A method to increase corn yield

Abstract

This invention discloses a method for increasing maize yield, belonging to the field of molecular genetics. This invention identifies a gene related to maize yield; editing this gene can reduce maize plant height and increase maize yield, possessing high industrial value.

Description

Technical Field

[0001] This invention discloses a method for increasing maize yield, belonging to the field of molecular genetics. Background Technology

[0002] Corn is an important food and feed crop, accounting for 40% of the total grain output (National Bureau of Statistics). Improving corn yield is of great significance. High corn yield is the result of the coordinated development of three yield factors: number of ears per unit area, number of kernels per ear, and kernel weight. Corn kernel shape, including kernel length, kernel width, kernel thickness, and other traits, is an important factor that determines kernel weight.

[0003] Flowering period is an important trait in the process of crop evolution and adaptation. Understanding the genetic basis of crop flowering period traits and cloning candidate genes can improve the environmental adaptability and plasticity of crops. This is of great significance for breeding superior crop varieties that are adapted to different ecological zones. At the same time, it will also promote the genetic improvement of important production traits closely related to flowering period, such as yield.

[0004] In addition, corn plant height also affects lodging resistance, photosynthetic efficiency, and harvest index, and is closely related to corn yield.

[0005] Therefore, the combined effect of numerous traits related to maize yield makes it more important to identify genes that regulate multiple yield-related traits, which will provide more guidance for improving maize yield. Summary of the Invention

[0006] To address the aforementioned issues, this invention analyzed an artificially synthesized maize population and, using association analysis, located the key gene ubi3 that affects yield. Furthermore, it was found that knocking out the ubi3 gene using gene editing altered the flowering period, plant height, ear weight, number of rows per ear, number of kernels per row, shelling percentage, and yield per plant, indicating that this gene has significant potential for increasing maize yield.

[0007] The purpose of this invention is to provide a method for increasing corn yield.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides the application of a gene in improving maize traits, characterized in that the gene is a B73 reference genome ID number of GRMZM2G014119 or Zm00001eb275020 or Zm00001d036717, and the trait is any one of the following or a combination thereof:

[0010] 1) Tasseling stage; 2) Silking stage; 3) Pollen shedding stage; 4) Plant height; 5) Ear height; 6) Ear weight; 7) Number of rows per ear; 8) Number of kernels per row; 9) Shelling percentage; 10) Number of kernels per ear; 11) Grain weight per ear; 12) Grain length; 13) Grain width; 14) Cob weight; 15) Cob diameter; 16) Ear diameter.

[0011] In some implementations, the above gene sequence is shown as either SEQ ID NO.1 or SEQ ID NO.2.

[0012] The present invention also provides a method for improving maize traits, characterized in that the expression and / or activity of the gene described in claim 1 is inhibited in the maize material to be improved, and maize plants with altered traits are selected.

[0013] The aforementioned traits include any one or a combination of the following:

[0014] 1) Earlier tasseling; 2) Earlier silking; 3) Earlier pollen shedding; 4) Lower plant height; 5) Lower ear height; 6) Increased ear weight; 7) Increased number of rows per ear; 8) Increased number of kernels per row; 9) Increased shelling percentage; 10) Increased number of kernels per ear; 11) Increased kernel weight per ear; 12) Increased kernel length; 13) Decreased kernel width; 14) Decreased cob weight; 15) Decreased cob diameter; 16) Increased ear diameter.

[0015] In some implementations, the methods for reducing gene expression and / or activity include any one of gene editing, RNA interference, or site-directed insertion of an inhibitory element.

[0016] In some implementations, the target sequence for the gene editing described above is shown in SEQ ID NO.3.

[0017] In some implementations, the gene editing described above uses the CRISPR / Cas9 method.

[0018] The present invention also provides a reagent kit, characterized in that it comprises any one of the following 1) to 3):

[0019] (1) An RNA molecule capable of recognizing the target sequence described in claim 4;

[0020] (2) A DNA molecule encoding the RNA described in (1);

[0021] (3) Vectors that express the RNA described in (1).

