Corn dwarf gene zm an1 mutant, primer pair for identifying the mutant, kit, detection method and application

Abstract

The present application relates to a maize dwarfing gene ZmAN1 mutant and its identification primer pair, kit, detection method and application, and belongs to the technical field of molecular genetics. The ZmAN1 mutant is obtained by single base mutation of the ZmAN1 gene, the gene number of the ZmAN1 gene is Zm00001d032961, and the gene mutants are an1-25, an1-65, an1-76, an1-75, an1-14 and an1-41. The present application also provides a primer pair for identifying the ZmAN1 mutant and its application. The present application creates and screens a series of different amino acid mutation sites distributed in the coding region of the ZmAN1 gene through EMS mutagenesis technology, significantly enriches the germplasm resource library of maize dwarfing breeding, effectively makes up for the current situation of the lack of allelic variation of the gene, and provides a basis for carrying out new high-quality dwarf material excavation.

Description

Technical Field

[0001] This invention relates to the technical field of molecular genetics, specifically to the ZmAN1 mutant of maize dwarfing gene and its identification primer pairs, kits, detection methods and applications. Background Technology

[0002] As one of the world's most important food crops, maize's yield and stability are crucial for ensuring food security. Plant height is a key agronomic trait affecting maize yield. Appropriately reducing plant height can significantly enhance maize's resistance to lodging, facilitate dense planting and mechanized harvesting, thus laying the foundation for increasing yield per unit area. Therefore, exploring and creating maize germplasm resources with different dwarfing degrees is of paramount importance for maize dwarfing breeding.

[0003] Traditional dwarfing breeding relies heavily on natural variation or large-scale phenotypic screening following physical or chemical mutagenesis. However, this method suffers from drawbacks such as long cycles, high workload, and difficulty in precisely locating key genes controlling plant height. With the development of molecular biology, gene editing technologies (such as CRISPR / Cas9) have made it possible to create mutants in a targeted manner. However, gene editing usually aims to completely knock out gene function, resulting in a limited number of allele types, which may lead to excessively low plant height or other unfavorable agronomic traits (such as severely reduced yield, loss of fertility, etc.), limiting its flexible application in breeding.

[0004] The ZmAN1 gene is a key enzyme in the gibberellin synthesis pathway, regulating plant height by modulating endogenous gibberellin levels. Existing research indicates that loss of function of this gene leads to dwarfing of maize plants. However, current understanding of the ZmAN1 gene is largely limited to its completely nonfunctional mutants. While these strong mutants exhibit significant dwarfing effects, they are often accompanied by severe yield losses or fertility defects, making them difficult to apply directly to breeding practices. Therefore, there is an urgent need in this field to discover a series of allelic mutations of the ZmAN1 gene with different functional intensities. These mutations could provide a continuous phenotype lineage of dwarfing from mild to severe, thus offering breeders diverse options to find the optimal balance between dwarfing and economic traits such as yield. Summary of the Invention

[0005] To address the shortcomings in existing research and application of the ZmAN1 gene for dwarfing breeding, this invention provides a ZmAN1 mutant of maize, its identification primer pairs, kits, detection methods, and applications to solve the aforementioned problems.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides a ZmAN1 mutant, which is obtained by a single-base mutation in the ZmAN1 gene, wherein the gene number of the ZmAN1 gene is Zm00001d032961, and the ZmAN1 mutant is selected from any of the following mutants: (1) an1-25, is a mutation from G to A at position 4197 of the AN1 gene DNA sequence starting from the start codon; (2) an1-65, a mutation from C to T occurs in the DNA sequence of the AN1 gene starting from the 3987th base from the start codon; (3) an1-76, a mutation from C to T occurs in the DNA sequence of the AN1 gene starting from the start codon at position 7132; (4) an1-75, a mutation from G to A occurs in the DNA sequence of the AN1 gene starting from the start codon at position 5555; (5) an1-14, a mutation from G to A occurs in the DNA sequence of the AN1 gene starting from the start codon at position 4199; (6) an1-41, a mutation from C to T occurs in the DNA sequence of the AN1 gene at position 5217, starting from the start codon.

[0007] Secondly, the present invention provides an application of the above-mentioned ZmAN1 mutant in maize dwarfing breeding.

