A wheat and barley material with early heading and its creation method and application

Abstract

The application discloses a wheat and barley material with early heading stage and a creating method and application thereof, and belongs to the technical field of plant biotechnology, and the target sequence of the material is that: by using single base editing, a T at the 17th base in a wheat TaPhyC gene sequence with a nucleotide sequence shown in SEQ ID NO. 1 and a barley HvPhyC gene sequence with a nucleotide sequence shown in SEQ ID NO. 2 is replaced by C, and the target sequences of the wheat and barley with early heading stage are created respectively. The application solves the problems of existing technology, such as extensive regulation, lack of precise editing and functional marker. Under the premise of maintaining the complete function of the gene, the editing states of A, B and D three subgenomic homologous copies of TaPhyC are independently identified and synchronously fixed, the conversion from the late heading genotype PhyC-l to the early heading genotype PhyC-e is realized, the heading stage is stably advanced, and the application can be used for wheat and barley heading stage improvement breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a wheat and barley material with an advanced heading period, its creation method, and its application. Background Technology

[0002] Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) are among the most important cereal crops globally, widely cultivated due to their versatility and adaptability to diverse climatic conditions. Originating in the Fertile Crescent during the Neolithic period, their cultivation has expanded to high-latitude, arid, and subtropical regions, reflecting the high plasticity of their heading and flowering times. Natural variations in photoperiod-responsive genes allow for the regulation of crop growth periods according to different seasonal conditions, thus driving their spread to diverse agricultural ecosystems.

[0003] The timing of crop heading and flowering is synergistically regulated by pathways such as photoperiod signaling, a mechanism crucial for successful reproductive growth. Identifying these key haplotypes is of great significance in the context of intensive agriculture and climate change. Currently, the regulation of heading time in wheat and barley mainly relies on natural variation or artificial modification of genes related to photoperiod and vernalization pathways. For example, allelic variations of genes such as VRN1, VRN2, and PPD1 have been widely used in breeding practices. However, these techniques often rely on natural variation or conventional hybridization, resulting in long breeding cycles and frequent linkage to unfavorable traits, making it difficult to achieve precise regulation of heading time. Furthermore, with the development of gene editing technology, methods such as CRISPR / Cas9 have been used for functional studies and trait improvement of genes related to crop heading time. Existing reports often employ gene knockout or large-fragment deletion to regulate flowering-related genes, but such knockout mutations often disrupt the overall function of the gene, easily leading to excessive trait amplitude, insufficient stability, and even adverse effects on yield and other agronomic traits. Therefore, their application in practical breeding remains limited.

[0004] Regarding research on the phytochrome C gene (PhyC), existing literature 1 (Pankin, A., Campoli, C., Dong, X., Kilian, B., Sharma, R., Himmelbach, A., Saini, R., Davis, SJ, Stein, N., Schneeberger, K., & von Korff, M. (2014). Mapping-by-SEDuencing identifies HvPHYTOCHROME C as a candidate gene for the early maturity locus modulating the circadian clock and photoperiodic flowering in barley. Genetics, 198(1), 383-396. DOI: Reference 10.1534 / genetics.114.165613 (https: / / academic.oup.com / genetics / article / 198 / 1 / 383 / 6556852) proposes that the red / far-red photoreceptor HvPHYC (barley phytochrome C) carries a mutation in a conserved region of the GAF domain, making it a potential candidate gene for the early-maturing 5 (eam5) locus in barley; however, this reference 1 is based only on specific materials, has not developed molecular markers, and its mechanistic analysis is not in-depth. Reference 2 (Nishida, H., Ishihara, D., Ishii, M. et al. Phytochrome C Is A Key Factor Controlling Long-Day Flowering in Barley. Plant Physiol 163, 804-814 (2013). https: / / doi.org / 10.1104 / pp.113.222570) discloses that the spring-type barley variety NIL flowers later than the winter-type barley variety HK2, and that this phenotypic difference is controlled by a single gene that co-segregates with HvPhyC. However, the conclusions of Reference 2 are mainly based on specific genetic materials, and no molecular markers that can be directly used for breeding have been developed.Furthermore, Reference 3 (Chinese invention patent application with publication number CN118813853A) developed an STS marker, primers, identification method, and its application in breeding that are completely linked to wheat heading date and plant height. The STS marker, completely linked to wheat heading date and plant height, is a G base insertion at nucleotide 68416769 on wheat chromosome 7D. This STS marker is a functional molecular marker associated with wheat heading date and plant height, and can be used for marker-assisted selection to screen wheat breeding materials. The sites identified by this functional STS marker in wild-type and mutant wheat can be used to improve wheat heading date and plant height through backcross selection. However, Reference 3 does not explicitly reveal the specific gene corresponding to the marker and its function, lacking biological and molecular mechanism explanations, and its application may be limited to specific genetic backgrounds. Summary of the Invention

