Multi-technology fusion rapid breeding method based on exogenous frame-free wheat homozygous mutant creation

Abstract

The invention relates to a multi-technology fusion rapid breeding method based on exogenous frame-free wheat homozygous mutants, and belongs to the technical field of wheat genetic breeding. The method comprises the following steps: determining a target gene (such as an LCY gene), creating a homozygous mutant by utilizing a CRISPR-Cas9 gene editing technology, separating an exogenous Cas9 editing frame through hybridization and PCR detection, developing a KASP molecular marker to track a mutation type, and performing phenotype analysis. According to the method, gene editing, cross breeding and molecular detection technologies are fused, the problems that traditional breeding is long in period and foreign genes are difficult to remove are solved, rapid and accurate wheat germplasm creation is achieved, and an efficient way is provided for functional gene utilization.

Description

A rapid breeding method based on multi-technology fusion of wheat homozygous mutants without exogenous frames Technical Field

[0001] This invention relates to the field of wheat genetics and breeding, specifically to a rapid breeding method based on the creation of homozygous mutant wheat without exogenous frames, incorporating multiple technologies. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of my country's three major grain crops and the most important staple food crop in northern my country. With global population growth, climate change, and decreasing arable land resources, improving wheat yield, stress resistance, and quality has become a key focus of agricultural scientific research. Gene editing, a new technology that relies on nucleases to modify endogenous genes in organisms through site-specific knock-in or knock-out, has been widely applied in gene function research and gene therapy.

[0003] In the field of wheat research, gene editing technology has demonstrated enormous application potential. Traditional wheat breeding methods mainly rely on hybridization and mutation breeding. While these methods have achieved some success in wheat variety improvement, they suffer from problems such as long cycles and low efficiency. The emergence of gene editing technology has brought new opportunities for wheat genetic improvement. Through gene editing technology, genes related to important agronomic traits in wheat can be precisely edited, thereby rapidly breeding new wheat varieties with superior traits. For example, in improving wheat disease resistance, researchers have successfully knocked out disease-susceptibility genes in wheat using gene editing technology, giving wheat broad-spectrum resistance to important diseases such as powdery mildew and stripe rust. In terms of increasing wheat yield, by editing yield-related genes, such as those regulating the number of grains per spike and grain weight, it is hoped that high-yielding wheat varieties can be bred. In addition, gene editing technology can also be used to improve wheat quality, such as regulating the content and composition of nutrients such as protein, starch, and fat in wheat grains to meet the needs of different consumers. With the rapid development of gene editing technology, the CRISPR-Cas9 system has become an important tool for plant gene function research and genetic improvement. Currently, the application of gene editing technology in wheat research has yielded a series of important results, and new research breakthroughs continue to emerge, bringing new hope and changes to wheat breeding and agricultural production.

[0004] Single nucleotide polymorphism (SNP) is a third-generation molecular marker developed based on next-generation sequencing (NGS). Variations in a single nucleotide lead to polymorphisms in the nucleic acid sequence, exhibiting characteristics such as good genetic stability, high density, and diallelicity, making it easy to achieve high-throughput and automated detection. Kompetitive allele-specific PCR (KASP) is a gel-free fluorescent polymerase chain reaction genotyping technique based on SNPs, widely used in animal and plant genetic breeding research. As a novel SNP genotyping method, KASP offers high accuracy, flexibility, and low cost. Furthermore, comparisons of TaqMan, KASP, and rhAmpSNP genotyping platforms in hexaploid wheat demonstrate that KASP is a time-saving and highly accurate method. Summary of the Invention

[0005] To address the shortcomings of existing breeding technologies, this invention provides a rapid breeding method that integrates multiple technologies based on the creation of homozygous wheat mutants without exogenous frames.

[0006] This invention addresses the problems of long breeding cycles, narrow genetic base, and scarce available gene resources in conventional wheat breeding. It utilizes gene editing technology to precisely improve target genes, and simultaneously establishes a precise and efficient gene breeding technology using conventional breeding methods and molecular biology detection techniques, providing an important material basis and effective technical means for the breeding of new wheat varieties.

