Maize dwarfing genes and their primer pair combinations, kits, detection methods and applications
By discovering and utilizing eleven new maize dwarfing genes, combined with specific primer detection and breeding methods, the problem of insufficient maize dwarfing gene pool has been solved, enabling precise regulation of maize plant type and yield improvement.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-04-03
AI Technical Summary
The existing maize dwarf gene pool is not rich enough, making it difficult to effectively improve maize's lodging resistance and yield per unit area through breeding methods.
Eleven new maize dwarfing genes (br2-6, br2-7, br2-70, br2-98, br2-100, br2-113, br2-115, br2-128, br2-129, br2-130, br2-131, etc.) were provided. These genes were detected by specific primer pair combinations and PCR amplification technology. Combined with hybridization and backcross breeding methods, new maize germplasm with dwarfing improvement was screened out.
It achieves precise control of maize plant type, increases planting density, enhances lodging resistance, and maintains or increases yield per plant while dwarfing, breaking the traditional limitations of tall maize on planting space.
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Figure CN121022878B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of maize breeding technology, specifically to maize dwarfing genes and their primer pair compositions, kits, detection methods, and applications. Background Technology
[0002] As an important crop for food, feed, and industrial raw materials, increasing maize yield is of immeasurable value in alleviating food crises, ensuring food supply, and expanding energy sources. Plant height, a key agronomic trait in maize, can be significantly enhanced by moderate dwarfing, which can effectively reduce the risk of yield reduction due to lodging. At the same time, dwarfing allows for higher planting density, making full use of land resources and thus increasing maize yield per unit area. Therefore, finding and utilizing dwarfing maize genes has become a research hotspot in the field of maize breeding.
[0003] The Brachytic2 (Br2) gene is a widely studied maize dwarfing gene. Its mutants, compared to the wild type, exhibit significantly reduced stem cell length, resulting in markedly shortened internodes. Br2 encodes a multidrug-resistant P-type glycoprotein containing two transmembrane domains (TMD1 and TMD2) and two nucleotide-binding domains (NBD1 and NBD2), regulating plant height by mediating the polar transport of auxin. The Br2 gene sequence is located on chromosome 1 and consists of five exons and four introns. Loss of function of the Br2 gene results in an extremely dwarfed maize plant phenotype; while a weak mutation in the fifth exon of the Br2 gene reduces plant height but does not significantly negatively impact other important agronomic traits.
[0004] Although many Br2 alleles have been identified, using new germplasm resources to find new dwarfing genes is of great theoretical and practical significance for enriching the maize dwarfing gene pool. Summary of the Invention
[0005] To address the technical problem of insufficient maize dwarfing gene library, this invention provides maize dwarfing genes and their primer pair compositions, kits, detection methods, and applications.
[0006] The technical solution of this invention is as follows:
[0007] In a first aspect, the present invention provides a maize dwarfing gene, which is obtained by a single-base mutation in the Br2 gene, the Br2 gene having the gene number Zm00001eb038710, and the maize dwarfing gene is as follows:
[0008] br2-6 is a mutation from G to A at position 4309 of the Br2 gene DNA sequence, starting from the start codon.
[0009] br2-7 is a mutation from G to A at position 575 of the Br2 gene DNA sequence, starting from the start codon.
[0010] br2-70 is a mutation from G to A at position 5736 of the Br2 gene DNA sequence, starting from the start codon.
[0011] br2-98 is a mutation from C to T at position 1749 of the Br2 gene DNA sequence, starting from the start codon.
[0012] br2-100 is a mutation from C to T at position 5719 of the Br2 gene DNA sequence, starting from the start codon.
[0013] br2-113 is a mutation from C to T at position 4924 of the Br2 gene DNA sequence, starting from the start codon.
[0014] br2-115 is a mutation from C to T at position 1731 of the Br2 gene DNA sequence, starting from the start codon.
[0015] br2-128 is a mutation from G to A at position 4895 of the Br2 gene DNA sequence, starting from the start codon.
[0016] br2-129 is a mutation from C to G in the 4991st base of the Br2 gene DNA sequence starting from the start codon;
[0017] br2-130 is a mutation from G to A at position 5332 of the Br2 gene DNA sequence, starting from the start codon.
[0018] Alternatively, br2-131, is a mutation from C to T at position 1086 of the Br2 gene DNA sequence, starting from the start codon;
[0019] The Br2 gene DNA sequence is shown in SEQ ID NO.1.
