Corn ZmRRP45B gene mutation SNP marker, dCAPS primer and application of corn ZmRRP45B gene mutation SNP marker

Abstract

The invention relates to a corn ZmRRP45B gene mutation SNP marker, a dCAPS primer and application of the corn ZmRRP45B gene mutation SNP marker and the dCAPS primer. Research finds that on the basis of a fourth version of a corn B73 reference genome, a G deletion exists at a chr5. S175415208 position, so that mutant grains become smaller, endosperm is not filled fully, the top collapses, and the hundred-grain weight is reduced; corresponding molecular markers are developed, development of corn embryos and endosperm can be regulated and controlled, high-yield or high-quality breeding materials of corn can be conveniently created, the breeding period of new crop varieties is shortened, the breeding cost is reduced, and the breeding efficiency is improved.

Description

[0001] This invention is a divisional application of the patent application with the application number 2023112022960, the application date of September 18, 2023, and the invention title of "Maize Kernel Development Regulatory Gene ZmRRP45B , Its Encoded Protein, SNP Marker, Functional Marker and Their Applications". Technical Field

[0002] This invention relates to the technical field of molecular genetic breeding, and specifically relates to a kind of maize ZmRRP45B gene mutation SNP marker, dCAPS primer and their applications. Background Art

[0003] Maize (Zea mays L. ) is an important food and feed crop, playing an irreplaceable role in ensuring national food security and national economic development. Kernels are the main organs for the yield source of maize, and the molecular mechanism of their formation and development has received extensive attention. Currently, although numerous genes regulating maize kernel development and filling have been cloned, due to the extremely complex biological process of maize embryo and endosperm development, the key genes and molecular mechanisms for precisely regulating kernel development and filling still need to be further analyzed.

[0004] RNA degradation plays an important role in maintaining the dynamic balance of the life activities of organisms and the quality control of gene expression. During the process of biological evolution, a precise RNA regulatory mechanism has been formed, which eliminates abnormal transcripts through degradation to maintain the normal development of biological organisms. The RNA degradation mechanism can also participate in the degradation process of normal mRNA, regulate the abundance of gene expression, and maintain the dynamic balance of transcripts. RNA degradation requires the action of 5’→3’ or 3’→5’ exonucleases, among which 3’→5’ RNA degradation is mainly accomplished by the exosome, playing an important role in aspects such as RNA maturation and quality control. Research shows that defects in the process of exosome-mediated RNA degradation lead to various physiological and reproductive defects in animals, plants, etc. However, the mechanism of action of plant RNA exosome regulating phenotypes still needs to be further studied.

[0005] The information disclosed in this background art section is only used to deepen the understanding of the background art of this disclosure, and should not be regarded as an admission or an implication in any form that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] This invention utilizes the natural kernel mutant discovered during the process of maize field breeding line selection l168 , and clones the key gene affecting RNA degradation ZmRRP45B , this gene encodes a core subunit of the RNA exosome complex; further research shows that,ZmRRP45B Genes regulate maize embryo and endosperm development, affect the accumulation of storage substances in grains, and can provide new gene resources for cultivating high-yield and high-quality maize varieties.

[0007] In the first aspect disclosed in this application, through phenotypic and cytological analyses, it was found that l168 The 100-grain weight of mutant grains was reduced by 75.75% compared with that of wild type, the embryo and endosperm development were abnormal, and the intravascular hyperplasia in the basal transfer layer cells of the endosperm decreased; and through genetic analysis and map-based cloning methods, the gene controlling this grain mutation was obtained, ZmRRP45B which has the CDS sequence shown in SEQ ID NO.1.

[0008] In the second aspect disclosed in this application, it relates to a coding protein of a gene regulating grain development, ZmRRP45B the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] In the third aspect disclosed in this application, it relates to a primer pair for amplifying the above ZmRRP45B gene, the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] In the fourth aspect disclosed in this application, it relates to a SNP marker for maize grain phenotypic mutation. Based on the fourth version of the maize B73 reference genome, there is a G deletion at the position of chr5.s_175415208.

