Corn ZmMORF3 gene, excellent haplotype thereof and application of corn ZmMORF3 gene in grain development regulation and molecular breeding

Abstract

The invention discloses a corn ZmMORF3 gene, an excellent haplotype of the corn ZmMORF3 gene and application of the corn ZmMORF3 gene in grain development regulation and molecular breeding. A key gene ZmMORF3 which affects mitochondrial gene RNA editing is cloned by using a grain mutant m497 found in a corn field breeding line selection process, and the gene encodes MORF protein and regulates assembly and activity of a mitochondrial compound I, so that corn grain development is affected; the excellent allelic variation genotype of the ZmMORF3 gene in the aspect of the row grain number is further excavated, and the row grain number can be increased by utilizing the ZmMORF3 gene, so that the yield is increased. In addition, distribution and utilization potentials of different haplotypes in a selfing line are researched, and technical support and excellent germplasm resources are provided for utilization of the gene.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, specifically to a type of maize. ZmMORF3 Genes, their superior haplotypes, and their applications in grain development regulation and molecular breeding. Background Technology

[0002] corn( Zea mays As one of the world's three major food crops, maize has wide applications in food, feed, and industrial raw materials, and plays an irreplaceable strategic role in ensuring global food security. Maize yield is mainly determined by traits such as the number of ears per unit area, the number of kernels per ear, and kernel size. Among these, the number of kernels per ear and kernel size (usually measured by weight per 100 kernels) are key traits that directly determine the final yield; both are complex quantitative traits controlled by multiple genes. Understanding their genetic basis and developing closely linked molecular markers is crucial for achieving genetic improvement of maize yield.

[0003] Currently, genetic studies on maize yield traits primarily employ quantitative trait mapping (QTL) techniques. For example, by constructing genetic populations such as recombinant inbred lines, genotyping can be performed using molecular markers distributed throughout the genome (such as SSRs or SNPs). Combined with multi-year, multi-location phenotypic data, QTL intervals associated with row number of kernels and kernel size can be identified. This QTL information provides a basis for understanding the genetic basis of yield traits. In breeding practice, for some QTLs with large effect values, indirect selection of target traits can be performed using linkage markers flanking them.

[0004] However, existing technical solutions have the following limitations: First, traditional QTL mapping has limited resolution and a wide confidence interval, typically involving hundreds of genes, making it difficult to pinpoint the true causal genes and loci, thus limiting its breeding application value. Second, many reported QTLs exhibit poor stability under different genetic backgrounds and environments, with effect values ​​prone to variation, leading to reduced marker effectiveness. Furthermore, negative correlations often exist between components of yield traits (such as row number and grain size), and existing markers struggle to effectively break this trade-off and achieve synergistic trait improvement.

[0005] Therefore, it is urgent to identify key functional genes that regulate maize kernel development, elucidate their molecular mechanisms of action, and explore superior allelic variations with breeding application value, so as to provide new theoretical basis and technical support for high-yield maize breeding.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The inventors of this application used a corn kernel mutant. m497 Using this as the basic material, its heterozygotes were crossed with the inbred line (W22) to construct the F2 segregating population. Phenotypic identification and cytological observation were performed on the offspring segregating population, and control was obtained through genetic analysis and map-based cloning. m497 Genes with mutant phenotypes ZmMORF3 Its encoded protein is located in mitochondria, and studies have shown that... ZmMORF3 It can affect the size of corn kernels, embryo and endosperm development; to clarify ZmMORF3 The study explored the utilization pathways of the gene, investigated the distribution and utilization potential of different haplotypes in inbred lines, and provided technical support and excellent germplasm resources for the utilization of this gene.

[0008] The first aspect disclosed in this application involves discovering, through phenotypic and cytological analysis... m497 The mutant kernels have a wrinkled and sunken top, smaller volume, and reduced kernel length, width, and thickness. Embryo and endosperm development in the mutant are both delayed compared to the wild type. Using genetic analysis and map-based cloning methods, a regulator of maize kernel development was obtained. ZmMORF3 The gene encodes the MORF protein, which participates in mitochondrial RNA editing and regulates the assembly and activity of mitochondrial complex I, thereby affecting maize kernel development; the CDS sequence of the gene is shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 90% sequence homology and the same function.

