SNP site related to ear position height of maize and reagent, kit for detecting the site and application

Abstract

The application provides a SNP site related to ear position height of corn, and reagents, kits and applications for detecting the site. Specifically, the SNP site is located at 123036564 of chromosome 8 by positioning with a corn genome B73v3 reference sequence genome. The SNP site is closely related to the ear position trait of a corn variety, and can be used as a powerful marker to predict the ear position height of corn. This provides a new screening method for breeding new corn varieties with low ear position, helps to accelerate the breeding process and improve the breeding efficiency, and the results of the research will provide important theoretical and practical guidance for agricultural production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a SNP site related to ear height in maize and reagents, kits, and applications for detecting this site. More specifically, this invention relates to uses, isolated nucleic acids, vectors, recombinant cells, transgenic maize varieties, methods for producing low-ear-position maize varieties, primer sets, kits, methods for identifying or assisting in the identification of ear height in maize varieties to be tested, and methods for breeding maize varieties with low ear positions. Background Technology

[0002] Maize (scientific name: Zea mays) is an important food crop and one of the most important crops globally. Belonging to the Poaceae family, maize is an annual herbaceous plant. It exhibits high adaptability and wide genetic diversity. A distinctive feature of maize is its inflorescence, also called an ear, which bears ears of caryopsis (fruits). Each ear typically contains numerous kernels, called maize seeds or maize kernels, which are the main edible part of the maize plant.

[0003] The ear position trait of maize varieties is crucial to the efficiency of photosynthesis, determining whether the maize plant can better receive sunlight and convert it into nutrients and energy. Sufficient sunlight promotes photosynthesis, thereby increasing plant growth rate and nutrient accumulation, ultimately increasing maize yield. Secondly, the ear position trait of maize varieties is an important trait for addressing lodging resistance; appropriately lowering the ear height can effectively improve the plant's resistance to lodging. However, the currently practiced high-density planting results in thinner maize stalks, higher ear heights, and reduced lodging resistance.

[0004] Therefore, there is an urgent need to develop a method for breeding low-ear maize, thereby enabling maize to achieve better lodging resistance. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0006] The inventors discovered that, at the genomic level, ear position in maize, as a quantitative trait, is influenced by multiple genes, and its genetic basis remains unclear. To overcome this problem, the inventors applied a novel marker, single nucleotide polymorphism (SNP), to the genetic study of maize ear position, thereby breeding maize varieties with low ear position and lodging resistance. This overcomes the shortcomings of conventional breeding methods, such as long cycles, large workload in later screening, and low efficiency in identifying varieties or lines.

[0007] Based on this, the first aspect of the present invention proposes the use of SNP loci in breeding maize varieties with low ear position, located on chromosome 8 at position 123036564 using the maize genome B73 v3 reference sequence. Through gene analysis of a large number of maize samples, the inventors discovered a close association between the nucleotide at position 123036564 on chromosome 8 of the maize genome B73 v3 reference sequence and the ear position trait of maize varieties. The genetic variation at this locus is closely related to differences in ear position traits, and therefore can serve as a powerful marker to predict ear height in maize varieties, overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workloads in later screening stages, and low efficiency in identifying varieties or lines. This provides a new screening method for breeding new maize varieties with low ear position, helping to accelerate the breeding process and improve breeding efficiency.

[0008] In a second aspect, this invention proposes the use of a reagent for detecting SNP loci in the breeding of low-ear-position maize varieties. The SNP locus is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP locus is closely associated with ear position traits in maize varieties. Therefore, a reagent for detecting this SNP locus can quickly and accurately predict ear height, thereby assisting in breeding and overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, heavy workload in later screening stages, and low efficiency in variety or line identification. This detection reagent can provide information on ear height of maize varieties in a short time, thus accelerating the screening process.

[0009] In a third aspect, this invention proposes the use of a reagent for mutating SNP sites in the breeding of low-ear maize varieties. The SNP site is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP site is closely related to the ear position trait of maize varieties. Therefore, a reagent for mutating this SNP site can transform ordinary or high-ear maize varieties into low-ear maize varieties, thereby breeding low-ear maize varieties and overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workload in later screening stages, and low efficiency in identifying varieties or lines.

