Haploid induction method based on brassica napus phospholipase gene BnaPLA2-a and application of haploid induction method

Abstract

The invention relates to a haploid induction method based on a brassica napus phospholipase gene BnaPLA2-a and application of the haploid induction method, the BnaPLA2-a gene in brassica napus is knocked out through a gene editing technology, a mutant material capable of inducing haploid generation is obtained, and the application blank of a phospholipase pathway in dicotyledon haploid induction is filled. Compared with a traditional microspore culture technology, the invention provides a brand-new haploid induction path which is derived from the rape and is used for the rape. The invention provides a brand new technical tool and germplasm resources for genetic breeding of brassica napus. Meanwhile, the invention discloses a gene, a mutant, a creation method and application in breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a haploid induction method based on the phospholipase gene BnaPLA2-a of Brassica napus and its application. Background Technology

[0002] Rapeseed (Brassica napus) is an important dicotyledonous oilseed crop in my country. Haploid breeding technology can obtain homozygous lines in just two generations, significantly shortening the time required for modern breeding, and is therefore now one of the important methods for crop genetic improvement. Currently, the haploid-induced genes BnaDMP and BnaGIG1 have been reported in Brassica napus.

[0003] Stock6 is the first naturally occurring haploid induction line in maize. Kelliher et al. located a pollen-specific phospholipase gene, MTL, in qhir1 and found that haploid induction was caused by a 4bp insertion into MTL. Subsequently, gene editing using Talen technology generated MTL mutants, which were found to possess haploid induction ability, with HIR (Haploid Induction Rate) ranging from 4.0% to 12.5%. In maize, omics analysis demonstrated that ROS bursts are key to HI (Haploid Induction), and by treating pollen in vitro with ROS inducers, they successfully induced 2.2%–17.2% haploids in maize under different environments. In wheat, this gene has three homologs: TapLA-A, TapLA-B, and TapLA-D, all located in the endoplasmic reticulum. Frameshift mutants of TapLA-A and TapLA-D showed normal pollen viability but significantly reduced seed set, with HIR ranging from 5.56% to 21.56%. The rice OspPLAIIφ / PLP1 gene is homologous to the maize MTL gene and is specifically expressed in pollen. Similar to maize, the pollen viability and pollen germination rate of the rice mutant are not different from those of the wild type, with an HIR of approximately 6%.

[0004] The technology for creating haploid inducible lines based on the phospholipase gene pathway is well-established in monocotyledonous crops, but it has not been proven in dicotyledonous crops. Based on the above analysis, it is essential to create in vivo haploid induction technology based on phospholipase gene mutants in dicotyledonous crops.

[0005] Currently, BnaDMP and BnaGIG1, published in Brassica napus, play completely different roles from phospholipases in crop reproductive development, and their induction mechanisms are also fundamentally different. Bioinformatics data analysis has revealed no homologous phospholipase genes in dicotyledonous crops. Exploring novel haploid induction techniques based on phospholipase genes in dicotyledonous crops would overcome the limitation that phospholipase-related gene induction can only be achieved in monocotyledonous plants, and would be of great significance to the development of dicotyledonous crop breeding technology. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing haploid induction techniques in Brassica napus, such as reliance on exogenous genes or complex operations, and to provide a highly efficient haploid induction method based on the phospholipase gene BnaPLA2-a of Brassica napus. By exploring the phospholipase pathway in the dicotyledonous plant Brassica napus and selecting some related genes for transgenic verification, it was finally discovered that the BnaPLA2-a gene has inducible ability. This invention is the first to identify and confirm that the mutation of the phospholipase gene BnaPLA2-a in the dicotyledonous plant Brassica napus has haploid induction function. It pioneers a new technical route for haploid induction based on phospholipase genes in Brassica napus, enabling efficient and convenient in vivo haploid induction in important dicotyledonous crops such as Brassica napus, accelerating the breeding process, and providing a new technical tool and germplasm resource for the genetic breeding of Brassica napus.

[0007] The present invention specifically adopts the following technical solution: I. Gene Identification and Core Discoveries This invention marks the first identification and cloning of the phospholipase gene BnaPLA2-a (nucleotide sequence shown in SEQ ID NO. 1-2, encoded amino acid sequence shown in SEQ ID NO. 3-4) in Brassica napus. Bioinformatics analysis revealed that the protein encoded by this gene contains a typical phospholipase structure, but shows no homology to key haploid-inducing genes such as ZmPLA1 in monocotyledonous plants.

[0008] II. Methods for Creating Haploid Inducible Lines 1. Gene editing target design: Brassica napus is an allotetraploid crop (AACC, 2n=38), and its genome has high redundancy. This invention innovatively designs sgRNAs that simultaneously target all BnaPLA2-a homologous genes on the A and C genomes (the target sequences are shown in SEQ ID NO.5-6). This multi-target editing strategy aims to ensure that all functionally redundant gene copies are completely knocked out, which is a key technical means to overcome the low induction efficiency.

[0009] 2. Construction of mutant vectors and genetic transformation Using the CRISPR / Cas9 system, specifically the vector PKSE401-GFP, the designed sgRNA was constructed into a plant expression vector. Through Agrobacterium-mediated genetic transformation, specifically using Agrobacterium GV3101 strain, the vector was introduced into the early-maturing Brassica napus variety Westar. After resistance selection and regeneration culture, T0 generation transgenic plants were obtained.

[0010] 3. Mutant screening and establishment of inducible lines Hi-TOM editing technology was used to detect T0 generation plants and positive plants with frameshift mutations or loss of function in the BnaPLA2-a gene were screened out. The T0 generation plants were then self-crossed, and plants without Cas9 transgenic elements but with homozygous mutations in the BnaPLA2-a gene at all genomic loci were screened out in the T1 generation population to obtain the botryoid inducible line of Brassica napus.