[0005] The purpose of this invention is to provide wheat and barley materials with advanced heading dates, their creation method, and applications. This invention solves the problems of extensive regulation and lack of precise editing and functional markers in existing technologies. While maintaining the complete function of the genes, it can achieve stable advancement of the heading dates of wheat and barley and obtain genetic materials suitable for breeding. These materials can be used for breeding to improve the heading dates of wheat and barley.

[0006] To achieve the above objectives, the present invention provides a target sequence for genetic modification of wheat materials that advances the heading date. This target sequence is obtained by replacing the 17th base T with C in the nucleotide sequence fragment shown in SEQ ID NO.1 of the wheat TaPhyC gene.

[0007] This invention provides a target sequence for genetic modification of barley materials with an advanced heading date. This target sequence is obtained by replacing the 17th base T with C in the nucleotide sequence fragment shown in SEQ ID NO.2 of the barley HvPhyC gene.

[0008] This invention provides primer combinations for detecting three homologous copies of the TaPhyC-e gene in the A, B, and D subgenomes of wheat with advanced heading date. The primer combinations are as follows: the nucleotide sequences of the wheat A subgenome TaPhyC-e gene-specific primer combination are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of the wheat B subgenome TaPhyC-e gene-specific primer combination are shown in SEQ ID NO.5 and SEQ ID NO.6; and the nucleotide sequences of the wheat D subgenome TaPhyC-e gene-specific primer combination are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0009] This invention provides a PCR reagent for detecting three homologous copies of the TaPhyC-e gene in the A, B, and D subgenomes of wheat with advanced heading date, comprising the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and SEQ ID NO.7 and SEQ ID NO.8.

[0010] This invention provides a primer combination that can distinguish any TaPhyC-l genotype fragment from three TaPhyC-l genotype fragments in the A, B, and D subgenomes of wild-type wheat and any TaPhyC-e genotype fragment from three TaPhyC-e homologous copy genes in the A, B, and D subgenomes of early-heading wheat. The primer combination consists of two genotype-specific forward primers with nucleotide sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10 and one universal reverse primer with nucleotide sequence as shown in SEQ ID NO. 11.

[0011] This invention provides a primer combination that can distinguish between the wild-type barley HvPhyC-l genotype fragment and the early-heading barley TaPhyC-e genotype fragment. The primer combination consists of one universal reverse primer with the nucleotide sequence as shown in SEQ ID NO.12 and two genotype-specific forward primers with the nucleotide sequences as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0012] This invention provides three TaPhyC-e homologous copy genes on the A, B, and D subgenomes of wheat that advance the heading stage. The nucleotide sequences of these three TaPhyC-e homologous copy genes are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively, and the encoded amino acid sequences are shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively.

[0013] This invention provides a barley HvPhyC-e gene with an advanced heading date. The nucleotide sequence of the barley HvPhyC-e gene is shown in SEQ ID NO.18, and the encoded amino acid sequence is shown in SEQ ID NO.22.