[0010] In a fourth aspect, this invention proposes the use of a reagent for overexpressing a SNP site in the breeding of low-ear maize varieties. The SNP site is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP site is closely associated with the ear position trait of maize varieties. Therefore, a reagent for overexpressing this SNP site can transform ordinary or high-ear maize varieties into low-ear maize varieties, thereby breeding low-ear maize varieties and overcoming the drawbacks of conventional breeding methods, such as long breeding cycles, large workload in later screening stages, and low efficiency in identifying varieties or lines.

[0011] In a fifth aspect, this invention proposes the use of the GRMZM2G179274 gene in the breeding of maize varieties with low ear positions. During the experiment, the inventors unexpectedly discovered that the aforementioned SNP site is closely linked to the GRMZM2G179274 gene. This discovery reveals the potential role of the GRMZM2G179274 gene in determining ear height in maize varieties. Therefore, in addition to this SNP site, the GRMZM2G179274 gene can also serve as another important marker for assessing ear height in maize varieties. This provides a powerful tool for rapidly screening low-ear-position maize varieties, thus overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workloads in later screening stages, and low efficiency in identifying varieties or lines.

[0012] In a sixth aspect, this invention proposes the use of a reagent for detecting the GRMZM2G179274 gene in the breeding of low-ear maize varieties. As mentioned earlier, the GRMZM2G179274 gene can also serve as another important marker for assessing the ear height of maize varieties. Therefore, a reagent for detecting this GRMZM2G179274 gene can also quickly and accurately predict the ear height of maize varieties. This detection method can provide information on ear height in a shorter time, thereby accelerating the screening process and overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workload in later screening stages, and low efficiency in identifying varieties or lines.

[0013] In a seventh aspect, the present invention provides an isolated nucleic acid. According to embodiments of the invention, the isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:1. Through gene analysis and ear height determination of a large number of maize samples, and using the maize genome B73 v3 reference sequence for genomic localization, the inventors discovered that when the nucleotide at position 123036564 of chromosome 8 is G, the maize variety has a lower ear height. This means that maize varieties containing the nucleic acid isolated according to the present invention have a lower ear height.

[0014] In an eighth aspect, the present invention provides a vector. According to an embodiment of the invention, the vector carries the isolated nucleic acid described in the seventh aspect of the invention. Therefore, the vector not only inherits the function of the isolated nucleic acid in reducing ear height in maize varieties, but also effectively introduces the isolated nucleic acid into plant cells, ensuring the stable transmission of its expression and function. This function makes the vector a potential tool in agricultural production for reducing ear height in maize varieties.

[0015] In a ninth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the isolated nucleic acid described in the seventh aspect of the invention or the vector described in the eighth aspect of the invention. Thus, the recombinant cell can reduce the ear height of maize varieties, providing a powerful tool for producing maize varieties with low ear height.

[0016] In a tenth aspect, the present invention provides a transgenic maize variety. According to embodiments of the invention, the transgenic maize variety carries the isolated nucleic acid described in the seventh aspect of the invention or the vector described in the eighth aspect of the invention. By introducing the isolated nucleic acid into the maize variety, the ear height of the maize variety can be reduced. Furthermore, the isolated nucleic acid can be stably present in the cells of the transgenic maize variety and continuously expressed. This ensures that offspring maize varieties can persistently possess the low ear height trait.

[0017] In an eleventh aspect, the present invention provides a method for producing low-ear maize varieties. According to an embodiment of the present invention, the method includes: transforming the recombinant cells described in the ninth aspect of the present invention into the maize variety to be constructed, or hybridizing the transgenic maize variety described in the tenth aspect of the present invention with the maize variety to be constructed. Using the method described in the present invention, maize varieties with the low-ear trait can be produced, providing an effective improvement strategy for breeding low-ear maize varieties.

[0018] In a twelfth aspect, the present invention provides a primer set. According to embodiments of the invention, the primer set includes a forward primer and a reverse primer; the forward primer has a nucleotide sequence as shown in SEQ ID NO:2; and the reverse primer has a nucleotide sequence as shown in SEQ ID NO:3. Using the primer set of the present invention, a high degree of matching can be achieved with the DNA sequence of maize varieties containing a G nucleotide at position 123036564 on chromosome 8, resulting in specific amplification. This allows the primers to accurately and reliably identify the nucleotide at this site, laying the foundation for screening maize varieties with low ear position traits.