[0011] 4. Verification of induction ability Using a newly created BnaPLA2-a gene mutant as the male parent, the line was crossed with different varieties of Brassica napus, including nuclear male sterile, cytoplasmic male sterile, and mosaic varieties, to test the effect on producing maternal haploids. Under different genetic backgrounds, this inducible line could stably induce maternal haploids, with induction rates ranging from 0.35% to 0.87%. This data provides the first quantitative experimental evidence for the haploid induction efficiency of the phospholipase pathway in dicotyledonous plants, demonstrating the feasibility of this technical approach.

[0012] Specifically, the present invention provides the application of the phospholipase gene BnaPLA2-a from Brassica napus in the preparation of haploid inducible lines of dicotyledonous plants and / or haploid breeding of dicotyledonous plants, wherein the amino acid sequence encoded by the phospholipase gene BnaPLA2-a is as shown in any one of SEQ ID NO. 3-4.

[0013] Furthermore, the nucleotide sequence of the phospholipase gene BnaPLA2-a is as shown in any one of SEQ ID NO.1-2.

[0014] Furthermore, by silencing or inhibiting the expression and / or activity of the phospholipase gene BnaPLA2-a or knocking out the phospholipase gene BnaPLA2-a, transgenic plants can be obtained, thus obtaining a haploid inducible line of dicotyledonous plants; or by further hybridizing the transgenic plants or their offspring with other dicotyledonous plants as the male parent to obtain hybrid offspring, thus obtaining a haploid line of dicotyledonous plants.

[0015] Furthermore, after obtaining transgenic plants, the transgenic plants are self-crossed, and plants with homozygous mutations in the BnaPLA2-a gene at all genomic loci are screened out to obtain stable haploid inducible lines of dicotyledonous plants.

[0016] Furthermore, by selecting haploid offspring from the hybrid offspring, a dicotyledonous haploid can be obtained.

[0017] Furthermore, the phospholipase gene BnaPLA2-a was edited using CRISPR / Cas9 system-mediated gene editing technology. The sgRNAs used included sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO.5 and the sequence of sgRNA2 is shown in SEQ ID NO.6.

[0018] Furthermore, the dicotyledonous plant is Brassica napus.

[0019] The present invention also provides a method for preparing a Brassica napus type oleoploid inducible line, comprising the following steps: obtaining transgenic plants by silencing or inhibiting the expression and / or activity of the phospholipase gene BnaPLA2-a or knocking out the phospholipase gene BnaPLA2-a, thereby obtaining a Brassica napus type oleoploid inducible line.

[0020] Furthermore, the phospholipase gene BnaPLA2-a was edited using CRISPR / Cas9 system-mediated gene editing technology. The sgRNAs used included sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO.5 and the sequence of sgRNA2 is shown in SEQ ID NO.6.

[0021] Furthermore, the method for editing the phospholipase gene BnaPLA2-a using the CRISPR / Cas9 system-mediated gene editing to obtain transgenic plants specifically includes: S1. Design and synthesize primers as shown in SEQ ID NO.7-10 to construct sgRNA1 and sgRNA2 into the plant expression vector PKSE401-GFP; S2. Using Agrobacterium-mediated genetic transformation, the PKSE401-GFP vector containing sgRNA1 and sgRNA2 was introduced into Brassica napus. S3. Transgenic plants were obtained through resistance screening and regeneration culture.

[0022] The present invention also provides a method for preparing a haploid oleifera of the Brassica napus type. The haploid induction line or its offspring prepared by the above method is used as the male parent and crossed with other rapeseeds to obtain hybrid offspring, thereby obtaining a haploid oleifera of the Brassica napus type.

[0023] Furthermore, by selecting haploid offspring from the hybrid offspring, a monoploid of the Brassica napus type is obtained.

[0024] This invention also provides any one of the following applications (A1)-A3): A1) Application of the Brassica napus type oil otoploid induction line prepared by the above method in the breeding of Brassica napus type oil otoploid; A2) Application of substances that silence or inhibit the expression and / or activity of the phospholipase gene BnaPLA2-a or knock out the phospholipase gene BnaPLA2-a in the preparation of oleostigma brassica oleifera induction lines and / or oleostigma brassica oleifera breeding. A3) Application of CRISPR / Cas9 systems containing sgRNA1 as shown in SEQ ID NO. 5 and / or sgRNA2 as shown in SEQ ID NO. 6 in the preparation of motile inducible lines of oleander-type oleander and / or motile breeding of oleander-type oleander. Beneficial effects: 1. Original innovation: This invention is the first to realize the haploid induction of phospholipase gene in the dicotyledonous plant Brassica napus, filling the technological gap in this field and representing a true breakthrough from "0" to "1".

[0025] 2. Sufficient universality verification: Successful verification on various special germplasm resources, such as nuclear male sterility and cytoplasmic male sterility, which are difficult to produce haploids through traditional methods, proves that the invention has broad applicability and provides an irreplaceable technical means for rapidly creating homozygotes of these precious materials.

[0026] 3. Commercial and legal value: The created induction lines and methods do not rely on any transgenic sequences from exogenous species, and the final products do not contain exogenous genes, which has significant advantages in terms of regulatory compliance and commercial promotion.

[0027] In summary, this invention not only provides a specific method for inducing otoploid oleifera in Brassica napus, but also pioneers a new technical path for genetic manipulation using the plant's own phospholipase genes in dicotyledonous plants, which is of milestone significance for the development of crop breeding technology. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a map of the vector PKSE401-GFP in an embodiment of the present invention.