[0014] This invention provides a method for creating wheat and barley materials with advanced heading dates, the method comprising: For wheat materials with an earlier heading date, the specific procedures are as follows: (1) Using the wheat TaPhyC genome as a template, PCR amplification was performed using primer combinations with nucleotide sequences as shown in SEQ ID NO.3~SEQ ID NO.8 to obtain PCR molecules specific to the A, B, and D subgenomes of wheat TaPhyC-e; wherein, the nucleotide sequences of the primers specific to the A subgenome of wheat TaPhyC-e are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of the primers specific to the B subgenome of wheat TaPhyC-e are shown in SEQ ID NO.5 and SEQ ID NO.6; and the nucleotide sequences of the primers specific to the D subgenome of wheat TaPhyC-e are shown in SEQ ID NO.7 and SEQ ID NO.8. (2) Using the subgenome-specific PCR molecules of wheat TaPhyC-e A, B, and D as templates, KASP labeling amplification was performed sequentially using primers with nucleotide sequences as shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11. Homozygous genotypes were determined by fluorescence signal typing to obtain subgenome-specific DNA molecules of wheat TaPhyC-e A, B, and D. The nucleotide sequence of the wheat TaPhyC-e A subgenome is shown in SEQ ID NO.15, the nucleotide sequence of the wheat TaPhyC-e B subgenome is shown in SEQ ID NO.16, and the nucleotide sequence of the wheat TaPhyC-e D subgenome is shown in SEQ ID NO.17. For barley materials with an early heading stage, the specific procedures are as follows: Using the barley HvPhyC gene as a template, KASP labeling amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively. Homozygous genotypes were determined by fluorescence signal typing to obtain the DNA molecule of the barley HvPhyC-e gene; the nucleotide sequence of the barley HvPhyC-e gene is shown in SEQ ID NO.18.

[0015] Preferably, for wheat, the fluorescence signal typing criteria are: obtaining a homozygous genotype with FAM fluorescence signal <0.75 and VIC fluorescence signal >2.2 at 35℃ for 30 sec; for barley, the fluorescence signal typing criteria are: obtaining a homozygous genotype with FAM fluorescence signal <0.5 and VIC fluorescence signal >2.5 at 35℃ for 30 sec.

[0016] This invention provides a target sequence for genetic modification of wheat materials with advanced heading date as described above, or the application of three TaPhyC-e homologous copies of wheat subgenomes A, B, and D with advanced heading date as described above in wheat breeding.

[0017] This invention provides a target sequence for genetic modification of barley materials with advanced heading date as described above, or the application of the barley HvPhyC-e gene with advanced heading date as described above in barley breeding.

[0018] This invention provides a wheat and barley material with an advanced heading period, its creation method, and its application. It solves the problems of extensive control methods and lack of precise editing and functional labeling in existing technologies, and has the following advantages: 1. This invention obtained the early flowering allelic variant PhyC-e in wheat and barley and its sequence. Through single-base editing technology, the 1139th base of the wheat and barley PhyC gene was precisely mutated from T to C, and the 380th amino acid was changed from phenylalanine to serine. With the primer combination and identification process provided by this invention, the sequence variation after editing the wheat and barley PhyC gene can be detected. Site-specific editing of PhyC copies in the three subgenomes of wheat (A, B, and D) and the H genome of barley can be performed to achieve the conversion from the late flowering haplotype PhyC-l to the early flowering haplotype PhyC-e, which can improve the heading time while maintaining the integrity of the overall gene structure and function.

[0019] 2. This invention, targeting the characteristics of wheat allohexaploids, provides three subgenome-specific PCR primers (A, B, and D) combined with KASP molecular markers to achieve independent identification and simultaneous fixation of the editing status of the homologous copies of TaPhyC in the three subgenomes (A, B, and D), thereby obtaining stable genetic material with the target mutation occurring in all three copies. Simultaneously, this invention is also applicable to the editing and identification of single-copy HvPhyC genes in the barley H genome, demonstrating cross-species universality. Attached Figure Description

[0020] Figure 1 This is a diagram showing the results of the specific amplification detection of TaPhyC on the three subgenomes A, B, and D of this invention.