[0009] A second aspect of this application discloses an isolated nucleic acid molecule, said nucleic acid molecule being selected from the group consisting of: (a) The nucleotide sequence encoding the MORF protein; (b) A nucleotide sequence complementary to (a); (c) A nucleotide sequence that hybridizes with (a) or (b) under stringent conditions and encodes a protein with the same function; (d) A nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 2 or a polypeptide having at least 95% sequence homology with it.

[0010] The third aspect disclosed in this application relates to the aforementioned gene. ZmMORF3 Or the application of the nucleic acid molecules in the preparation of reagents that regulate the size traits of maize kernels, embryo and / or endosperm development.

[0011] The fourth aspect disclosed in this application provides a combination of molecular markers related to the kernel row number trait of maize, which is... ZmMORF3 The haplotype of the 5' UTR region of a gene contains a combination of the following SNP sites: Allele C or T at the B73V4.chr10.s_143307834 locus; Allele C or T at the B73V4.chr10.s_143309089 locus; Among them, the haplotype Hap4, with two alleles of C and T, was significantly associated with a higher number of rows.

[0012] The fifth aspect disclosed in this application relates to the use of the molecular marker combination in at least one of the following (a) to (c): (a) Identification ZmMORF3 Gene haplotype or preparation identification ZmMORF3 Reagent for genotyping; (b) Improving the kernel number trait in maize or preparing reagents for improving the kernel number trait in maize; (c) Breeding varieties / lines of maize with row kernel number or preparing reagents for breeding varieties / lines of maize with row kernel number.

[0013] The sixth aspect of this application discloses a kit for detecting the molecular marker combination, comprising: (a) Primer pairs capable of specifically amplifying DNA fragments containing the chr10.s_143307834 and chr10.s_143309089 sites; (b) A probe or restriction endonuclease capable of detecting the genotype at the said site.

[0014] The seventh aspect disclosed in this application provides a non-naturally occurring corn plant material, which in ZmMORF3 The 5'UTR region of the gene contains the haplotype Hap4, defined by a combination of the following SNP sites: The C allele at the chr10.s_143307834 locus; and The T allele at the chr10.s_143309089 locus; The maize plant material was obtained through molecular marker-assisted selection breeding technology.

[0015] The eighth aspect of this application discloses a breeding method for improving the row kernel number trait of maize, comprising the following steps: (a) Genotyping of maize germplasm resources using the aforementioned molecular marker combination; (b) Select maize germplasm carrying the haplotype Hap4 as parental material; (c) The haplotype Hap4 is introduced into a target maize variety by hybridization, backcrossing or gene introgression to obtain a maize line with increased row kernel number.

[0016] The ninth aspect of this application discloses a method for identifying maize kernel development-related mutants, including detecting the... ZmMORF3The expression level or functional activity of the gene or the nucleic acid molecule, when the loss of gene function or the significantly reduced expression level is detected, is identified as a grain development defect mutant, which is characterized by wrinkled and sunken top of the grain, reduced volume, and reduced number of starch grains and protein bodies.

[0017] The ninth aspect of this application discloses a method for improving maize kernel traits, comprising: introducing the aforementioned method into a maize plant. ZmMORF3 Genes or said nucleic acid molecules are used to obtain maize plants with improved grain traits, said grain traits being at least one of grain length, grain width, grain thickness, grain volume, grain shape, endosperm translucency, alcohol-soluble protein content, non-alcohol-soluble protein content, starch content, and germination rate.

[0018] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: 1. This invention reveals for the first time the key regulatory role of MORF family proteins in maize kernel development, filling a gap in research on the association between mitochondrial RNA editing mechanisms and kernel development. The selective regulatory mechanism of ZmMORF3 protein on specific mitochondrial gene editing provides a new perspective for understanding organelle gene expression regulatory networks. Experiments demonstrate that loss of ZmMORF3 function leads to a significant decrease in the abundance of mitochondrial complex I, directly linking abnormal mitochondrial energy metabolism to kernel development defects, establishing a complete regulatory pathway of "gene editing-complex assembly-energy supply-kernel development," and deepening our understanding of the molecular mechanisms of maize kernel development.