[0030] Figure 2This is a schematic diagram of the BnaPLA2-a target point in rapeseed in an embodiment of the present invention.

[0031] Figure 3 This refers to the editing type of Brassica napus BnaPLA2-a in this embodiment of the invention.

[0032] Figure 4 This invention relates to the screening and flow cytometry identification of SSR molecular markers in embodiments of the present invention.

[0033] Figure 5 This is for the identification of the rapeseed 50K chip in the embodiments of the present invention.

[0034] Figure 6 This is a diagram showing the haploid induction efficiency in an embodiment of the present invention.

[0035] Figure 7 This is a haploid phenotypic diagram from an embodiment of the present invention. Detailed Implementation

[0036] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0037] The plant materials used in this invention are: the early-maturing Brassica napus variety Westar, and the hybrid parent materials are cytoplasmic male sterility material ganA, nuclear male sterility material S45A, and mosaic material HY. All materials used in the experiments were subjected to an environment of 22 ℃ and 100 µmol / m² light. -2 s -1 They were grown in a plant culture room with a photoperiod of 16 h light / 8 h darkness.

[0038] Example 1. Identification of the BnaPLA2-a gene This invention explores the phospholipase pathway in the dicotyledonous plant Brassica napus and selects some related genes for transgenic verification, specifically including genes BnaPLA2-a, BnaMCTP12, and BnaDALL2. Ultimately, it was found that only the BnaPLA2-a gene has haploid induction ability.

[0039] This invention marks the first identification and cloning of the phospholipase gene BnaPLA2-a in Brassica napus. Bioinformatics analysis revealed that the protein encoded by this gene contains a typical phospholipase structure, but it shows no homology to key haploid-inducing genes such as ZmPLA1 in monocotyledonous plants.

[0040] In Brassica napus, there are two BnaPLA2-a genes: BnaPLA2-a.A05 (BnaA05G0158800WE) and BnaPLA2-a.C06 (BnaC06G0115000WE), whose nucleotide sequences are shown in SEQ ID NO.1-2 and whose encoded amino acid sequences are shown in SEQ ID NO.3-4. BnaPLA2-a.A05: ATGGCGGCTCCGATCATACTTTCCTGTTTCCTTTTCTTATTCTTCTTCTGTCTCCGTCTCTGCTCTTAACGTCGGTGTTCAGCTCACACATCCCACCGTTTCTCTGAGCAAAGAATGTAGCCGGAAATGTGAATCAGAGTTCTGTTCAGTGCCTCCACTTTTGAGGTATGGAAAGTACTGTGGACTATTGTACAGTGGATGTCCTGGAGAGAGACCCTGTGATGGT CTTGACTCATGTTGCATGAAGCATGATGCTTGTGTCCAATCCAAGAATAATGATTATCTAAGCCAAGAGTGTAGTCAGAAGTTCATAAACTGCATGAACAATTTCAGCAATACGAAGCAACCAACGTTTAATGGTAACACATGCGATCCCGATGAAGTCATTGATGTCATCTCTATTGTCATGGACGCCGCTCTTATCGCCGGCAGAGTCTTCCGGAAACCCTAA (SEQ ID NO.1) BnaPLA2-a.C06: ATGGCGGCTCCGATCATACTTTCCTGTTTTTTTTTGTCATTCTTCTTCTCTGTCTCCGTCTCTGCTCTTAACGTCGGTGTTCAGCTCACTCATCCCACCGTTTCTCTGAGCAAAGAATGTAGCCGGAAATGTGAATCAGAGTTCTGTTCAGTGCCTCCACTTTTGAGGTATGGAAAGTACTGTGGACTATTGTACAGTGGATGTCCTGGAGAGAGACCTTGCGATGGTCTTGATTCATGTTGCATGAAGCATGATGCTTGTGTCCAATCCAAGAATAATGATTATCTAAGCCAAGAGTGTAGTCAGAAGTTCATAAACTGCATGAACAATTTCAGCAATACGAAGCAACCAACGTTTAATGGTAACACATGCGATCCCGATGAAGTCATTGATGTCATCTCCATTGTCATGGACGCCGCTCTTATCGCCGGCAGAGTCTTCCGGAAACCCTAA (SEQ ID NO.2) BnaPLA2-a.A05 protein: MAAPIILSCFLFLFFFSVSVSALNVGVQLTHPTVSLSKECSRKCESEFCSVPPLLRYGKYCGLLYSGCPGERPCDGLDSCCMKHDACVQSKNNDYLSQECSQKFINCMNNFSNTKQPTFNGNTCDPDEVIDVISIVMDAALIAGRVFRKP (SEQ ID NO.3) BnaPLA2-a.C06 protein: MAAPIILSCFFLSFFFSVSVSALNVGVQLTHPTVSLSKECSRKCESEFCSVPPLLRYGKYCGLLYSGCPGERPCDGLDSCCMKHDACVQSKNNDYLSQECSQKFINCMNNFSNTKQPTFNGNTCDPDEVIDVISIVMDAALIAGRVFRKP(SEQ ID NO.4) 2. Construction of BnaPLA2-a gene knockout vector (1) Primer design Brassica napus is an allotetraploid crop (AACC, 2n=38), and its genome has high redundancy. This invention uses the CRISPR / Cas9 system and the online tool CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to predict the sgRNA sequence of the target gene. Based on factors such as the GC content of the target site, two suitable sgRNA sequences with the highest knockout efficiency were selected (as shown in SEQ ID NO. 5-6). sgRNA1:CCCACCGTTTCTCTGGTTAGAT (SEQ ID NO.5) sgRNA2: ATCTAACCAGAGAAACGGTGGG (SEQ ID NO.6) This invention innovatively designs a multi-target editing strategy using sgRNAs that simultaneously target all BnaPLA2-a homologous genes on both the A and C genomes. This strategy aims to ensure the complete knockout of all functionally redundant gene copies, and is a key technical means to overcome low induction efficiency. The target pattern diagram is shown below. Figure 2 As shown.

