopsis thaliana after maturation. [4] These seeds are placed on a medium containing a specific antibiotic for growth screening, and positive plantlets are obtained.

[0071] 4. Cultivation of genetically modified strains The genetically modified materials are grown in light-illuminated incubation boxes. Light treatment is 15 hours, with an average temperature of 24°C; dark treatment is 9 hours, with an average temperature of 22°C. The average soil humidity is 75%, with a ratio of nutrient soil:bentonite=1:1. Nutrient solution is allowed to seep through the bottom of the tray every 3-4 days.

[0072] 5. Testing for genetically modified strains Primers are designed on the CDS sequence, and the primers span the intron region, and these primers are used as detection primers for gene recombination positive strains. For the test system, please refer to Table 8 in Example 1. For the amplification process, please refer to Table 9 in Example 1.

[0073] 6. Characterize the phenotype Phenotypic characteristics include plant shape and maturity rate.

[0074] 6.1To investigate the plant type of the T2 generation plants (Figure 8), mutant and wild-type Arabidopsis were selected for comparative observation (n=3).

[0075] 6.2 Statistics of maturity rate: Collect mature but not yet cracked wild-type and mutant Arabidopsis fruits and bleach them to transparency in bleaching solution (ethanol:acetic acid = 3:1). When the seeds are clearly visible inside the fruits, remove them and statistics of seed maturity rate under a microscope.

[0076] 7. Flow cytometry polyploidy test Use flow cytometry technology to check the cell ploidy of plants. Please refer to Example 1 for the method of flow cytometry ploidy test.

[0077] Experimental Example 4 1. Materials: See Example 1.

[0078] 2. Construction of ectopic expression vector: See Example 1.

[0079] 3. Construction of multigene knockout vectors The CRISPR-Cas9 multi-gene knockout system was used to simultaneously knock out three endogenous rice genes OsOSD1, OsPAIR1 and OsREC8 to construct MiMe materials. The main steps are as follows (the operation details are also described in CN201510485573.2): 3.1 Design target sequences and primers (1) Design of target sequence: Find a NNNNNNNNNNNNNNNNNNN (NGG) specific sequence with GC content of 35%-75% at the target position of the gene. The following four sites were selected as the sites for the CRISPR-Cas9 gene editing system to knock out OsOSD1, OsPAIR1, and OsREC8, respectively (PAM sequences are underlined): Knockout site of OsOSD1 gene: CTGCCGCCGACGAGCAACA AGG Knockout site of OsPAIR1 gene: AAGCAACCCAGTGCACCGC TGG Knockout site of OsREC8 gene: CGGAGAGCCTTAGTGCCAT GGG

[0080] (2) Primer design: add four GGCA nucleotides before the forward target sequence and add four AAAC nucleotides before the reverse target sequence, and name them as g++ and g--, respectively, for primer synthesis.

[0081] 3.2 Construction of intermediate vectors (1) The intermediate vector SK-gRNA is cleaved using AarI enzyme to form sticky ends. The cleavage scheme is shown in Table 11. TIFF2024545555000014.tif45170

[0082] (2) Mix 20 μL each of 100 μM g++ and g-- primers in equal proportions and incubate at 100° C. for 5 minutes to form sticky ends after denaturation and annealing.

[0083] (3) Ligation using T4 DNA ligase and allowing to ligate for 1 hour at room temperature. The ligation scheme is shown in Table 12. TIFF2024545555000015.tif47170

[0084] (4) Conversion of ligated products: See Example 1.

[0085] 3.3 Constructing the final vector Using the properties of BamHI and BglII as isocaudarners, polymerization of three SK-gRNA intermediate vectors was completed using T4 DNA ligase. The final vector was digested with KpnI and BamHI, the provided fragments were enzymatically cleaved with KpnI and BglII, and the ligation between the SK-gRNAs was performed using two pairs of isocaudarners NheI / XbaI and SalI / XhoI. The scheme for ligating multiple intermediate vectors is shown in Table 13. TIFF2024545555000016.tif58170

[0086] 4. Constructing a Binary Fusion Expression Vector The ectopic expression vector and the multi-gene knockout vector are combined to construct a binary fusion expression vector, in which the ectopic expression vector provides the required fragment and the multi-gene knockout vector serves as the backbone vector for enzymatic cleavage.