[0011] In the fifth aspect disclosed in this application, it relates to dCAPS primers for identifying phenotypic mutants, including primer pairs shown in SEQ ID NO.5 and SEQ ID NO.6; and / or primer pairs shown in SEQ ID NO.7 and SEQ ID NO.8.

[0012] In the sixth aspect disclosed in this application, it relates to a molecular marker for identifying ZmRRP45B overexpression materials, including primer pairs shown in SEQ ID NO.9 and SEQ ID NO.10.

[0013] In yet another aspect disclosed in this application, the above-mentioned maize grain development regulating gene, ZmRRP45B the amplification primers, the maize grain phenotypic mutation SNP marker or the dCAPS primers are applied to the regulation of grain size and / or maize embryo and endosperm development traits.

[0014] Another aspect disclosed in the present application relates to an SNP marker significantly associated with the corn kernel type and storage substance content. Based on the fourth version of the maize B73 reference genome, A / T, A / C, and G / A mutations exist at positions chr5.s_175413975, chr5.s_175413829, and chr5.s_175418484, respectively.

[0015] Another aspect disclosed in the present application relates to an identification gene ZmRRP45B dCAPS primer for excellent haplotypes, including primer pairs shown in SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, and SEQ ID NO.17 and SEQ ID NO.18.

[0016] In yet another aspect disclosed in the present application, the maize kernel development regulatory gene ZmRRP45B 、the SNP marker or the dCAPS primer is applied to the selection of inbred lines for maize kernel yield and quality traits.

[0017] In yet another aspect disclosed in the present application, a method for identifying genes ZmRRP45B excellent haplotypes is provided, including the following steps: Using the extracted DNA of the material to be identified as a template, specifically PCR amplify the PCR products containing the chr5.s_175413829 and chr5.s_175418484 SNP sites with the outer primer pairs; then use the inner primer pairs and the diluted PCR products as templates for the second round of amplification. For the PCR products obtained in the second round, use restriction endonucleases Sac I and Spe I for digestion, and perform gel electrophoresis detection. The AAA and TAA haplotypes are two higher bands; both markers of the ACG and TCG haplotypes are lower bands; the ACA and TCA haplotypes are lower bands after digestion with Sac I, Spe I for digestion are higher bands, the AAG and TAG haplotypes are higher bands after digestion with Sac I, Spe I for digestion are lower bands; select the AAA and TAA haplotypes as breeding improvement materials for starch content.

[0018] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages: 1. Positional cloning ZmRRP45BGenes regulate the development of maize embryos and endosperms, affect the accumulation of storage substances in grains, and have important application values for improving maize grain yield and quality.

[0019] 2. ZmRRP45B The gene has the following mutation sites: Based on the fourth version of the maize B73 reference genome, there is a deletion of G at the position of chr5.s_175415208, resulting in smaller mutant grains, incomplete endosperm filling, collapse at the top, and a decrease in 100-grain weight. Description of the Drawings

[0020] Figure 1 In one embodiment of this application l168 Phenotypic analysis of mutant grains; in the figure, A: Grain phenotypes on a mature separated ear; B: Comparison of wild-type and mutant grains on the same ear 7 days after pollination; C: Comparison of wild-type and mutant grains on the same ear 15 days after pollination; D: Comparison of wild-type and mutant grains 20 days after pollination; E: Comparison of longitudinal sections of wild-type and mutant grains; F: Comparison of phenotypes of grain length, grain width, and grain thickness of wild-type and mutant grains; G: Difference in 100-grain weight between wild-type and mutant grains; H: Statistical difference in grain length, grain width, and grain thickness between wild-type and mutant grains; I: Difference in germination between wild-type and mutant grains; J: Representative plants of wild-type and mutant at the adult stage; K: Difference in germination rate between wild-type and mutant grains; L: Difference in starch content between wild-type and mutant grains.

[0021] Figure 2 Cytological analysis of wild-type and mutant grains in one embodiment of this application; in the figure, the first row shows a comparison of paraffin sections of mutant and wild-type grains 8 - 15 days after pollination; the second row shows a comparison of embryos of mutant and wild-type grains; the third row shows a comparison of the basal transfer layer of mutant and wild-type endosperms.