[0007] This invention establishes a rapid breeding method that integrates gene editing, conventional hybridization, and molecular biological detection. Specifically, the technical solution involves creating an LCYe gene-edited mutant using gene editing technology, hybridizing the mutant material with the recipient parent to obtain the F1 generation, using PCR to detect the segregation of edit frames in the F1 generation, using Hi-TOM to detect mutant types without edit frames, designing KASP markers based on mutant types and tracking mutant types, and investigating phenotypes using observation and measurement methods.

[0008] This invention is achieved through the following technical solution:

[0009] Identify target genes: Based on breeding objectives (such as improving quality or stress resistance), select key genes (such as the LCY gene involved in carotenoid metabolism) as editing targets.

[0010] Creating gene-edited mutants: Using gene-editing technologies such as CRISPR-Cas9, vectors are constructed and transformed into wheat varieties (such as Jimai 1803), and T2 generation homozygous mutant single plants are obtained through self-pollination purification.

[0011] Hybridization and segregation of exogenous edit frames: The homozygous mutant was hybridized with the original parent to generate the F1 generation population. PCR technology was used to detect the segregation of exogenous Cas9 edit frames to ensure that exogenous frame-free materials were obtained.

[0012] KASP markers are used to track mutant types: KASP molecular markers are designed for homozygous mutation sites to perform high-throughput genotyping of hybrid offspring, replacing cumbersome sequencing methods and enabling rapid genotyping.

[0013] Phenotypic analysis: Field trait surveys (such as endosperm color and agronomic traits) were conducted on the selected lines to verify the actual effect of gene editing.

[0014] The beneficial effects of this invention are as follows: Mutants of the target gene LCYe are created using gene editing technology; the resulting mutants are used as the male parent and the recipient parent for hybridization; negative single plants isolated from the exogenous frame are obtained using PCR detection technology; the editing type of the negative single plants is identified using Hi-TOM detection technology; KASP markers are designed based on the mutants and mutation types are tracked; finally, LCYe gene mutants without exogenous frames and with distinct phenotypes are obtained. The success of this method provides a refined and rapid approach for the utilization of wheat functional genes. Attached Figure Description

[0015] Figure 1 is an electrophoresis image of the separation of the Cas9 edit box in PCR detection, where M is a DL2000bp marker;

[0016] Figure 2 shows the editing types of the mutants, where A represents the wild type (unedited); B represents the paternal T2-11-1 edited type; C represents the T2-11-1 F1 offspring edited type; D represents the paternal T2-17-4 edited type; E represents the T2-17-4 F1 offspring edited type; F represents the paternal T2-18-9 edited type; and G represents the T2-18-9 F1 offspring edited type.

[0017] Figure 3 shows the KASP marker genotyping diagram of the LCY-F1 strain. Blue dots represent alleles with the FAM fluorescent group; red dots represent alleles with the HEX fluorescent group; green dots represent heterozygous genotypes; black dots represent blank controls.

[0018] Figure 4 shows a photograph of the LCYe gene mutant F1, displaying the endosperm color.

[0019] Figure 5 is a technical roadmap for a multi-technology fusion rapid breeding method based on homozygous wheat mutants without exogenous frames. Detailed Implementation

[0020] The embodiment describes the construction of a multi-technology fusion rapid breeding method based on the creation of homozygous wheat mutants without exogenous frames.

[0021] 1. Materials and Methods

[0022] 1.1 Materials

[0023] Using the new wheat variety Jimai 1803 as the recipient, gene-edited mutants of the LCYe gene were created, resulting in LCY-T2 mutant plants. Three LCY-T2 lines with different editing types were used as male parents and crossed with the recipient parent Jimai 1803. The resulting hybrid seeds were planted at the transgenic base of the Crop Research Institute of the Shandong Academy of Agricultural Sciences, forming the LCY-F1 population, comprising more than 200 lines.