[0008] Furthermore, the maize germplasm to be improved is hybridized, backcrossed, and self-crossed with maize germplasm containing the above-mentioned ZmAN1 mutant, and its dwarfing trait is introduced into superior maize inbred lines or hybrids to obtain new dwarfed and improved maize germplasm.

[0009] Thirdly, the present invention provides a primer pair for detecting the above-mentioned ZmAN1 mutant, the primer pair comprising: (1) The primer pairs used to detect the an1-25 mutation site are as follows: Forward primer: 5'-ctacgaccgcatgatcaacac-3' (SEQ ID NO.3); Reverse primer: 5'-cgaccggataaacattgggga-3' (SEQ ID NO.4).

[0010] (2) The primer pairs used to detect the an1-65 mutation site are as follows: Forward primer: 5'-tgcaattgggtgccgaaaaa-3' (SEQ ID NO.5); Reverse primer: 5'-gagggtgttgatcatgcggt-3' (SEQ ID NO.6).

[0011] (3) The primer pairs used to detect the an1-76 mutation site are as follows: Forward primer: 5'-tcttgcagtaagttatccccact-3' (SEQ ID NO.7); Reverse primer: 5'-acaaggtttacctgggacagt-3' (SEQ ID NO.8).

[0012] (4) The primer pairs used to detect the an1-75 mutation site are as follows: Forward primer: 5'-atcgactcaagccgtcactg-3' (SEQ ID NO.9); Reverse primer: 5'-gaggcatcctgccaaaaacg-3' (SEQ ID NO.10).

[0013] (5) The primer pairs used to detect the an1-14 mutation site are as follows: Forward primer: 5'-ctgcagccacttatcgacca-3' (SEQ ID NO.11); Reverse primer: 5'-gtaagcggtagccgaaggag-3' (SEQ ID NO.12).

[0014] (6) The primer pairs used to detect the an1-41 mutation site are as follows: Forward primer: 5'-gacacagctatggctttccga-3' (SEQ ID NO.13) Reverse primer: 5'-acaccgtctaattttcgcctcta-3' (SEQ ID NO.14).

[0015] Fourthly, the present invention provides a method for detecting the genotype of the ZmAN1 gene carried by maize using the above-mentioned primer pair.

[0016] Furthermore, the method includes: using the genomic DNA of the maize to be tested as a template, performing PCR amplification with the above-mentioned primer pair sequence as primers, performing Sanger sequencing on the PCR amplification product, and determining the genotype of the ZmAN1 gene carried by the maize to be tested based on the bases at the mutation site.

[0017] Fifthly, the present invention provides a kit comprising the above-described primer pairs.

[0018] The beneficial effects of this invention are as follows: (1) This invention provides a series of six alleles with different intensities, with dwarfing levels ranging from mild (9.5%) to moderate (13.6%~45.4%) to extreme (69.5%), forming a continuous phenotypic gradient. The most suitable dwarfing level can be precisely selected according to specific needs to significantly enhance lodging resistance.

[0019] (2) The key finding of this invention is that the degree of dwarfing and yield loss are not simply proportional. For example, the an1-65 mutant achieved a golden balance of "moderate dwarfing and high yield," with a plant height reduction of 13.6% (which effectively enhanced lodging resistance), but a yield reduction of only 18.2%. In contrast, the less dwarfed an1-25 (reduced by 9.5%) resulted in a greater yield loss (40.6%). This discovery breaks the conventional wisdom that "dwarfing inevitably leads to a significant reduction in yield" and provides valuable germplasm resources that can be directly used to breed commercial varieties.

[0020] (3) This invention can introduce dwarfing genes of different intensities into different backbone inbred lines (paternal / maternal parents) to finely regulate the final plant height of hybrids. Two moderately effective dwarfing genes (such as an1-65 and an1-75) can be aggregated into one variety, which may produce additive or super-additive effects, creating new plant height types. If an allele exhibits an unknown defect under specific conditions, it can be quickly switched to another allele in the same series without starting from scratch, reducing breeding risks.

[0021] (4) This invention provides six precise molecular marker sites, namely six specific single nucleotide mutations. Multiple high-throughput molecular marker detection systems can be developed simultaneously to screen dwarf genotypes of different intensities in parallel in the early stages of breeding. This enables parallel manipulation of the entire dwarf lineage, and compared with the traditional scheme that can only track one genotype at a time, the breeding efficiency is increased by orders of magnitude.