[0019] In a thirteenth aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises the primer set described in the twelfth aspect of the invention. Using the kit described in this invention, a high degree of matching with the DNA sequence of maize varieties containing a G nucleotide at position 123036564 on chromosome 8 is achieved, resulting in specific amplification. This enables the kit to accurately and reliably identify the nucleotide at this site, thereby screening for maize varieties with low ear position traits.

[0020] In a fourteenth aspect, this invention provides a method for identifying or assisting in the identification of ear height in a maize variety. According to an embodiment of the invention, the method includes: performing PCR amplification on the DNA of the maize variety to be tested using the primer set described in the twelfth aspect of the invention or the kit described in the thirteenth aspect of the invention; sequencing the amplified product and locating it using the maize genome B73 v3 reference sequence to obtain the nucleotide type located at position 123036564 on chromosome 8 of the maize variety to be tested. By using the method described in this invention, the ear height of a maize variety to be tested can be effectively and rapidly identified.

[0021] In a fifteenth aspect, this invention provides a method for breeding maize varieties with low ear height. According to an embodiment of the invention, the method includes: performing PCR amplification on the DNA of the maize variety to be bred using the primer set described in the twelfth aspect of the invention or the kit described in the thirteenth aspect of the invention; sequencing the amplified product and locating it using the maize genome B73 v3 reference sequence to obtain the nucleotide type at position 123036564 of chromosome 8. By employing the method described in this invention, maize varieties with low ear height can be effectively and rapidly screened.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0024] Figure 1 This is a diagram showing the ear height of the corn material in Embodiment 3 of this application. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0027] The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0028] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0030] This invention proposes the uses of SNP sites and the GRMZM2G179274 gene, isolated nucleic acids, vectors, recombinant cells, transgenic maize varieties, methods for producing low-ear maize varieties, primer sets, kits, methods for identifying or assisting in the identification of ear height in maize varieties to be tested, and methods for breeding maize varieties with low ear height. These will be described in detail below.

[0031] use

[0032] In a first aspect, this invention proposes the use of SNP loci in breeding maize varieties with low ear position, located on chromosome 8 at position 123036564 using the maize genome B73 v3 reference sequence. Through gene analysis and protein content determination of a large number of maize samples, the inventors discovered a close association between the nucleotide at position 123036564 on chromosome 8 of the maize genome B73 v3 reference sequence and the ear position trait of maize varieties. Genetic variation at this locus is closely related to differences in ear position, thus serving as a powerful marker for predicting maize ear height, overcoming the drawbacks of conventional breeding methods such as long cycles, heavy workload in later screening stages, and low efficiency in variety or line identification. This provides a new screening method for breeding new maize varieties with low ear position, helping to accelerate the breeding process and improve breeding efficiency.

[0033] According to an embodiment of the present invention, the SNP site is closely linked to the GRMZM2G179274 gene.

[0034] According to an embodiment of the present invention, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

[0035] According to an embodiment of the present invention, "ear position" refers to the height of the corn ear on the plant.

[0036] In a second aspect, this invention proposes the use of a reagent for detecting SNP loci in the breeding of low-ear-position maize varieties. The SNP locus is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP locus is closely associated with ear position traits in maize varieties. Therefore, a reagent for detecting this SNP locus can quickly and accurately predict ear height in maize varieties, thereby assisting in breeding and overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workloads in later screening stages, and low efficiency in identifying varieties or lines. This detection reagent can provide information on ear height in maize varieties in a shorter time, thus accelerating the screening process.

[0037] According to an embodiment of the present invention, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

[0038] In a third aspect, this invention proposes the use of a reagent for mutating SNP sites in the breeding of low-ear maize varieties. The SNP site is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP site is closely related to the ear position trait of maize varieties. Therefore, a reagent for mutating this SNP site can transform ordinary or high-ear maize varieties into low-ear maize varieties, thereby breeding low-ear maize varieties and overcoming the shortcomings of conventional breeding methods, such as long breeding cycles, large workload in later screening stages, and low efficiency in identifying varieties or lines.

