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A METHOD FOR INDUCING HAPLOIDS AND ITS APPLICATION IN PLANT BREEDING

20240409591 ยท 2024-12-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to the field of biotechnology and plant breeding, and in particular relates to a method using plant cell proliferation regulator gene to induce haploid, and a use thereof in plant breeding. In the present invention, expression profiles of unfertilized egg cells of rice and rice embryos 5 days after fertilization are analyzed, and 2 genes are discovered to have relatively high expression, OsCPRO1 and OsCPRO2. The research shows that these two genes can be utilized to induce the production of haploid descendants. Specifically, a promoter specifically expressed in egg cells controls the gene to convert a plant, obtaining induced haploid plant material of a positive transgenic plant. Furthermore, the induced haploid plant material can be combined with a MiMe system to obtain apomixes material, and thereby generate clone seeds.

    Claims

    1. A protein with haploid induction ability, among which are the POX superfamily domain and the Homeobox domain, specifically, POX, the superfamily domain containing BELL domain and SKY domain: SKYLKAAQELLDEVVSV; preferably it is a OsCPRO1 or OsCPRO2 protein, amino acid sequences are shown as in SEQ ID NO: 3 and SEQ ID NO: 6, respectively, or their orthologous genes derived from the following species, optimally greater than 90%, 95%, more than 98% or 99%: rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana(Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata), pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia (Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinesecabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina), day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Allium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba), green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinifera), pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), tulip (Tulipa gesneriana).

    2. The gene encoding a protein as described in claim 1, which is either OsCPRO1 or OsCPRO2, where the gene symbol of OsCPRO1 is LOC_Os03g47740, the gene symbol of OsCPRO2 is LOC_Os12g43950; Or BEL1-like homeodomain protein 6 derived from maize, Sequence ID: PWZ21223.1; Or BEL1-like homeodomain protein 7 derived from wheat, Sequence ID: XP_044365192.1; Or BEL1-like homeodomain protein 1 derived from soybean, Sequence ID: XP_003543416.1; Or BEL1-like homeodomain protein 1 from groundpeanut, Sequence ID: XP_016170518.1; Or at least 90%, 95% of the above genes, more preferably more than 98%, or 99% of the homologous genes derived from the same species, and still has the haploid induction ability.

    3. The gene with haploid induction ability as described in claim 2 is characterized by the nucleotide sequence of OsCPRO1 as shown in SEQ ID NO: 1, or more than 90%, 95%, 98% and 99% of homologous genes derived from rice and still have haploid induction ability; the nucleotide sequence of OsCPRO2 as shown in SEQ ID NO: 4, preferably more than 98% and 99% of homologous genes derived from rice, and still has haploid induction ability.

    4. The gene having the haploid induction ability as described in claim 2, it is characterized by the fact that, the CDS nucleotide sequence of OsCPRO1 is as shown in SEQ ID NO: 2, or is derived from rice that still has a haploid-inducing nucleotide sequence with greater than 98% or 99% identity, and still has the haploid induction ability; the CDS nucleotide sequence of OsCPRO2 is as shown in SEQ ID NO: 5, or derived from rice still has haploid induction ability with more than 98% and 99% identity.

    5. An expression cassette, a recombinant vector, a recombinant cell, or a host cell containing a gene according to claim 2.

    6. The protein according to claim 1, wherein the plant is selected from the group consisting of rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana (Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata), pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia(Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinesecabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina), day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Allium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba), green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinfera), pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), and tulip (Tulipa gesneriana).

    7. A method of inducing plant haploids using a gene according to claim 1, comprising the steps of including the gene to obtain a positive transgene plant controlled by the promoter for specific expression in egg cells of the gene and, preferably, the plant including a monocot and a dicotyledon; preferred of all, the plant was selected from rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana(Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata), pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia(Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinesecabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina), day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Ailium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba), green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinifera), pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), tulip (Tulipa gesneriana).

    8. The method of claim 7, wherein the promoter for egg specific expression is a promoter of AtDD45 egg specific expression; preferably with the nucleotide sequence shown in SEQ ID NO: 7; the import is an Agrobacterium-mediated or gene gun or gene editing method.

    9. The method of claim 7, comprising the step of taking transgenic positive material to obtain haploid material.

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

    Description

    ATTACHED FIGURE DESCRIPTION

    [0018] FIG. 1. Plant type of the T.sub.0generation OsCPRO series of haploid-inducing materials. Where, CY84: control; OsCPRO-ee: haploid induction material; percentage: seed set; scale bar=10 cm.

    [0019] FIG. 2. Head pattern of the T.sub.0generation OsCPRO series of haploid-inducing material. Where, CY84: control; OsCPRO-ee: haploid induction material; percentage: seed set; scale bar=5 cm.

    [0020] FIG. 3. Genotyping of the Indel markers used for haploid detection.

    [0021] FIG. 4. Detection of Indel markers in T.sub.1generation haploid and double haploid material. Where, 16A, C84 and CY84: parental control; numbers for haploid and double haploid material.

    [0022] FIG. 5. Flow cytometric ploidy detection of T.sub.1 generation haploid and double haploid materials. Where, A: haploid and diploid control; B: haploid and double haploid material.

    [0023] FIG. 6. Comparison of haploid material with wild-type CY84, where A: comparison of plant type, scale bar=10 cm; B: comparison of spike type, scale bar=5 cm.

    [0024] FIG. 7. Trait comparison of double-haploid material with wild-type CY84. A: comparison of plant type, scale=10 cm; B: comparison of ear type, scale=5 cm.

    [0025] FIG. 8. Trait comparison between haploid material and Col-0 ecotype wild Arabidopsis. A: wild-type, B: haploid, scale bar=1 cm.

    [0026] FIG. 9. Overview of the technical route of the present invention.

    SPECIFIC IMPLEMENTATION METHODS

    [0027] The present invention is further explained in combination with the embodiments. These embodiments are used only to illustrate the invention and not to limit the scope of the invention.

    Example 1: Discovery and Characteristics of OsCPRO1 and OsCPRO2

    [0028] Through the analysis of expression profiles of rice unfertilized egg cells and embryos 5 days after fertilization, two genes with high expression were found, and two new genes with haploid induction ability were found in rice, named OsCPRO1 (CELL PROLIFERATION1) and OsCPRO2 (CELL PROLIFERATION2) respectively, the gene symbol of OsCPRO1 was LOC 0s03g47740, and the gene symbol of OsCPRO2 was LOC_Os12g43950.

    1. OsCPRO1 (LOC_Os03g47740)

    [0029] (1) Gene sequence of OsCPRO1 (LOC 0s03g47740), as shown in SEQ ID NO: 1. [0030] (2) The OsCPRO1 (LOC 0s03g47740) CDS sequence, as shown in SEQ ID NO: 2. [0031] (3) The amino acid sequence of OsCPRO1 (LOC 0s03g47740), as shown in SEQ ID NO: 3. [0032] (4) OsCPRO1 (LOC 0s03g47740) important domains: [0033] {circle around (1)} POX superfamily domains (pfam07526): a family of protein domains associated with nuclear function. Contains the -BELL domain: LQNKMAKLMAMLDEVDRKYKHYYHQMQIVVSSFDMVAGSGAAKPYTAVALQTISKHF RCLKDAINDQINVIRKKLGEEESSSGKEGKLTRLRYIDQQLRQQRAFQQYGLLQ and the SKY domain: SKYLKAAQELLDEVVSV. {circle around (2)} Homeobox domain (pfam05920): this is a homeodomain transcription factor KN domain conserved from fungi to humans and plants. They were originally identified in eukaryotes as the TALE homeobox genes (including the KNOX and MEIS genes).

