Method for increasing corn yield

Abstract

The invention discloses a method for increasing corn yield, and belongs to the field of molecular genetics. The gene related to the corn yield is located, the corn plant height can be reduced and the corn yield can be increased by editing the gene, and the gene has very high industrial value.

Description

Technical Field

[0001] The invention discloses a method for increasing corn yield and belongs to the field of molecular genetics. Background Art

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

[0003] Flowering period is an important trait in the evolution and adaptation of crops. Understanding the genetic basis of crop flowering period traits and cloning candidate genes can improve the environmental adaptability and plasticity of crops, which is of great significance for breeding excellent crop varieties adapted to different ecological zones. 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 will also affect corn's lodging resistance, photosynthetic efficiency, and harvest index, and are closely related to corn yield.

[0005] Therefore, as a result of the combined effects of many traits of corn yield, obtaining genes that have regulatory effects on multiple yield-related traits has more important guiding significance for improving corn yield. Summary of the invention

[0006] To solve the above problems, the present invention locates the key gene ubi3 that affects yield by analyzing an artificially synthesized positioning population of corn in combination with an association analysis method, and finds that after knocking out the ubi3 gene using a gene editing method, the flowering period, plant height, ear weight, number of ear rows, number of grains per row, seed rate, and single plant yield of corn all change, indicating that this gene has great application prospects in increasing corn yield.

[0007] The object of the present invention is to provide a method for increasing corn yield.

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

[0009] The present invention provides an application of a gene in improving corn traits, characterized in that the gene is a gene with 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 ear rows; 8) number of grains per row; 9) seed yield rate; 10) number of grains per ear; 11) grain weight per ear; 12) grain length; 13) grain width; 14) ear axis weight; 15) ear axis thickness; 16) ear thickness.

[0011] In some embodiments, the gene sequence is shown in any one of SEQ ID NO.1 or SEQ ID NO.2.

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

[0013] Wherein, the properties include any one or a combination of the following:

[0014] 1) Early tasseling; 2) Early silking; 3) Early pollen shedding; 4) Reduced plant height; 5) Reduced ear height; 6) Increased ear weight; 7) Increased number of ear rows; 8) Increased number of grains per row; 9) Increased seed yield; 10) Increased number of grains per ear; 11) Increased grain weight per ear; 12) Increased grain length; 13) Reduced grain width; 14) Reduced ear cob weight; 15) Reduced ear cob thickness; 16) Increased ear thickness.

[0015] In some embodiments, the above-mentioned method for reducing gene expression and / or activity comprises any one of gene editing, RNA interference, and site-directed insertion of an inhibitory element.

[0016] In some embodiments, the target sequence of the above-mentioned gene editing is shown as SEQ ID NO.3.

[0017] In some embodiments, the above-mentioned gene editing adopts the CRISPR / Cas9 method.

[0018] The present invention also provides a 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) A vector for expressing the RNA described in (1).

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

[0023] In some embodiments, the above-mentioned kit also includes Cas9 protein.

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

[0025] The present invention also provides the use of the above method, kit, or mutant gene in improving corn traits;

[0026] The above-mentioned properties include any one or a combination of the following:

[0027] 1) Early tasseling; 2) Early silking; 3) Early pollen shedding; 4) Reduced plant height; 5) Reduced ear height; 6) Increased ear weight; 7) Increased number of ear rows; 8) Increased number of grains per row; 9) Increased seed yield; 10) Increased number of grains per ear; 11) Increased grain weight per ear; 12) Increased grain length; 13) Reduced grain width; 14) Reduced ear cob weight; 15) Reduced ear cob thickness; 16) Increased ear thickness.

[0028] Compared with the existing technology, the beneficial effect of the present invention is that the function of the ubi3 gene provided by the present invention to control various yield-related traits has not been reported in previous public materials. The present invention can advance the flowering period, reduce plant height, increase ear weight, number of ear rows, number of grains per row, seed yield, and ultimately increase single plant yield by knocking out the ubi3 gene through gene editing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Ubi3 positioning result diagram.