[0022] In summary, compared with existing technologies, this invention systematically solves the fundamental contradiction in dwarfing breeding—the contradiction between the urgent need for precise control of plant height and the extreme phenotypes and poor traits of existing germplasm resources. This invention not only provides new germplasm but also offers a completely new, more precise, and more efficient solution for dwarfing breeding. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1It is the phenotype of B73 wild type and various an1 mutants.

[0025] In the figure, A is a plant morphology diagram of wild-type B73 and various an1 mutants, scale bar = 30 cm; B is a schematic diagram of the intersegmental phenotypes of B73 wild-type and various an1 mutants, scale bar = 5 cm; C is a bar chart showing the plant height statistics for B73 wild-type and various an1 mutants; D is a bar chart showing the statistical data on ear height for the B73 wild type and various an1 mutants; E is a bar chart showing the statistical data on the number of internodes in the B73 wild-type and various an1 mutants; F is the phenotypic diagram of the ear of B73 wild type and various an1 mutants, scale bar = 5 cm; G is a bar chart showing the yield statistics of individual plants for the B73 wild type and various an1 mutants.

[0026] Figure 2 This is a bar chart showing the phenotypic analysis of F1 plants from wild-type B73 and different an1 mutants and allelic tests, as well as the statistical results of plant height.

[0027] Figure 3 These are Sanger sequencing results for different ZmAN1 mutants. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0029] The gene number of the ZmAN1 gene involved in the following embodiments of the present invention is Zm00001d032961, and its DNA sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1: SEQ ID NO. 2: MKLLSPAAAPSSSPLFPPRIVEAAARQSGPCRIRIRIRGKAAAAGGGGGAGATGPRGSLRLAGWWRAQQQAPATATTTQQPDNVSSAKVFQTSRVETESEIAKWPGKPQDLEDEHQAEEAELQPLIDQVRAMLRSMNDGDTSASAYDTAWVAMVPKVGGDGGAQPQFPATVRWIVDHQLPDGSWGDSALFSAYDRMINTVACVVALTKWSLEPARCEAGLSFLHENMWRLAEEEAESMPIGFEIAFPSLIQTARDLGVVDFPYGHPALQSIYANREVKLKRIPRDMMHRVPTSILHSLEGMPDLDWPRLLNLQSCDGSFLFSPSATAYALMQTGDKKCFEYIDRIVKKFNGGVPNVYPVDLFEHIWVVDRLERLGISRYFQREIEQCMDYVNRHWTEDGICWARKSNVKDVDDTAMAFRLLRLHGYNVSPSVFKNFEKDGEFFCFVGQSTQAVTGMYNLNRASQISFQGEDVLHRARVFSYEFLRQREEQGMIRDKWIVAKDLPGEVQYTLDFPWYASLPRVEARTYLDQYGGKDDVWIGKTLYRMPLVNNDTYLELAIRDFNHCQALHQLECNGLQTWYKDNCLDAFGVEPQDVLRSYFLAAACIFEPSRAAERLAWARTSMIANAISTHLRDISEDKKRLECFVHCLYEENDVSWLKRNPNDVILERALRRLINLLAQEALPIHEGQRFIHSLLSLAWTEWMLQKANKEENKYHKCSGIEPQYMVHDRQTYLLLVQVIEICAGRIGEAVSMINNKDNDWFIQLTCATCDSLNHRMLLSQDTMKNEARINWIEKEIELNMQELAQSLLLRCDEKTSNKKTKKTLWDVLRSLYYATHSPQHMIDRHVSRVIFEPV* Example 1 Screening of mutation sites for reducing maize plant height This invention utilizes EMS mutagenesis and next-generation sequencing technology to construct a maize EMS mutant library (http: / / maizeems.qlnu.edu.cn / ), which is used for... ZmAN1 Screening of all mutation sites in the coding region of gene (Zm00001d032961) that can alter amino acid coding revealed 6 mutation sites that can reduce maize plant height to varying degrees. Specific mutation sites and phenotypic changes are as follows: (1) an1-25 is caused by a mutation from G to A at position 4197 of the AN1 gene DNA sequence starting from the start codon; resulting in the amino acid at position 300 changing from glycine G (codon GGG) to arginine R (codon AGG). (2) an1-65 is caused by a mutation from C to T at position 3987 of the AN1 gene DNA sequence starting from the start codon; resulting in the amino acid at position 148 changing from threonine T (codon ACG) to methionine M (codon ATG). (3) an1-76 is caused by a C to T mutation at position 7132 of the AN1 gene DNA sequence, starting from the start codon, resulting in the amino acid at position 731 changing from glutamine Q (codon CAA) to the stop codon (TAA). (4) an1-75 is caused by a mutation from G to A at position 5555 of the AN1 gene DNA sequence starting from the start codon, resulting in the amino acid at position 538 changing from tryptophan W (codon TGG) to the stop codon (TGA). (5) an1-14 is caused by a mutation from G to A at the 4199th base of the AN1 gene DNA sequence starting from the start codon, resulting in the 219th amino acid changing from glycine G (codon GGG) to arginine R (codon AGG). (6) an1-41 is caused by a C to T mutation at position 5217 of the AN1 gene DNA sequence starting from the start codon, resulting in the amino acid at position 463 changing from serine S (codon TCT) to phenylalanine F (TTT).