[0022] Figure 3 In one embodiment of this application ZmRRP45B Map-based cloning, gene structure, and function verification of the gene; in the figure, A: ZmRRP45B Map-based cloning of the gene; B: ZmRRP45B Schematic diagram of the gene structure; C: ZmRRP45B Schematic diagram of the protein structure; D: ZmRRP45B Functional complementation verification of

[0023] Figure 4 In one embodiment of this application, the molecular marker maps of dCAPS1-F1 / R1 and dCAPS1-F2 / R2 and their digested products, and the verification map of the mutation site in the natural population, A: Electrophoresis bands of the dCAPS1-F2 / R2 PCR product digested by the restriction endonuclease NdeI in a 10% nucleic acid PAGE gel after digestion, B: 178 natural populations atZmRRP45B ( rrp45b ) Verification of the locus chr5.s_175415208 on the gene.

[0024] Figure 5 This is the result of the association analysis of grain traits in an embodiment of the present application; in the figure, the upper part is ZmRRP45B Manhattan plot of the association analysis between SNPs and grain traits; the middle part is ZmRRP45B Schematic diagram of the gene structure; the lower part is the LD block diagram of SNPs; 100KW represents the 100-kernel weight trait, KW represents the grain width trait, KNPE represents the kernel number per ear trait, Oil represents the grain oil content trait, PC represents the grain protein content trait, and SC represents the grain starch content trait.

[0025] Figure 6 This is the molecular marker for haplotype utilization in an embodiment of the present application; in the figure, A: Agarose gel electrophoresis bands of the PCR product of dCAPS2-F1R1 (chr5.s_175413829); B: Agarose gel electrophoresis bands of the PCR product of dCAPS2-F2R2; C: Electrophoresis bands of the PCR product of dCAPS2-F2R2 after digestion with Sac I enzyme on a 10% nucleic acid PAGE gel; D: Agarose gel electrophoresis bands of the PCR product of dCAPS3-F1R1 (chr5.s_175418484); E: Agarose gel electrophoresis bands of the PCR product of dCAPS3-F2R2; F: Electrophoresis bands of the PCR product of dCAPS3-F2R2 after digestion with Spe I enzyme on a 10% nucleic acid PAGE gel; Note: Lanes 1 (CF3), 2 (B110), and 3 (CIMBL50) are Hap1; Lanes 4 (GEMS50), 5 (CHUAN48-2), and 6 (BY809) are Hap7; Lanes 7 (CIMBL39), 8 (SY1077), and 9 (CIMBL11) are Hap5 / 6; Lanes 10 (CIMBL70), 11 (CIMBL99), and 12 (CML50) are Hap3 / 4. Detailed implementation methods

[0026] In the following examples, the instruments and equipment involved are all conventional instruments and equipment unless otherwise specified; the reagents and materials involved are all commercially available conventional products unless otherwise specified; the experimental and detection methods involved (such as extraction of genomic DNA, extraction and reverse transcription of RNA, RT-PCR, real-time fluorescence quantitative PCR, BN-PAGE gel electrophoresis, Westen blot, maize grain paraffin section, etc.) are all conventional methods unless otherwise specified.

[0027] Example 1: l168 Phenotypic analysis of mutants l168 The mutant (Tang Jihua, collected from the Yuanyang Science and Education Park of Henan Agricultural University in 2020) is a grain mutant discovered during the process of selecting lines for field breeding. Observation of mature wild-type and mutant grains on the same ear showed that, compared with the wild-type, l168 the mutant grains were smaller, the endosperm was not fully filled and slightly sunken at the top, resulting in a significant decrease in the 100-grain weight of the mutant (75.75% lower than that of the wild-type) (see Figure 1 A-H); most mutant grains could not germinate normally, and the growth of the mutant seedlings was weak after emergence (see Figure 1 I-J), and its germination rate (24.8%) was lower than that of the wild-type (91.7%) (see Figure 1 K), and the starch content of the mutant decreased by 8.98% under the same dry weight (see Figure 1 L).

[0028] Paraffin sections of mutant grains and wild-type grains at 8-15 days after pollination (8-15 DAP) were observed, and it was found that the embryo development of the mutant was delayed and the proliferation in the cells of the endosperm basal transfer layer decreased (see l168 ). Figure 2 ).