    2. OsCPRO2 (LOC_Os12g43950)

    [0034] (1) Gene sequence of OsCPRO2 (LOC_Os12g43950), as shown in SEQ ID NO: 4. [0035] (2) The OsCPRO2 (LOC_Os12g43950) CDS sequence, as shown in SEQ ID NO: 5. [0036] (3) The amino acid sequence of OsCPRO2 (LOC_Os12g43950), as shown in SEQ ID NO: 6. [0037] (4) OsCPRO2 (LOC_Os12g43950) important domains: [0038] {circle around (1)}POX superfamily domains (pfam07526): a family of protein domains associated with nuclear function. Contains the BELL domain: LQNKMAKLMAMLDEVDRKYKHYYHQMQTVVSSFDVVAGPGSAKPYTAVALQTISRHF RCLKDAINDQINVIRKKLGEEENSSGKEGKLTRLRYIDQQLRQQRAFQQYGMIP and the SKY domain: SRYLKAAQELLDEVVSV. [0039] {circle around (2)}Homeobox domain (pfam05920): this is a homeodomain transcription factor KN domain conserved from fungi to humans and plants. They were originally identified in eukaryotes as the TALE homeobox genes (including the KNOX and MEIS genes).
    3. The Species where the OsCPRO1 and OsCPRO2 Homologs are Located

    [0040] Homology alignment of amino acid sequences was performed using the NCBI database, including maize, wheat, soybean and groundnut. The results of amino acid sequence alignment of OsCPRO1 and OsCPRO2 homologous are as follows:

    TABLE-US-00001 Maize[Zeamays],BEL1-likehomeodomainprotein6,SequenceID:PWZ21223.1Length: 643NumberofMatches:1,Range1:1to634. Score Expect Method Identities Positives Gaps 955bits(2469) 0.0 Compositionalmatrixadjust. 499/642(78%) 559/642(87%) 15/642(2%) Query1 MATYYSSPGNERDSQAMYPADSGNSSYPVPSAIGNMLYPGNGSSGPYTEFSGIIQHQQNF 60 Sbjct1 MATYYSSPDSERDSQTMYSTESGNASYPVPSALGNFLYLNSASSGPYTEFNGIVQSQQNF 60 Query61 MELPGHPTAISQDSSSRE-PNMVASYMDQRSFGPAKDMRNEMLMHLMDGAHNAGADLIHN 119 Sbjct61 MELTGHPSAISHDSSSNEATNIGTSLTEQRSFGPLKDMRNEMLMHLMDGAHSSGSDLIHN 120 Query120 DTHSSAQIEFGLLNNHNSMSVAPAPGQGLSLSLNTHILAPSYPYWSAKTELLTPHSYHGD 179 Sbjct121 DDHSTAQLEFGMLNNHNSTSLPSASGQGLSLSLNTHILAPSYPYWSAKQDLLTPNSYQGD 180 Query180 DNRMKNMQSEASQAIRNSKYLKAAQELLDEVVSVWKSIKQKAQKDQAEAGKSDNKEAEGG 239 Sbjct181 DNRMKNMQSEASQAIRNSKYLKAAQELLDEIVSVWKSVKQKTDKGPSEAGKSDGKETDGG 240 Query240 SKGEGVSSNPQESTANAAPEISAAEKQELQNKMAKLMAMLDEVDRKYKHYYHQMQIVVSS 299 Sbjct241 TKSEGVSFDPQESGANTAAELSTAEKQELQNKMAKLMAMLDEVDRKYKHYYHRMQLVMSS 300 Query300 FDMVAGSGAAKPYTAVALQTISKHFRCLKDAINDQINVIRKKLGEEESSSGKEGKLTRLR 359 Sbjct301 FDMVAGSGAAKPYTAVALQTISRHFRCLKDAINDQISVIRKKLGEDDDASGKEGKLIRLR 360 Query360 YIDQQLRQQRAFQQYGLLQQNAWRPQRGLPENSVSILRAWLFEHFLHPYPKDSEKLMLAR 419 Sbjct361 YIDQQIRQQRAFQQYGMLQQNAWRPQRGLPENSVSILRAWLFEHFLHPYPKDSEKLMLSR 420 Query420 QTGLTRSQISNWFINARVRLWKPMIEDMYKEEI--GEADLDSNSSSDNVPRSKDKIATSE 477 Sbjct421 QTGLTRSQISNWFINARVRLWKPMIEDMYKEEIGEGEAELDSNSSSDNVQRNKDKPPSSE 480 Query478 DKEDLKSSMSQTYQPSQLGESKANI-GMMSLGGAPA-GFHNEGNQDDSFMNLMLKDQRPG 535 Sbjct481 EKEDHKTSTSQVCQTSQLGESKSNIGGLMSFSGAPAGGFHNDVNPDDSFMSLMLKAQRPG 540 Query536 EAEGS-LLHDAVAHHSDENARFMAYHLSGLGRYGNSNVSLTLGLQHPDNRLSVQNTHQPG 594 Sbjct541 ETDGSGLLHDAVAHHSDESARFMAYHLTEFGRYGNNNVSLTLGLQHAENT-------QPG 593 Query595 FAGA-GEEIYNSTASLGVAAASSSDYESTNQIDQRQRSSCRI 635 Sbjct594 FPGVRDQDIYNSTAPLNV-TSTSSEYDSASQIDQQQRQRFEV 634

    TABLE-US-00002 Wheat[Triticumaestivum],BEL1-likehomeodomainprotein7,SequenceID: XP_044365192.1Length:638NumberofMatches:1,Range1:1to626. Score Expect Method Identities Positives Gaps 944bits(2441) 0.0 Compositionalmatrixadjust. 503/636(79%) 548/636(86%) 16/636(2%) Query1 MATYYSSPGNERDSQAMYPADSGNSSYPVPSAIGNMLYPGNGSSGPYTEFSGIIQHQQNF 60 Sbjct1 MSNYYSSPGDERDPQTMYSPDTGNASYPVPSALGNLLYSNNASSGPYTEFSGIIQPQQNF 60 Query61 MELPGHPTAISQDSSSREP-NMVASYMDQRSFGPAKDMRNEMLMHLMDGAHNAGA--DLI 117 Sbjct61 MELHGHP---SEHSSSREPPNMVTSLTEQSSFAPVKDMRNEMLMHFMDGAQSGGGGGDLI 117 Query118 HNDTHSSAQIEFGLLNNHNSMSVAPAPGQGLSLSLNTHILAPSYPYWSAKTELLTPHSYH 177 Sbjct118 HNDAHSSAQLDFGLLNNPSSASVPSAPGQGLSLSLNTHILAPSYPYWSPKPDLLTTQSYQ 177 Query178 GDDNRMKNMQSEASQAIRNSKYLKAAQELLDEVVSVWKSIKQKAQKDQAEAGKSDNKEAE 237 Sbjct178 GDENGMKNMQSEASRAIRNSKYLKAAQELLDEIVSVWKSIKQNAQKEKAEAGKMDGKDAD 237 Query238 GGSKGEGVSSNPQESTANAAPEISAAEKQELQNKMAKLMAMLDEVDRKYKHYYHQMQIVV 297 Sbjct238 EVLKSEGVSSNPQESTANAEAEISAAEKQELQNKMAKLLAMLDEVDRKYKHYFHQMQIVV 297 Query298 SSEDMVAGSGAAKPYTAVALQTISKHFRCLKDAINDQINVIRKKLGEEESSSGKEGKLTR 357 Sbjct298 SSEDMIAGSGAAKPYTAVALQTISRHFRCLKDAINDQVNVIRKKLGEEDSSSGREGKLTR 357 Query358 LRYIDQQLRQQRAFQQYGLLQQNAWRPQRGLPENSVSILRAWLFEHFLHPYPKDSEKLML 417 Sbjct358 LRYIDQQLRQQRAFQQYGMLQQNAWRPQRGLPENSVSILRAWLFEHFLHPYPKDSEKLML 417 Query418 ARQTGLTRSQISNWFINARVRLWKPMIEDMYKEEIGEADLDSNSSSDNVPRSKDKIATSE 477 Sbjct418 ARQTGLTRSQISNWFINARVRLWKPMIEDMYKEETGEAELDSNSSSDNLPRSKDKMASCE 477 Query478 DKEDLKSSMSQTYQPSQLGESKANIGMMSLGGAPAGFHNEGNQDDSFMNLMLKDQRPGEA 537 Sbjct478 DKEDLKCSMSQG-QAYQTSEFKANMEMAGLTGAPSSFHNEANSDDGFMNLLLKDQRPGEA 536 Query538 EGSLLHDAVAHHSDENARFMAYHLSGLGRYGNSNVSLTLGLQHPDNRLSVQNTHQPGFAG 597 Sbjct537 DGSLL------HGDESARFMAYHLAELGGYQNSNVSLTLGLQHTENSLSAPNAHRPGFTA 590 Query598 AGEE-IYNSTASL--GVAAASSSDYESTNQIDQRQR 630 Sbjct591 AGEEDIYNTTAANPGGGAAVASSDYESTNQLDQRQQ 626

