Molecular marker of wheat taBTB11 gene and application thereof
By developing molecular markers for the wheat TaBTB11 gene and using CAPS markers to detect TaBTB11-B gene mutations, the problem of key gene discovery for wheat root hair growth and development was solved, molecular marker-assisted breeding was realized, wheat root hair length and stress resistance were improved, and the breeding process was promoted.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2026-04-24
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to effectively identify key genes for wheat root hair growth and development, which affects root growth and wheat growth and development, resulting in insufficient yield and stress resistance.
Molecular markers for the wheat TaBTB11 gene were developed. CAPS markers were used to detect the mutation at position 2143 of the TaBTB11-B gene. The TaBTB11-BI and TaBTB11-B-II genotypes were distinguished by PCR amplification and restriction endonuclease Mbol digestion for use in marker-assisted breeding.
It provides effective genetic resources and molecular markers, significantly improves the breeding process, accelerates the breeding speed, promotes the improvement of wheat root hair length, and enhances yield and stress resistance.
Smart Images
Figure CN122104999A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to molecular markers and their applications, specifically to molecular markers of the wheat TaBTB11 gene and their application in assisted breeding, belonging to the field of crop selection and breeding technology. Background Technology
[0002] Wheat (Triticum aestivum L.) is one of the world's three major food crops, providing essential dietary calories and protein for approximately 20% of the global population. As the primary organ influencing wheat growth and development, the interaction between its structure and function is crucial for plant survival, yield, and stress resistance. Root hairs significantly increase the contact area between the root and soil, serving as the main structure for efficient water and mineral absorption. Studies have shown that maintaining or enhancing root hair development effectively improves the plant's water status and nutrient absorption capacity, thus playing a vital role in promoting wheat growth and development. Therefore, identifying key genes in wheat root hair growth and development not only helps reveal the regulatory mechanisms of wheat root growth and development but also has positive implications for cultivating high-yielding, stable-yielding, and high-quality new wheat varieties through genetic improvement. Summary of the Invention
[0003] The purpose of this invention is to discover key genes for wheat root hair growth and development, develop related molecular markers, and provide applications of related molecular markers in wheat-assisted breeding, aiming to provide effective gene resources and molecular markers for crop genetic improvement and molecular marker-assisted selection.
[0004] To achieve the above objectives, the present invention adopts the following technical solution: Application of molecular markers in the wheat TaBTB11 gene for predicting wheat root hair length. The TaBTB11 gene contains two genotypes: TaBTB11-BI and TaBTB11-B-II. In the TaBTB11-BI genotype, the base at position 2143 of the TaBTB11-B gene is C, and in the TaBTB11-B-II genotype, the base at position 2143 of the TaBTB11-B gene is A. The molecular marker is a CAPS marker. Using the wheat genomic DNA to be tested as a template, the upstream primer shown in SEQ ID NO: 10 and SEQ ID NO: 10 were used. After PCR amplification using the downstream primers shown in IDNO: 11, the resulting PCR product is digested with the restriction endonuclease Mbol to obtain the CAPS marker. If the PCR product is digested into two DNA fragments with lengths of 296 bp and 369 bp, respectively, the genotype of the wheat to be tested is TaBTB11-BI. If the length of the PCR product is still 665 bp, the genotype of the wheat to be tested is TaBTB11-B-II. The root hair length of wheat with genotype TaBTB11-B-II is significantly lower than that of wheat with genotype TaBTB11-BI.
[0005] The advantages of this invention are: (1) This invention has discovered a new key gene for wheat root hair growth and development—TaBTB11 gene, which provides an effective gene resource for crop genetic improvement and molecular marker-assisted selection; (2) For the TaBTB11 gene, this invention has developed a CAPS molecular marker related to the gene—CAPS-TaBTB11-B. This molecular marker can be widely used in molecular marker-assisted breeding of wheat root hair length to accelerate the breeding process. Attached Figure Description
[0006] Figure 1 This is a diagram showing the root hair phenotype results of wheat Fielder wild-type and TaBTB11 gene mutant seeds on the second day after germination. Figure 2 This is a statistical result of root hair length on the second day after germination of wheat Fielder wild-type and TaBTB11 gene mutant seeds; Figure 3 This is a CAPS molecular marker gel electrophoresis result of TaBTB11-B gene in some wheat varieties. In the image, M is a 2000bp DNA ladder marker, C indicates that the base at position 2143 of TaBTB11-B gene is C, and A indicates that the base at position 2143 of TaBTB11-B gene is A. Figure 4 This is a graph showing the statistical results of root hair length in wheat of different genotypes. *** indicates P < 0.001. Detailed Implementation
[0007] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.
[0008] I. Cloning of TaBTB11 genomic DNA DNA was extracted from the wheat variety Fielder using the CTAB method. Based on the TaBTB11 gene sequence (reference sequence) downloaded from the Ensembl Plants website, the TaBTB11 gene DNA sequence was amplified using Fielder DNA as a template.