[0087] 4.1 Obtaining fragments required for constructing double-element fusion expression vectors Amplify the AtDD45 egg cell specific expression promoter from the ectopic expression vector to drive the OsCPRO1 and OsCPRO2 gene CDS sequences, completing the overall fragment of the ectopic expression process, including the AtDD45 promoter fragment, the OsCPRO1 and OsCPRO2 gene CDS sequences and the terminator sequence. The amplification primers are shown in Table 14, and the lower case letters represent the adapter primers. TIFF2024545555000017.tif27170

[0088] 4.2 Enzyme-cleaved multi-gene knockout vector Use PmeI endonuclease to synthesize pC1300-Cas9-gRNA OSD1 -gRNA PAIR1 -gRNA REC8 Digest the multigene knockout vector. The enzyme digestion system is shown in Table 15. TIFF2024545555000018.tif49170

[0089] 4.3 Ligating amplified fragments into multi-gene knockout vector The ligation system is shown in Table 16 and after making a homogenous system, ligation is carried out for 15 minutes at 50° C. After ligation, the product is kept on ice. TIFF2024545555000019.tif60170

[0090] 4.4 Converting the ligation product: See Example 1.

[0091] 5. Obtain the genetically modified strain: See Example 1.

[0092] 6. Planting the genetically modified strain: See Example 1.

[0093] 7. Testing for genetically modified strains 7.1 Positive test for genetically modified strains: See Example 1.

[0094] 7.2 Inspection of knockout status at target sites (1) Design target site inspection primers to determine the knockout status of OsOSD1, OsPAIR1, and OsREC8 genes. Primer information is shown in Table 17. Lowercase letters indicate primers. TIFF2024545555000020.tif50170(2) Sequence analysis: The PCR amplified products were sent to the China Rice Research Institute, and data analysis was performed using the Hi-TOM (http: / / www.hi-tom.net / hi-tom / ) platform to obtain mutation information of the three genes.

[0095] 8. Investigate the phenotype of the field: See Example Experiment 1.

[0096] 9. Flow cytometry polyploidy test 9.1 Material preparation The material with successful binary fusion expression was selected, i.e., the AtDD45 promoter drives the expression of OsCPRO1 / OsCPRO2 gene CDS sequence well, and three genes, OsOSD1, OsPAIR1, and OsREC8, were well knocked out (MiMe). Seeds of this material were germinated and cultivated (first generation of offspring).

[0097] 9.2 Flow cytometry testing Using flow cytometry analysis, the ploidy of the cells can be sorted and the progeny plants with the same ploidy as the mother plant can be selected to obtain apomictic material.

[0098] 10. Whole genome sequencing analysis: See Example 1.

[0099] Experimental Example 5 1. Materials: See Example 1.

[0100] 2. Construction of ectopic expression vector: See Example 2.

[0101] 3. Construct a multi-gene knockout vector. See Example 4.

[0102] 4. Constructing a Binary Fusion Expression Vector The ectopic expression vector and the multi-gene knockout vector are combined to construct a binary fusion expression vector, in which the ectopic expression vector provides the required fragment and the multi-gene knockout vector serves as the backbone vector for enzymatic cleavage.

[0103] 4.1 Obtaining fragments required for constructing double-element fusion expression vectors Amplify the promoter of AtDD45 egg-specific expression from the ectopic expression vector, driving the genomic sequence of OsCPRO1 and OsCPRO2 genes, and complete the entire fragment of the ectopic expression process, including the AtDD45 promoter fragment, the genomic sequence of OsCPRO1 and OsCPRO2 genes, and the terminator sequence. The amplification primers are shown in Table 14 of Example 3.

[0104] 4.2 Enzyme-cleaved multi-gene knockout vector: See Example 4.

[0105] 4.3 Integrating amplified fragments into a multi-gene knockout vector: See Example 4.

[0106] 4.4 Converting the ligation product: See Example 1.

[0107] 5. Obtain the genetically modified strain: See Example 1.

[0108] 6. Planting the genetically modified strain: See Example 1.

[0109] 7. Testing for genetically modified strains 7.1 Positive test for genetically modified strains: See Example 2.

[0110] 7.2 Testing for knockout status at target site: See Example 4.

[0111] 8. Investigate the phenotype of the field: See Example Experiment 1.

[0112] 9. Flow cytometry polymorphism test: See Example 4.

[0113] 10. Whole genome sequencing analysis: See Example Experiment 1.