    TABLE-US-00003 Soybean[Glycinemax],BEL1-likehomeodomainprotein1,SequenceID:XP_003543416.1Length: 702NumberofMatches:1,Range1:166to489. Score Expect Method Identities Positives Gaps 273bits(697) 1e-74 Compositionalmatrixadjust. 162/359(45%) 218/359(60%) 46/359(12%) Query413 MDSGGRKHLASSSYSGPSGTAGSSNHISASKFLRSAQAILNEVCRVTPLKRPPKSVRSSD 472 Sbjct166 LDVAGQGHVAGIGNSPMSASIGVSGVIMGSKYLKAAQELLDEVVNVG------KGIY--- 216 Query473 QQHWSMAGGSSTSVDANLTYNGREERSGMLAGEVDSARDPASFvttsslvtvsQVPLESE 532 Sbjct217 -------------------KEEK------FSEKVKANRESTNSGAAGDGGDGSSGGGENS 251 Query533 MIQGLAEAARCESRDDLELKKQKLSLMLDEVEARYRRYCDHLQLVITGFNSQAGPNTATP 592 Sbjct252 AGKQVVELSTAQ-RQELQMKKSKLVTMLDEVEQRYRQYHHQMQIVVSSFEQAAGYGAAKS 310 Query593 YTILALQAMSRHFRCLKDAIGSQLRIVKRTLGEDDRTG-QGETSRLRYVDqqirqqralq 651 Sbjct311 YTALALKTISKQFRCLKDAISAQIKATSKTLGEDDCLGVKVEGSRLRFVDHHLRQQRALQ 370 Query652 qlGMLQQHAWRPQRGLPERAVSVLRAWLFEHFLHPYPKDVDKLSLAKQTGLTRSQVSNWF 711 Sbjct371 QLGMIQPNAWRPQRGLPERAVSILRAWLFEHFLHPYPKDSDKVMLAKQTGLARSQVSNWF 430 Query712 INARVRLWKPMVEEMYVEEQKEY----------SEDHSTALAQSERMARDQVEIENNTY 760 Sbjct431 INARVRLWKPMVEEMYLEEIKEHEQGNGSENTKSKESSKELASTANVALDHLQSKHESF 489

    TABLE-US-00004 [Arachisipaensis],BEL1-likehomeodomainprotein1,SequenceID:XP_016170518.1Length: 733NumberofMatches:1,Range1:278to488. Score Expect Method Identities Positives Gaps 269bits(687) 4e-73 Compositionalmatrixadjust. 139/211(66%) 162/211(76%) 1/211(0%) Query546 RDDLELKKQKLSLMLDEVEARYRRYCDHLQLVITGFNSQAGPNTATPYTILALQAMSRHF 605 Sbjct278 RQELQMKKSKLVCMLDEVEQRYRQYHHQMQVVISSFEQAAGFGAAKSYTSLALKTISKQF 337 Query606 RCLKDAIGSQLRIVKRTLGEDDRTG-QGETSRLRYVDqqirqgralqqlGMLQQHAWRPQ 664 Sbjct338 RCLKDAISSQIRATSKTLGEDDCLGAKVEGSRLRYVDHHLRQQRALQQLGMIQPNAWRPQ 397 Query665 RGLPERAVSVLRAWLFEHFLHPYPKDVDKLSLAKQTGLTRSQVSNWFINARVRLWKPMVE 724 Sbjct398 RGLPERAVSILRAWLFEHFLHPYPKDSDKVMLAKQTGLTRSQVSNWFINARVRLWKPMVE 457 Query725 EMYVEEQKEYSEDHSTALAQSERMARDQVEI 755 Sbjct458 EMYMEEVKEQEINNNNNNNGSERSKESSKEL 488

    [0041] After verification, homologous genes in species include but are not limited to: rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexilis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana(Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata),pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia(Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinese cabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina), day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Allium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba), green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinifera), pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), tulip (Tulipa gesneriana).

    Example 2: A Method of Inducing Haploids Using OsCPRO1 and OsCPRO2

    [0042] The method of inducing haploid by OsCPRO1 and OsCPRO2 is summarized as follows (FIG. 9): {circle around (1)} selects the AtDD45 promoter specifically expressed in egg cells, and the nucleotide sequence, as shown in SEQ ID NO: 7, drives the CDS sequence or genome sequence of OsCPRO1 and OsCPRO2 genes respectively to construct ectopic expression vector; {circle around (2)} uses Agrobacterium-mediated method to obtain transgenic-positive material; {circle around (3)} investigates field phenotypic traits of wild-type CY84 and T.sub.0 transgenic-positive plants; {circle around (4)} Indel markers designed for haploid detection; {circle around (5)} statistics seed setting rate of T.sub.0trans-positive material and germination treatment to obtain T.sub.1generation of transgenic plants; {circle around (6)} using the Indel markers against T.sub.1generation of gene positive material; {circle around (7)} statistics haploid induction rate of T.sub.1transgenic plants; {circle around (8)} combines haploid induction material with the MiMe system to obtain apomixis material. The technical route profile is shown in FIG. 9.

    Embodiment 1

    1. Experimental Materials

    [0043] In this embodiment, the indica japonica hybrid rice Chunyou 84 (CY84) was selected as the genetic transformation material, CY84 is a hybrid breeding of the japonica sterile line Chunjiang 16A as the female parent and the indica restoring line C84 as the male parent, the hybrid rice has a longer growth period and is suitable for single season late rice cultivation. It has strong growth and large panicles with many grains. Due to its thick and robust stem, it has strong lodging resistance.