[0029] Example 2: ZmRRP45B Map-based cloning and functional verification of genes Using l168 the F2 segregation population derived from the cross with the elite inbred line Zheng 58 as the material, through genetic analysis and map-based cloning, the target gene was gradually mapped to a physical interval of about 500 kb on chromosome 5 of maize (see Figure 3 A).

[0030] The linkage markers and their sequences used in the map-based cloning process are shown in Table 1. Gene annotation and sequencing analysis within the candidate region showed that Zm00001d016770 the gene (the CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) is a potential candidate gene, which contains 9 exons. In the mutant l168 there Zm00001d016770 was a deletion of a G at +85 bp in the 7th exon of the gene, resulting in a mutation of the 302nd amino acid encoded by it from valine to a stop codon (see Figure 3 B).

[0031] Table 1 ZmRRP45B Primers and sequences used for cloning .

[0032] Zm00001d016770The method for cloning the gene is as follows: ① Using a plant polysaccharide-polyphenol RNA extraction kit, extract the RNA of the mutant and wild-type endosperm 10 days after pollination according to the kit instructions. Dissolve the total RNA thoroughly with RNase-free double-distilled water and remove possible DNA residues with DNaseI; ② Initially detect the RNA quality by agarose gel electrophoresis, and use a NanoDrop instrument to detect the RNA concentration and the optical absorption values of the RNA at 260 nm and 280 nm. Determine the integrity and purity of the RNA according to whether OD 260 / 280 is between 1.8 and 2.2 (28S:18S>1.0); ③ Use the obtained RNA as a template for reverse transcription, and store the obtained cDNA in aliquots at -20 °C in a refrigerator for later use; ④ According to the Zm00001d016770 transcript sequence in the maize B73 reference genome, design amplification primers: F: 5’-ATGGAGCACCTCCGGTG-3’ (SEQ ID No.3); R: 5’-CTAACTCCTGTCACTTTTCTTCTT-3’ (SEQ ID No.4).

[0033] To verify the candidate gene and further clarify its biological function, an overexpression vector of Zm00001d016770 was constructed, transformed into maize immature embryos, and transgenic T0 seeds were obtained. Hybridize the overexpression positive single plants with + / rrp45b , perform genotype identification on the F1 ears obtained from the hybridization, and obtain the grains that are overexpression positive and + / rrp45b for self-crossing to obtain F2. Perform phenotypic observation and genotype identification on the segregating ears of F2. When the l168 mutation site is rrp45b / rrp45b , the transgenic positive single plants show the normal grain phenotype, while the transgenic negative single plants show the mutant grain phenotype. This indicates that Zm00001d016770 can complement the l168 grain mutant phenotype, is the functional gene responsible for the l168 grain mutant phenotype, and is named ZmRRP45B .

[0034] ZmRRP45B The transgenic detection primers for are as follows: F: 5’- GCCCTGCCTTCATACGCTA-3’ (SEQ ID No.9);

[0035] Example 3: Development of molecular markers for the ZmRRP45B mutation site in maize Develop an easily detectable PCR molecular marker dCAPS1 for single-base mutations in ZmRRP45B as follows: l168 The actual position of the mutation site in the fourth version of the maize B73 reference genome is 175,415,208 on chromosome 5. Design the following dCAPS primers: dCAPS1-F1: 5’-AGGAAGCCACTATCACTATGGTTG-3’ (SEQ ID No.5); dCAPS1-R1: 5’-AACAGTACCCGGCAAATCAG- 3’ (SEQ ID No.6); dCAPS1-F2: 5’ -CCTTTTTATACTTTGCTGGTTCT -3’ (SEQ ID No.7); dCAPS1-R2: 5’ -CCTCAGAAAATTAATGTCACATAT -3’ (SEQ ID No.8).

[0036] Among them, dCAPS1-F1R1 is the outer primer, specifically amplifying the ZmRRP45B gene sequence; dCAPS1-F2R2 is the inner primer, and the PCR product diluted 10 times by dCAPS1-F1R1 is used as the template for the second round of amplification.