[0020] Furthermore, the maize dwarfing gene is preferably br2-6 or br2-115.
[0021] Secondly, the present invention provides an application of the above-mentioned maize dwarfing gene in maize breeding, wherein the maize germplasm to be improved is hybridized and backcrossed with maize germplasm containing the above-mentioned maize dwarfing gene, and offspring containing the relevant traits of the maize germplasm to be improved and containing the maize dwarfing gene are screened to obtain new maize germplasm with dwarfing improvement.
[0022] Thirdly, the present invention provides a primer pair composition for detecting the above-mentioned maize dwarfing gene, the primer pair composition comprising:
[0023] Primer pairs that specifically amplify br2-6, br2-113, or br2-128 consist of a forward primer F-1 and a reverse primer R-1.
[0024] Forward primer F-1: 5'-CCCTGGTCAAACTCAAACACTC-3' (SEQ ID NO.3).
[0025] Reverse primer R-1: 5'-CCGATGAGCAGGTAGCAGTATT-3' (SEQ ID NO.4);
[0026] The primer pair for specific amplification of br2-7 consists of forward primer F-2 and reverse primer R-2.
[0027] Forward primer F-2: 5'-TGCTTTGCTCTGCCACTCTGCT-3' (SEQ ID NO.5).
[0028] Reverse primer R-2: 5'-CGGTCCACATCCAGCAAGAGAT-3' (SEQ ID NO.6);
[0029] Primer pairs specifically amplifying br2-98 or br2-115 consist of forward primer F-3 and reverse primer R-3.
[0030] Forward primer F-3: 5'-CACCTACTTCACCGTCTTCTGC-3' (SEQ ID NO.7).
[0031] Reverse primer R-3: 5'-TCTACCCAGCAATGGCAGTTGG-3' (SEQ ID NO.8);
[0032] The primer pair specifically amplifying br2-70 and br2-100 consists of forward primer F-4 and reverse primer R-4.
[0033] Forward primer F-4: 5'-CAAGATCACGGGGCTGTTCGA-3' (SEQ ID NO.9).
[0034] Reverse primer R-4: 5'-CGTACGCGATGTTCTCGTGGAT-3' (SEQ ID NO.10);
[0035] Primer pairs specifically amplifying br2-129 or br2-130 consist of forward primer F-5 and reverse primer R-5.
[0036] Forward primer F-5: 5'-GACTTCTCCACCTCCGACTTCAC-3' (SEQ ID NO.11)
[0037] Reverse primer R-5: 5'-CTCGAACAGCCCCGTGATCTTG-3' (SEQ ID NO.12);
[0038] The primer pair for specific amplification of br2-131 consists of forward primer F-6 and reverse primer R-6.
[0039] Forward primer F-6: 5'-TCGTGGGCAGGTACGCTATCCCT-3' (SEQ ID NO.13).
[0040] Reverse primer R-6: 5'-CTGCACGCGATCCACAGATACC-3' (SEQ ID NO.14).
[0041] Fourthly, the present invention provides the application of the above-mentioned primer pair composition in the preparation of a kit for detecting maize dwarfing genes.
[0042] Fifthly, the present invention provides a kit comprising the above-described primer pair composition.
[0043] In a sixth aspect, the present invention provides a method for detecting maize dwarfing genes, which involves PCR amplification of the genomic DNA of the maize to be tested using the above-mentioned primer pair composition, sequencing the PCR amplification product, and determining the genotype of the maize dwarfing gene carried by the maize to be tested based on the bases at the mutation sites.
[0044] The mutation site is selected from one of the following:
[0045] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at position 4309 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.
[0046] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at position 575 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.
[0047] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at the 5736th base from the start codon. The mutation site has a base G, indicating that the genotype is wild-type, and a mutation site has a base A, indicating that the genotype is mutant.
[0048] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C mutation at position 1749 from the start codon, indicating a wild-type genotype, and a T mutation at position 1749, indicating a mutant genotype.
[0049] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C mutation at position 5719 from the start codon, indicating a wild-type genotype, and a T mutation at position 5719, indicating a mutant genotype.
[0050] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at the 4924th base from the start codon. The mutation site has a base C, indicating that the genotype is wild-type, and a mutation site has a base T, indicating that the genotype is mutant.
[0051] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C mutation at position 1731 from the start codon, indicating a wild-type genotype, and a T mutation at position 1731, indicating a mutant genotype.