    2. Construction of the Ectopic Expression Vector

    [0044] Select AtDD45 oocyte specific expression promoter to drive the CDS sequences of OsCPRO1 and OsCPRO2 genes, respectively, to complete the ectopic expression process.

    2.1 Get the Fragments Required for the Ectopic Expression Vector Constructs

    [0045] (1) Arabidopsis leaf genomic DNA was used as a template to amplify the AtDD45 promoter sequence.

    [0046] {circle around (1)} DNA extraction method: take about 2 cm of rice leaves into a 2 mL centrifuge tube with steel beads, 500 L of CTAB extract was added; {circle around (2)} Transfer the centrifuge tube to the histiocyte shatter for shock grinding, 60 Hz/sec, 2 min; {circle around (3)} Place the grinding samples in a 65 C. water bath for 30 min, during this period, flip up and down for 10 min/time; {circle around (4)} After taking out the sample, centrifuge at 12000 rpm for 30 seconds, then add 300 L of chloroform to the fume hood and mix upside down. {circle around (5)} i Centrifuge again at 12000 rpm for 8 minutes. After centrifugation, take 350 L of the supernatant and place it into a 1.5 mL sterilized centrifuge tube. Add 700 L of precooled anhydrous ethanol, shake well, and transfer to a 20 C. freezer for 15 minutes. {circle around (6)} After allowing the sample to stand still, centrifuge at 12000 rpm for 10 minutes. Discard the supernatant and air dry at room temperature. {circle around (7)} Add 100 L of ddH.sub.2O for dissolution, gently shake, and store at 4 C. in the refrigerator for later use.

    [0047] (2) RNA was extracted from CY84 and subsequently reverse transcribed into cDNA to provide a template for amplification of the CDS sequences of genes OsCPRO1 and OsCPRO2.

    [0048] Methods for RNA extraction: {circle around (1)} Put 3-4 cm young rice panicles into a sterilized mortar pre cooled with liquid nitrogen, grind clockwise until they turn into a white green powder, and quickly transfer them to an EP tube without RNase containing 1 mL of Trizol. Vortex and shake for 1 minute to fully mix; {circle around (2)} Incubate at room temperature for 5 minutes, add 200 L chloroform to the EP tube, vortex for 15 seconds, and let it stand on ice for 2 minutes; {circle around (3)} centrifuge at 12000 rpm at 4 C. for 15 minutes; {circle around (4)} Take 500 L of supernatant, add 100 L of chloroform, vortex for 15 seconds, and let it stand on ice for 2 minutes; {circle around (5)} i centrifuge at 12000 rpm at 4 C. for 15 minutes; {circle around (6)} Take 400 L of the supernatant into a 1.5 mL EP tube without RNase, add 500 L of isopropanol, invert and mix well; {circle around (7)} Let it stand on ice for 10 minutes, centrifuge at 12000 rpm at 4 C. for 10 minutes, and discard the supernatant; {circle around (8)} Add 1 mL of 75% ethanol, wash the precipitate with vortex shaking, centrifuge at 12000 rpm at 4 C. for 10 minutes, discard the supernatant and repeat the previous step; {circle around (10)} Retain the RNA precipitate at the bottom of the tube, remove the residual liquid, simply air dry, add 100 L of RNase free water, dissolve with light tap, and test the concentration and purity. Store at 80 C. for later use.

    [0049] Methods for RNA reverse transcription into cDNA: preparation of cDNA by HiFiScript cDNA Synthesis Kit (CW2569M). {circle around (1)} The reagent and RNA template provided in the kit were dissolved on ice for standby; {circle around (2)} mixed the reagent according to RNA reverse transcription system (Table 1), briefly centrifuged the liquid at the bottom of the tube; {circle around (3)} Subsequently, the reaction procedure was carried out, with 15 minutes at 42 C. followed by 5 minutes at 85 C.; {circle around (4)} Cool the cDNA on ice after the reaction for later use.

    TABLE-US-00005 TABLE 1 System in which the RNA is reverse-transcribed into cDNA Reagent Addition RNA template 3 g Primer Mix 2 L dNTP Mix 4 L DDT 2 L 5 RT Buffer 4 L HiFiScript 1 L RNase-Free Water Fill it up to 20 L

    [0050] {circle around (3)} Design of the primers for amplification. The primers for the AtDD45 promoter sequence and the CDS sequences of the OsCPRO1 and OsCPRO2 genes are shown in Table 2, with lower case letters indicating adaptor primers.

    TABLE-US-00006 TABLE2 Primersfortheectopicexpressionvectorconstructs Primername Sequence AtDD45-F tacgaattcgagctcggtacAAATGTTCCTCGCTGACGTAAGAAG AtDD45-R ACTTGTGTTAGAAGCCATTATTC CPRO1-F gaataatggcttctaacacaagtATGGCTACTTACTACTCGAGCCCTGGCAATG CPRO1-R catgcctgcaggtcgactctagagTCATCGAACCCACAGAGAAGCCATAG CPRO2-F gaataatggcttctaacacaagtATGGCTACTTACTATTCAAGCCCTGGTAGCG CPRO2-R catgcctgcaggtcgactctagagTCAGGCCACAAAATCATGCAGAAGAGGTG

    [0051] {circle around (4)} The promoter sequences and the gene CDS sequences were amplified using the amplification primers. The amplification system is shown in Table 3, and the amplification procedure is shown in Table 4.

    TABLE-US-00007 TABLE 3 Amplification system of the fragments required for the vector construction Reagent Addition KOD FX buffer 25 L dNTP 10 L KOD FX 1 L DNA template 2 L Primer-F (10 M) 1.5 L Primer-R (10 M) 1.5 L ddH.sub.2O 9 L

    TABLE-US-00008 TABLE 4 Amplification procedure of the fragments required for the vector construction Cycle index Temperature setting and reaction time 1 94 C. 2 min 35 98 C. 10 sec, 60 C. 30 sec, 68 C. 1 min/kb 1 68 C. 5 min [0052] {circle around (5)} 1% agarose gel electrophoresis, cut the gel and save it at 20 C.

    2.2 Skeleton Vector Enzyme Digestion

    [0053] The pCAMBIA1300-ACTIN skeleton vector was digested with KpnI-HF and Bam HI-HF endonuclease, removing the original ACTIN promoter sequence, forming a gap at the sticky end, and retaining all other sequences including the original terminator. The restriction system is shown in Table 5.

    TABLE-US-00009 TABLE 5 Enzyme digestion system of skeleton vector Reagent Addition pCAMBIA1300-ACTIN 2 g Cutsmart buffer 5 L Kpn I-HF 0.5 L Bam HI-HF 0.5 L ddH.sub.2O Fill it up to 50 L
    2.3 the Recovered Amplified Fragments were Ligated to the Skeleton Vector

    [0054] The ligation system is shown in Table 6. The mixing system is placed at 50 C. for 15 min. After ligation, the product is placed on ice.