[0024] 1.2 DNA Extraction

[0025] DNA was extracted using a high-salt, low-pH method. 0.1 g of leaf tissue was ground with liquid nitrogen, and 900 μL of extraction buffer (Tris 6.05 g, KCl 37.275 g, 0.5 mol / L EDTA 10 mL, pH adjusted to 9.5, volume brought to 500 mL) was added. The mixture was incubated at 65 ℃ for 1 h, then 300 μL of potassium acetate solution (147 g potassium acetate, 57.5 mL glacial acetic acid, pH adjusted to 5.8, volume brought to 500 mL) was added and thoroughly mixed. The mixture was allowed to stand for 30 min. After centrifugation at 12,000 r / min for 10 min, 900 μL of the supernatant was aspirated, and 0.6 times the volume of isopropanol was added and mixed. The mixture was centrifuged at 12,000 r / min for 1 min, washed twice with 70% ethanol, dried, and then 100 μL of water was added.

[0026] 1.3 PCR Amplification

[0027] Primers were designed based on the gene and vector sequences (see Table 1). PCR amplification was performed using these primers to identify whether the Cas9 edit frames were separated in the mutants. The PCR reaction system was as follows: Genomic DNA (100 ng / μL): 1.5 μL; Primer F (10 μM): 0.5 μL; Primer R (10 μM): 0.5 μL; 2 × Taq PCR Mix with Loading Dye (Genesand): 10 μL; ddH2O: 7.5 μL. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, annealing for 15 s (annealing temperature depends on the primers), 72℃ extension for 40 s, 35 cycles; 72℃ final extension for 40 s. Based on the differences in amplified fragment length, 1%–2% agarose gel electrophoresis was used to detect fragment length polymorphism.

[0028] Table 1 Primer Information for Detecting Wheat Gene-Editing Mutants

[0029] 1.4 Gel electrophoresis

[0030] Gel preparation: Add 1.5 g of agarose powder to 100 mL of 0.5×TAE solution, microwave for 3 min, and after slightly cooling, add 10 μL of nucleic acid dye, shake well, and pour onto a gel casting tray with combs. PCR amplification products were electrophoresed on a 1.5% agarose gel at 180 V for 8 min. After electrophoresis, gel electrophoresis images were obtained using a gel imaging system.

[0031] 1.5 Hi-TOM Detection

[0032] Hi-TOM stands for "High-Throughput and Absolute Quantification of Targeted Oligonucleotides via Microfluidics," meaning high-throughput and absolute quantitative analysis of targeted oligonucleotides using microfluidics. The HI-TOM reaction system consists of: 2.0 μL genomic DNA (100 ng / μL); 0.8 μL Primer F (10 μM); 0.8 μL Primer R (10 μM); 10 μL 2 × Taq PCR Mix with LoadingDye (Genesand); and 6.4 μL ddH2O. The PCR reaction program is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 62℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 5 min.

[0033] After PCR amplification, the amplification effect is detected using gel electrophoresis. If the amplification effect is good, the sample is sent to the company for sequencing, and the sequence type is edited based on the sequence analysis.

[0034] 1.6 KASP Tag Detection

[0035] KASP (Kompetitive Allele-Specific PCR) is a fluorescence-based SNP genotyping technique. Three primers were designed targeting the SNP / Indel site: two forward-specific primers and one reverse universal primer. The 5' ends of the two KASP forward primer sequences were augmented with adapter sequences containing either FAM (6-carboxy-fluorescein) or HEX (Hexachloro-fluoresein) fluorescent groups (primer sequences are shown in Table 1). The PCR reaction mixture was 5 μL, comprising 1.0 μL DNA (100 ng•μL⁻¹), 2.5 μL KASP Master Mix (LGC Genomics, Hoddeston, UK), 0.07 μL mixed primers, and 1.43 μL ddH₂O. ddH₂O was used as a blank control. PCR reactions were performed on an ABI PCR instrument. The PCR program was as follows: 95°C pre-denaturation for 15 min; 95°C denaturation for 20 s, 65°C annealing and extension for 25 s, 10 cycles; 57°C annealing and extension for 60 s, 30 cycles. After the reaction, fluorescence data were read using a Pherastar multi-mode microplate reader, and genotyping maps were generated using KlusterCaller software (LGC Genomics, Hoddeston, UK).