[0052] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at position 4895 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.
[0053] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C base at position 4991 from the start codon, indicating a wild-type genotype, and a G base at position G, indicating a mutant genotype.
[0054] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at position 5332 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype.
[0055] The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C mutation at position 1086 from the start codon, indicating a wild-type genotype, and a T mutation at position 1086, indicating a mutant genotype.
[0056] Maize plants carrying the mutant dwarfing gene are shorter than those carrying the wild-type dwarfing gene.
[0057] The beneficial effects of this invention are as follows:
[0058] This invention identifies eleven novel single-base mutation sites in the maize Br2 gene (gene number Zm00001eb038710). Mutations at these sites can effectively reduce maize plant height by regulating gene function, providing precise and efficient targets for maize plant architecture improvement and molecular-assisted breeding. Based on these sites, maize plant architecture improvement and molecular-assisted breeding can produce dwarf maize plants. Simultaneously, the dwarf plant architecture breaks the limitations of traditional tall maize on planting space, allowing for a reasonable increase in maize planting density, thus contributing to increased yield per unit area. In particular, two of these mutation sites, compared to wild-type B73 maize, achieve dwarfing and lodging resistance without a significant decrease in average yield per plant, demonstrating higher application value in breeding practice. Attached Figure Description
[0059] Figure 1 This is a phenotypic analysis of wild-type B73 and different dwarfing mutants; where A is the whole plant phenotypic analysis, scale bar = 30 cm; B is the internode phenotypic analysis, scale bar = 5 cm; C is the plant height statistics; D is the ear height statistics; E is the number of aboveground internodes statistics; F is the ear phenotypic analysis, scale bar = 5 cm; G is the yield statistics per plant. Figure 1 middle,** P <0.01.
[0060] Figure 2 This is a phenotypic analysis of F1 plants from wild-type B73, different dwarfing mutants, and allelic tests; where A is the whole plant phenotypic analysis, scale bar = 50 cm; and B is the plant height statistics. Figure 2 middle,** P <0.01.
[0061] Figure 3 These are Sanger sequencing results for different mutation sites in Br2. Detailed Implementation
[0062] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.
[0063] The maize Br2 gene involved in the following embodiments of the present invention has the gene number Zm00001eb038710, and its DNA sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.
[0064] SEQ ID NO.1:
[0065]
[0066] SEQ ID NO.2:
[0067]
[0068] Example 1
[0069] The applicant constructed a maize EMS mutant library (http: / / maizeems.qlnu.edu.cn / ) using EMS mutagenesis and next-generation sequencing technology. From this library, mutation sites related to the Br2 gene were screened, and eleven mutation sites that could reduce maize plant height to varying degrees were identified. Specifically:
[0070] The DNA sequence of the maize Br2 gene has a mutation site at position 4309 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a mutation site has a base of A, indicating a mutant genotype. This mutant gene is named br2-6. The single-base mutation causes the amino acid at position 599 to change from glycine G (codon GGC) to aspartic acid D (codon GAC).
[0071] The DNA sequence of the maize Br2 gene has a mutation site at position 575 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a mutation site has a base of A, indicating a mutant genotype. This mutant gene is named br2-7. The single-base mutation causes the amino acid at position 192 to change from glycine (G, codon GGA) to glutamic acid (GAA).
[0072] The DNA sequence of the maize Br2 gene has a mutation site at position 5736 from the start codon. A mutation site with a base of G indicates a wild-type genotype, while a mutation site with a base of A indicates a mutant genotype. This mutant gene is named br2-70. A single base mutation causes the amino acid at position 1075 to change from glutamic acid E (codon GAG) to lysine K (codon AAG).
[0073] The DNA sequence of the maize Br2 gene has a mutation site at position 1749 from the start codon. The mutation site has a base C, indicating a wild-type genotype, and a base T, indicating a mutant genotype. This mutant gene is named br2-98. The single-base mutation causes the amino acid at position 504 to change from threonine T (codon ACG) to methionine M (codon ATG).
[0074] The DNA sequence of the maize Br2 gene has a mutation site at position 5719 from the start codon. The mutation site has a base of C, indicating a wild-type genotype, and a base of T, indicating a mutant genotype. This mutant gene is named br2-100. The single-base mutation causes the amino acid at position 1069 to change from serine S (codon TCC) to phenylalanine F (codon TTC).