[0031] Twenty maize seeds each from the different mutants and 20 wild-type B73 maize seeds were selected and sown simultaneously in the experimental field during the normal growing season. Agronomic traits were investigated after all mutants and wild-type maize had shed pollen. After harvest, the whole plant phenotype, ear phenotype, and yield per plant were assessed. The results are as follows: Figure 1 As shown.

[0032] (1) The average plant height of the an1-25 mutant (190.5 cm) was 9.5% lower than that of the wild type B73 (210.67 cm), and its average yield per plant (69.37 g) was 40.6% lower than that of the wild type B73 (116.78 g); (2) The average plant height of the an1-65 mutant (182 cm) was 13.6% lower than that of the wild type B73 (210.67 cm), and its average yield per plant (95.58 g) was 18.2% lower than that of the wild type B73 (116.78 g); (3) The average plant height of the an1-76 mutant (128.75 cm) was 38.9% lower than that of the wild type B73 (210.67 cm), and its average yield per plant (69.675 g) was 40.3% lower than that of the wild type B73 (116.78 g); (4) The average plant height of the an1-75 mutant (125.17 cm) was 40.6% lower than that of the wild-type B73 (210.67 cm), and its average yield per plant (86.64 g) was 30.1% lower than that of the wild-type B73 (116.78 g); (5) The average plant height of the an1-14 mutant (115cm) was 45.4% lower than that of the wild type B73 (210.67cm), and its average yield per plant (66.675g) was 42.9% lower than that of the wild type B73 (116.78g); (6) The average plant height of the an1-41 mutant (64.33 cm) was 69.5% lower than that of the wild type B73 (210.67 cm). Because its male inflorescence does not shed pollen, it cannot produce seeds.

[0033] To further confirm that these mutation sites in the AN1 gene are functional sites affecting maize plant height, the an1-75 mutant was crossed with other an1 mutants to obtain different hybrid maizes, namely an1-75 / an1-25, an1-75 / an1-65, an1-75 / an1-76, an1-75 / an1-14, and an1-75 / an1-41; the results are as follows: Figure 2 As shown in the figure, the height of the hybrid maize plant was significantly lower than that of the wild-type B73, consistent with the parental phenotype. This indicates that the mutation site is the functional site affecting maize plant height. In summary, these mutants all reduced maize plant height to varying degrees, and the plant height of these mutants decreased by 9.5% to 69.5%, providing important germplasm resources for dwarfing breeding using different allelic mutants of this gene.

[0034] Example 2 PCR primer design for detecting different mutants Based on the different mutation sites in Example 1, this invention designs specific primer pairs, as follows: (1) The primer pairs used to detect the an1-25 mutation site are as follows: Forward primer: 5'-ctacgaccgcatgatcaacac-3' (SEQ ID NO.3); Reverse primer: 5'-cgaccggataaacattgggga-3' (SEQ ID NO.4).