    TABLE-US-00010 TABLE 6 Ligation system of the overexpression vectors Reagent Addition Products digested from pCAMBIA1300-ACTIN 2 L AtDD45 promoter of the amplified fragments 0.2 g Gene fragments amplified from the CDS sequences 0.4 g Gibson Assembly Master Mix (2) 10 L ddH.sub.2O Fill it up to 20 L

    2.4 Transformation of the Ligation Products

    [0055] Method of transforming the ligation products: {circle around (1)} Remove the competent cells stored in the 80 C. refrigerator (provided by the laboratory) and put them on ice for dissolution; {circle around (2)} When the competent cell (50 L100 L) reaches the molten state, add the ligation products immediately; {circle around (3)} Set still on the ice for 30 min; {circle around (4)} 42 C. water bath heat shock 90 sec; {circle around (5)} i 500 L of liquid LB medium was added; {circle around (6)} 37 C. shaker culture for 1 h; {circle around (7)} 4000 rpm, 3 min, slowly centrifuge and apply onto LB solid culture medium with Kan resistance; {circle around (8)} Incubate overnight at 37 C. and perform colony PCR detection after the plaque grows. Select a single colony and culture it in Kan resistant liquid medium on a constant temperature shaker at 200 rpm and 37 C. for 14 hours. Extract plasmids and perform Sanger sequencing.

    3. Transgenic Plants Obtaining

    [0056] The ectopic expression vector was transferred into Agrobacterium tumefaciens strain EHA105 by electroshock and transferred into CY84 in hybrid rice using Agrobacterium-mediated method.

    [0057] Specific method of transformation: {circle around (1)} Sterilize the embryos of hybrid rice CY84 seeds; {circle around (2)} Inoculate into the culture medium for inducing callus tissue; {circle around (3)} After one week of cultivation, select embryogenic callus tissue that is vigorous in growth, light yellow in color, and relatively loose as the receptor for transformation; {circle around (4)} Infect rice callus tissue with EHA105 strains containing pC1300-AtDD45P-OsCPRO1 and pC1300-AtDD45P-OsCPRO2 plasmids respectively; {circle around (5)} Incubate in the dark at 25 C. for 3 days; {circle around (6)} Screening resistant callus tissues and transgenic plants using a selective culture medium containing 50 mg/L hygromycin; {circle around (7)} Select the transgenic plants for normal growth.

    4. Plant the Transgenic Plants

    [0058] The transgenic plants are planted in transgenic experimental fields in summer and in greenhouses in winter. The average temperature during the day in the greenhouse is 34 C., the average temperature at night is 25 C., there is a 12 hour/12 hour dark cycle, and the relative humidity is 75%.

    5.Transgenic Plants Detection

    5.1 Design of the Primers for Detection

    [0059] Primers designed for the AtDD45 promoter sequence and the gene CDS sequence (spanning the intron region) were also used as detection primers for transgenic positive plants. The specific primer information is shown in Table 7.

    TABLE-US-00011 TABLE7 Primersforthedetectionofthetransgenicplants CDS DNA amplification amplification Primername Sequence length length AtDD45-F2 CTGATCTAGATGATGGTTATAGACTG 588bp AtDD45-R2 ACTTGTGTTAGAAGCCATTATTC CPRO1-F1 CAATCTGAGGCCTCACAGGCAATCAG 398bp 1154bp CPRO1-R1 GTCTGAAGGGCCACTGCAGTATAAGGC CPRO2-F1 GATGAGGTCGTGAGTGTTTGGAAGAGC 690bp 1121bp CPRO2-R1 GACACGGGCATTTATGAACCAATTC

    5.2 the Detection System is Shown in Table 8 and the Detection Procedure is Shown in Table 9.

    TABLE-US-00012 TABLE 8 Detection system of the transgenic plants Reagent Addition 2 Rapid Taq Master Mix 7.5 L DNA template 1 L Primer-F (10 M) 0.5 L Primer-R (10 M) 0.5 L ddH.sub.2O 5.5 L

    TABLE-US-00013 TABLE 9 Detection procedures for the transgenic plants Cycle index Temperature setting and reaction time 1 95 C. 3 min 35 95 C. 15 sec, 60 C. 15 sec, 72 C. 15 sec 1 72 C. 5 min

    6.Investigation of the Field Phenotype

    [0060] Field phenotype survey includes: plant type, ear type and seed set rate.

    6.1 Mutant strain of plant type (FIG. 1) and spike type (FIG. 2) versus wild-type CY84 (n=3).
    6.2 Statistics of seed set rate: 3 rice ears per plant were selected for statistics, and the calculation formula: seed set rate=(real grain number/total grain number) 100%.

    7.Haploidsscreened Using the Indel Markers

    [0061] According to the whole genome sequence of hybrid rice CY84 and its two parents (16A and C84), a pair of Indel markers were designed on each chromosome of rice, a total of 12 pairs of primer markers, primer information is shown in Table 10, genotyping is shown in FIG. 3 for detection of haploid induction rate in T.sub.1 generation transgenic materials. If all 12 pairs of Indel markers show the same single band genotype as the parent 16A or C84, rather than the same double band genotype as the heterozygous CY84, the probability of being haploid or double haploid can reach 99.98%, calculated as follows: 1(1/2).sup.12=99.98%. The Indel marker detection of T.sub.1 haploid and double haploid materials is shown in FIG. 4.

    TABLE-US-00014 TABLE10 Indelmarkersusedtodetecthaploids Amplification Chromosome Primername Sequence length Chr.01 C01-4.122-F GTGGTCAGGTGGTGATGGTGTT 156bp C01-4.122-R AAAGAAACACGCAAATAAAAGC Chr.02 C02-3.797-F AACCTACCACTGCCATTGC 202bp C02-3.797-R GGCATTATCCATACCAGCAG Chr.03 C03-25.115-F ACATGGCCTTGTAGTAGACGAGAG 204bp C03-25.115-R ACGCTGTGGCTATGCCTTTGG Chr.04 C04-1.866-F ACCATGCCTCATGACATGTGG 128bp C04-1.866-R TGGTTTTGTGTAGCTCTGTCGG Chr.05 C05-27.299-F ACAGCGATAATAACACGCACAA 163bp C05-27.299-R TCAAGTGCTATACTTGACACGG Chr.06 C06-9.538-F CCATAAGATGCAGGCCGTTGT 129bp C06-9.538-R CAGCTTTGGTCAGATGGTCAC Chr.07 C07-6.338-F GATTTATAGTTTGAGTGTTTGC 125bp C07-6.338-R CTTGGTTAGTTTCTACCCTGCT Chr.08 C08-22.188-F CATGCAGATAGCTCGCTTGT 143bp C08-22.188-R CACCTCTCAGGACAACTGTA Chr.09 C09-11.923-F TTCATCCCAGCCTACCTCCT 174bp C09-11.923-R GCTTAATCCCGTAGTCTTCAA Chr.10 C10-13.555-F GCACATGGTGAGACGTCCTC 103bp C10-13.555-R AAGTCCTGTAGTAGGTCACACCG Chr.11 C11-9.573-F GGCATCATTAAGGCTTGT 160bp C11-9.573-R CTGGCGATCTCTGTGAGG Chr.12 C12-2.460-F GAGCAGATCACCCCTAAATTATG 150bp C12-2.460-R GATTCATTCATCTTTCGAAGAG

    8. Flow-Cytometric Ploidy Detection

    [0062] The haploid or diploid plants screened by Indel markers were further confirmed for cell ploidy using flow cytometry (FIG. 5). The comparison of traits between haploid materials and wild-type CY84 is shown in FIG. 6, while the comparison of traits between double haploid materials and wild-type CY84 is shown in FIG. 7.