[0075] The DNA sequence of the maize Br2 gene has a mutation site at position 4924 from the start codon. The mutation site has a base of C, indicating a wild-type genotype, and a mutation site has a base of T, indicating a mutant genotype. This mutant gene is named br2-113. A single base mutation causes the amino acid at position 804 to change from serine S (codon TCC) to phenylalanine F (codon TTC).
[0076] The DNA sequence of the maize Br2 gene has a mutation site at position 1731 from the start codon. The mutation site has a base C, indicating a wild-type genotype, and a mutation site has a base T, indicating a mutant genotype. This mutant gene is named br2-115. A single base mutation causes the amino acid at position 498 to change from serine S (codon TCC) to phenylalanine F (codon TTC).
[0077] The DNA sequence of the maize Br2 gene has a mutation site at position 4895 from the start codon. The mutation site has a base of G, indicating a wild-type genotype, and a base of A, indicating a mutant genotype. This mutant gene is named br2-128. The single-base mutation causes the amino acid at position 794 to change from tryptophan W (codon TGG) to the stop codon (TGA).
[0078] The DNA sequence of the maize Br2 gene has a mutation site at position 4991 from the start codon. The mutation site has a base C, indicating a wild-type genotype, and a base G, indicating a mutant genotype. This mutant gene is named br2-129. A single base mutation causes the amino acid at position 826 to change from tyrosine Y (codon TAC) to a stop codon (TAG).
[0079] The DNA sequence of the maize Br2 gene has a mutation site at position 5332 from the start codon. A mutation site with a base of G indicates a wild-type genotype, while a mutation site with a base of A indicates a mutant genotype. This mutant gene is named br2-130. A single base mutation causes the amino acid at position 940 to change from tryptophan W (codon TGG) to the stop codon (TAG).
[0080] The DNA sequence of the maize Br2 gene has a mutation site at position 1086 from the start codon. The mutation site has a base of C, indicating a wild-type genotype, and a base of T, indicating a mutant genotype. This mutant gene is named br2-131. A single base mutation causes the amino acid at position 315 to change from glutamine Q (codon CAG) to the stop codon (TAG).
[0081] Twenty seeds each of the mutant and wild-type B73 were selected and sown simultaneously in the experimental field during the normal growing season. Agronomic traits were investigated after all the mutant and wild-type seeds had shed pollen. The phenotype of the ear was investigated after harvest.
[0082] The results are as follows Figure 1 As shown, the average plant height of maize with the br2-6 genotype was 128 cm, which was 32.2% lower than that of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-6 genotype was 126 g, which was not significantly different from that of wild-type B73 (129.3 g).
[0083] The average plant height of br2-7 genotype maize is 104.7 cm, which is 42.5% lower than the average plant height of wild type B73 (188.8 cm). The average yield per plant of br2-7 genotype maize is 91.5 g.
[0084] The average plant height of maize with the br2-70 genotype is 143.3 cm, which is 24.1% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-70 genotype is 82.5 g.
[0085] The average plant height of maize with the br2-98 genotype is 124.3 cm, which is 34.2% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-98 genotype is 88.8 g.
[0086] The average plant height of maize with the br2-100 genotype is 87.3 cm, which is 53.8% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-100 genotype is 60.3 g.
[0087] The average plant height of maize with the br2-113 genotype is 158.3 cm, which is 16.2% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-113 genotype is 93.7 g.
[0088] The average plant height of maize with the br2-115 genotype was 125 cm, which was 33.8% lower than that of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-115 genotype was 128.3 g, which was not significantly different from that of wild-type B73 (129.3 g).
[0089] The average plant height of maize with the br2-128 genotype is 99.2 cm, which is 47.5% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-128 genotype is 71.8 g.
[0090] The average plant height of maize with the br2-129 genotype is 110.8 cm, which is 41.3% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-129 genotype is 69.7 g.
[0091] The average plant height of maize with the br2-130 genotype is 104.7 cm, which is 44.6% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-130 genotype is 69.7 g.
[0092] The average plant height of maize with the br2-131 genotype is 97 cm, which is 48.6% lower than the average plant height of wild-type B73 (188.8 cm). The average yield per plant of maize with the br2-131 genotype is 77.7 g.
[0093] To further confirm that these mutation sites in the Br2 gene are functional sites affecting maize plant height, allelic mutants of different Br2 mutation sites were crossed to obtain F1 plants, such as... Figure 2 As shown, the height of F1 plants was significantly lower than that of wild-type B73, consistent with the parental phenotype, indicating that this mutation site is the functional site affecting maize plant height.