[0041] The designed sgRNA was constructed into the plant expression vector PKSE401-GFP (vector map shown below). Figure 1 (As shown), primers were synthesized according to the vector usage instructions. The primer sequences are shown in SEQ ID NO.7-10: BnaPLA2-a DT1-BsF (SEQ ID NO.7): ATATATGGTCTCGATTGATCTAACCAGAGAAACGGTGTT BnaPLA2-a DT1-F0 (SEQ ID NO.8): TGATCTAACCAGAGAAACGGTGTTTTAGAGCTAGAAATAGC BnaPLA2-a DT2-R0 (SEQ ID NO.9): AACGCGATAAGAGCGGCGTCCACAATCTCTTAGTCGACTCTAC BnaPLA2-a DT2-BsR (SEQ ID NO.10): ATTATTGGTCTCGAAACGCGATAAGAGCGGCGTCCACAA (2) PCR amplification Phanta Uc Super-Fidelity DNA Polymerase (P507-01) from Vazyme was used. The pCBC vector diluted 100-fold was used as a template. -BsF / -BsR were the normal primer concentrations, and -F0 / -R0 were diluted 20-fold for four-primer PCR amplification. The amplification system consisted of: pCBC 2 µL, BsF 1 µL, BsR 1 µL, F0 1 µL, R0 1 µL, Mix 1 µL, dNTP 1 µL, Buffer 25 µL, and ddH2O 17 µL. The reaction conditions were: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 45 s, 35 cycles; 72 °C final extension for 10 min; and cooling at 25 °C for 1 min.

[0042] The amplified fragment was 726 bp in length. After amplification, it was detected by electrophoresis using a 1% agarose gel, and the product was recovered using the gel DNA recovery kit (2001050 / 2001250SK8132) from Newview Biotech.

[0043] (3) Enzyme digestion and ligation The enzyme digestion and ligation system consisted of 2 µL PCR product, 2 µL PKSE401 vector, 1 µL BsaI, 1.5 µL 10 × Cutsmart Buffer, 1 µL T4 Ligase, 1.5 µL 10 × T4 Ligase Buffer, and 6 µL ddH2O. Enzyme digestion and ligation were performed in a PCR instrument (reaction program: 37 ℃ for 5 h, 50 ℃ for 5 min, and 80 ℃ for 10 min). After enzyme digestion and ligation, the reaction system was cooled at 4 ℃ or on ice.

[0044] (4) Product cloning The enzyme digestion and ligation products were transformed into DH5α competent cells for cloning.

[0045] a) Take 20-50 µL of DH 5α competent cells into a sterile centrifuge tube, add 10 µL of ligation product, gently tap the tube wall to mix, and place on ice for 20-30 min. b) After incubating the centrifuge tubes in a water bath at 42 °C for 90 s, immediately place them on ice to cool for 2 min, then add 200 µL LLB liquid culture medium (without antibiotics), and shake at low speed (150 g) at 37 °C for 1 h. c) Spread 100 µL of bacterial culture evenly on a plate containing 100 mg / mL kanamycin sulfate and incubate at 37 °C for 12 h-16 h.

[0046] d) Positive clones detected by colony PCR, amplified to a size of 726 bp. The colony PCR primers are as follows: U626p-F: TGTCCCAGGATTAGAATGATTAGGC U629p-R: AGCCCTCTTTCTTTCGATCCATCAAC e) Positive clones were selected and sequenced at Quintiles (Wuhan) Biotechnology Co., Ltd., using U626p-F as the sequencing primer.

[0047] 3. Genetic transformation of hypocotyls in Brassica napus After successful vector sequencing, the plasmid was extracted and transferred into Agrobacterium tumefaciens strain GV3101 (Video Biotechnology CAT#: AC1001) for further genetic transformation experiments. The Agrobacterium tumefaciens strain GV3101-mediated genetic transformation method was used to transform the early-maturing Brassica napus variety Westr. After resistance selection and regeneration culture, T0 generation gene-edited material was obtained. The specific transformation method is as follows: (1) Soak rapeseed seeds in 75% alcohol for 1 minute, pour the soaked seeds into a sterile culture box, rinse with sterile water, add an appropriate amount of disinfectant HgCl2 (0.1%-0.2%), and sterilize for 15 minutes. For heavily contaminated seeds, the sterilization time can be extended to 20 minutes; (2) After surface sterilization of the seeds, rinse the seeds with sterile water 4-5 times; (3) Sterilize the tweezers with high temperature, cool them, and then sow them. Sow the sterilized seeds onto M0 medium, with 30-40 seeds in each sowing box. Place the sterile culture box in a dark environment at 24 ℃ for 5-6 days. (4) One day before infection, Agrobacterium was added to LB liquid medium containing the corresponding antibiotic at a volume ratio of 1:1000 and cultured at 28 °C for 12-16 h until the OD of the bacterial culture was reached. 600 =0.3-0.4; (5) Centrifuge the cultured Agrobacterium at 3500 g for 10 min and collect the bacterial cells. Discard the supernatant and resuspend the cells in 10 mL of DM liquid medium for later use; (6) Use sterile forceps and a scalpel to cut the hypocotyls of etiolated seedlings 6 days after sowing. The cut hypocotyls are the explant material at the time of infection. Each explant is 0.8-1.0 cm long. (7) Place the cut explants into a sterilized glass dish and infect them with Agrobacterium tumefaciens solution containing DM liquid medium for 10-15 min, shaking once every 5 min. (8) After infection, pour off the bacterial solution, use sterile filter paper to absorb the remaining bacterial solution, and dry the explants in a clean bench. Then transfer the explants to M1 solid culture medium and place them in the dark at 24 ℃ for 36-48 h. (9) Transfer the explants on M1 medium to M2 medium and add the appropriate antibiotics for screening; (10) After culturing on M2 medium for 3 weeks, the explants were transferred to M3 differentiation medium and subcultured every 2-3 weeks until green shoots appeared. After the green shoots appeared, the green shoots containing the growth points were cut off with a scalpel and placed into M4 rooting medium. After the roots grew strong, the transgenic positive plants were transplanted to a greenhouse or transgenic experimental field.