[0019] 2. This invention successfully discovered... ZmMORF3 The superior haplotype Hap4, carrying a specific SNP combination (CT), is significantly positively correlated with the row grain number trait. Association analysis confirmed that materials carrying the Hap4 haplotype had an average row grain number of 22.0702, a 5.22% increase compared to materials carrying the unfavorable haplotype Hap1 (average 20.9765). This finding provides a precise target for marker-assisted selection, which can significantly improve the efficiency and accuracy of high-yield maize breeding. Since Hap4 has a low frequency in natural populations (14.59%), targeted selection and aggregation breeding can rapidly enhance the yield potential of maize varieties.

[0020] 3. The haplotype information provided by this invention can be directly applied to maize breeding practices. Based on ZmMORF3Molecular markers developed from two key SNP sites in the 5'UTR region of a gene offer advantages such as simple detection, low cost, and reliable results, making them suitable for large-scale germplasm resource screening and purity identification in hybrid production. Based on haplotype information, parental materials carrying the Hap4 haplotype can be preferentially selected for hybridization, or superior haplotypes can be introduced into excellent inbred lines through backcrossing, achieving targeted improvement of yield traits. Practice has shown that using this invention for molecular-assisted selection can shorten the breeding cycle by 30-40%, significantly improving breeding efficiency.

[0021] 4. This invention provides novel genetic resources and theoretical support for high-yield maize breeding. ZmMORF3 Genes and their regulatory networks can serve as important targets for creating new germplasm with improved grain development through biotechnological means such as gene editing, overexpression, or tissue-specific expression. In particular, precise regulation of the key link of mitochondrial energy metabolism can effectively solve practical production problems such as insufficient grain filling and low seed setting rate.

[0022] In summary, this invention, through in-depth analysis ZmMORF3 By studying the functional mechanisms of genes and exploring their superior haplotype variations, a complete technical system from basic research to breeding applications has been established. Significant results have been achieved in three aspects: theoretical innovation, technological breakthroughs, and industrial applications, providing new strategic resources and technical pathways for high-yield maize breeding. Attached Figure Description

[0023] Figure 1 Wild type and m497 Phenotypic and biochemical analysis of mature seeds of mutants; In the figure, A: m497 Heterozygous ear phenotype, red arrows pointing to mutant kernels, scale bar, 1 cm; BD: wild type and m497 Comparison of grain length, width, and thickness of mutant grains, scale bar, 1 cm; E: wild type at maturity and m497 Comparison of mutant grain appearance on a lightbox, scale bar, 1 cm; F: wild type and m497 Longitudinal section of mutant grain, em represents embryo, en represents endosperm, scale bar, 0.5 cm; G: wild type and m497 Mutant germination test, scale bar, 1 cm; HK: wild type and m497 SDS-PAGE (HJ) and quantitative analysis (K) of prolysin and non-prolysin per unit dry matter of mutant endosperm; L: wild type and m497 Starch content per unit dry mass of mutant endosperm; * p <0.001、 *** p <0.05, Student'st test.

[0024] Figure 2 Wild type and m497 Comparison of paraffin sections of mutant seeds; In the figure, A: wild type and [other types] at 9, 12, and 15 days post-pollination. m497 Comparison of paraffin sections of mutant seeds; B: Wild type and mutant seeds 15 days after pollination. m497 Comparison of embryos in mutant seeds; C: Wild type and... 10 days post-pollination m497 Comparison of the endosperm basal transfer layer in mutant grains.

[0025] Figure 3 As shown in one embodiment of this application ZmMORF3 Map-based cloning of genes, gene structure and function verification; in the diagram, A: ZmMORF3 Map-based cloning of genes; B: ZmMORF3 Schematic diagram of gene structure; C: Wild type and mutant ZmMORF3 Comparison of gene transcripts; D: ZmMORF3 Phenotypic and linkage verification of 8-base deletion mutants.

[0026] Figure 4 Wild type and m497 Comparison of mitochondrial complex accumulation and activity in mutants; in the figure, A: wild type and... m497 Comparison of mitochondrial complex accumulation in mutants; BC: wild type and m497 Comparison of the activity of mitochondrial complex I in mutants.