[0094] In summary, these mutants all reduced maize plant height to varying degrees. Furthermore, the br2-6 and br2-115 mutation sites significantly reduced maize plant height without causing a decrease in yield, an advantage not previously observed in the Br2 allelic mutants. In addition, while the remaining nine mutation sites negatively impacted yield, their stalk-reducing effect was stronger than that of the previously reported Br2 allelic mutants. This provides important germplasm resources for dwarfing breeding of different allelic mutants of this gene.
[0095] Example 2
[0096] Using the genomic sequences flanking the mutation site in Example 1 as templates for primer synthesis, forward and reverse primers were designed to the left and right of the mutation site using Primer Premier5. Primer design principles: Tm value around 60℃, product size 250-550 bp, and primer length 21-24 bp.
[0097] The primer pairs for each maize dwarfing gene were designed as follows:
[0098] Primer pairs that specifically amplify br2-6, br2-113, or br2-128 consist of a forward primer F-1 and a reverse primer R-1.
[0099] Forward primer F-1: 5'-CCCTGGTCAAACTCAAACACTC-3' (SEQ ID NO.3).
[0100] Reverse primer R-1: 5'-CCGATGAGCAGGTAGCAGTATT-3' (SEQ ID NO.4);
[0101] The primer pair for specific amplification of br2-7 consists of forward primer F-2 and reverse primer R-2.
[0102] Forward primer F-2: 5'-TGCTTTGCTCTGCCACTCTGCT-3' (SEQ ID NO.5).
[0103] Reverse primer R-2: 5'-CGGTCCACATCCAGCAAGAGAT-3' (SEQ ID NO.6);
[0104] Primer pairs specifically amplifying br2-98 or br2-115 consist of forward primer F-3 and reverse primer R-3.
[0105] Forward primer F-3: 5'-CACCTACTTCACCGTCTTCTGC-3' (SEQ ID NO.7).
[0106] Reverse primer R-3: 5'-TCTACCCAGCAATGGCAGTTGG-3' (SEQ ID NO.8);
[0107] Primer pairs specifically amplifying br2-70 or br2-100 consist of forward primer F-4 and reverse primer R-4.
[0108] Forward primer F-4: 5'-CAAGATCACGGGGCTGTTCGA-3' (SEQ ID NO.9).
[0109] Reverse primer R-4: 5'-CGTACGCGATGTTCTCGTGGAT-3' (SEQ ID NO.10);
[0110] Primer pairs specifically amplifying br2-129 or br2-130 consist of forward primer F-5 and reverse primer R-5.
[0111] Forward primer F-5: 5'-GACTTCTCCACCTCCGACTTCAC-3' (SEQ ID NO.11)
[0112] Reverse primer R-5: 5'-CTCGAACAGCCCCGTGATCTTG-3' (SEQ ID NO.12);
[0113] The primer pair for specific amplification of br2-131 consists of forward primer F-6 and reverse primer R-6.
[0114] Forward primer F-6: 5'-TCGTGGGCAGGTACGCTATCCCT-3' (SEQ ID NO.13).
[0115] Reverse primer R-6: 5'-CTGCACGCGATCCACAGATACC-3' (SEQ ID NO.14).
[0116] The primer pairs mentioned above were synthesized by Nanjing Qingke Biotechnology Co., Ltd.
[0117] On the one hand, the above primer pair combination can be used to detect the maize dwarfing gene in Example 1. The genomic DNA of the maize to be tested was amplified by PCR using the above primer pair composition. The total volume of the PCR system was 25 μL, including 1 μL each of the forward and reverse primers (10 μmol / L), 1 μL of genomic DNA, 12.5 μL of 2× Flash HS PCR Master Mix enzyme, and ddH2O to a final volume of 25 μL. The PCR reaction program was: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 5 s, 58℃ annealing for 5 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ over-extension for 2 min; and storage at 16℃. After sequencing the PCR amplification products, the PCR amplification products were then separated by 1% agarose gel electrophoresis. PCR products matching the target fragment size were sent to the company for Sanger sequencing. The sequencing peak diagram was submitted to SnapGene software for analysis. The results ( Figure 3 The results show that wild-type and mutant plants are inconsistent at the mutation sites. This indicates that the marker can effectively distinguish between wild-type and mutant genes.