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

[0031] Figure 3 Plant height and flowering period after ubi3 editing.

[0032] Figure 4 The particle size display after editing with ubi3. DETAILED DESCRIPTION

[0033] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public 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 present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the inventive method, step or condition, all belong to the scope of the present application. If not otherwise specified, the embodiments are according to conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions of the manufacturer's instructions. If not otherwise specified, the chemical reagents used in the embodiments are conventional commercial reagents, and the technical means used in the embodiments 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 corn backbone materials commonly used in breeding in my country, through two rounds of complete diallel hybridization, six generations of open pollination and six generations of continuous selfing, to create a corn artificial synthetic CUBIC population containing 1404 selfing families with high genetic diversity, unclear population structure and sufficient recombination. By performing low-coverage whole-genome sequencing on CUBIC parents and offspring, a total of 14 million high-quality SNP variations were obtained, and a high-density recombination map was constructed based on this (Liu et al., Genome Biology, 2020).

[0037] On this basis, in order to systematically analyze the hybrid vigor of corn, the inventors created a large-scale F1 population with the corn CUBIC population and its 24 parents as maternal parents and 30 excellent inbred lines with foreign blood as paternal parents, including 42,820 F1 hybrids. 8,652 representative F1 samples were selected, and phenotypic tests were carried out in five provinces and cities across the country for two years. About 2.5 million phenotypic data of 23 agronomic traits were obtained through manual investigation. 4.5 million high-quality SNP variants were screened using the whole genome resequencing data of the CUBIC population and 30 paternal parents, and the genotype of F1 was inferred based on this. With 4.5 million whole genome variants and machine learning algorithms, a genotype-phenotype prediction model (G2P) was built to efficiently and cost-effectively predict the phenotypic values ​​of about 34,188 other F1s. The whole genome association analysis was performed under a mixed linear model using the genotype and plant height phenotype data predicted by F1, and a significant SNP site Chr6:95877243 was associated with chromosome 6 of the maize MG1544 hybrid. This position has an annotated gene ubi3 in the B73 reference genome, with LOC numbers GRMZM2G014119 or Zm00001eb275020 or Zm00001d036717. The genome sequence of ubi3 in B73 is shown in SEQ ID NO.1. The inventors also determined the genome 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 investigate the performance of corn traits after knockout (inhibiting gene expression, reducing protein expression or activity).

[0040] Gene editing adopts conventional operations commonly used in the art, wherein the selected editing target sequence is shown in SEQ ID NO.3, and the corn receptor for the gene editing operation is 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 line (KO) were planted in Sanya, Hainan Province, with 14 and 11 rows for each genotype, and five agronomic traits were measured for each plant, including: tasseling period (the interval from sowing to the day of tasseling), pollen shedding period (the interval from sowing to the day of complete pollen shedding), silking period (the interval from sowing to the day of silking), plant height (the vertical height from the ground to the top of the male spike), and ear height (the vertical height from the ground to the node where the top ear appears). In 2022, the isolated wild type (NT) and knockout line (KO) were planted in Sanya, Hainan Province, with 16 rows for each genotype, and 11 ear traits were measured for each plant, including: ear weight, ear thickness, ear length, number of ear rows, number of grains per row, number of grains per ear, grain weight per ear, grain length, grain width, ear axis weight, and ear axis thickness. The seed yield rate was calculated according to “seed yield rate = grain weight per ear / ear weight”.