[0114] Example 3 1. Phenotypic characteristics of T0 generation OsCPRO series haploid derivatives For Experimental Examples 1 and 2 in Example 2, a field phenotypic survey of transgenic plants showed that there was no significant difference in vegetative growth of T0 generation transgenic plants compared to wild-type CY84, and the maturity rate was reduced. For five change ranges of 80-100%, 50-80%, 30-50%, 10-30%, and 0-10%, a summary of plant types is shown in Figure 1, and a summary of spike types is shown in Figure 2.

[0115] 2. Gene classification of 12Indel marks As shown in FIG. 3, in Experiments 1 and 2 of Example 2, 12 pairs of Indel marks for selecting haploids and double haploids have obvious polymorphisms and can be used for genotyping of genetically modified materials.

[0116] 3. Genotyping and ploidy testing of haploid and double haploid material For the haploid or double haploid materials screened in Experimental Examples 1 and 2 in Example 2, the results of the ectopic expression of the CDS sequence of OsCPRO2 by the egg cell-specific expression promoter AtDD45 in Experimental Example 1 are taken as an example: among the genetically modified materials, 12 pairs of Indel-marked sites in four strains were screened, all of which showed a single-band genotype (Figure 4), indicating that they were haploid or double haploid materials. Furthermore, using flow cytometry, two strains, CPRO2-22-1 and CPRO2-39-1, were confirmed to have haploid (n) genomes and were haploid materials. The other two strains (CPRO2-32-1 and CPRO2-32-2) had a double (2n) genome and were double haploid materials (Figure 5).

[0117] 4. Characterization of haploid and double haploid material For the haploid and double haploid materials screened in Experimental Examples 1 and 2 of Example 2, the AtDD45 follicle-specific expression promoter in Experimental Example 1 drives OsCPRO2 to complete ectopic expression. The haploid materials are significantly affected in both vegetative and reproductive growth compared to wild-type CY84: plant height is reduced, ear length is shortened, culms are small, and fertility is completely lost (Figure 6). There is no significant difference in both vegetative and reproductive growth compared to wild-type CY84 (Figure 7). The haploid materials selected in Experimental Example 3 of Example 2 are significantly affected in both vegetative and reproductive growth compared to wild Arabidopsis of Col-0 ecotype: plant type is smaller, leaves are thinner (Figure 8).

[0118] 5. Testing status of CPRO series haploid derived materials and apomixis materials Testing the T1 generation transgenic plants of Experimental Examples 1, 2, and 3 in Example 2 revealed that the maturation rate of the haploid inducible materials ranged from 18.98±2.14% to 50.88±0.74%. The induction rate ranged from 0.44% to 7.14%, and the specific statistics are shown in Table 18. Testing the T1 generation transgenic plants of Experimental Examples 4 and 5 in Example 2 revealed that the apomixis materials produced by ectopic expression of OsCPRO1 and OsCPRO2 genes in egg cells combined with the MiMe system had a clone seed rate of 0.95% to 7.92%, and the specific statistics are shown in Table 19.

[0119] TIFF2024545555000021.tif190170 Wild-type CY84 was used as a control, data were t-tested, error bars represent standard deviation (n=3), **P<0.01.

[0120] TIFF2024545555000022.tif128170 Wild-type CY84 was used as a control, and data were t-tested; error bars represent standard deviation (n=3), **P<0.01.