[0035] (2) The primer pairs used to detect the an1-65 mutation site are as follows: Forward primer: 5'-tgcaattgggtgccgaaaaa-3' (SEQ ID NO.5); Reverse primer: 5'-gagggtgttgatcatgcggt-3' (SEQ ID NO.6).

[0036] (3) The primer pairs used to detect the an1-76 mutation site are as follows: Forward primer: 5'-tcttgcagtaagttatccccact-3' (SEQ ID NO.7); Reverse primer: 5'-acaaggtttacctgggacagt-3' (SEQ ID NO.8).

[0037] (4) The primer pairs used to detect the an1-75 mutation site are as follows: Forward primer: 5'-atcgactcaagccgtcactg-3' (SEQ ID NO.9); Reverse primer: 5'-gaggcatcctgccaaaaacg-3' (SEQ ID NO.10).

[0038] (5) The primer pairs used to detect the an1-14 mutation site are as follows: Forward primer: 5'-ctgcagccacttatcgacca-3' (SEQ ID NO.11); Reverse primer: 5'-gtaagcggtagccgaaggag-3' (SEQ ID NO.12).

[0039] (6) The primer pairs used to detect the an1-41 mutation site are as follows: Forward primer: 5'-gacacagctatggctttccga-3' (SEQ ID NO.13) Reverse primer: 5'-acaccgtctaattttcgcctcta-3' (SEQ ID NO.14).

[0040] The primer pairs were amplified by PCR and then genotyped using Sanger sequencing. These primer pairs can effectively detect wild-type and mutant types of different mutation sites in the Zm00001d032961 gene, and can be used as an auxiliary selection method for breeding new dwarf maize varieties. 。 The results are as follows Figure 3 As shown.

[0041] Example 3 A method for identifying the wild-type and mutant genotype (plant dwarfing) of the maize dwarfing gene Zm00001d032961 in hybrid progeny using the above-mentioned PCR primers, taking the an1-25 mutation site as an example, is as follows: (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.3 and SEQ ID NO.4 as primers; (2) The PCR products obtained in step (1) were subjected to Sanger sequencing, and the peak diagram was interpreted using SnapGene software. If the mutation site is base G, the maize material to be tested is a wild-type genotype; if the mutation site is base A, the maize material to be tested is a material with a homozygous mutant genotype and shorter plant height; if the mutation site is G / A, the maize material to be tested is a material with a heterozygous genotype and the same plant height as the wild-type material.

[0042] Example 4 Application in molecular marker-assisted selection breeding The dwarfing characteristics of the an1-65 mutant were introduced into superior maize inbred lines. Using the an1-65 mutant as the dwarfing donor parent, and superior maize inbred lines with higher plant height requiring improvement (such as the two parents of "Xianyu 335") as recurrent parents, the specific procedures are as follows: (1) The an1-65 mutant was crossed with the recurrent parent to obtain F1 generation seeds.

[0043] (2) Plant the F1 generation and let it self-pollinate to produce the F2 segregating population.

[0044] (3) During the F2 seedling stage, genotyping was performed using molecular markers at the an1-65 mutation site to screen out single plants carrying homozygous an1-65 mutant alleles.

[0045] (4) The selected single plants were backcrossed with the recurrent parents for 3-4 generations. Molecular marker-assisted selection was used in each generation to quickly restore the genetic background of the recurrent parents.

[0046] (5) The last generation of self-crossing, combined with molecular marker selection, yielded a new dwarf inbred line that was homozygous for carrying the an1-65 mutation and whose genetic background was highly consistent with the recurrent parent.

[0047] (6) The two improved dwarf inbred lines were crossbred to produce hybrid varieties.

[0048] Although the present invention has been described in detail with reference to the accompanying drawings and 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 ZmAN1 mutant in maize dwarfing breeding, characterized in that, The ZmAN1 mutant is an1-65, which is a mutation from C to T in the DNA sequence of the AN1 gene starting from the 3987th base from the start codon; the gene number of ZmAN1 is Zm00001d032961, and its DNA sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The maize germplasm to be improved is hybridized, backcrossed, and self-crossed with maize germplasm containing the above-mentioned ZmAN1 mutant. The dwarfing trait is then introduced into superior maize inbred lines or hybrids to obtain new maize germplasm with dwarfing improvement.

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