[0021] Figure 2 This image shows the results of detecting fluorescence values ​​of wheat A, B, and D subgenome TaPhyC gene-edited materials using KASP molecular markers, as per the present invention.

[0022] Figure 3 This image shows the results of detecting fluorescence values ​​in barley HvPhyC gene-edited materials using KASP molecular markers, as presented in this invention.

[0023] Figure 4 This figure shows the genotype and heading phenotype identification results of homozygous lines in which all copies of the TaPhyC gene in the three subgenomes of wheat (A, B, and D) have point mutations.

[0024] Figure 5This figure shows the genotype and heading phenotype identification results of the homozygous HvPhyC point mutant barley lines according to the present invention. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: A method for creating barley seedlings with advanced heading stage, utilizing single-base editing technology to obtain the created barley seedlings, the method comprising: The experimental material was the common barley variety “Vlamingh”. References (Zong, Y., Song, Q., Li, C., Jin, S., Zhang, D., Wang, Y., Qiu, JL, & Gao, C. (2018). Efficient C-to-Tbase editing in plants using a fusion of nCas9 and human APOBEC3A. Naturebiotechnology, 10.1038 / nbt.4261. https: / / doi.org / 10.1038 / nbt.4261; Xin, X., Li, J., Zhao, D., Li, S., Xie, Q., Li, Z., Fan, F., Bi, C., & Zhang, X. (2019). Double-Check Base Editing for Efficient A to G Conversions. ACS syntheticbiology, 8(12), 2629–2634.) (https: / / doi.org / 10.1021 / acssynbio.9b00284) Using single-base editing technology, the 1139th base of PhyC was mutated from T to C (i.e., the 17th base in the sequence shown in SEQ ID NO.2 was mutated from T to C). Correspondingly, the 380th amino acid changed from phenylalanine to serine, resulting in barley seedlings with an earlier heading date. The nucleotide sequence of the sgRNA target sequence of the barley PhyC gene edit is shown in SEQ ID NO.2: ccgaggttcgtccctttcccgct.

[0027] A method for creating wheat seedlings with advanced heading date, utilizing single-base editing technology to obtain the created wheat seedlings, the method comprising: The experimental material was the common wheat variety “Zhengmai 1862”. References: (Zong, Y., Song, Q., Li, C., Jin, S., Zhang, D., Wang, Y., Qiu, JL, & Gao, C. (2018). Efficient C-to-Tbase editing in plants using a fusion of nCas9 and human APOBEC3A. Naturebiotechnology, 10.1038 / nbt.4261. https: / / doi.org / 10.1038 / nbt.4261; Xin, X., Li, J., Zhao, D., Li, S., Xie, Q., Li, Z., Fan, F., Bi, C., & Zhang, X. (2019). Double-Check Base Editing for Efficient A to G Conversions. ACS syntheticbiology, 8(12), 2629–2634.) (https: / / doi.org / 10.1021 / acssynbio.9b00284) Using single-base editing technology, the 1139th base of PhyC in subgenomes A, B, and D was mutated from T to C (i.e., the 17th base T in the sequence shown in SEQ ID NO.1 was mutated to C), and the corresponding amino acid changed from phenylalanine to serine, resulting in wheat seedlings with an earlier heading date. The nucleotide sequence of the sgRNA target sequence of the wheat PhyC gene editing is shown in SEQ ID NO.1: ccgaggttcgtccccttcccgct.

[0028] The PCR amplification and gel electrophoresis detection method for the TaPhyC-specific fragments on the A, B, and D subgenomes of wheat seedlings mentioned above includes the following steps: Step 1: Extract genomic DNA from wheat seedlings after single-base editing using the CTAB method. The specific steps are as follows: S1. Prepare a 2 mL centrifuge tube containing 2 glass beads (Sigma, 18406-500G, 5 mm). Collect 100-200 mg of fresh young leaves (from a single wheat seedling) and place them in the test tube. Immediately immerse the tube in liquid nitrogen for freezing. Grind the tissue 2-3 times using a tissue homogenizer, 1 minute each time. Add 1 mL of 2× CTAB buffer and mix thoroughly.