[0009] Based on the TaBTB11 gene sequence (reference sequence) downloaded from the Ensembl Plants website, specific primers for amplifying three partial homologous genes of TaBTB11—TaBTB11-A (TraesFLD7A01G350200), TaBTB11-B (TraesCS7B02G220300), and TaBTB11-D (TraesCS7D02G316000)—were designed using DNAMAN software. The DNA sequences of the three partial homologous genes TaBTB11-A, TaBTB11-B, and TaBTB11-D were then amplified.
[0010] The PCR reaction procedure is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 56℃ annealing for 20 s, 72℃ extension for 1 min / 1 kb, 35 cycles; store at 4℃ protected from light.
[0011] The amplified PCR products were detected using 1% agarose gel electrophoresis. The electrophoresis apparatus was set to 150V for 15 minutes. After the electrophoresis, the products were observed under a gel imaging system. The clear, bright bands of the correct length were cut off and purified using the FastPure Gel DNA Extraction Mini Kit from Nanjing Vazyme Biotechnology Co., Ltd.
[0012] After purification, the PCR products were ligated into the pCE2 TA / Blunt-Zero vector using the 5min TA / Blunt-ZeroCloning Kit from Nanjing Vazyme Biotechnology Co., Ltd.
[0013] Subsequent transformation experiments were performed using Fast-T1 competent E. coli cells, followed by single-clone sequencing.
[0014] The genomic sequences of the three partially homologous genes of TaBTB11 obtained by cloning, TaBTB11-A, TaBTB11-B, and TaBTB11-D, are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
[0015] The specific primers for amplifying the three partial homologous genes TaBTB11—TaBTB11-A, TaBTB11-B, and TaBTB11-D—are as follows: TaBTB11-AF: 5'-ATGGTGAGGAAGAGGAAGGAGC-3' (SEQ ID NO: 4); TaBTB11-AR: 5'-GGCACGTCCGTAATCAG-3' (SEQ ID NO: 5); TaBTB11-BF: 5'-CCATACGACATCCAGAGCTAAAGC-3' (SEQ ID NO: 6); TaBTB11-BR: 5'-CATCCAATGAAGGGCCATAAGC-3' (SEQ ID NO: 7); TaBTB11-DF: 5'-ATGGTGAGGAAGAGGAAGGAGC-3' (SEQ ID NO: 8); TaBTB11-DR: 5'-GGCACGTCCGTAATCAG-3' (SEQ ID NO: 9).
[0016] II. Verification of the function of the TaBTB11 gene The TaBTB11 gene was knocked out using the CRISPER / Cas9 gene editing system, resulting in the TaBTB11 mutants Tabtb11-AB and Tabtb11-ABD. Root hair phenotypes were identified in the wheat CRISPER / Cas9 gene editing recipient material Fielder and the TaBTB11 mutants.
[0017] Wheat Fielder wild-type and TaBTB11 mutant seeds treated with sodium hypochlorite were cultured at 4℃ for 2 days, then placed in darkness for 1 day. Seeds with uniform growth were selected and sown on a medium containing 1% agar. The seeds were then cultured in a greenhouse under the following conditions: 24℃ for 16 hours of light, 18℃ for 8 hours of darkness, and 75% relative humidity. Root hair length differences were observed and photographed using a stereomicroscope (M205C, Leica Microsystems CMS GmbH). Root hair length was measured using Photoshop software, and the data were used for subsequent analysis. At least 10 seedlings of each variety were selected for measurement. Statistical analysis of phenotypic data was performed using one-way ANOVA to test for significant differences. The statistical analysis was conducted using SPSS statistical software package (Version 26.0, IBM Corporation, USA).
[0018] The root hair phenotype results of wheat Fielder wild-type and TaBTB11 gene mutant seeds on the second day after germination are shown in the figure. Figure 1 The statistical results of root hair length are shown in [link to data]. Figure 2 .
[0019] Depend on Figure 1 and Figure 2 It can be seen that, compared with the wild-type wheat Fielder, the root hair length of the TaBTB11 gene mutant is significantly shortened, the number of root hairs is greatly reduced, and the root hair density is significantly decreased.
[0020] III. Development of molecular markers for the TaBTB11-B gene By performing PCR amplification and resequencing on the TaBTB11-B gene of wheat varieties with different genetic backgrounds, a substitution mutation (substitution of C and A bases) was detected at 2143 bp in the TaBTB11-B genome of different wheat varieties. Targeting this mutation site, corresponding primer pairs were designed, and a CAPS molecular marker (CAPS-TaBTB11-B) was developed for the detection and identification of the wheat TaBTB11-B gene.
[0021] The primer pair designed for this variant site is as follows: CAPS-TaBTB11-BF: 5'-CCTGTTTCTATGTGTAGGACTAC-3' (SEQ ID NO: 10); CAPS-TaBTB11-BR: 5'-GAGGCTGCAGTTGTAACAG-3' (SEQ ID NO: 11).
[0022] The wheat genomic DNA to be tested was extracted using conventional methods in the field.