[0027] Figure 5 This is the result of a row particle number correlation analysis in one embodiment of this application; in the figure, A: ZmMORF3 A: Association analysis of SNPs with row number traits; B: Haplotype analysis of significantly associated sites. Detailed Implementation

[0028] The specific implementation of this application will be described below with reference to the embodiments. However, the following embodiments are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0029] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the biological reagents and raw materials involved are all commercially available conventional products unless otherwise specified; and the experimental methods involved are all conventional methods unless otherwise specified.

[0030] Example 1: m497 Phenotypic analysis of mutants corn m497The mutant (collected by Zhiyuan Fu in August 2022 at the Xinxiang Yuanyang Experimental Base of Henan Agricultural University) is a naturally occurring variant discovered in the field. Observations of mature wild-type and mutant grains from the same ear revealed that, compared to the wild-type, the mutant… m497 The seeds are smaller, with a sunken top, lighter in color, and their length, width, and thickness are all reduced compared to the wild type. Figure 1 AD). The mutant endosperm has reduced light transmittance and an increased proportion of powdery endosperm. Figure 1 EF). The mutant embryo develops abnormally, and the mutant grains fail to germinate at all. Figure 1 FG). Protein and starch content analysis of mature grain endosperm showed that the mutant had lower prolysin content and higher non-prolysin and starch content compared to the wild type. Figure 1 HL). Observe wild-type and HL 9-15 days after pollination. m497 Paraffin sections of the grains showed that the embryo and endosperm development of the mutant were delayed compared to the wild type. The mutant embryo showed no obvious differentiation, and the proliferation within the basal transfer layer of the endosperm was reduced. Figure 2 ).

[0031] Example 2: Corn ZmMORF3 Map cloning use m497 The F2 segregating population, formed by mating heterozygotes with the inbred line W22, was used to locate the target gene within a physical region of approximately 1.2 Mb on chromosome 10 of maize through genetic analysis and map-based cloning. Figure 3 A). The linkage markers and their sequences used in map-based cloning are shown in Table 1. Gene annotation and sequencing analysis within candidate regions indicate that... Zm00001d026307 The gene (CDS sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) is a potential candidate gene containing 4 exons. In the mutant, Zm00001d026307 The gene exhibits transcriptional abnormalities, with exon sequences 2, 3, and 4 missing from the transcript. Therefore, this gene is listed as a candidate gene. Figure 3 BC).

[0032] Table 1 ZmMORF3 Primers and sequences used in map-based cloning

[0033] To validate this candidate gene, a sample was purchased from the Miwei Maize mutant library (Miwei Bio). Zm00001d026307 Knockout mutant materials ( morph3-2 ). + / morph3-2 The self-pollinated fruit clusters showed the appearance of... m497 Similar grain mutant phenotypes, and this mutant phenotype is linked to the deletion of 8 bases. Figure 3 D). Indicates Zm00001d026307It is very likely that m497 The functional genes of the mutant phenotype were identified and named. ZmMORF3.

[0034] Example 3: ZmMORF3 Gene Functional Analysis Subcellular localization indicates ZmMORF3 The encoded protein is located in the mitochondria ( Figure 4 A). Mitochondrial complex analysis of wild-type and mutant endosperm 15 days post-pollination showed that the abundance of complex I was decreased in the mutant. Complex I activity staining results indicated decreased activity of complex I in the mutant. Figure 4 BC).

[0035] Example 4: ZmMORF3 Candidate gene association analysis To clarify ZmMORF3 The gene utilization pathway used a single nucleotide polymorphism (SNP) (B73V4.chr10: 143302255-143309390) in 507 maize inbred lines (from Yan Jianbing's research group, Huazhong Agricultural University, www.maizego.org) as the genotype. Candidate gene association analysis was performed based on the grain phenotype, population structure (Q), and phylogenetic relationships (K) of this population. The study found that... ZmMORF3 The chr10.s_143307834 and chr10.s_143309089 of the 5'UTR of the gene were significantly associated with the grain number per ear trait. p <0.0052356, see Figure 5 A).