[0037] For the molecular marker detection of l168 the mutation site, it includes the following steps: (1) The reaction system is: DNA template (30 - 50 ng / μL) 1 μL; Left primer (10 μM) 0.5 μL; Right primer (10μM) 0.5 μL; ddH2O 2 μL; 2×PCR Mix 5 μL; (2) The PCR amplification program of dCAPS1-F1R1 is: 1 cycle 95℃ pre-denaturation for 3 min; 35 cycles 95℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 30 s; Delay 72℃ for 5 min; Soak at 25℃; (3) The PCR amplification program of dCAPS1-F2R2 is: 1 cycle 95℃ pre-denaturation for 3 min; 35 cycles 95℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 15 s; Delay 72℃ for 5 min; Soak at 25℃.

[0038] The PCR product of dCAPS1-F2R2 is diluted 10 times and digested with restriction endonucleaseNde Ⅰ digestion, and then detected by 10% nucleic acid PAGE gel. After digestion, the wild-type material showed smaller bands (see Figure 4 D).

[0039] Example 4: ZmRRP45B Candidate gene association analysis To clarify ZmRRP45B the utilization pathway of the p gene, 129 SNPs (single nucleotide polymorphism sites) (B73V4: Chr5: 175412532 - 175422968) of this gene in 471 inbred lines were used as genotypes, and candidate gene association analysis was performed based on the grain phenotypes, population structure (Q) and kinship (K) of this population. Taking p <7.75E-03 (1 / 129) as the threshold, a total of 45 SNPs significantly associated with grain width, grain thickness, 100-grain weight, kernel number per row, grain starch, grain protein and grain oil content were detected (see Table 2).

[0040] Table 2 ZmRRP45B Results of candidate gene association analysis .

[0041] Example 5: ZmRRP45B Identification of excellent haplotypes of Among the 45 significantly associated SNPs, chr5.s_175413975 in the 5'UTR of the ZmRRP45B gene, chr5.s_175413829 and chr5.s_175418484 in the 3'UTR are the 3 SNPs most significantly associated with grain traits (p < 4.41E-03, see Figure 5 ). Based on these 3 SNPs, haplotype analysis was performed. There are 7 combinations (Hap1 AAA, Hap2 TAA, Hap3 ACA, Hap4 TCA, Hap5 AAG, Hap6 TAG, Hap7 ACG) in 471 inbred lines, and the differences in significantly associated traits all reach an extremely significant level. Taking grain starch content as an example, the mean grain starch contents of Hap2 and Hap3, Hap5 and Hap6 are close, and they are calculated as one type of haplotype respectively ( p= 1.49E-08) (see Table 2): Hap1 (4 copies), Hap2 / 3 (195 copies), Hap4 (2 copies), Hap5 / 6 (69 copies), Hap7 (146 copies). Among them, the average starch content of the excellent haplotype Hap1 with excellent starch content is 66.567%, and its distribution frequency in inbred lines is relatively low (2 / 471), belonging to a rare haplotype and having potential for utilization in breeding improvement; the average starch content of the unfavorable haplotype Hap4 is 62.338%, and its distribution frequency in inbred lines is extremely low (2 / 471), belonging to a rare haplotype; most inbred lines are haplotypes with a relatively high distribution frequency after selection, and their average starch content is between 65% and 66% (see Table 3).

[0042] Table 3 Haplotype analysis 。

[0043] Example 6: ZmRRP45B Development of molecular markers for excellent haplotypes Based on the clear excellent haplotypes of starch content, two simple and easy-to-operate PCR markers, dCAPS2 and dCAPS3, were developed for the two SNPs of chr5.s_175413829 and chr5.s_175418484.