[0042] By analyzing the specific traits of the edited material, it was found that the flowering period (male drawing, pollen shedding, silking) of the ubi3 knockout line KO was significantly advanced by 1-2 days compared with the wild-type NT, the plant height and ear height were significantly reduced by 12cm-16cm, and the single ear weight increased by nearly 11g. In addition, the inventors found that a series of changes occurred in the ear and grain traits after ubi3 knockout. Compared with the wild type, the number of ear rows, the number of grains per row, and the ear thickness of the ubi3 knockout line increased significantly, the grain length increased but the grain width decreased significantly. Therefore, the increase in the number of ear rows and the number of grains per row is the reason for the increase in yield after ubi3 knockout. In addition, after ubi3 knockout, the axis thickness and axis weight decreased, and the seed rate of the ear increased. These results prove that ubi3 increases the yield of corn per ear by increasing the number of grains per row, the number of ear rows, and the seed rate, and has significant breeding application value.

[0043] Table 1. Traits of gene-edited materials

[0044]

[0045]

[0046] “*” indicates the degree of significant difference.

[0047] The sequence of the ubi3 gene after mutation is shown in SEQ ID NO. 5. The mutant gene can be introduced into other corn varieties to improve their flowering period, plant height, ear row number, row kernel number, seed yield, single plant yield and other traits, achieving the technical effects of 1) early tasseling; 2) early silking; 3) early pollen shedding; 4) reduced plant height; 5) reduced ear height; 6) increased ear weight; 7) increased ear row number; 8) increased row kernel number; 9) increased seed yield; 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 thickness; 16) increased ear thickness.

[0048] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of a gene in improving corn traits, characterized in that: The gene ID number is GRMZM2G014119 or Zm00001eb275020 or Zm00001d036717, and the trait is any one of the following or a combination thereof: 1) tasseling stage; 2) silking stage; 3) pollen shedding stage; 4) plant height; 5) ear height; 6) ear weight; 7) number of ear rows; 8) number of grains per row; 9) seed yield rate; 10) number of grains per ear; 11) grain weight per ear; 12) grain length; 13) grain width; 14) ear axis weight; 15) ear axis thickness; 16) ear thickness. Optionally, the gene sequence is shown in any one of SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for improving corn traits, characterized in that: Inhibiting the expression and / or activity of the gene described in claim 1 in the corn material to be improved, and selecting corn plants with altered traits; Wherein, the properties include any one or a combination of the following: 1) Early tasseling; 2) Early silking; 3) Early pollen shedding; 4) Reduced plant height; 5) Reduced ear height; 6) Increased ear weight; 7) Increased number of ear rows; 8) Increased number of grains per row; 9) Increased seed yield; 10) Increased number of grains per ear; 11) Increased grain weight per ear; 12) Increased grain length; 13) Reduced grain width; 14) Reduced ear cob weight; 15) Reduced ear cob thickness; 16) Increased ear thickness.

3. The method according to claim 2, characterized in that: The method for reducing gene expression and / or activity includes any one of gene editing, RNA interference, and site-directed insertion of an inhibitory element.

4. The method according to claim 3, characterized in that: The target sequence for gene editing is shown in SEQ ID NO.

3.

5. The method according to claim 3, characterized in that: The gene editing adopts the CRISPR / Cas9 method.

6. A kit, characterized in that: Including any of the following 1) to 3): (1) An RNA molecule capable of recognizing the target sequence described in claim 4; (2) a DNA molecule encoding the RNA described in (1); (3) A vector for expressing the RNA described in (1).

7. The kit according to claim 6, characterized in that: The sequence of the RNA molecule is shown in SEQ ID NO.

4.

8. The kit according to claim 6, characterized in that: The kit also includes Cas9 protein.

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

5.

10. Use of the method according to any one of claims 2 to 5, or the kit according to any one of claims 6 to 8, or the mutant gene according to claim 9 in improving corn traits; in, The properties include any one or a combination of the following: 1) Early tasseling; 2) Early silking; 3) Early pollen shedding; 4) Reduced plant height; 5) Reduced ear height; 6) Increased ear weight; 7) Increased number of ear rows; 8) Increased number of grains per row; 9) Increased seed yield; 10) Increased number of grains per ear; 11) Increased grain weight per ear; 12) Increased grain length; 13) Reduced grain width; 14) Reduced ear cob weight; 15) Reduced ear cob thickness; 16) Increased ear thickness.

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