[0029] S2. Incubate in a 65℃ constant temperature mixer for 30 minutes.

[0030] S3. Add 600 μL of pre-cooled chloroform / isoamyl alcohol (v / v, 24:1), shake vigorously for 30 seconds to mix thoroughly. Centrifuge at 13000 rpm for 15 minutes.

[0031] S4. Take 650 µL of supernatant and transfer it to a new 1.5 mL centrifuge tube.

[0032] S5. Add 5 µL RNase A (10 mg / mL, ribonuclease A), invert to mix, and incubate at 37°C for 15 minutes.

[0033] S6. Add 650 µL of isopropanol, invert the centrifuge tube 3 times to mix thoroughly, and precipitate the DNA.

[0034] Centrifuging at 13,000 rpm for 10 minutes at 4°C (S7) usually results in a clearly visible white DNA precipitate.

[0035] S8. Add 800 µL of 70% ethanol and rinse at room temperature for 2 minutes. Centrifuge at 13000 rpm for 5 minutes, and aspirate the supernatant. Handle with care to avoid aspirating DNA attached to the tube wall.

[0036] S9. Open the centrifuge tube cap and let it dry at room temperature for 5-10 minutes.

[0037] S10. Add 100 µL of TE buffer (Tris-EDTA buffer) to dissolve the DNA and incubate at 55°C for 10 minutes. Detect the DNA concentration using a Nanodrop micro-spectrophotometer.

[0038] The solvent used for DNA extraction is: 2× CTAB (store at room temperature) 2% CTAB (Cetyltrimethylammonium bromide) 200 mM Tris / HCl (pH 8.0) (Tris(hydroxymethyl)aminomethane / hydrochloric acid) 20 mM EDTA (ethylenediaminetetraacetic acid) 1.4 M NaCl (sodium chloride) 1.0% PVP (Polyvinylpyrrolidone) 20 mL β-mercaptoethanol TE-buffer (pH 8.0) (store at room temperature) 10 mM Tris / HCl pH 8.0 (Tris(hydroxymethyl)aminomethane / hydrochloric acid) 1 mM EDTA (ethylenediaminetetraacetic acid) Step 2: Using wheat genomic DNA as a template, PCR amplification was performed using three subgenome-specific primer pairs (A, B, and D) to obtain the subgenome-specific TaPhyC sequences, i.e., the wheat subgenome-specific PCR molecules. The PCR reaction system consisted of 20 µL: 2 µL DNA template (30 ng / µL), 10 µL 2× Phanta Max buffer, 0.4 µL 10 mM dNTP mix, 0.4 µL each of the 10 µM primer pairs, 0.2 µL Phanta Max Super-Fidelity DNA polymerase (Nanjing Novizan Biotechnology Co., Ltd.; P505; 1 U / µL), and 6.6 µL ddH2O. The PCR reaction procedure is detailed in Table 1.

[0039] The primers for subgene A are: SEQ ID NO.3 and SEQ ID NO.4; The primers for the B subgene are: SEQ ID NO.5 and SEQ ID NO.6; The primers for the D subgene are: SEQ ID NO.7 and SEQ ID NO.8.

[0040] SEQ ID NO. 3: gatgatatgtgattgtgctgcaag.

[0041] SEQ ID NO. 4: ataaaaggaccagattcaagacatag.

[0042] SEQ ID NO. 5: atgatatgtgattgtgctgccag.

[0043] SEQ ID NO. 6: caacgtgtcacaaaaaaaaacca.

[0044] SEQ ID NO. 7: ctcatgattcccgccgatt.

[0045] SEQ ID NO. 8: ggaccagattcaagacatggtg.