[0048] 4. Hi-TOM high-throughput sequencing determined the mutant genotype of the edited single plant. To enable high-throughput detection of gene mutations in transgenic positive single plants and their offspring, two rounds of overlap PCR were performed according to the method of Liu et al., followed by high-throughput sequencing of self-built libraries (Liu et al 2019).

[0049] The principle of Hi-TOM is to construct a library and sequence it after two rounds of PCR: (1) Use site-specific primers (SSP) to perform the first round of PCR amplification on the DNA fragment containing the target site; (2) Use a set of universal barcoding primers for the second round of amplification; (3) Mix equal amounts of the second round amplification products from different monocultures and perform gel recovery, and send the recovered products to Nanjing Paisennong Biotechnology Co., Ltd. for second-generation sequencing of 150 bp at the paired ends; (4) Decode and analyze the mutation sequence through the online Hi-TOM website (http: / / www.hi-tom.net / hi-tom / ) to determine the mutation type of each monoculture at each target site.

[0050] (1) Design principles of primers for the first round of PCR Since the effective length of paired-end sequencing is 150 bp, and there is an adapter sequence of about 30 bp between the sequencing primers and the designed specific primers (SSP), the distance between the designed specific primers and the target site should ideally be 20-80 bp, and should not exceed 120 bp, to ensure that paired-end sequencing can detect the target site. If the amplified fragment contains multiple target sites, all target sites must be within the range covered by paired-end sequencing.

[0051] For genes with multiple copies, if the first-round specific primers specifically amplify each copy, subsequent sequencing data analysis can be performed directly using online platforms. If the first-round specific primers can amplify multiple copies simultaneously, the primer design must ensure that there are SNP / INDEL sequence differences within the amplified region. This ensures that the sequenced sequences can be distinguished by these SNP / INDEL sequence differences. Before using online platforms for analysis, the sequenced sequences must be classified by copy and then analyzed separately. The designed specific primers need to have adapter sequences added to their 5′ ends. The adapter sequences and the first-round PCR primers are shown below: Add the following to the 5′ end of primer F: 5′-ggagtgagtacggtgtgc-3′ Add the following to the 5′ end of the R primer: 5′-gagttggatgctggatgg-3′ HI-A05-2-F:ggagtgagtacggtgtgcTCATACTTTCCTGTTTCCTTTTCTT HI-A05-2-R:gagttggatgctggatggCAATCTTTTGATTCATATCAAAACGAAA HI-A05-11-F:ggagtgagtacggtgtgcAATGGTAACACATGCGATC HI-A05-11-R:gagttggatgctggatggGAGAAACAATTAACTCAAACTATAGAAG HI-C06-2-F:ggagtgagtacggtgtgcCCATTATTAACTAGTTTTTATCTCTGTT HI-C06-2-R:gagttggatgctggatggCATACTTTCCTGTTTTTTTTTGTC HI-C06-11-F:ggagtgagtacggtgtgc GAGAAACAATTAACTCAAACTATAGAAA HI-C06-11-R: gagttggatgctggatgg AAGTCATTGATGTCATCTCC (2) First round of PCR amplification The first round of PCR amplification was performed using CWBIO 2 × Taq Master Mix (Dye Plus) (CW6090). The amplification system consisted of: DNA 1 µL, Primer F 0.5 µL, Primer R 0.5 µL, 2 × Taq Master Mix 5 µL, and ddH2O 3 µL. The reaction conditions were: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 56 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for 32 cycles; 72 ℃ final extension for 10 min; and cooling at 25 ℃ for 1 min.

[0052] The amplification product sizes of BnaPLA2-a.A05 were 156 bp and 137 bp, respectively, and those of BnaPLA2-a.C06 were 191 bp and 110 bp, respectively. 5 µL of the first-round amplification product was subjected to electrophoresis to determine its amplification specificity and amplification intensity.

[0053] (3) Second round of PCR amplification The first-round PCR product, diluted 10-40 times, was used as the DNA template and universal primers for amplification. The universal primers consisted of 12 F primers (F1-F12), 8 R primers (RA-RH), and a pair of index primers. The 12 F primers (F1-F12) and 8 R primers (RA-RH) were mixed according to the corresponding primer combinations for a 96-well plate to prepare the Primer mix. The preparation method was as follows: 1. Add 6 µL each of 10 µM F1-F12 to each row of the PCR plate; 2. Add 66 µL each of 10 µM RA-RH to each column of the PCR plate; 3. Add 78 µL of ddH2O to each well, mix well, centrifuge, and store at -20 ℃ for later use.

[0054] The second round of PCR amplification was performed using CWBIO 2 × Taq Master Mix (Dye Plus) (CW6090). The amplification system consisted of: DNA 1 µL, Index Primer F 0.2 µL, Index Primer R 0.2 µL, Primer Mix 3 µL, 2 × Taq Master Mix 5 µL, and ddH2O 0.6 µL. The reaction conditions were: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 30 s, for 32 cycles; 72 °C final extension for 10 min, and cooling at 25 °C for 1 min.