[0022] In some embodiments, the sequence of the RNA molecule described above is shown in SEQ ID NO.4.

[0023] In some implementations, the kit described above also includes the Cas9 protein.

[0024] The present invention also provides a mutant gene, characterized in that: the sequence of the mutant gene is shown in SEQ ID NO.5.

[0025] The present invention also provides the above-described method, or kit, or mutant gene, for use in improving maize traits;

[0026] The aforementioned traits include any one or a combination of the following:

[0027] 1) Earlier tasseling; 2) Earlier silking; 3) Earlier pollen shedding; 4) Lower plant height; 5) Lower ear height; 6) Increased ear weight; 7) Increased number of rows per ear; 8) Increased number of kernels per row; 9) Increased shelling percentage; 10) Increased number of kernels per ear; 11) Increased kernel weight per ear; 12) Increased kernel length; 13) Decreased kernel width; 14) Decreased cob weight; 15) Decreased cob diameter; 16) Increased ear diameter.

[0028] Compared with existing technologies, the beneficial effects of this invention are that the function of the ubi3 gene in controlling various yield-related traits provided by this invention is not reported in previous publications. This invention, through gene editing technology, knocks out the ubi3 gene, which can advance the flowering period, reduce plant height, and increase ear weight, number of rows per ear, number of grains per row, shelling percentage, and ultimately increase yield per plant. Attached Figure Description

[0029] Figure 1 ubi3 localization result image.

[0030] Figure 2 ubi3 gene structure, editing target, and post-editing genotype.

[0031] Figure 3 The plant height and flowering period after editing with ubi3.

[0032] Figure 4 The grain shape after editing with ubi3. Detailed Implementation

[0033] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0035] Example 1: Maize Yield Gene Mapping Process

[0036] The applicant previously used 24 core maize breeding materials commonly used in my country to create an artificially synthesized maize CUBIC population containing 1404 inbred families with high genetic diversity, indistinct population structure, and sufficient recombination through two rounds of complete diallel crosses, six generations of open pollination, and six generations of continuous self-pollination. Low-coverage whole-genome sequencing of the CUBIC parents and progeny yielded 14 million high-quality SNP variants, from which a high-density recombination map was constructed (Liu et al., Genome Biology, 2020).

[0037] Building upon this foundation, to systematically analyze heterosis in maize, the inventors created a large-scale F1 population containing 42,820 F1 hybrids, using the maize CUBIC population and its 24 parents as maternal parents and 30 superior inbred lines with foreign lineages as paternal parents. Representative F1 samples were selected and subjected to a two-year phenotypic trial in five provinces and cities across China, with approximately 2.5 million phenotypic data points for 23 agronomic traits obtained through manual surveys. Using whole-genome resequencing data from the CUBIC population and the 30 paternal parents, 4.5 million high-quality SNP variants were identified, from which the genotypes of the F1 generation were inferred. Based on these 4.5 million whole-genome variants and machine learning algorithms, a genotype-phenotype prediction model (G2P) was built, efficiently and cost-effectively predicting the phenotypic values ​​of approximately 34,188 other F1 hybrids. Genome-wide association analysis (GWAS) was performed using F1-predicted genotype and plant height phenotype data under a mixed linear model. A significant SNP locus, Chr6:95877243, was associated on chromosome 6 of the maize MG1544 hybrid. This location corresponds to an annotated gene, ubi3, in the B73 reference genome, with LOC numbers GRMZM2G014119, Zm00001eb275020, or Zm00001d036717. The genomic sequence of ubi3 in B73 is shown in SEQ ID NO.1. The inventors also determined the genomic sequence of this gene in KN5585, as shown in SEQ ID NO.2.

[0038] Example 2 Gene Function Verification

[0039] To further verify the function of the candidate gene, the inventors used CRISPR-Cas9 gene editing technology to knock out the ubi3 gene and investigated the phenotypic performance of maize after knockout (suppressing gene expression, reducing protein expression or activity).