    [0063] The method of flow cytometry ploidy detection: {circle around (1)} Cut about 3 cm of fresh young leaves and place them in sterilized glass petri dishes. Place the petri dishes in a tray filled with ice, and perform the following on ice; {circle around (2)} Add 1 mL of plant lysis buffer (LB01) to the petri dish, cut the leaves with a blade, and immerse the tissue cells of the leaves into the lysis buffer; {circle around (3)} Extract the lysis solution from the petri dish, filter it with 50 m nylon mesh into a 1.5 mL sterile centrifuge tube, and store it on ice. Centrifuge at 1200 rpm at 4 C. for 5 minutes; {circle around (5)} i Discard the supernatant and add pre cooled 450 L of LB01, 25 L of PI (1 mg/mL), and 25 L of RNaseA (1 mg/mL); {circle around (6)} Staining on ice in the dark for 10 minutes; {circle around (7)} Detecting with BD Accuri C6 machine.

    9. Whole-Genome Sequencing Analysis

    [0064] Select the parents 16A and C84 of hybrid rice CY84, and CY84, as well as the leaves of haploid plants, and extract DNA for whole genome sequencing. The whole genome sequencing results show that there are many different homozygous genotypes between 16A and C84; The genotype of CY84 at these loci is a heterozygous genotype that includes both the 16A genotype and the C84 genotype; while the haploid plant at these loci with homozygous genotypes of 16A or C84.

    Embodiment 2

    [0065] 1. Experimental materials: see embodiment 1 [0066] 2. Ectopic expression vector construction

    [0067] The AtDD45 egg specific expression promoter was selected to drive the genomic sequence of OsCPRO1 and OsCPRO2 genes respectively to complete the ectopic expression process. [0068] 2.1 Get the fragments required for the ectopic expression vector constructs [0069] (1) Genomic Arabidopsis leaf DNA was used as a template to amplify the AtDD45 promoter sequence. For DNA extraction, refer to the described in embodiment 1. [0070] (2) Genomic DNA extracted from rice leaves was used as a template to amplify the genomic sequence of the OsCPRO1 and OsCPRO2 genes. [0071] (3) The amplification primers were designed and used to amplify the AtDD45 promoter sequence and the OsCPRO1 and OsCPRO2 genomic sequences. [0072] (4) 1% agarose gel electrophoresis, cut the gel and save it at 20 C. [0073] 2.2 Skeleton vector enzyme digestion: see embodiment 1. [0074] 2.3 Ligate the recovered amplified fragment to the skeleton vector: see embodiment 1. [0075] 2.4 Transformation and ligation products: see embodiment 1. [0076] 3. Get transgenic plants: see embodiment 1. [0077] 4. Plant transgenic plants: see embodiment 1. [0078] 5. Transgenic plants detection

    [0079] Primers were designed on the AtDD45 promoter sequence for detection of transgenic-positive plants. See primer information in Table 7 in embodiment 1, Table 8 in embodiment 1 for detection system, and Table 9 in embodiment 1 for amplification procedures. [0080] 6. Investigate field phenotypes: see embodiment 1. [0081] 7. Haploid screening using Indel markers: see embodiment 1. [0082] 8. Flow cytometric ploidy detection: see embodiment 1. [0083] 9. Whole-genome sequencing analysis: see embodiment 1.

    Embodiment 3

    1. Experimental materials: wild Arabidopsis Columbia (Col-0) ecotype was used as the experimental materials.
    2. Ectopic expression vector construction

    [0084] The egg specific expression promoter of AtDD45 was selected to drive the CDS sequences of AtCPRO1 and AtCPRO2 genes respectively to complete the ectopic expression process.

    2.1 Get the Fragments Required for the Ectopic Expression Vector Constructs

    [0085] (1) Arabidopsis leaf Genomic DNA was used as a template to amplify the AtDD45 promoter sequence. For DNA extraction, described in embodiment 1. [0086] (2) RNA was extracted from young flowers of the Arabidopsis Columbia ecotype and subsequently reverse transcribed into cDNA to provide a template for the amplification of the CDS sequences of the genes AtCPRO1 and AtCPRO2. Methods for RNA extraction and RNA reverse transcription into cDNA see embodiment 1. [0087] (3) The amplification primers were designed and amplified using the AtDD45 promoter sequence and the CDS sequences of the AtCPRO1 and AtCPRO2 genes. The amplification system is shown in Table 3 in embodiment 1 and the amplification procedure is shown in Table 4 in embodiment 1. [0088] (4) 1% agarose gel electrophoresis, cut the gel and save it at 20 C. [0089] 2.2 Skeleton vector enzyme digestion: see embodiment 1. [0090] 2.3 Ligate the recovered amplified fragment to the skeleton vector: see embodiment 1. [0091] 2.4 Transformation and ligation products: See embodiment 1. [0092] 3. Transgenic plants obtaining

    [0093] The ectopic expression vector was transferred into Agrobacterium tumefaciens strain GV 310 by electroshock and transferred into Col-0 ecotype wild A. thaliana using Agrobacterium-mediated dipping method.

    [0094] Specific method of transformation: {circle around (1)} Immerse the inflorescence of T.sub.0 generation Arabidopsis in a suspension of Agrobacterium tumefaciens strain GV310 containing the target transformation plasmid at a certain concentration; {circle around (2)} Cultivate under certain conditions; {circle around (3)} Collect seeds of T.sub.0 generation Arabidopsis after maturity; {circle around (4)} Place these seeds on a culture medium containing specific antibiotics for growth screening and obtain positive plants.

    4. Plant the Transgenic Plants

    [0095] The transgenic material was grown in a light incubator. Light treatment for 15 hours, with an average temperature of 24 C.; dark treatment for 9 hours, with an average temperature of 22 C. The average humidity of the soil is 75%, and the nutrient soil: vermiculite=1:1. Water the nutrient solution every 3-4 days and immerse it from the bottom of the plate.

    5. Transgenic Plants Detection

    [0096] Design primers on the CDS sequence of genes, spanning intron regions, and use this primer as a detection primer for transgenic positive plants. The detection system can be found in Table 8 of embodiment 1, and the amplification program can be found in Table 9 of embodiment 1.

    6. Investigation of the Phenotypic Traits

    [0097] Phenotypic trait surveys included plant type and seed set rate.

    6.1 Investigation of the plant type of the T.sub.2 generation (FIG. 8), selecting mutant plants and wild-type Arabidopsis for comparative observation (n=3).
    6.2 Statistics of Seed Setting Rate: Collect mature but not yet cracked wild type and mutant Arabidopsis longhorned fruits, put them into a decolorization solution (ethanol: acetic acid=3:1) for transparent decolorization, and when the seeds in the pods can be clearly seen, take them out and place them under a microscope to calculate the seed setting rate.

    7. Flow-Cytometric Ploidy Detection

    [0098] Confirm the cell ploidy of plants using flow cytometry technology, and refer to embodiment 1 for the method of flow cytometry ploidy detection.

    Embodiment 4

    1. Experimental materials: see embodiment 1.
    2. Construct an ectopic expression vector: see embodiment 1.

    3. Multigene Knockout Vectors Construction

    [0099] MiMe material was constructed by using the CRISPR-Cas 9 multigene knockout system to simultaneously knockout OsOSD1, OsPAIR1 and OsREC8 rice endogenous genes, with the main steps as follows (see CN201510485573.2 for specific operational details):

    3.1 Design of the Target Sequences and the Primers

    [0100] (1) Design of target sequence: search for NNNNNNNNNNNNNNNNNNN (NGG) specific sequence at the target position of the gene, with GC content between 35% and 75%. The following four sites were selected as sites for knockout OsOSD1, OsPAIR1 and OsREC8 by CRISPR-Cas 9 gene editing system (underlined PAM sequence): [0101] Knockdown site of the OsOSD1 gene: CTGCCGCCGACGAGCAACA AGG [0102] Knockdown site of the OsPAIR1 gene: AAGCAACCCAGTGCACCGC TGG [0103] Knockdown site of the OsREC8 gene: CGGAGAGCCTTAGTGCCAT GGG

    [0104] (2) Primer design: add four bases of GGCA before the forward target sequence and four bases of AAAC before the reverse target sequence, respectively named g.sup.++ and g.sup., performed the primer synthesis.