[0039] According to an embodiment of the present invention, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

[0040] In a fourth aspect, this invention proposes the use of a reagent for overexpressing a SNP site in the breeding of low-ear maize varieties. The SNP site is located at position 123036564 on chromosome 8, using the maize genome B73 v3 reference sequence. As mentioned earlier, this SNP site is closely associated with the ear position trait of maize varieties. Therefore, a reagent for overexpressing this SNP site can transform ordinary or high-ear maize varieties into low-ear maize varieties, thereby breeding low-ear maize varieties and overcoming the drawbacks of conventional breeding methods, such as long breeding cycles, large workload in later screening stages, and low efficiency in identifying varieties or lines.

[0041] According to an embodiment of the present invention, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

[0042] In a fifth aspect, this invention proposes the use of the GRMZM2G179274 gene in the breeding of maize varieties with low ear position. During the experiment, the inventors unexpectedly discovered that the aforementioned SNP site is closely linked to the GRMZM2G179274 gene. This discovery reveals the potential role of the GRMZM2G179274 gene in determining ear height in maize varieties. Therefore, in addition to this SNP site, the GRMZM2G179274 gene can also serve as another important marker for assessing ear height in maize varieties, providing a powerful tool for rapidly screening for maize varieties with low ear position.

[0043] In a sixth aspect, the present invention proposes the use of a reagent for detecting the GRMZM2G179274 gene in the breeding of low-ear maize varieties. As mentioned above, the GRMZM2G179274 gene can also serve as another important marker for assessing the ear height of maize varieties; therefore, a reagent for detecting this GRMZM2G179274 gene can also quickly and accurately predict the ear height of maize varieties. This detection method can provide information on ear height in a shorter time, thereby accelerating the screening process.

[0044] Isolated nucleic acids, vectors, recombinant cells, and transgenic maize varieties

[0045] In a seventh aspect, the present invention provides an isolated nucleic acid. According to embodiments of the invention, the isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:1. Through gene analysis and ear height determination of a large number of maize samples, and using the maize genome B73 v3 reference sequence for genomic localization, the inventors discovered that when the nucleotide at position 123036564 of chromosome 8 is G, the maize variety has a lower ear height. This means that maize varieties containing the nucleic acid isolated according to the present invention have a lower ear height.

[0046] According to embodiments of the present invention, an isolated polypeptide is provided. According to embodiments of the present invention, the isolated polypeptide has the amino acid sequence shown in SEQ ID NO:4. Therefore, maize varieties expressing the isolated polypeptide of the present invention have lower ear heights.

[0047] The "isolated" nucleic acid / peptide molecules described in this invention are artificially separated from their natural environment and are therefore not natural products. The isolated nucleic acid / peptide molecules can exist in purified form or in non-natural environments, such as recombinant cells or transgenic plants.

[0048] In this invention, "natural" refers to genes present in the genome of untransformed cells.

[0049] The "nucleic acid molecule" or "nucleic acid sequence" described in this invention can be a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of this invention, preferably, the nucleic acid molecule is a DNA fragment.

[0050] In an eighth aspect, the present invention provides a vector. According to an embodiment of the invention, the vector carries the isolated nucleic acid described in the seventh aspect of the invention. Therefore, the vector not only inherits the function of the isolated nucleic acid in reducing ear height in maize varieties, but also effectively introduces the isolated nucleic acid into plant cells, ensuring the stable transmission of its expression and function. This function makes the vector a potential tool in agricultural production for reducing ear height in maize varieties.

[0051] The term "vector" as used in this invention refers to a transformation vector that can be used for plant transformation, which is known to those skilled in the art of plant transformation, and the nucleic acid molecules of this invention can be used in conjunction with any such vector. The selection of the vector will depend on the preferred transformation technology used for transformation and the target plant species.

[0052] The term "plant" as used in this invention can refer to any plant at any stage of development.

[0053] The "plant cell" described in this invention is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell can be an isolated single cell or a cultured cell, or as a higher organized unit, such as a plant tissue, plant organ, or part of a whole plant.

[0054] The "protoplast" described in this invention is a separated plant cell that has no cell wall or only a partial cell wall.

[0055] In a ninth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the isolated nucleic acid described in the seventh aspect of the present invention or the vector described in the eighth aspect of the present invention. Thus, the recombinant cell can reduce the ear height of maize varieties, providing a powerful tool for producing maize varieties with low ear height. It should be noted that the recombinant cell described in the present invention is not particularly limited, as long as it can transform the target plant; for example, the recombinant cell can be selected from Agrobacterium.