[0046] Table 1 PCR reaction procedure ; Step 3: Perform gel electrophoresis using 1% (w / v) agarose gel, and the results are as follows. Figure 1The figure shows the results of specific amplification detection of TaPhyC in the three subgenomes A, B, and D of this invention. The sample order is as follows: 1 represents wild-type "Zhengmai 1862"-1; 2 represents wild-type "Zhengmai 1862"-2; 3 represents homozygous triple-mutation editing line-1; 4 represents homozygous triple-mutation editing line-2; 5 represents heterozygous triple-mutation editing line-1; 6 represents heterozygous triple-mutation editing line-2; 7 represents template-free negative control. Figure 1 It is known that the primer sequences SEQ ID NO.3 and NO.4, NO.5 and NO.6, NO.7 and NO.8 provided by the present invention can specifically amplify the PhyC gene fragments on the three subgenomes of wheat, namely A, B and D.

[0047] Example 2: A method for detecting KASP markers of TaPhyC editing fragments in wheat subgenomes A, B, and D, comprising: Step 1: First, configure KASP Primer Mix.

[0048] The details of the KASP Primer Mix system are shown in Table 2.

[0049] Table 2. KASP Primer Mix System ; SEQ ID NO.9: gaaggtgaccaagttcatgctcccgaggttcgtcccctt.

[0050] SEQ ID NO. 10: gaaggtcggagtcaacggattcccgaggttcgtcccctc.

[0051] SEQ ID NO. 11: cctttgcctgagaagcaagtt.

[0052] Step 2: Using the wheat subgenome-specific PCR molecules obtained in Example 1 as templates, DNA molecules were amplified. The KASP reaction system consisted of 4 µL, 1.95 µL of PCR amplification product template (1:100 dilution), 2 µL of HiGeno 2×Probe Mix A (Beijing Jiacheng Biotechnology Co., Ltd.; E02 / 2019), and 0.05 µL of the KASP Primer Mix prepared in Step 1. The KASP amplification reaction procedure is detailed in Table 3.

[0053] Table 3 KASP amplification reaction procedure ; Step 3: The DNA molecules amplified by KASP markers in Step 2 were read for fluorescence values ​​at 35°C for 30 seconds using a QuantStudio 7 Flex real-time PCR instrument (Applied Biosystems). FAM fluorescence signal represents wild type, VIC signal represents homozygous editing line, and FAM / VIC signal represents heterozygous editing line. This method was used to identify editing lines in wheat subgenomes A, B, and D where the target mutation of the TaPhyC homologous copy occurred.

[0054] like Figure 2 As shown in the figure, this invention utilizes KASP molecular markers to detect the fluorescence values ​​of TaPhyC gene-edited materials from wheat subgenomes A, B, and D. Wild-type TTC shows a FAM fluorescence signal, homozygous edited line TCC shows a VIC fluorescence signal, heterozygous edited line TTC / TCC shows a FAM / VIC heterozygous signal, and the template-free negative control has a fluorescence signal value close to 0. Figure 2 It is known that the primer sequence combination SEQ ID NO.9, NO.10 and NO.11 provided by the present invention can accurately identify the PhyC gene editing sites on the three subgenomes of wheat A, B and D.

[0055] Example 3: A method for detecting KASP markers in barley HvPhyC edited fragments is described below: Step 1: First, configure KASP Primer Mix.

[0056] The details of the KASP Primer Mix system are shown in Table 4.

[0057] Table 4. KASP Primer Mix System ; SEQ ID NO. 12: ggagacactggggagtgacca.

[0058] SEQ ID NO. 13: gaaggtgaccaagttcatgctgcaagcatacctgagcggga.

[0059] SEQ ID NO. 14: gaaggtcggagtcaacggattgcaagcatacctgagcgggg.

[0060] Step 2: Using barley genomic DNA as a template, the KASP reaction system and amplification procedure are the same as in Example 2.

[0061] Step 3, consistent with Example 2, yields the following results: Figure 3 As shown.

[0062] like Figure 3 As shown in the figure, this invention utilizes KASP molecular markers to detect the fluorescence values ​​of barley HvPhyC gene-edited materials. Wild-type TTC represents the FAM fluorescence signal, homozygous edited line TCC represents the VIC fluorescence signal, heterozygous edited lines TTC / TCC represent the FAM / VIC heterozygous signal, and the template-free negative control has a fluorescence signal close to 0. Figure 3 It is known that the primer sequence combination SEQ ID NO.12, NO.13 and NO.14 provided by the present invention can accurately identify the PhyC gene editing site in barley plants.