    3.2 Construction of the Intermediate Vector

    [0105] (1) The Aar I enzyme was used to digest the intermediate vector SK-gRNA to form the sticky ends, and the digestion system is shown in Table 11.

    TABLE-US-00015 TABLE 11 Enzyme digestion system of the intermediate vector SK-gRNA Reagent Addition SK-gRNA 2 L 10 buffer Aar I 5 L 50 oligonucleotide 1 L Aar I 1 L ddH.sub.2O 41 L

    [0106] (2) Mix 20 L of 100 M g++ and g primers in equal proportions, and incubate at 100 C. for 5 minutes to form sticky ends after denaturation annealing.

    [0107] (3) Ligating via T4 DNA ligase and ligate at room temperature for 1 hour. The ligation system is shown in Table 12.

    TABLE-US-00016 TABLE 12 The ligation system of the DNA ligase Reagent Addition SK-gRNA/Aar I 20-50 ng The primer was annealed to the product 7 L 10 T.sub.4ligase buffer 1 L T.sub.4DNA ligase 1 L ddH.sub.2O Fill it up to 10 L
    (4) Transformation ligation products: see embodiment 1.

    3.3 Construct the Final Vector

    [0108] Utilizing the properties of BamHI and BglII being homologous enzymes, the polymerization of three SK-gRNA intermediate vectors was completed using T4 DNA ligase. As the final vector, KpnI and BamHI were used for digestion, while KpnI and BglII were used for digestion of the fragments provided. The ligation between SK-gRNAs was completed using two pairs of homologous enzymes NheI/XbaI and SalI/XhoI. The system with multiple intermediate vectors ligated is shown in Table 13.

    TABLE-US-00017 TABLE 13 Ligation systems of multiple intermediate vectors Reagent Addition pC1300-Ubi-Cas9/Kpn I + Bam HI 50 ng gRNA1/Kpn I + Sal I 8 ng gRNA2/XhoI + NheI 8 ng gRNA3/XbaI + BgIII 8 ng 10 T.sub.4ligase buffer 1 L T.sub.4DNA ligase 0.5 L ddH.sub.2O Fill it up to 10 L

    4. Binary Fusion Expression Vector Construction

    [0109] The ectopic expression vector and the multigene knockout vector were combined to construct a binary fusion expression vector. Among them, the ectopic expression vector provided the desired fragment, and the multigene knockout vector was used as the backbone vector for enzyme digestion.

    4.1 Get the Fragments Required for the Construction of the Binary Fusion Expression Vector

    [0110] The whole fragment of the ectopic expression process was amplified from the ectopic expression vector, including three parts: AtDD45 promoter fragment, OsCPRO1 and OsCPRO2 gene CDS sequence and terminator sequence. The amplification primers are shown in Table 14 and lower letters indicate the adaptor primers.

    TABLE-US-00018 TABLE14 Amificationprimersfortheconstructionof binaryfusionexpressionvector Primer name Sequence CPRO-F gattgtcgtttcccgccttcagtttAAATGTTCCTCGCTG CPRO-R ACGTAAGAAGCGCCAATATATCCTGTCAAACACTGATAGT TT

    4.2 Digestion of Multiple Gene Knockout Vectors

    [0111] Using PmeI endonuclease, the pC1300-Cas9-gRNAOSD1-gRNAPAIR1-gRNAREC8 multi gene knockout vector was digested. The digestion system is shown in Table 15.

    TABLE-US-00019 TABLE 15 Multigene knockout vector enzyme digestion system Reagent Addition pC1300-Cas9-gRNA.sup.OSD1-gRNA.sup.PAIR1-gRNA.sup.REC8 20 ng Cutsmart buffer 5 L Pme I 1 L ddH.sub.2O Fill it up to 50 L

    4.3 the Amplified Fragment was Attached to the Multigene Knockout Vector

    [0112] The ligation system is shown in Table 16. The mixing system is placed at 50 C. for 15 min, and the product is placed on ice after the ligation.

    TABLE-US-00020 TABLE 16 Ligation system of the binary fusion expression vectors Reagent Addition pC1300-Cas9-gRNA.sup.OSD1-gRNA.sup.PAIR1-gRNA.sup.REC8/Pme I 2 L AtDD45 Promoter-OsCPRO1(CDs)/OsCPRO2(CDs)- 4 L terminator amplified fragment Gibson Assembly Master Mix (2) 10 L ddH.sub.2O Fill it up to 20 L
    4.4 Transformation of the ligation products: See embodiment 1.
    5. Get transgenic plants: see embodiment 1.
    6. Plant transgenic plants: see embodiment 1.
    7. Transgenic plants detection
    7.1 Positive detection of transgenic plants: see embodiment 1.

    7.2 Detection of the Knockdown of the Target Sites

    [0113] (1) Primers were designed for target site detection to determine the knockdown of OsOSD1, OsPAIR1 and OsREC8 genes. The primer information is shown in Table17. Lower case letters indicate the joint primers.

    TABLE-US-00021 TABLE17 PrimersforOsOSD1,OsPAIR1andOsREC8knockdown Primer name Sequence OSD1-F ggagtgagtacggtgtgcTATCAGGAGGACGACGTCGCCG OSD1-R gagttggatgctgagtggCTCCTCCTCTTGGGTGTAGC PAIR1-F ggagtgagtacggtgtgcCTTCTTGCGCGCGAGAAGAGTCTC PAIR1-R gagttggatgctgagtggGAGATGTAGTGCGTGGGTCTTG REC8-F ggagtgagtacggtgtgcTTGGGTTAGTGAGGAGAT REC8-R gagttggatgctgagtggTGCGATCGGAACTATGGAGAC [0114] (2) sequencing analysis. The PCR amplification products were sent to the China Rice Research Institute for second-generation sequencing, using Hi-TOM (http://www.hi-tom. The net/hi-tom/) platform performed data analysis to obtain mutational information for the three genes.
    8. Investigate field phenotypes: see embodiment 1.

    9. Flow-Cytometric Ploidy Detection

    9.1 Material Preparation

    [0115] The materials with successful binary fusion expression were selected, that is, when the AtDD45 promoter drives the successful expression of the CDS sequence of the OsCPRO1/OsCPRO2 gene, OsOSD1, OsPAIR1 and OsREC8 were successfully knocked out (MiMe). The seeds of this material are germinated and planted (first filial generation).

    9.2 Flow Cytometry

    [0116] We screened the cell ploidy by flow cytometry to select the first filial generation plants with consistent cell ploidy and the parent plant as apomixis material.

    10. Analysis of whole-genome sequencing: see embodiment 1.

    Embodiment 5

    1. Experimental materials: see embodiment 1.
    2. Construction of an ectopic expression vector: see embodiment 2.
    3. Construction of multigene knockout vectors: see embodiment 4.
    4. Construct a binary fusion expression vector

    [0117] The ectopic expression vector and the multigene knockout vector were combined to construct a binary fusion expression vector. Among them, the ectopic expression vector provided the desired fragment, and the multigene knockout vector was used as the backbone vector for enzyme digestion.