Claims

1. A haploid inducing protein having a POX domain and a homeobox, specifically, the POX domain includes a TELL domain and a SKY domain: SKYLKAAQELLDEVVSV, and is preferably OsCPRO1 or OsCPRO2 protein, the amino acid sequences of which are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Proteins derived from the homologous genes of the following species, preferably 90%, 95% or more, more preferably 98%, 99% or more of which are the homologous genes of the following species: rice (Oryza sativa), Oryza meyeriana var, maize (Zeamays), wheat (Triticuma estivum), hard wheat (Triticum turgidum subsp.), wild two-grain wheat (Triticum dicoccoides), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), Aegilopstauschii, rye (Secalecereale), oats (Avenasativa), buckwheat (Fagopyrum esculentum), naked barley (Hordeum vulgare), pearl barley (Coixlacryma-jobi), foxtail millet (Setaria italica), foxtail millet (Fagopyrum esculentum), foxtail millet (Fagopyrum vulgare), foxtail millet (Fagopyrum esculentum ... Digitariaexilis, Sorghumbicolor, Panicummiliaceum, Zizaniaalatifolia, Marshmallow, Eleusinecoracana, Yam, Manihotesculenta, Solanumtuberosum, Dioscoreapolystachya, Peanut, Arachishypogaea, Arachisduranensis, Soybean, Glycinemax, Wild Soybean, Vignaangularis, Rice Bean, Vignaumbellata, Lentil, Vignaradiata, Cowpea, Vignaunguiculata,Phaseolus vulgaris, broad bean, pea, canavalia gladiata, pigeon pea, lupinus albus, mucuna pruriens, lupinus angustifolius, calopogonium mucunoides, sesame, mindicum), flax (Linumusitatissimum), rapeseed (Brassicanapus), sunflower (Helianthusannuus), castor bean (Ricinuscommunis), sugarcane (Carthamus tinctorius), white beet (Betavulgaris), sugarcane (Saccharum officinarum), cotton (Gossypiumspp), cannabis (Cannabissativa), Selaginella moellendorffii, lemon eucalyptus Corymbia citriodora, Tetracentronsinense, Prosopisalba, Taxus chinensis, Oleaeuropaea, Abrusprecatorius, Panicum virgatum, Rhamnella rubrinervis, Caryai illinensis, llinoinensis, Camellia sinensis, Nicotianatabacum, Pennisetum purpureum, Lolium rigidum, Miscanthus lutarioriparius, Setariaviridis, Pennisetumalopecuroides, Sorghum sudanense,Thalictrum thalictroides, Eragrostis curvula, Brachypodium distachyon, Lotus corniculatus L, Arabidopsis thaliana, Physcomitrium patens, Ceratodon purpureus, Brachypodium distachyon, Brassica oleracea, Brassica juncea, Brassica rapa, Brassica oleracea, Lactucasativa, Spinacia oleracea, Raphanus sativus, Brassica arapa, Cucurbita pepo, Apium graveolens raveolens, Allium tuberosum, Daucus carota, Solanum melongena, Lycopersicon esculentum, Cucumissativus, Benincasa hisspida, Cucurbitamoschata, Luffacylindrica, Cucumis melo smelo), Potentilla anserina, Hemerocalliscitrina, Lactucasativa, Asparagus officinalis, Capsicum annuum, Allium sativum, Allium fistulosum, Helianthus tuberosus),Coriander (Coriandrum sativum), Watermelon (Citrullus lanatus), Pear (Pyrus spp), Peach (Prunus persica), Apricot (Armeniacavulgaris), Plum (Prunus salicina), Jujube (Ziziphus jujuba), Vaticamangachapoi, Apple (Maluspumila), Coral Apple (Malus asiatica), Cherry (Cerasus spp), Walnut (Juglansregia), Strawberry (Fragaria × ana nassa), grape (Vitis vinifera), pineapple (Ananascomosus), Coptis chinensis, burdock (Astragalussinicus), ginseng (Panaxginseng), angelica (Angelicasinensis), honeysuckle (Lonicera japonica), red root (Artemisiaargyi), mint (Menthacanadensis), orchid (Cymbidiumssp), lily (Liliumbrownii), tulip (Tulipagesneriana).

2. encoding OsCPRO1 or OsCPRO2, where the gene symbol for OsCPRO1 is LOC_Os03g47740 and the gene symbol for OsCPRO2 is LOC_Os12g43950, or BEL1-like homeodomain protein 6 from maize (Sequence ID: PWZ21223.1), or BEL1-like homeodomain protein 7 from wheat (Sequence ID: XP_044365192.1), or BEL1-like homeodomain protein 1 from soybean (Sequence ID: XP_003543416.1), or BEL1-like homeodomain protein 1 from peanut (Sequence ID: XP_016170518.1), or a gene that is at least 90%, 95% or more, more preferably 98% or 99% or more of the same species as the above gene and still has the ability to induce haploidy and encodes the protein described in claim 1.

3. The gene according to claim 2, characterized in that the nucleotide sequence of OsCPRO1 is set forth in SEQ ID NO:1, and is at least 90%, 95% or more, more preferably 98% or 99% of the homologous gene derived from rice, and still has the ability to induce haploidy; the nucleotide sequence of OsCPRO2 gene is set forth in SEQ ID NO:4, and is preferably greater than 98%, 99% or more of the homologous gene derived from rice, and still has the ability to induce haploidy.

4. The gene described in claim 2, characterized in that the CDS nucleotide sequence of OsCPRO1 is set forth in SEQ ID NO: 2 or is a nucleotide sequence derived from rice, which still has haploid inducibility and has an identity of more than 98% and greater than 99% thereto, and still has haploid inducibility, and the CDS nucleotide sequence of OsCPRO2 is set forth in SEQ ID NO: 5 or is a nucleotide sequence derived from rice, which still has haploid inducibility and has an identity of more than 98% and greater than 99% thereto.