[0063] Example 4 A method for identifying lines with simultaneous mutations in all three copies of the TaPhyC gene in the A, B, and D subgenomes of wheat, and with homozygous mutations in the HvPhyC gene in the barley H genome, is disclosed. The experimental method is the same as that described in Examples 2 and 3. This invention employs single-base gene editing technology to perform single-base editing on the target sequences of the TaPhyC gene in the wheat variety “Zhengmai 1862” and the HvPhyC gene in the barley variety “Vlamingh,” respectively. Combined with marker-assisted selection, homozygous lines with target mutations in the TaPhyC gene in all three subgenomes of wheat (A, B, and D), and homozygous barley lines with target mutations in the HvPhyC gene were obtained.

[0064] The nucleotide sequences of the sgRNA target sequences edited from the mutated PhyC gene in wheat and barley are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the nucleotide sequences of subgenomic A, B, and D of wheat TaPhyC-e are shown in SEQ ID NO.15, SEQ ID NO.16, or SEQ ID NO.17, respectively; the nucleotide sequence of barley HvPhyC-e is shown in SEQ ID NO.18; the amino acid sequences of subgenomic A, B, and D of wheat TaPhyC-e are shown in SEQ ID NO.19, SEQ ID NO.20, or SEQ ID NO.21, respectively; and the amino acid sequence of barley HvPhyC-e is shown in SEQ ID NO.22.

[0065] wheat TaPhyC-e The specific nucleotide sequence of subgenomic A, SEQ ID NO. 15, is as follows:

[0066] wheat TaPhyC-e The specific nucleotide sequence of the B subgenome (SEQ ID NO. 16) is as follows:

[0067] wheat TaPhyC-e The D subgenome nucleotide sequence SEQ ID NO.17 is as follows:

[0068] barley HvPhyC-e The nucleotide sequence SEQ ID NO.18 is as follows:

[0069] wheat TaPhyC-e The amino acid sequence of subgenomic A, SEQ ID NO. 19, is as follows:

[0070] wheat TaPhyC-e The amino acid sequence of the B subgenome (SEQ ID NO. 20) is as follows:

[0071] wheat TaPhyC-e The D subgenomic amino acid sequence SEQ ID NO.21 is as follows:

[0072] barley HvPhyC-e The amino acid sequence SEQ ID NO.22 is as follows:

[0073] In the autumn of 2024 at the Shunyi Experimental Base of the Chinese Academy of Agricultural Sciences in Beijing, and in the spring of 2025 in the greenhouse of the Beipu Experimental Farm of the Chinese Academy of Agricultural Sciences, the wheat TaPhyC three-copy homozygous target mutant material obtained in Example 4 was planted and compared with the wheat wild-type material (without single-base editing). Simultaneously, the barley HvPhyC homozygous target mutant material was planted and compared with the barley wild-type material (without single-base editing). In the field trials, three 1.5 m × 0.5 m replicate plots were set up for each genotype material, and in the greenhouse cultivation, 12 replicate single plants were set up for each genotype material. The heading date survey results showed that the heading date of both the wheat three-copy homozygous mutant line and the barley homozygous mutant line was significantly earlier than that of the wild type, as shown in the figure below. Figure 4 and Figure 5 As shown.

[0074] like Figure 4 The figure shows the genotype and heading phenotype identification results of homozygous lines with point mutations in all copies of the TaPhyC gene in wheat subgenomes A, B, and D. A represents the sgRNA nucleotide sequence of PhyC in wheat “Zhengmai 1862”; horizontal lines indicate PAM sequences, and triangles indicate target bases; B represents the phenotype of the homozygous triple-mutant edited line of wheat TaPhyC at the heading stage; the wild type is the wild-type line without single-base editing; the homozygous edited line, homozygous edited line #1, and homozygous edited line #2 are all the same lines as in Example 4; scale bar = 10 cm.