[0036] 2. Results and Analysis

[0037] 2.1 Cas9 Edit Box Separation Detection

[0038] Three LCY-T2 lines, LCY-T2-11-1, LCY-T2-17-4, and LCY-T2-18-9, were selected as male parents and crossed with the recipient parent, Jimai 1803. The editing types and Cas9 segregation of the LCY-F1 population of hybrid offspring were analyzed.

[0039] The separation of Cas9 edit frames was detected by PCR. The results of gel electrophoresis are shown in Figure 1. The Cas9 edit frames with bands were not separated, and the Cas9 edit frames without bands were successfully separated. The segregation of Cas9 edit frames in the F1 populations after pollination with recipient parents of three different types of paternal lines is shown in Table 2. Of the 79 LCY-F1 individuals obtained using the LCY-T2-11-1 line as the paternal line, 40 showed no detectable Cas9 edit frames, resulting in a segregation rate of 50.63%. Of the 104 LCY-F1 individuals obtained using the successfully segregated Cas9 LCY-T2-17-4 line as the paternal line, 102 showed no detectable Cas9 edit frames, resulting in a segregation rate of 98.08%. Of the 101 LCY-F1 individuals obtained using the Cas9-segregated LCY-T2-18-9 line as the paternal line, 12 showed no detectable Cas9 edit frames, resulting in a segregation rate of 88.12%. Recipient parents served as controls, showing no edit frames due to their negative results. Significant differences in the segregation rates of Cas9 edit frames were observed in the offspring of hybrids from different types of mutant lines.

[0040] Table 2. Statistical table of segregation ratio of Cas9 edit frames in F1 individuals of the LCYe gene.

[0041] 2.2 Analysis of Mutant Editing Types

[0042] Hi-TOM analysis was performed on individual plants successfully isolated from the Cas9 edit frame. The Hi-TOM results showed that the edit types of LCY-F1 were basically consistent with those of the parent lines (see Figure 2). The F1 lines with the parent LCY-T2-11-1 had edit types A and B (unedited) and D d2 (heterozygous), with LCY-F1-1-7 exhibiting an abnormal edit type. The F1 lines with the parent LCY-T2-17-4 had edit types A and B (unedited) and Di2 (heterozygous), with LCY-F1-7-13 being unedited and wild-type. The F1 lines with the parent LCY-T2-18-9 all had edit types B (unedited), A d4 (heterozygous), and Di1 (heterozygous). The recipient parent, Jimai 1803, was wild-type and unedited.

[0043] 2.3 KASP Tag Tracking

[0044] KASP marker primers were designed based on the editing types of the LCYe mutant lines LCY-T2-17-4 and LCY-T2-18-9 (see Table 1). KASP detection was performed on homozygous T2-17-4 and T2-18-9 lines and their F1 progeny using primers LCY3D+1 and LCY3A-4, respectively. Figure 3 shows the KASP detection results for homozygous and heterozygous mutants. The KASP detection results for the LCY-F1 genotype were consistent with the Hi-TOM detection results, indicating that the faster and more efficient KASP detection can replace the Hi-TOM detection in tracking progeny editing types.

[0045] 2.4 Mutant Phenotypic Analysis

[0046] The LCYe gene is involved in the carotenoid metabolic pathway. Phenotypic surveys and endosperm color observations were conducted on the LCY-F1 population. The plant height, flag leaf length, awn length, and spike length of different lines did not vary much, but the endosperm color showed differences (see Figure 4).

[0047] 3. A rapid breeding method integrating multiple technologies based on the creation of homozygous wheat mutants without exogenous frames.

[0048] This invention analyzes the segregation of Cas9 edit frames and the inheritance of edit types in the F1 generation of LCY gene-editing mutants and their crosses with recipient parents. The aim is to clarify the segregation patterns of Cas9 edit frames and the inheritance patterns of mutation types in gene-editing mutants. The results show that when using the mutants T2-17-4 and T2-18-9, whose Cas9 edit frames have been successfully removed, as male parents, the negative rates of individual F1 offspring were 98.08% and 88.12%, respectively. When using the mutant line T2-11-1, whose Cas9 edit frames were not removed, as male parents, the negative rate of the F1 offspring was 50.63%.