[0023] Using wheat genomic DNA as a template, PCR amplification was performed using the above primers with CAPS-TaBTB11-BF / R. The PCR amplification system was 30 μL, specifically containing: 3 μL of wheat genomic DNA (template) at a concentration of 20 ng / μL-100 ng / μL, 15 μL of 2.5 U Taq enzyme, 0.6 μL of 10 μM forward primer, 0.6 μL of 10 μM reverse primer, and 10.8 μL of sterile water. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for 34 cycles; final extension at 72℃ for 5 min; and storage at 4℃.
[0024] After PCR amplification, the PCR product was digested with the restriction endonuclease Mbol. Specifically, 25 μL of the PCR amplification system was taken, and then 5 μL of 10× NE buffer, 1 μL of restriction endonuclease Mbol, and 19 μL of nuclease-free water were added sequentially to construct a 50 μL digestion reaction system. The digestion reaction was then carried out at 30℃ for 1 h, and finally inactivated at 65℃ for 20 min to obtain the digested product.
[0025] The restriction endonuclease Mbol recognizes the GATC restriction site sequence. The PCR amplification product is 665 bp in length. If the base at position 2143 of the TaBTB11-B gene is C (genotype denoted as TaBTB11-BI), the GATC sequence is formed. This site is recognized by the restriction endonuclease Mbol, which digests the PCR amplification product into two DNA fragments with lengths of 296 bp and 369 bp, respectively. If the base at position 2143 of the TaBTB11-B gene is A (genotype denoted as TaBTB11-B-II), the GATA sequence is formed. The GATC sequence cannot be formed, and therefore the restriction endonuclease Mbol cannot recognize it. After the digestion reaction, the DNA fragment length remains 665 bp.
[0026] The CAPS molecular marker gel electrophoresis results of the TaBTB11-B gene in some wheat varieties are shown below. Figure 3 .
[0027] IV. Association Analysis of the TaBTB11-B Gene Molecular Marker CAPS-TaBTB11-B with Root Hair Length The wheat samples tested were 197 wheat varieties with different genetic backgrounds (Tables 1-1 to 1-4).
[0028] Genomic DNA was extracted from young leaves of wheat using the CTAB method.
[0029] PCR amplification of 197 wheat genomic DNA samples was performed using primer pair CAPS-TaBTB11-BF / R, following the method described above.
[0030] The PCR products were digested with enzymes according to the method described above, and the wheat genotype was analyzed.
[0031] The root hair length of the wheat seeds was measured on the second day after germination using the method described above. The root hair length was analyzed using SPSS software, and the significance of the differences in the data was tested using one-way ANOVA.
[0032] The genotyping results of the tested wheat are shown in Tables 1-1 to 1-4. The statistical results of root hair length of different genotypes of the tested wheat are shown in... Figure 4 .
[0033] Table 1-1 Genotyping results of 197 wheat samples (Part 1)
[0034] Table 1-2 Genotyping results of 197 wheat samples (Part II)
[0035] Table 1-3 Genotyping results of 197 wheat samples (Part III)
[0036] Table 1-4 Genotyping results of 197 wheat samples (Part 4)
[0037] Depend on Figure 4 It can be seen that the average root hair length of wheat with genotype TaBTB11-B-II is 1.452 cm, and the average root hair length of wheat with genotype TaBTB11-BI is 2.257 cm. The root hair length of wheat with genotype TaBTB11-B-II is significantly lower than that of wheat with genotype TaBTB11-BI.
[0038] Wheat varieties with the genotype TaBTB11-B-II, i.e., wheat varieties with nucleotide A at position 2143 of the TaBTB11-B gene, have shorter root hairs, while wheat varieties with the genotype TaBTB11-BI, i.e., wheat varieties with nucleotide C at position 2143 of the TaBTB11-B gene, have relatively longer root hairs.
[0039] Correlation analysis showed that the CAPS molecular marker CAPS-TaBTB11-B developed in this invention was significantly correlated with the root hair length trait of wheat.
[0040] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.
Claims
1. A molecular marker for the wheat TaBTB11 gene, characterized by, The molecular marker is a CAPS marker. Using wheat genomic DNA as a template, PCR amplification is performed using the upstream primer shown in SEQ ID NO: 10 and the downstream primer shown in SEQ ID NO:
11. The resulting PCR product is then digested with the restriction endonuclease Mbol to obtain the CAPS marker.
2. The application of the molecular marker of the wheat TaBTB11 gene as described in claim 1 in predicting wheat root hair length, characterized in that, The TaBTB11 gene contains two genotypes: TaBTB11-BI and TaBTB11-B-II. When the CAPS marker is two DNA fragments of 296bp and 369bp, the genotype of the wheat to be tested is TaBTB11-BI. When the CAPS marker is a DNA fragment of 665bp, the genotype of the wheat to be tested is TaBTB11-B-II. The root hair length of wheat with genotype TaBTB11-B-II is significantly lower than that of wheat with genotype TaBTB11-BI.