[0056] According to an embodiment of the present invention, the recombinant cells are selected from Agrobacterium.

[0057] In a tenth aspect, the present invention provides a transgenic maize variety. According to embodiments of the invention, the transgenic maize variety carries the isolated nucleic acid described in the seventh aspect of the invention or the vector described in the eighth aspect of the invention. By introducing the isolated nucleic acid into maize, the ear height of the maize variety can be reduced. Furthermore, the isolated nucleic acid can be stably present in the cells of the transgenic maize variety and continuously expressed. This ensures that offspring maize varieties can persistently possess the low ear height trait.

[0058] In this invention, "transformed / genetically modified / recombinant" refers to a host organism, such as bacteria or plants, in which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome or may exist as an extrachromosomal molecule. This extrachromosomal molecule can replicate autonomously. Transformed cells, tissues, or plants are understood to include not only the final products of the transformation process but also their transgenic progeny.

[0059] Methods for producing low-ear maize varieties

[0060] In an eleventh aspect, the present invention provides a method for producing low-ear maize varieties. According to an embodiment of the present invention, the method includes: transforming the recombinant cells described in the ninth aspect of the present invention into the maize variety to be constructed, or hybridizing the transgenic maize variety described in the tenth aspect of the present invention with the maize variety to be constructed. Using the method described in the present invention, maize varieties with the low-ear trait can be produced, providing an effective improvement strategy for breeding low-ear maize varieties.

[0061] Primer set

[0062] In a twelfth aspect, the present invention provides a primer set. According to embodiments of the invention, the primer set includes a forward primer and a reverse primer; the forward primer has a nucleotide sequence as shown in SEQ ID NO:2; and the reverse primer has a nucleotide sequence as shown in SEQ ID NO:3. Using the primer set of the present invention, a high degree of matching can be achieved with the DNA sequence of maize varieties containing a G nucleotide at position 123036564 on chromosome 8, resulting in specific amplification. This allows the primers to accurately and reliably identify the nucleotide at this site, laying the foundation for screening maize varieties with low ear position traits.

[0063] Reagent test kit

[0064] In a thirteenth aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises the primer set described in the twelfth aspect of the invention. Using the kit described in this invention, a high degree of matching with the DNA sequence of maize varieties containing a G nucleotide at position 123036564 on chromosome 8 is achieved, resulting in specific amplification. This enables the kit to accurately and reliably identify the nucleotide at this site, thereby screening for maize varieties with low ear position traits.

[0065] Methods for identifying or assisting in the identification of ear height in maize varieties under test

[0066] In a fourteenth aspect, this invention provides a method for identifying or assisting in the identification of ear height in a maize variety. According to an embodiment of the invention, the method includes: performing PCR amplification on the DNA of the maize variety to be tested using the primer set described in the twelfth aspect of the invention or the kit described in the thirteenth aspect of the invention; sequencing the amplified product and locating it using the maize genome B73 v3 reference sequence to obtain the nucleotide type located at position 123036564 on chromosome 8 of the maize variety to be tested. By using the method described in this invention, the ear height of a maize variety to be tested can be effectively and rapidly identified.

[0067] According to an embodiment of the present invention, the ear position of the maize variety to be tested, which has nucleotide G at position 123036564 of chromosome 8, is lower than that of the maize variety to be tested, which has nucleotide A at position 123036564 of chromosome 8.

[0068] According to an embodiment of the present invention, the nucleotide at position 123036564 of chromosome 8 being G is an indicator that the maize variety under test has a low ear position.

[0069] Methods for breeding maize varieties with low ear position

[0070] In a fifteenth aspect, this invention provides a method for breeding maize varieties with low ear height. According to an embodiment of the invention, the method includes: performing PCR amplification on the DNA of the maize variety to be bred using the primer set described in the twelfth aspect of the invention or the kit described in the thirteenth aspect of the invention; sequencing the amplified product and locating it using the maize genome B73 v3 reference sequence to obtain the nucleotide type at position 123036564 of chromosome 8. By employing the method described in this invention, maize varieties with low ear height can be effectively and rapidly screened.

[0071] According to an embodiment of the present invention, the nucleotide at position 123036564 of chromosome 8 is G, which is an indicator that the maize variety to be bred has a low ear position.