[0049] The results showed that the offspring of different editing types had the same editing type as their parents, were heterozygous, and conformed to Mendel's laws of inheritance. Only LCY-F1-1-7 showed an editing type inconsistent with its parents.

[0050] To achieve simplicity, efficiency, and cost-effectiveness, this study also designed KASP markers based on mutation type for homozygous gene-editing lines. KASP marker detection was performed on homozygous mutant lines and F1 hybrids using these markers. The results were consistent with Hi-TOM detection results, indicating that the method of using KASP markers to detect mutants with clearly defined editing types is feasible. This invention provides a high-throughput detection method for tracking the editing type of gene-editing mutant offspring.

[0051] (1) The combination of hybridization and PCR detection is an effective method for isolating Cas9 edit frames from gene-editing mutants. To establish an effective method for isolating edit frames from gene-editing mutants, LCYe gene-editing mutant lines T2-11-1, T2-17-4, and T2-18-9 were hybridized with recipient parents to obtain F1 populations. The segregation of Cas9 edit frames in the offspring populations was detected using PCR technology. The negative rates of Cas9 edit frames in the three mutant lines were 50.63%, 98.08%, and 88.12%, respectively. The results indicate that the segregation rate of Cas9 edit frames in the F1 offspring of different wheat mutant lines varies, and hybridization technology is an effective means of isolating Cas9 edit frames from gene-editing mutants.

[0052] (2) Mutations created using gene editing technology can be stably inherited by offspring. Using Hi-TOM to detect the editing type of F1 individual plants of hybrids successfully isolated from Cas9 edit frames, it was found that the gene editing type of hybrid offspring of different editing type mutants was consistent with that of the parent, indicating that the wheat LCYe gene mutation type created using gene editing technology can be stably inherited by hybrid offspring.

[0053] (3) KASP markers designed based on mutant sites can effectively track mutation types. KASP markers were designed based on the mutation types of stable homozygous wheat LCYe gene, and KASP typing was performed on the mutation types. The KASP marker detection results were consistent with the Hi-TOM detection results, indicating that KASP markers can be used to track wheat mutation types.

[0054] Based on the above research, a multi-technology fusion rapid breeding method based on the creation of wheat homozygous mutants without exogenous frames was constructed. The technical route includes five steps (see Figure 5).

[0055] Step 1: Identify the target gene

[0056] To provide precise molecular targets for gene editing and ensure the directionality of subsequent work.

[0057] Based on breeding objectives (such as improving quality and enhancing stress resistance), key genes related to the target trait are selected as editing targets. For example, in the study mentioned above, the LCY gene, which is involved in the carotenoid metabolic pathway, was selected.

[0058] Step 2: Creating gene-edited mutants

[0059] Obtain primary materials with a relatively stable genetic background and where the target gene has undergone the expected editing.

[0060] (1) Vector construction and genetic transformation: A CRISPR-Cas9 system was designed for the selected LCY gene and transformed into a superior wheat variety (such as "Jimai 1803") to obtain T0 generation mutant plants.

[0061] (2) Self-pollination purification and screening: T0 generation plants were self-pollinated to obtain T1 generation, and then self-pollinated again to obtain T2 generation population. Through molecular detection, single plants with homozygous target gene editing type were screened in T2 generation.

[0062] Step 3: Hybridization and Separation Cas9 Edit Box

[0063] Through genetic isolation, wheat materials containing only the target mutation and without exogenous Cas9 genes were obtained, thus meeting biosafety requirements.

[0064] (1) Hybridization design: The T2 generation homozygous mutant obtained in step 2 (regardless of whether it contains an exogenous Cas9 edit frame) is used as the father and hybridized with the original recipient parent (Jimai 1803) to produce the F1 generation population.

[0065] (2) PCR detection and isolation verification: PCR detection was performed on F1 generation plants using specific primers to determine whether the exogenous Cas9 edit frame was successfully isolated.

[0066] As shown in Figure 1, agarose gel electrophoresis reveals that plants without bands were successfully isolated by the Cas9 edit box (negative).

[0067] As shown in Table 2, the segregation rate of Cas9 edit frames differs among F1 populations from different paternal sources (e.g., from 50.63% to 98.08%), demonstrating that hybridization is an effective means of eliminating exogenous genes.