[0036] Example 5: ZmMORF3 Excellent haplotype identification Haplotype analysis showed that among the 507 maize inbred lines, 425 inbred lines with valid data had four combinations, with significant differences in row and seed number. p =0.00692): Hap1 (CC 316 copies), Hap2 (TC 28 copies), Hap3 (TT 19 copies), Hap4 (CT 62 copies) (see Table 2). Among them, the superior haplotype Hap4 has an average row number of 22.0702 and a low distribution frequency in inbred lines (62 / 425), belonging to a rare haplotype, which has the potential for utilization in breeding improvement; the unfavorable haplotype Hap1 has an average row number of 20.9765 and a high distribution frequency in inbred lines (316 / 425) (see Table 2). Figure 5 B).

[0037] Table 2 Haplotype analysis

[0038] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A method for regulating maize kernel development ZmMORF3 Genes, characterized by, The gene encodes the MORF protein, which participates in mitochondrial RNA editing and regulates the assembly and activity of mitochondrial complex I, thereby affecting maize kernel development; the CDS sequence of the gene is shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 90% sequence homology and the same function.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecules are selected from the following group: (a) The nucleotide sequence encoding the MORF protein of claim 1; (b) A nucleotide sequence complementary to (a); (c) A nucleotide sequence that hybridizes with (a) or (b) under stringent conditions and encodes a protein with the same function; (d) A nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 2 or a polypeptide having at least 95% sequence homology with it.

3. The gene according to claim 1 ZmMORF3 Or the use of the nucleic acid molecule described in claim 2 in the preparation of reagents for regulating the size traits of maize kernels, embryo and / or endosperm development.

4. A molecular marker combination related to the kernel row number trait of maize, characterized in that, It is ZmMORF3 The haplotype of the 5' UTR region of a gene contains a combination of the following SNP sites: Allele C or T at the B73V4.chr10.s_143307834 locus; Allele C or T at the B73V4.chr10.s_143309089 locus; Among them, the haplotype Hap4, with two alleles of C and T, was significantly associated with a higher number of rows.

5. The use of the molecular marker combination of claim 4 in at least one of (a) to (c): (a) Identification ZmMORF3 Gene haplotype or preparation identification ZmMORF3 Reagent for genotyping; (b) Improving the kernel number trait in maize or preparing reagents for improving the kernel number trait in maize; (c) Breeding varieties / lines of maize with row kernel number or preparing reagents for breeding varieties / lines of maize with row kernel number.

6. A kit for detecting the molecular marker combination of claim 4, comprising: (a) Primer pairs capable of specifically amplifying DNA fragments containing the chr10.s_143307834 and chr10.s_143309089 sites; (b) A probe or restriction endonuclease capable of detecting the genotype at the said site.

7. A non-naturally occurring corn plant material, characterized in that, Its in ZmMORF3 The 5'UTR region of the gene contains the haplotype Hap4, defined by a combination of the following SNP sites: The C allele at the chr10.s_143307834 locus; and The T allele at the chr10.s_143309089 locus; The maize plant material was obtained through molecular marker-assisted selection breeding technology.

8. A breeding method for improving the row kernel number trait of maize, characterized in that, Includes the following steps: (a) Genotyping of maize germplasm resources using the molecular marker combination described in claim 4; (b) Select maize germplasm carrying the haplotype Hap4 as parental material; (c) The haplotype Hap4 is introduced into a target maize variety by hybridization, backcrossing or gene introgression to obtain a maize line with increased row kernel number.

9. A method for identifying mutants related to maize kernel development, characterized in that, Including the detection described in claim 1 ZmMORF3 The expression level or functional activity of the gene or the nucleic acid molecule of claim 2, when the loss of function of the gene or the significantly reduced expression level is detected, is identified as a grain development defect mutant, which is characterized by wrinkling and depression at the top of the grain, reduced volume, and reduced number of starch grains and protein bodies.

10. A method for improving the traits of maize kernels, comprising: Introducing the substance of claim 1 into maize plants ZmMORF3 The gene or the nucleic acid molecule of claim 2 is used to obtain maize plants with improved grain traits, wherein the grain traits are at least one of the following: grain length, grain width, grain thickness, grain volume, grain shape, endosperm translucency, alcohol-soluble protein content, non-alcohol-soluble protein content, starch content, and germination rate.

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