[0055] After amplification, 5 µL of the second-round PCR product was run on a gel to determine its amplification specificity and intensity. If the intensity of each well was similar, 1 µL of amplified product from each well on each PCR plate was mixed thoroughly. If the intensity of individual wells differed significantly, adjustments needed to ensure equal mixing of PCR product from each well. 50 µL of the mixture was then purified and recovered via gel electrophoresis. The recovered product was sent to Nanjing Paiseno Biotechnology Co., Ltd. for next-generation sequencing. Typically, the data size for each sample on each plate is 1 GB.

[0056] (4) Sequencing data analysis By submitting paired-end sequencing files and reference sequence files online through the Hi-Tom website (http: / / www.hi-tom.net / hi-tom / ), the mutation status of individual plants was analyzed, and the final editing status of individual plants is as follows: Figure 3 As shown, the results indicate that the BnaPLA2-a gene has a 2bp deletion on chromosome A05, which corresponds to GT; and a 2bp deletion on chromosome C06, which corresponds to AC.

[0057] This invention successfully obtained positive plants with frameshift mutations or loss of function in the BnaPLA2-a gene. The T0 generation plants were self-crossed, and in the T1 generation population, plants without Cas9 transgenic elements but with homozygous mutations in the BnaPLA2-a gene at all genomic loci were screened out. This yielded a haploid inducible line of the male parent of Brassica napus.

[0058] 4. Haploid identification (1) GFP fluorescence screening Potential haploids in the hybrids of the bnapla2-a inducible line and wild-type material, denoted by GFP, were screened using a LUYOR-3415RG dual-wavelength fluorescent protein excitation light source from LUYOR (USA). Seeds were laid flat in petri dishes containing moistened filter paper and germinated at room temperature in the dark. After approximately 30 hours, the radicle emerged from the seed coat. The radicle was then irradiated with a GFP excitation light source; those showing fluorescence were hybrids, while those without fluorescence were potential haploid plants. Non-fluorescent seeds were placed in germination boxes containing Hogland nutrient solution for subsequent screening experiments.

[0059] (2) SSR molecular marker screening The SSR molecular markers used for haploid selection are co-dominant markers; if the plant is haploid, the SSR band should be a single band. By performing marker-assisted selection on the hybrid progeny, heterozygous plants are discarded, and the remaining plants are used for further screening experiments. The primer sequences are as follows: C09-2-F:GGTGCCCACTTTCCTCCG C09-2-R:ACTCCTCCAATGACCAAGTTT C02-4-F:TGGGACAGTGGATAGCCCA C02-4-R:TGAGACAGCCTACGATCATCT C09-1-F:TCATGTGCCCTCAGGACA C09-1-R: GCGAAGTTGAACTTGCAGCA A09-5-F:CACCAAGCGAGCTCTGGA A09-5-R: CGAGCGTTCCAACAGGGT The results are as follows Figure 4 As shown in the right figure, the first column is the 2000 Marker, the second column is the paternal band pattern, the third column is the maternal band pattern, the fourth column is the induced maternal haploid, which is consistent with the maternal haploid in size, and the last column is the diploid, which is mixed with the band pattern of the parents. Using the above SSR molecular markers, haploid materials with only a single band can be successfully screened.

[0060] (3) Flow cytometry for cell ploidy identification Pluripotency of potential haploid plants was determined using a Sysmex CyStain UV Precise P (05-5002). Approximately 0.5 cm of sample was taken. 2 The leaf samples were placed in a culture dish, and 0.5 mL of Nuclei Extraction Buffer was added. The leaves were chopped using a blade and incubated at room temperature for 1 min. The samples were then filtered into sample tubes (04-2000) using a 50 µm CellTrics® filter (04-0042-2317). 2.0 mL of Staining Buffer was added to each sample tube, and the samples were incubated at room temperature for 1 min. Samples were loaded and analyzed using a Sysmex CyFlow Ploidy Analyser flow cytometer, and the data were plotted using FlowJo v10 software. The results are shown below. Figure 4 The left figure shows the graph, where the horizontal axis (DNA : DNA) represents the relative DNA content, reflecting the ploidy of DNA within the cell, and the vertical axis (Count) represents the number of cells, i.e., the number of cells corresponding to the DNA content. The blue peak represents diploid cells, whose DNA content is at an intermediate level; the peak height indicates a relatively large number of cells of this ploid type. The red peak represents haploid cells, whose DNA content is approximately half that of diploid cells; the peak height indicates a considerable number of haploid cells as well. The results show that haploid plants were successfully identified using flow cytometry.

[0061] (4) Rapeseed 50K chip typing The Bnapus50K SNP chip technology of Brassica napus was used to genotype hybrid parents and haploid plants, and to identify the genetic background of haploid plants (Xiao Qing 2022).

[0062] a) Rapeseed 50K chip typing process Reagents: DNA (50 ng / µL, 4 µL), NaOH (0.1 mol / L, NaOH), isopropanol, 95% formamide / 1 mM EDTA, MA1, MA2, MSM, FMS, PM1, PB1, PB2, RA1, XC1, XC2, XC3, XC4, TEM, ATM, STM.

[0063] Tools: MSA3 plate, hybridization furnace, rack, 24-hole SNP chip.