[0040] The gene editing was performed using standard procedures common in the field, with the selected editing target sequence shown in SEQ ID NO.3, and the maize receptor for the gene editing operation being KN5585.

[0041] The edited genotypes of the obtained transformation materials were identified, and a gene-edited material with a 6bp deletion was screened. In 2021, the isolated wild-type (NT) and knockout lines (KO) were planted in Sanya City, Hainan Province, with 14 and 11 rows for each genotype, respectively. Five agronomic traits were measured on each plant, including: tasseling date (the interval from sowing to the date of tasseling), pollen shedding date (the interval from sowing to the date of complete pollen shedding), silking date (the interval from sowing to the date of silking), plant height (vertical height from the ground to the top of the tassel), and ear height (vertical height from the ground to the node where the top ear appears). In 2022, the isolated wild-type (NT) and knockout lines (KO) were planted in Sanya City, Hainan Province, with 16 rows for each genotype. Eleven ear traits were measured on each plant, including: ear weight, ear diameter, ear length, number of rows per ear, number of kernels per row, number of kernels per ear, kernel weight per ear, kernel length, kernel width, cob weight, and cob diameter. The shelling rate is calculated based on the formula: "Seed yield = Grain weight per ear / Ear weight".

[0042] Analysis of the specific traits of the edited material revealed that the KO line of the Ubi3 knockout strain had a significantly earlier flowering period (tasseling, pollen shedding, silking) by 1-2 days compared to the wild-type NT line, significantly lower plant height and ear height by 12-16 cm, and an increased ear weight by nearly 11 g. Furthermore, the inventors found a series of changes in ear and kernel traits after Ubi3 knockout. Compared to the wild type, the Ubi3 knockout line showed a significant increase in the number of rows of kernels per row, the number of kernels per row, and ear diameter, while kernel length increased but kernel width decreased significantly. Therefore, the increase in the number of rows of kernels per row is the reason for the increased yield after Ubi3 knockout. In addition, Ubi3 knockout resulted in a decrease in cob diameter and cob weight, and an increase in ear shelling percentage. These results demonstrate that Ubi3 increases maize ear yield by improving the number of kernels per row, the number of rows of kernels per ear, and shelling percentage, demonstrating significant breeding application value.

[0043] Table 1. Phenotypic characteristics of gene-edited materials

[0044]

[0045]

[0046] "*" indicates the degree of significance of the difference.

[0047] The sequence of the mutated ubi3 gene is shown in SEQ ID NO.5. This mutant gene can be introduced into other maize varieties to improve their traits such as flowering time, plant height, number of rows per ear, number of kernels per row, shelling percentage, and yield per plant, achieving the following technical effects: 1) earlier tasseling; 2) earlier silking; 3) earlier pollen shedding; 4) reduced plant height; 5) reduced ear height; 6) increased ear weight; 7) increased number of rows per ear; 8) increased number of kernels per row; 9) increased shelling percentage; 10) increased number of kernels per ear; 11) increased kernel weight per ear; 12) increased kernel length; 13) reduced kernel width; 14) reduced cob weight; 15) reduced cob diameter; and 16) increased ear diameter.

[0048] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A mutant gene, characterized in that: The mutated gene sequence is shown in SEQ ID NO.

5.

2. The application of the mutant gene as described in claim 1 in improving maize traits; in, The trait includes any one or a combination of the following: 1) Earlier tasseling; 2) Earlier silking; 3) Earlier pollen shedding; 4) Lower plant height; 5) Lower ear height; 6) Increased ear weight; 7) Increased number of rows per ear; 8) Increased number of kernels per row; 9) Increased shelling percentage; 10) Increased number of kernels per ear; 11) Increased kernel weight per ear; 12) Increased kernel length; 13) Decreased kernel width; 14) Decreased cob weight; 15) Decreased cob diameter; 16) Increased ear diameter.

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