[0118] Maize plants carrying the mutant dwarfing gene are shorter than those carrying the wild-type dwarfing gene.
[0119] Taking the br2-7 mutation site as an example, the genotype detection method is as follows:
[0120] (1) Using the genomic DNA of the maize material to be tested as a template, PCR amplification was performed using the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 above as primers;
[0121] (2) The PCR products obtained in step (1) were subjected to Sanger sequencing, and then the peak diagram was interpreted using SnapGene software. If the mutation site is base G, the maize material to be tested is wild-type; if the mutation site is base A, the maize material to be tested is a material with homozygous mutant genotype and shorter plant height; if the mutation site is G / A, the maize material to be tested is a material with heterozygous genotype and plant height is consistent with wild-type material.
[0122] On the other hand, the combination of the above primer pairs can also be used to prepare a kit containing the above six primer pairs, which can detect maize dwarfing genes.
[0123] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.
Claims
1. The application of a maize dwarfing gene in maize breeding, characterized in that, The maize germplasm to be improved is hybridized and backcrossed with maize germplasm containing maize dwarfing genes. Offspring containing the relevant traits of the maize germplasm to be improved and also containing maize dwarfing genes are screened to obtain new maize germplasm with dwarfing improvement. The maize dwarfing gene was obtained by a single-base mutation in the Br2 gene, the gene number of which is Zm00001eb038710. The single-base mutation is as follows: br2-6 is a mutation from G to A at position 4309 of the Br2 gene DNA sequence, starting from the start codon. Alternatively, br2-115, is a mutation from C to T at position 1731 of the Br2 gene DNA sequence, starting from the start codon; The Br2 gene DNA sequence is shown in SEQ ID NO.
1.
2. The application of a primer pair composition in the preparation of a kit for detecting maize dwarfing genes, characterized in that, The maize dwarfing gene was obtained by a single-base mutation in the Br2 gene, the gene number of which is Zm00001eb038710. The single-base mutation is as follows: br2-6 is a mutation from G to A at position 4309 of the Br2 gene DNA sequence, starting from the start codon. Alternatively, br2-115, is a mutation from C to T at position 1731 of the Br2 gene DNA sequence, starting from the start codon; The Br2 gene DNA sequence is shown in SEQ ID NO.1; Maize plants carrying the mutant dwarfing gene are shorter than those carrying the wild-type dwarfing gene. The primer pair composition includes: The primer pair for specific amplification of br2-6 consists of forward primer F-1 and reverse primer R-1. The nucleotide sequence of the forward primer F-1 is shown in SEQ ID NO.
3. The nucleotide sequence of the reverse primer R-1 is shown in SEQ ID NO.4; The primer pair for specific amplification of br2-115 consists of forward primer F-3 and reverse primer R-3. The nucleotide sequence of the forward primer F-3 is shown in SEQ ID NO.
7. The nucleotide sequence of the reverse primer R-3 is shown in SEQ ID NO.
8.
3. A method for detecting maize dwarfing genes, characterized in that, The genomic DNA of the maize to be tested was amplified by PCR using primer pair composition. After sequencing the PCR amplification product, the genotype of the maize dwarfing gene carried by the maize to be tested was determined based on the bases at the mutation site. The mutation site is selected from one of the following: The DNA sequence of the maize Br2 gene Zm00001eb038710 has a mutation site at position 4309 from the start codon. The mutation site has a base G, indicating a wild-type genotype, and a mutation site has a base A, indicating a mutant genotype. The DNA sequence of the maize Br2 gene Zm00001eb038710 has a C mutation at position 1731 from the start codon, indicating a wild-type genotype, and a T mutation at position 1731, indicating a mutant genotype. The DNA sequence of the Br2 gene Zm00001eb038710 is shown in SEQ ID NO.1; Maize plants carrying the mutant dwarfing gene are shorter than those carrying the wild-type dwarfing gene. The primer pair composition includes: The primer pair for specific amplification of br2-6 consists of forward primer F-1 and reverse primer R-1. The nucleotide sequence of the forward primer F-1 is shown in SEQ ID NO.
3. The nucleotide sequence of the reverse primer R-1 is shown in SEQ ID NO.4; The primer pair for specific amplification of br2-115 consists of forward primer F-3 and reverse primer R-3. The nucleotide sequence of the forward primer F-3 is shown in SEQ ID NO.
7. The nucleotide sequence of the reverse primer R-3 is shown in SEQ ID NO.8.