[0072] The sequences in this invention are shown in the table below:

[0073] sequence list

[0074]

[0075]

[0076] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0077] Example 1

[0078] To study the genotypic differences between maize with low ear position and ordinary maize, the inventors conducted gene analysis and ear height measurements on a large number of superior maize varieties with low ear position, and screened out SNP loci closely related to ear height. The specific experimental process is as follows:

[0079] 1. Test materials

[0080] The maize materials used in this invention are all from the public germplasm resources of the Shenzhen National Gene Bank Germplasm Resource Bank. The materials and the original sequencing data are from Li C, Guan H, Jing X, Li Y, Wang B, Li Y, Liu X, Zhang D, Liu C, Xie X, Zhao H, Wang Y, Liu J, Zhang P, Hu G, Li G, Li S, Sun D, ​​Wang X, Shi Y, Song Y, Jiao C, Ross-Ibarra J, Li Y, Wang T, Wang H. Genomic insights into historical improvement of heterotic groups during modern hybrid maize breeding. NatPlants. 2022 Jul;8(7):750-763. doi:10.1038 / s41477-022-01190-2. Epub 2022 Jul18.PMID:35851624.

[0081] 2. Measurement of ear height in maize varieties

[0082] The ear position of 1600 maize samples was measured with a measuring tape at maturity. Three plants were selected from each sample, and the measurement was performed three times. The average value was taken as the ear position of each sample.

[0083] 3. WGS (Whole Genome Sequencing) Library Construction and Resequencing

[0084] Fresh young leaves (0.5g each) were harvested from 1600 maize samples collected in step 2. Genomic DNA was extracted from each sample using the CTAB (hexadecyltrimethylammonium bromide) method and its quality was assessed. Sequencing libraries were generated using the TruSeq NanoDNAHT Sample Preparation Kit (Illumina USA), and indexes were added to each sample. The genomic DNA samples were fragmented to approximately 350 base pairs (bp) using sonication. The DNA fragments were then end-repaired, A-tailed, ligated with full-length adapters, and further amplified by PCR. The PCR products were then purified (using the AMPure XP bead system), and the size distribution of the libraries was analyzed using an Agilent 2100 bioanalyzer. Quantification was performed using real-time quantitative PCR. Subsequently, sequencing was performed using the Illumina HiSeq X platform, yielding raw sequences with paired-end reads of 150 bp.

[0085] 4. Mutation detection

[0086] Variation detection used the maize B73 reference genome v3 version. Quality control, alignment, deduplication, and variation detection were performed using the Mega BOLT full workflow developed by BGI Genomics Co., Ltd. Genotype files for each sample were obtained and merged to obtain a total file containing SNP variation information from all 1600 materials. Plink (v1.9) was used to filter SNP sites with a minor allele frequency less than 0.01 and a deletion rate greater than 0.6.

[0087] 5. Genome-wide association analysis of maize ear position

[0088] Genome-wide association analysis (GEMMA) was performed using genotype files from 1600 maize materials as input. First, the G matrix of the 1600 materials was calculated using the `-gk 2` command. Then, GEMMA was used with the G matrix as a covariate to eliminate errors caused by kinship. Finally, the `-lmm 1` model was used to perform the GEMMA analysis on the 1600 maize materials.

[0089] 6. Gene screening and mining

[0090] Using 0.05 / number of valid SNPs as the significance threshold and 0.01 / number of valid SNPs as the highly significant threshold, the p_wald values ​​in the genome-wide association analysis output files were screened. The results showed that the SNP site POS123036564 on chromosome 8 of the maize B73 reference genome, with a mutation to A / G, was highly significantly associated with maize ear position and tightly linked to the GRMZM2G179274 gene.

[0091] Table 1

[0092] chromosome Location Alleles P_wald Gene annotation 8 123036564 A / G <![CDATA[3.0213e -08 ]]> GRMZM2G179274

[0093] Example 2

[0094] Using samtools (v1.3.1), a fAI index file was created for the FASTA file corresponding to the maize B73 reference genome v3 version, and sequences of 250 bp upstream and downstream of the target SNP, i.e., positions 123036314 to 123036814 on chromosome 8, were extracted. The extracted sequences were imported into Primer Premier 5 software to design primers, setting the primer amplification result fragment to include the target SNP at position 251, and selecting the primer pair with the highest score and the fewest hairpin and other special structures in the software design.