5. A cassette, recombinant protein, recombinant or host cell comprising a gene according to any one of claims 2 to 4.

6. Use of the protein according to claim 1 or the gene according to any one of claims 2 to 4 in inducing plant haploidy, preferably in the plant, wherein the plant is one of the following: rice (Oryza sativa), Oryza meyeriana var, maize (Zeamays), wheat (Triticuma estivum), hard wheat (Triticum turgidum subsp.), wild two-grain wheat (Triticum dicoccoides), Urartu wheat (Triticum u rartu), Barley (Hordeumvulgare), Aegilopstauschii, Rye (Secalecereale), Oats (Avenasativa), Buckwheat (Fagopyrum esculentum), Barley (Hordeumvulgare), Coixlacryma-jobi, Foxtail millet (Setaria italica), Fornio (Digitariaexilis), Sorghum (Sorghum bicolor), Millet (Panicummi liaceum), Zizania alatifolia, Zizania palustris, Eleusine coracana, Zizania palustris, Manihotesculenta, Solanum tuberosum, Dioscorea polystachya, Arachis hypogaea, Arachis duranensis, Glycine max, Wild Soybean (Glycinesoja), Cosmic grass (Vigna angularis), Rice bean (Vignaumbellata), Lentil (Lensculinaris), Green bean (Vignaradiata), Cowpea (Vignaunguiculata), Common bean (Phaseolusvulgaris), Broad bean (Viciafaba), Pea (Pisumsativum), Jack bean (Canavalia gladiata), Pigeon pea (Cajanus cajan), Lupinus rubus (Lupinus albus),Mucuna pruriens, Lupinus angustifolius, Calopogonium mucunoides, Cicerarietinum, Sesamumindicum, Linumusitatissimum, Brassicanapus, Sunflower, Ricinus communis, Carthamus tinctorius ), White beet (Betavulgaris), Sugarcane (Saccharum officinarum), Cotton (Gossypium spp), Cannabis (Cannabissativa), Selaginella moellendorffii, Lemon eucalyptus (Corymbia citriodora), Tetracentronsinense, Prosopisalba, Taxus chinensis, Olive Oleaeuropaea, Abrusprecatorius, Panicumvirgatum, Rhamnellarubrinervis, Caryaillinoinensis, Camellia sinensis, Nicotianatabacum, Pennisetumpurpureum, Italian ryegrass (L. olium rigidum), Miscanthus lutarioriparius, Setariaviridis, Pennisetum alopecuroides, Sorghum sudanense, Thalictrum thalictroides, Eragrostis curvula, Brachypodium distachyon,LotuscorniculatusL, Arabidopsisthaliana, Physcomitriumpatens, Ceratodonpurpureus, Brassicaoleracea, Brassicajuncea, Brassicaarapa, Brassicaoleracea, Lactuca sativa Lactuca sativa, Spinacia oleracea, Raphanus sativus, Brassicarapa, Cucurbita pepo, Apium graveolens, Allium tuberosum, Daucus carota, Solanum melongena, Lycopersicon esculentum onesculentum, Cucumissativus, Benincasa hisspida, Cucurbitamoschata, Luffacylindrica, Cucumismelo, Potentilla anserina, Hemerocalliscitrina, Lactucasativa, Asparagus officinalis gusofficinalis), Capsicum annuum, Allium sativum, Allium fistulosum, Helianthus tuberosus), Coriandrum sativum, Watermelon, Pear, Peach, Armenia cavulgaris, Plum,Jujube (Ziziphus jujuba), Vaticamangachapoi (Vaticamangachapoi), Apple (Maluspumila), Coral Apple (Malus asiatica), Cherry (Cerasus spp), Walnut (Juglansregia), Strawberry (Fragaria × ananassa), Grape (Vitis vinifera), Pineapple (Ananascomosus), Coptis chinensis, Purple berry (Astragalus sinicus), Ginseng (Panax ginseng), Angelica (Angelicasinensis), Honeysuckle (Lonicera japonica), Red root (Artemisia argyi), Mint (Menthacanadensis), Orchid (Cymbidium ssp), Lily (Lilium brownii), Tulip (Tulipagesneriana). ,