    4.1 Get the Fragments Required for the Construction of the Binary Fusion Expression Vector

    [0118] The genomic sequence of the AtDD45 egg-specific expression promoter driving the OsCPRO1 and OsCPRO2 genes, completing the whole fragment of the ectopic expression process, was amplified from the ectopic expression vector, containing three parts: the AtDD45 promoter fragment, the genomic sequence of the OsCPRO1 and OsCPRO2 genes, and the terminator sequence. The amplification primers are shown in Table 14 in embodiment 3.

    4.2 To digest multiple gene knockout vector: see embodiment 4.
    4.3 Attach the amplified fragments to the multigene knockout vector: see embodiment 4.
    4.4 Transformation of the ligation products: see embodiment 1.
    5. Get transgenic plants: see embodiment 1.
    6. Plant transgenic plants: see embodiment 1.
    7. Transgenic plants detection
    7.1 Positive detection of transgenic plants: see embodiment 2.
    7.2 Detection of target site knockout: see embodiment 4.
    8. Investigate field phenotypes: see embodiment 1.
    9. Flow cytometric ploidy detection: see embodiment 4.
    10. Analysis of whole-genome sequencing: see embodiment 1.

    Example 3

    1. Phenotypic Traits of T.SUB.0.generation OsCPRO Series Haploid-Inducing Material

    [0119] For embodiment 1 and 2 in Example 2, investigation of field phenotypes of T.sub.0transgenic positive plants revealed that compared with the wild-type CY84, the transgene positive material had no significant difference in vegetative growth, and the seed set rate was reduced to different degrees, including 80-100%, 50-80%, 50-30-50%, 10-30%, and 0-10%. The plant type was summarized in FIG. 1 and the spike type was shown in FIG. 2.

    2. Genotyping of the 12 Pairs of Indel Markers

    [0120] For embodiment 1 and 2 in Example 2, 12 pairs of Indel markers used to screen haploids and double haploids showed obvious polymorphism and could be used for genotype identification of transgenic materials, as shown in FIG. 3.

    3. Genotyping and Ploidy Detection for Haploid and Double Haploid Material

    [0121] For screening the haploid material or double haploid material in Example 2, the test results of the CDS sequence of OsCPRO2 in Example 1:12 Indel marker sites showed single band genotype (FIG. 4), which is haploid material or double haploid material. Two ploidy (CPRO 2-22-1 and CPRO 2-39-1) showed haplotype (n) genome and 2 (CPRO 2-32-1 and CPRO 2-32-2) showed diplotype (2n) genome as double haploid material (FIG. 5).

    4. Trait Examination of Haploid and Double-Haploid Materials

    [0122] For the haploid and double haploid materials selected from embodiment 1 and 2 in Example 2, taking the detection results of AtDD45 oocyte specific expression promoter driving OsCPRO2 to achieve ectopic expression in embodiment 1 as an example, compared with the wild-type CY84, haploid materials were significantly affected in terms of nutritional and reproductive growth: plant height decreased, spike length shortened, glume reduced, and fertility completely lost (FIG. 6). Compared with the wild-type CY84, there was no significant difference in nutritional and reproductive growth between the double haploid material (FIG. 7). Compared with the Col-0 ecotype wild Arabidopsis thaliana, the haploid material obtained from embodiment 3 in Example 2 was significantly affected in both nutritional and reproductive growth: the plant size decreased and the leaves narrowed (FIG. 8).

    5. Detection of the CPRO Series of Haploid Induced Materials and Apomixis Materials

    [0123] For the T1 generation transgenic positive materials of embodiment 1, 2, and 3 in Example 2, it was found that the materials with haploid induction ability had a variation range of 18.982.14%-50.880.74% in seed setting rate and 0.44%7.14% in induction rate. The specific statistical information is shown in Table 18. Testing was conducted on the T1 generation transgenic positive materials of embodiment 4 and 5 in Example 2, and it was found that the fusion free reproductive materials produced by ectopic expression of OsCPRO1 and OsCPRO2 genes in oocytes combined with the MiMe system ranged in proportion to cloned seeds from 0.95% to 7.92%. The specific statistical information is shown in Table 19.

    TABLE-US-00022 TABLE 18 CPRO detection of haploid-induced materials in the series Haploid or double haploid/total Experimental Plant plant number example Genotype number Setting percentage (induction rate) Rice wild WT CY84 82.12 4.02% 0 type Experiment The CDS sequences of 4 22.48 1.50%** 8/112 (7.14%) case 1 the ectopically expressed OsCPRO1 17 24.91 1.93%** 5/240 (2.08%) gene 20 27.35 3.02%** 4/69 (5.80%) The CDS sequences of 22 28.83 0.62%** 1/76 (1.32%) the ectopically 32 19.94 1.94%** 4/153 (2.61%) expressed OsCPRO2 39 25.13 4.74%** 1/226 (0.44%) gene Experiment Genomic sequence of 19 25.73 1.97%** 2/115 (1.74%) case 2 the ectopically 35 23.56 2.32%** 5/102 (4.90%) expressed OsCPRO1 36 28.87 0.92%** 7/111 (6.31%) gene Genomic sequence of 2 24.82 1.98%** 2/95 (2.11%) the ectopically 11 18.98 2.14%** 3/162 (1.85%) expressed OsCPRO2 23 30.35 1.04%** 2/326 (0.61%) gene Wild type of WT Col-0 84.25 3.23% 0 Arabidopsis thaliana Experimental The CDS sequences of 1 41.15 1.48%** 1/100 (1.00%) case 3 the ectopically 9 32.08 1.76%** 1/102 (0.98%) expressed At CPRO1 21 25.40 2.39%** 2/96 (2.08%) gene The CDS sequences of 3 33.34 1.57%** 1/95 (1.05%) the ectopically 7 27.69 3.85%** 1/84 (1.19%) expressed At CPRO2 14 50.88 0.74%** 1/116 (0.86%) gene Using wild-type CY84 as a control, the data were t-test, error lines indicate the standard deviation (n = 3), ** P < 0.01.

    TABLE-US-00023 TABLE 19 Detection of CPRO series Number of cloned seeds/ Experimental Plant total grains example Genotype number Setting percentage (induction rate) Control WT CY84 82.12 4.02% 0 materials Experimental The CDS sequence of the 5 29.20 1.67%** 3/96 (3.13%) case 4 ectopically expressed 11 19.84 2.43%** 8/101 (7.92%) OsCPRO1 genes is bound 23 25.81 1.87%** 3/86 (3.49%) to MiMe The CDS sequence of the 2 18.92 1.09%** 3/80 (3.75%) ectopically expressed 21 21.45 0.56%** 2/144 (1.39%) OsCPRO2 genes is bound 22 26.55 1.73%** 2/211 (0.95%) to MiMe Experimental The genomic sequence of 17 22.75 1.18%** 4/105 (3.81%) case 5 ectopically expressed 32 28.90 0.44%** 5/111 (4.50%) OsCPRO1 genes binds to 35 27.33 1.54%** 2/104 (1.92%) MiMe The genomic sequence of 24 21.78 1.20%** 1/65 (1.54%) ectopically expressed 31 19.65 2.28%** 2/88 (2.27%) OsCPRO2 genes binds to 34 25.98 3.01%** 1/94 (1.06%) MiMe Using wild-type CY84 as a control, the data were t-test, error lines indicate the standard deviation (n = 3), ** P < 0.01.