[0095] Example 3

[0096] VCFtools (v0.1.16) was used to extract the SNP types at position 123036564 on chromosome 8 from 1600 maize samples. Undetected types and heterozygotes were ignored. The ear height phenotype of samples homozygous at position 123036564 on chromosome 8 (i.e., all A or all G nucleotides) was statistically analyzed and plotted. Figure 1 As shown, the spike height of the sample with nucleotide type G at position 123036564 on chromosome 8 is lower than that of the sample with nucleotide type A.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0098] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of SNP loci in breeding maize varieties with low ear position, located using the maize genome B73 v3 reference sequence, wherein the SNP locus is located at position 123036564 on chromosome 8.

2. The use according to claim 1, characterized in that, The SNP site is closely linked to the GRMZM2G179274 gene; Optionally, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

3. The use of reagents for detecting SNP sites in the breeding of low-ear maize varieties, with the SNP site located at position 123036564 on chromosome 8, based on the maize genome B73 v3 reference sequence. Optionally, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

4. The use of reagents for mutating SNP sites in the breeding of low-ear maize varieties, with the SNP site located at position 123036564 on chromosome 8, based on the maize genome B73 v3 reference sequence. Optionally, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

5. The use of reagents that overexpress SNP sites in the breeding of low-ear maize varieties, with the SNP site located at position 123036564 on chromosome 8, based on the maize genome B73 v3 reference sequence. Optionally, the nucleotide at position 123036564 is G, which is an indication that the maize variety has a low ear position.

6. The use of the GRMZM2G179274 gene in the breeding of maize varieties with low ear position.

7. Application of reagents for detecting the GRMZM2G179274 gene in the breeding of low-ear maize varieties.

8. An isolated nucleic acid, characterized in that, The isolated nucleic acid has a nucleotide sequence as shown in SEQ ID NO:

1.

9. A carrier, characterized in that, Carrying the isolated nucleic acid as described in claim 8.

10. A recombinant cell, characterized in that, Carrying the isolated nucleic acid as described in claim 8 or the vector as described in claim 9; Optionally, the recombinant cells are selected from Agrobacterium.

11. A genetically modified maize variety, characterized in that, Carrying the isolated nucleic acid as described in claim 8 or the vector as described in claim 9.

12. A method for producing low-ear maize varieties, characterized in that, include: The recombinant cells described in claim 10 are transformed into the maize variety to be constructed, or the transgenic maize variety described in claim 11 is hybridized with the maize variety to be constructed.

13. A primer set, characterized in that, Including forward primers and reverse primers; The forward primer has a nucleotide sequence as shown in SEQ ID NO:2; The reverse primer has a nucleotide sequence as shown in SEQ ID NO:

3.

14. A reagent kit, characterized in that, Includes: the primer set as described in claim 13; Optionally, the kit further comprises at least one of the following components: dNTPs, Taq enzyme, and PCR reaction buffer.

15. A method for identifying or assisting in the identification of ear height of a maize variety, characterized in that, include: The DNA of the maize variety to be tested was amplified by PCR using the primer set described in claim 13 or the kit described in claim 14. The amplification product was sequenced and located using the B73 v3 reference sequence of the maize genome to obtain the nucleotide type located at position 123036564 on chromosome 8 in the maize variety to be tested.

16. The method according to claim 15, characterized in that, The ear position of the maize variety to be tested, which has nucleotide G at position 123036564 on chromosome 8, is lower than that of the maize variety to be tested, which has nucleotide A at position 123036564 on chromosome 8. Optionally, the nucleotide at position 123036564 of chromosome 8 being G is an indicator that the maize variety being tested has a low ear position.

17. A method for breeding maize varieties with low ear position, characterized in that, include: The DNA of the maize variety to be bred was amplified by PCR using the primer set described in claim 13 or the kit described in claim 14. The amplified product was sequenced and located using the B73 v3 reference sequence of the maize genome to obtain the nucleotide type at position 123036564 on chromosome 8. Optionally, the nucleotide at position 123036564 of chromosome 8 is G, which is an indicator that the maize variety to be bred has a low ear position.

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