7. A method for inducing plant haploidy using the gene according to any one of claims 2 to 4, comprising the steps of: introducing a reconstructed expression vector containing a predetermined gene into a plant to obtain a positive transgenic line, the gene being controlled by an egg cell-specific expression promoter, preferably the plant includes monocotyledonous and dicotyledonous plants, preferably the plant is one of the following: rice (Oryza sativa) sativa, Oryza meyeriana subsp. (Oryza meyeriana var), Maize (Zeamays), Wheat (Triticuma estivum), Hard wheat (Triticum turgidum subsp.), Wild wheat (Triticum dicoccoides), Urartu wheat (Triticum urartu), Barley (Hordeum vulgare), Aegilopstauschii, Rye (Secalecereale), Oats (Avenasativa), Buckwheat (Fagopyrum esculentum), Naked barley (Hordeum vulgare), Job's tears (Coixlacryma-jobi), Foxtail millet (Setaria italica), Fornio rice (Digitaria exilis), Sorghum (Sorghum bicolor), Millet (Panicum miliac eum), Zizania alatifolia, Zizania palustris, Eleusine coracana, Zizania yams, Manihotesculenta, Solanum tuberosum, Dioscorea polystachya, Arachis hypogaea, Arachis duranensis, Glycine max, Glycinesoja, Vigna angularis, Vigna umbellata, Lentils, Vigna radiata, Vigna unguiculata, Phaseolus vulgaris, Broad beans, Viciafaba,Pea (Pisum sativum), Jack bean (Canavalia gladiata), Pigeon pea (Cajanus cajan), Lupinus albus, Mucuna pruriens, Lupinus angustifolius, Calopogonium mucunoides, Cicerarietinum, Sesamumindicum, Flax (Linumus itatissimum), Brassica napus, sicanapus), sunflower (Helianthusannuus), castor bean (Ricinuscommunis), sugar beet (Carthamus tinctorius), white beet (Betavulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), cannabis (Cannabissativa), Selaginella moellendorffii, lemon eucalyptus (Corymbia citriodora), Tetracentron sinense (Tetrac entronsinense, Prosopisalba, Taxus chinensis, Oleaeuropaea, Abrusprecatorius, Panicum virgatum, Rhamnella rubrinervis, Caryaillinoinensis, Camellia sinensis , Nicotianatabacum, Pennisetumpurpureum, Loliumrigidum, Miscanthuslutarioriparius, Setariaviridis, Pennisetumalopeculoides, Sorghumsudanense, Thalictrumthalictroides,Eragrostiscurvula, Brachypodium distachyon, LotuscorniculatusL, Arabidopsisthaliana, Physcomitriumpatens, Ceratodonpurpureus, Brassicaoleracea, Brassicajuncea sicajuncea, Brassica rapa, Brassica oleracea, Lactucasativa, Spinacia oleracea, Raphanus sativus, Brassicarapa, Cucurbita pepo, Apium graveolens, Allium tuberosum, Daucus carota sativa, Daucus carota, Solanum melongena, Lycopersicone sculentum, Cucumissativus, Benincasa hisspida, Cucurbitamoschata, Luffacylindrica, Cucumis melo, Potentilla anserina, Hemerocallis serrata Hemerocalliscitrina, Lactucasativa, Asparagusofficinalis, Capsicumannuum, Alliumsativum, Alliumfistulosum, Helianthustuberosus, Coriandrumsativum, Citrulluslanatus,Pear (Pyrusspp), Peach (Prunuspersica), Apricot (Armeniacavulgaris), Plum (Prunussalicina), Jujube (Ziziphusjujuba), Vaticamangachapoi, Apple (Maluspumila), Coral Apple (Malusasiatica), Cherry (Cerasusspp), Walnut (Juglansregia), Strawberry (Fragaria×ananassa), Grape (Vitisvinifera ), Pineapple (Ananascomosus), Coptischinensis, Astragalussinicus, Panax ginseng (Panaxginseng), Angelica (Angelicasinensis), Honeysuckle (Lonicerajaponica), Artemisiaargyi, Mint (Menthacanadensis), Orchid (Cymbidiumssp), Lily (Liliumbrownii), Tulip (Tulipagesneriana).,

8. 8. The method according to claim 7, characterized in that the egg-specific expression promoter is the AtDD45 egg-specific expression promoter, preferably the nucleotide sequence of which is shown in SEQ ID NO:7, and said introduction is performed by Agrobacterium-mediated method or gene shot method or gene editing method.

9. 9. The method according to claim 7 or 8, further comprising the step of conducting a haploid test from a first generation progeny plant of the genetically modified positive material to obtain haploid material.

10. 10. The method of claim 9, further comprising combining haploid derived material with a MiMe system to obtain apomixis material.

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