[0068] Step 4: KASP markers track mutant types

[0069] To establish a rapid, low-cost, high-throughput method for tracking and screening target mutant genotypes in large-scale populations, replacing the relatively cumbersome sequencing methods.

[0070] (1) Marker development: KASP molecular markers were developed for the identified homozygous mutation sites (see the primer sequences in Table 1 of the document, such as LCY3D+1 and LCY3A-4).

[0071] (2) High-throughput genotyping: The developed KASP marker was used to perform genotyping detection on the hybrid offspring (F1).

[0072] (3) Validation and substitution: The KASP typing results were compared with the accurate Hi-TOM sequencing results.

[0073] As shown in Figure 3, the KASP detection results (showing FAM and HEX fluorescence signals) are highly consistent with the Hi-TOM results, proving the reliability of KASP labeling.

[0074] Step 5: Phenotypic Analysis

[0075] Based on molecular-level confirmation, the actual effects of gene editing are verified, and genotype is linked to phenotype.

[0076] For the strains selected through the above steps that do not contain exogenous frames and have clearly defined mutant genotypes, field agronomic trait surveys (such as plant height, flag leaf length, etc.) and observations and analyses of specific traits are conducted.

[0077] As shown in Figure 4, observation of the endosperm color of the LCY gene mutant revealed significant phenotypic differences (such as the endosperm turning yellow), confirming that the gene editing successfully affected the target trait.

[0078] Final result: A new wheat germplasm was obtained that is free of exogenous genes, homozygous and stable with the target mutant genotype, and has excellent target traits.

[0079] The core of this technical approach lies in the organic integration of five key stages: target gene identification, gene editing, hybridization and segregation, molecular marker tracking, and phenotypic identification, forming a rapid, precise, and efficient breeding process. It effectively solves the critical challenges of exogenous gene knockout and rapid screening and identification of mutants in gene-editing breeding, significantly shortening the breeding cycle.

[0080] The above description is only a preferred embodiment of this patent. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of this patent, and these improvements and substitutions should also be considered within the scope of protection of this patent.

Claims

1. A rapid breeding method integrating multiple technologies based on the creation of homozygous wheat mutants without exogenous frames, characterized in that, Includes the following steps: (1) Identify target genes: Based on the breeding objectives, select key genes related to the target traits as editing targets; (2) Creating gene-edited mutants: Using gene editing technology, the selected target gene is precisely edited, and through genetic transformation and self-pollination purification, a single wheat mutant plant with the target gene editing type is obtained; (3) Hybridization to isolate exogenous editing frames: The homozygous mutant obtained in step (2) is used as the male parent and hybridized with the original recipient parent to produce the F1 generation population. The exogenous Cas9 editing frames are isolated by PCR detection. (4) KASP marker tracking of mutant types: KASP molecular markers are developed for homozygous mutation sites. High-throughput genotyping is performed on the hybrid offspring to track the target mutant genotype. (5) Phenotypic analysis: Agronomic traits and specific phenotypes are observed for the lines selected in step (4) that do not contain exogenous frames and have clear mutant genotypes to verify the gene editing effect.

2. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The target gene mentioned in step (1) is the LCY gene, which is involved in the carotenoid metabolic pathway.

3. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The gene editing technology described in step (2) is the CRISPR-Cas9 system.

4. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The self-pollination purification described in step (2) includes self-pollinating T0 generation plants to obtain T1 generation, then self-pollinating to obtain T2 generation population, and screening homozygous single plants by molecular detection.

5. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The PCR detection described in step (3) uses specific primers, and the separation of the Cas9 edit frame is judged by agarose gel electrophoresis. No band indicates successful separation.

6. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The KASP marker detection described in step (4) uses primers labeled with fluorescent groups to distinguish genotypes by fluorescence signals and verify consistency with Hi-TOM sequencing results.

7. The rapid breeding method integrating multiple technologies according to claim 1, characterized in that, The phenotypic analysis described in step (5) includes observation of endosperm color and investigation of agronomic traits such as plant height, flag leaf length, awn length, and ear length.

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