[0075] like Figure 5 The figure shows the genotype and heading phenotype identification results of the homozygous HvPhyC point mutant lines of barley in this invention. A is the sgRNA nucleotide sequence of PhyC in barley “Vlamingh”; the horizontal line marks the PAM sequence and the triangle marks the target base; B is the phenotype of the homozygous edited barley PhyC line at the heading stage; the wild type is the wild type line without single-base editing; the homozygous edited line, homozygous edited line #1 and homozygous edited line #2 are all the same lines as in Example 4.

[0076] Depend on Figure 4 and Figure 5 It is evident that the edited material is clearly distinguishable from the wild type in the KASP typing and exhibits a stable phenotype with significantly earlier heading date under the same field conditions. This invention represents a clear technological advancement over existing technologies in improving heading date.

[0077] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A target sequence for genetic modification of wheat materials that advances the heading date, characterized in that: The target sequence was obtained by replacing the 17th base T with C in the nucleotide sequence fragment shown in SEQ ID NO.1 of the wheat TaPhyC gene.

2. A target sequence for genetic modification of barley materials with advanced heading date, characterized in that: The target sequence was obtained by replacing the 17th base T with C in the nucleotide sequence fragment shown in SEQ ID NO.2 of the barley HvPhyC gene.

3. A primer combination for detecting three homologous copies of the TaPhyC-e gene in the A, B, and D subgenomes of wheat with advanced heading date, characterized in that: The nucleotide sequences of the wheat subgenome A TaPhyC-e gene-specific primer combinations are shown in SEQ ID NO. 3 and SEQ ID NO. 4, the nucleotide sequences of the wheat subgenome B TaPhyC-e gene-specific primer combinations are shown in SEQ ID NO. 5 and SEQ ID NO. 6, and the nucleotide sequences of the wheat subgenome D TaPhyC-e gene-specific primer combinations are shown in SEQ ID NO. 7 and SEQ ID NO.

8.

4. A PCR reagent for detecting three homologous copies of the TaPhyC-e gene in the A, B, and D subgenomes of wheat with advanced heading date, characterized in that: It includes the nucleotide sequences as described in claim 3, such as those shown in SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.

8.

5. A primer combination capable of distinguishing any TaPhyC-l genotype fragment from three TaPhyC-l genotype fragments in the A, B, and D subgenomes of wild-type wheat and any TaPhyC-e genotype fragment from three TaPhyC-e homologous copy genes in the A, B, and D subgenomes of early-heading wheat, characterized in that: The primer combination consists of two genotype-specific forward primers with nucleotide sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10, and one universal reverse primer with nucleotide sequence as shown in SEQ ID NO.

11.

6. A primer combination capable of distinguishing between the HvPhyC-l genotype fragment of wild-type barley and the TaPhyC-e genotype fragment of early-heading barley, characterized in that: The primer combination consists of one universal reverse primer with nucleotide sequences as shown in SEQ ID NO. 12 and two genotype-specific forward primers with nucleotide sequences as shown in SEQ ID NO. 13 and SEQ ID NO.

14.

7. A wheat subgenome containing three homologous copies of TaPhyC-e gene at subgenomes A, B, and D that advances the heading date, characterized by: The nucleotide sequences of the three TaPhyC-e homologous copies are shown in SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively, and the encoded amino acid sequences are shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively.

8. A barley HvPhyC-e gene for advanced heading date, characterized by: The nucleotide sequence of the barley HvPhyC-e gene is shown in SEQ ID NO.18, and the encoded amino acid sequence is shown in SEQ ID NO.

22.

9. The application of a target sequence for genetic modification of wheat materials with advanced heading date as described in claim 1, or the three TaPhyC-e homologous copies of wheat subgenomes A, B, and D with advanced heading date as described in claim 7, in wheat breeding.

10. The application of a target sequence for genetic modification of barley materials with advanced heading date as described in claim 2 or the application of the barley HvPhyC-e gene with advanced heading date as described in claim 8 in barley breeding.

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