[0064] 1) DNA quantification and DNA amplification: Add 20 µL MA1, 4 µL DNA and 4 µL 0.1 mol / L NaOH to the MSA3 plate, shake at 1600 g for 1 min, centrifuge at 280 g for 1 min, and let stand for 10 min; then add 34 µL MA2 and 38 µL MSM, shake at 1600 g for 1 min, centrifuge at 280 g for 1 min; place in a hybridization oven at 37 ℃ for 20-24 h; 2) DNA fragmentation: Preheat the dry bath to 37 °C; remove the FMS from the freezer to thaw, gently invert to mix, and centrifuge at 280 g for 1 min; remove the MSA3 plate from the hybridization oven and centrifuge at 50 g for 1 min; add 25 µL of FMS, cover, vortex at 1600 g for 1 min, centrifuge at 22 °C for 1 min at 50 g, and dry bath at 37 °C for 1 h; 3) DNA enrichment: Add 50 µL PM1 to the MSA3 plate, cover, shake at 1600 g for 1 min, dry in a 37 ℃ bath for 5 min, centrifuge at 22 ℃ for 1 min at 50 g; add 155 µL isopropanol, cover with a new lid, invert 10 times; place in a 4 ℃ refrigerator for 30 min, centrifuge at 4 ℃ for 20 min at 2200 g; remove the lid, pour out the liquid, invert on clean filter paper, and dry at room temperature for 1 h; 4) DNA fragment recovery: Adjust the hybridization oven temperature to 48 ℃, preheat the sealing machine for 20 min, thaw RA1 at room temperature, and gently invert to dissolve; add 23 µL RA1 to the MSA3 plate; seal with aluminum foil and incubate at 170 ℃ for 5 s; incubate in the hybridization oven at 48 ℃ for 1 h, shake at 1600 g for 1 min, and centrifuge at 280 g for 1 min; 5) DNA denaturation: Denature the MSA3 plate in a 95 ℃ dry bath for 20 min, then cool to room temperature for 30 min; 6) DNA Hybridization with Chips: Prepare the hybridization box and chips. Place a gasket in the hybridization box, add 400 µL of PB2 to each of the 8 small slots in the hybridization box, and close the lid. Take the chips out of the 4 ℃ freezer and place them in the corresponding positions in the hybridization box, ensuring that the barcode positions on the chips correspond to the barcode positions in the hybridization box. Apply 12 µL of sample to each well of each chip, carefully applying the sample vertically with a multi-channel pipette, avoiding air bubbles, and ensuring that the DNA covers the magnetic bead stripe area. 7) Resuspension using XC4 reagent: Place the hybridization box with the spotted sample in a hybridization oven at 48 ℃ for 16-24 h, and adjust the speed to 5; add 330 mL of anhydrous ethanol to XC4, shake vigorously for 15 s, and let it stand overnight at room temperature until ready for use; 8) Cleaning the chips: Remove the hybridization box from the 48 ℃ hybridization oven and let it cool to room temperature for 25 min. Adjust the water circulation system to 44 ℃. Add 200 mL of PB1 to two glass tanks, and place a washing rack in one of them. Remove the chips from the box and, wearing powder-free gloves, peel off the chip seal along the diagonal. Quickly place the chips in the washing rack and soak them in PB1, keeping the chip surface away from you. Carefully lift the rack up and down for 1 min, and wash the other rack for 1 min in the same way. 9) Install the flow chamber: Add 150 ml of PB1 to the installation box and place the black frame inside; remove the chip and place it back into the black frame in the installation box, ensuring the barcode on the chip is aligned with the barcode on the installation box, so that the chip is completely submerged in PB1; remove the white protective layer from the surface of the plastic gasket, place the plastic gasket on the chip and fit it onto the corresponding protrusion in the installation box; place the positioning rod, place the cleaning slide on the chip, with one sloping end facing the barcode and the slope facing down, forming a sample loading groove with the chip, and secure it with metal clips; remove the chip from the installation box and assemble it, trimming off any excess gasket at both ends with scissors; clean the PB1 in the box with ddH2O; 10) Single base extension: After installation, place the tube in a 44 ℃ circulating water bath. Add the following reagents to the flow chamber sequentially: 150 µL RA1, incubate for 30 s, repeat 5 times for a total of 6 times; 450 µL XC1, incubate for 10 min; 450 µL XC2, incubate for 10 min; 200 µL TEM, incubate for 15 min; 450 µL 95% formamide / 1 mM EDTA, incubate for 1 min; 450 µL 95% formamide / 1 mM EDTA, incubate for 6 min; adjust the circulating water bath temperature to 32 ℃ according to the temperature on the STM tube; 450 µL XC3, incubate for 1 min; 450 µL XC3, incubate for 1 min; wait for the circulating water bath to reach 32 ℃. 11) Chip staining: Add the following reagents sequentially to the flow cell: 250 µL STM for 10 min; 450 µL XC3 for 1 min; 450 µL XC3 for 6 min; 250 µL ATM for 10 min; 450 µL XC3 for 1 min; 450 µL XC3 for 6 min; 250 µL STM for 10 min; 450 µL XC3 for 1 min; 450 µL XC3 for 6 min; 250 µL ATM for 10 min; 450 µL XC3 for 1 min; 450 µL XC3 for 6 min; 250 µL STM for 10 min; 450 µL XC3 for 1 min; 450 µL XC3 for 6 min; quickly remove the flow cell and incubate at room temperature. 12) Washing and embedding: Add 310 mL of PB1 (8 chips) to the washing box and insert the staining rack with the locking arm facing you; remove the two metal clips, remove the slide, carefully remove the gasket, avoiding the magnetic bead area, remove the chip, and place it in the staining rack with the chip surface away from you; lift the staining rack up and down 10 times; soak for 5 minutes, shake the XC4 vigorously to resuspend it, and let it stand to allow the air bubbles to disappear; add the XC4 to another box and let it stand for no more than 10 minutes; transfer the staining rack into the XC4, lift the staining rack up and down 10 times, and soak for 5 minutes; place the staining rack on the test tube rack with the chip horizontal and the barcode facing upwards; place the chip in the test tube and put it in the vacuum pump for 1 hour; store the chip at room temperature for 72 hours (it can be stored for about 15 days at 4 ℃ in the dark), clean and dry each device, or blot dry with absorbent paper, and place it in its original place.