[0044] dCAPS primers: dCAPS2-F1: 5’ -CCGTGTCGTAAATGGGCCTT-3’ (SEQ ID No.11); dCAPS2-R1: 5’ -TTTGTATGCCCCAGGTGAGT-3’ (SEQ ID No.12); dCAPS2-F2: 5’ -ATGTATGGCACCCCGGATGAGAGCT-3’ (SEQ ID No.13); dCAPS2-R2: 5’ -GATTGCGCATGCTACAACTGTATG-3’ (SEQ ID No.14); dCAPS3-F1: 5’ -AACTCGGTGAATATGGCCAGC-3’ (SEQ ID No.15); dCAPS3-R1: 5’ -GCCCAAAAGGTAGACGCAAT-3’ (SEQ ID No.16); dCAPS3-F2: 5’ -TTTCACATCCAAACCGGAAAAACTA-3’ (SEQ ID No.17); dCAPS3-R2: 5’ -GCTTCACTCTTGCATATTTCGAAC-3’ (SEQ ID No.18).

[0045] Among them, dCAPS2-F1R1 and dCAPS3-F1R1 are outer primer pairs, which can specifically amplify genomic sequences containing two SNPs (see Figure 6 A and Figure 6 D); dCAPS2-F2R2 and dCAPS3-F2R2 are inner primer pairs for chr5.s_175413829 and chr5.s_175418484 respectively. The PCR products of dCAPS2-F1R1 and dCAPS3-F1R1 are diluted 10 times as templates for the second-round amplification.

[0046] For the molecular marker detection of excellent haplotypes, it includes the following steps: (1) Reaction system: 1 μL of DNA template (30 - 50 ng / μL); 0.5 μL of Left primer (10 μM); 0.5 μL of Right primer (10 μM); 2 μL of ddH2O; 5 μL of 2×PCR Mix; (2) The PCR amplification program of dCAPS2-F1R1 and dCAPS3-F1R1 is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s; Delay at 72°C for 5 min; Soak at 25°C; (3) The PCR amplification program of dCAPS2-F2R2 is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 15 s; Delay at 72°C for 5 min; Soak at 25°C; (4) The PCR amplification program of dCAPS3-F2R2 is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 15 s; Delay at 72°C for 5 min; Soak at 25°C.

[0047] The PCR products of dCAPS2-F2R2 and dCAPS2-F3R3 are digested with restriction enzymes Sac I and Spe I respectively, and then detected by 10% acrylamide gel electrophoresis. Hap1 and Hap2 are two higher bands; both markers of Hap7 are lower bands; Hap3 and Hap4 are lower bands after digestion with Sac I, Spe I are higher bands after digestion, and Hap5 and Hap6 are in SacAfter digestion with Enzyme I, it shows a higher band. Spe After digestion with Enzyme I, it shows a lower band (see Figure 6 ).

[0048] Although some preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present disclosure without departing from the spirit and scope of its inventive concept. Thus, if these modifications and variations to the present disclosure fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A kind of corn ZmRRP45B The gene mutation SNP marker, based on the fourth version of the maize B73 reference genome, has a deletion of a G at the position chr5.s_175415208, resulting in smaller mutant kernels, incomplete endosperm filling, collapse at the top, and reduced 100-kernel weight.

2. An identification ZmRRP45B dCAPS primer for gene mutation, including an outer primer pair shown in SEQ ID NO.5 and SEQ ID NO.6; and / or an inner primer pair shown in SEQ ID NO.7 and SEQ ID NO.

8.

3. The corn according to claim 1 ZmRRP45B Use of the gene mutation SNP marker or the dCAPS primer according to claim 2 in at least one of the following (1) to (9): (1) Regulating the maize grain size trait or preparing a reagent for regulating the maize grain size trait; (2) Regulating the maize embryo development trait or preparing a reagent for regulating the maize embryo development trait; (3) Regulating the maize endosperm development trait or preparing a reagent for regulating the maize endosperm development trait; (4) Regulating the maize grain yield trait or preparing a reagent for regulating the maize grain yield trait; (5) Regulating the maize grain quality trait or preparing a reagent for regulating the maize grain quality trait; (6) Selecting inbred lines with maize grain size traits; (7) Selecting inbred lines with maize embryo or endosperm development traits; (8) Selecting inbred lines with maize grain yield traits or maize grain quality traits; (9) Identification ZmRRP45B Identification of gene mutant type or preparation ZmRRP45B Reagent for gene mutant type The maize grain yield traits include at least one of grain width, grain thickness, 100-grain weight, and number of grains per row; the maize grain quality traits include at least one of grain starch content, grain protein content, and grain oil content.

Images