[0065] b) Chip scanning and data analysis The back of the chip was wiped clean with a lint-free soft cloth dampened with anhydrous ethanol and placed in Illumina's (www.illumina.com) dedicated chip scanner, HiScan. A laser was used to excite the fluorescent groups of the single-base extension products on the chip, and the scanner captured the fluorescence emitted by these groups, generating a high-resolution image and exporting the scan results. The scanned data was imported into GenomeStudio 2.0.4 software (Version 2015, Illumina Inc.) (Staaf et al. 2008) to extract the chip data and perform genotyping, obtaining SNP genotyping data for each sample.

[0066] The results are as follows Figure 5As shown, the four rows represent the induced line F, the wild-type maternal parent M, the haploid H, and the diploid D, respectively. We use gray to represent the induced male parent and red to represent different wild-type female parents. In their F1 generation, the induced maternal haploids are red, matching the color of the maternal parent, while the diploids are blue, representing the two different genotypes. The results show that haploid plants were successfully identified using the rapeseed 50K microarray genotyping.

[0067] (5) Haploid detection results This invention obtained BnaPLA2-a gene mutant plants through gene editing. These mutant plants were then crossed with four other Brassica napus varieties (cytoplasmic male sterility material ganA, nuclear male sterility material S45A, and mosaic material HY). The offspring were identified, and haploid plants were successfully screened from all of them. In the offspring flowering in March 2024, the induction rate of the maternal parent ganA was 0.87%, and in the offspring flowering in March 2025, the induction rate of the maternal parent S45A was 0.38%, and the induction rate of the maternal parent mosaic material HY was 0.35%. This indicates that the efficiency of the BnaPLA2-a gene as the paternal parent in inducing haploids is between 0.35% and 0.87%. Figure 6 Haploid plants were observed using materials hybridized with the maternal parent S45A. Figure 7 As shown, haploid plants develop slowly, with small and shriveled flower buds, petals that are significantly smaller than those of diploid plants, and male sterility, which is consistent with the characteristics of haploid plants.

[0068] In summary, this invention is the first to identify and clone the phospholipase gene BnaPLA2-a in rapeseed, and the first to demonstrate that mutating this gene in rapeseed can yield a haploid inducible line. When this inducible line was used as the male parent and crossed with various rapeseed varieties, it consistently induced maternal haploids under different genetic backgrounds, with induction rates ranging from 0.35% to 0.87%. This invention pioneers a novel haploid induction technique based on the phospholipase gene BnaPLA2-a in rapeseed, enabling efficient and convenient in vivo haploid induction in important dicotyledonous crops such as rapeseed, accelerating the breeding process, and providing a new technical tool and germplasm resource for the genetic breeding of rapeseed, with broad application prospects.

[0069] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Use of the Brassica napus phospholipase gene BnaPLA2-a for the preparation of a dicotyledon haploid inducer line and / or a dicotyledon haploid breeding, characterized in that, The amino acid sequence encoded by the phospholipase gene BnaPLA2-a is shown in any one of SEQ ID NO. 3-4.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the phospholipase gene BnaPLA2-a is shown in any one of SEQ ID NO. 1-2.

3. Use according to claim 1, characterized in that, By silencing or inhibiting the expression and / or activity of the phospholipase gene BnaPLA2-a or knocking out the phospholipase gene BnaPLA2-a, a transgenic plant is obtained, i.e. a haploid induction line of a dicotyledonous plant; or further crossing the transgenic plant or its offspring as a male parent with other dicotyledonous plants, a hybrid offspring is obtained, i.e. a haploid of a dicotyledonous plant.

4. Use according to claim 1, characterized in that, The sgRNA used in the gene editing technology mediated by the CRISPR / Cas9 system for editing the phospholipase gene BnaPLA2-a includes sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO. 5 and the sequence of sgRNA2 is shown in SEQ ID NO.

6.

5. Use according to any one of claims 1 to 4, characterized in that, The dicotyledonous plant is Brassica napus.

6. A method for preparing a Brassica napus haploid-inducing line, characterized in that, The method comprises the following step: by silencing or inhibiting the expression and / or activity of the phospholipase gene BnaPLA2-a or knocking out the phospholipase gene BnaPLA2-a, a transgenic plant is obtained, i.e. a haploid induction line of Brassica napus.

7. The method of claim 6, wherein, The sgRNA used in the gene editing technology mediated by the CRISPR / Cas9 system for editing the phospholipase gene BnaPLA2-a includes sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO. 5 and the sequence of sgRNA2 is shown in SEQ ID NO.

6.

8. A method for the production of a Brassica napus haploid, characterized in that, The haploid induction line or its offspring prepared by the method of any one of claims 6-7 is crossed with other Brassica napus as a male parent, a hybrid offspring is obtained, i.e. a haploid of Brassica napus.

9. The following A1)-A3) in any one of the following A1)-A3): A1) The use of the haploid induction line of Brassica napus prepared by the method of any one of claims 6-7 in haploid breeding of Brassica napus; A2) The use of a substance for silencing or inhibiting the expression and / or activity of the phospholipase gene BnaPLA2-a or knocking out the phospholipase gene BnaPLA2-a in the preparation of a haploid induction line of Brassica napus and / or in the haploid breeding of Brassica napus; A3) The use of a CRISPR / Cas9 system containing sgRNA1 shown in SEQ ID NO. 5 and / or sgRNA2 shown in SEQ ID NO. 6 in the preparation of a haploid induction line of Brassica napus and / or in the haploid breeding of Brassica napus.

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