SNP markers, dCAPS primers significantly associated with maize kernel type and storage material content and application thereof

Abstract

The present application relates to a kind of corn kernel type and the SNP marker of significant correlation of storage material content, dCAPS primer and its application.The present application research finds that, located ZmRRP45B The chr5.s_175413975 of 5'UTR and the chr5.s_175413829 and chr5.s_175418484 of 3'UTR of gene are the 3 most significant SNPs associated with kernel traits;The corresponding molecular marker is developed from this, and the identification of excellent haplotype, the creation of corn high yield or high quality breeding material can realize ZmRRP45B Excellent haplotype identification, corn high yield or high quality breeding material creation, and then shorten the breeding cycle of new crop varieties, reduce the breeding cost, improve the breeding efficiency.

Description

[0001] The application is a divisional application of patent application No. 2023112022960, filed on September 18, 2023, and entitled “Maize kernel development regulation gene ZmRRP45B , its encoded protein, SNP marker, functional marker and application”. TECHNICAL FIELD

[0002] The application relates to the technical field of molecular genetic breeding, in particular to a SNP marker significantly associated with maize kernel type and storage material content, a dCAPS primer and application thereof. BACKGROUND

[0003] Maize (Zea mays L. is an important food and feed crop, and plays an irreplaceable role in ensuring national food security and economic development. The kernel is the main organ of maize yield source, and its formation and development mechanism has attracted widespread attention. At present, although a large number of genes regulating maize kernel development and filling have been cloned, due to the extremely complex biological process of maize embryo and endosperm development, the node genes and molecular mechanisms for accurately regulating kernel development and filling still need to be further analyzed.

[0004] RNA decay plays an important role in maintaining the dynamic balance of biological life activities and quality control of gene expression. The biological evolution process has formed a precise RNA monitoring mechanism, which eliminates abnormal transcripts through degradation to maintain the normal development of the organism. RNA degradation mechanism can also be involved in the degradation process of normal mRNA, regulating the abundance of gene expression and maintaining the dynamic balance of transcripts. RNA degradation requires the action of 5'→3' or 3'→5' exonuclease, and 3'→5' RNA degradation mainly relies on EXOSOME to complete, which plays an important role in RNA maturation and quality control. Studies have shown that defects in EXOSOME-mediated RNA degradation processes lead to various physiological and reproductive defects in animals and plants. However, the mechanism of plant RNA EXOSOME in regulating phenotypes still needs further research.

[0005] The information disclosed in this section of the background art is only intended to deepen the understanding of the background art of the present disclosure, and should not be regarded as admitting or implying in any form that this information constitutes prior art known to those skilled in the art. SUMMARY

[0006] The application utilizes a natural grain mutant l168 discovered in the process of maize field breeding selection lines, clones a key gene affecting RNA degradation ZmRRP45B , which encodes a core subunit of the RNA EXOSOME complex; further research shows that,ZmRRP45B The gene regulates the development of maize embryo and endosperm, affects the accumulation of grain storage material, and can provide new gene resources for breeding high-yield and high-quality maize varieties.

[0007] In a first aspect of the present disclosure, it is found through phenotype and cytology analysis that l168 The 100-grain weight of the mutant grain is reduced by 75.75% compared with the wild type, the development of embryo and endosperm is abnormal, and the hyperplasia in the basal transfer layer cells of endosperm is reduced; and a gene controlling the grain mutation is obtained through genetic analysis and map-based cloning method. ZmRRP45B The gene has a CDS sequence as shown in SEQ ID NO. 1.

[0008] In a second aspect of the present disclosure, it relates to a coding protein of a grain development regulating gene ZmRRP45B , and the amino acid sequence of the coding protein is as shown in SEQ ID NO. 2.

[0009] In a third aspect of the present disclosure, it relates to a primer pair for amplifying the ZmRRP45B gene, and the sequences of the primer pair are as shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0010] In a fourth aspect of the present disclosure, it relates to a maize grain phenotype mutant SNP marker, and there is a G deletion at the position of chr5.s_175415208 based on the fourth version of maize B73 reference genome.

[0011] In a fifth aspect of the present disclosure, it relates to a dCAPS primer for identifying a phenotype mutant, which includes a primer pair as shown in SEQ ID NO. 5 and SEQ ID NO. 6; and / or a primer pair as shown in SEQ ID NO. 7 and SEQ ID NO. 8.

[0012] In a sixth aspect of the present disclosure, it relates to a primer for identifying ZmRRP45B overexpression material, which includes a primer pair as shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0013] In still another aspect of the present disclosure, the maize grain development regulating gene ZmRRP45B , the amplification primer, the maize grain phenotype mutant SNP marker or the dCAPS primer is applied in the regulation of grain size or / and maize embryo and endosperm development traits.

[0014] In another aspect of the present disclosure, it relates to a SNP marker significantly associated with maize kernel type and storage material content, and there are A / T, A / C, G / A mutations at the positions of chr5.s_175413975, chr5.s_175413829, chr5.s_175418484 based on the fourth version of maize B73 reference genome.

[0015] Yet another aspect of the present disclosure relates to a method for identifying a gene ZmRRP45B The dCAPS primers of the superior haplotype include primer pairs as shown in SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and SEQ ID NO. 15 and SEQ ID NO. 16, and SEQ ID NO. 17 and SEQ ID NO. 18.

[0016] Still another aspect of the present disclosure, the maize kernel development regulatory gene ZmRRP45B The SNP marker or the dCAPS primer is applied in the inbred line selection of maize kernel yield and quality traits.

[0017] Still another aspect of the present disclosure, a method for identifying a gene ZmRRP45B The method for identifying a superior haplotype comprises the following steps:

[0018] The DNA of the extracted material to be identified is used as a template, and the PCR products containing the chr5.s_175413829 and chr5.s_175418484 SNP sites are amplified by the outer primer pairs respectively; the second round of amplification is performed by using the inner primer pairs and the diluted PCR products as templates, and the second round of PCR products are digested by restriction enzymes Sac I and Spe I, and gel electrophoresis detection is performed, the AAA and TAA haplotypes are two higher bands; the two markers of the ACG and TCG haplotypes are lower bands; the ACA and TCA haplotypes are lower bands after Sac I digestion, Spe I digestion, the AAG and TAG haplotypes are higher bands after Sac I digestion, Spe I digestion; the AAA and TAA haplotypes are selected as the breeding improvement materials of starch content.

[0019] The one or more technical solutions provided in the embodiments of the present disclosure have at least any of the following technical effects or advantages:

[0020] 1. The ZmRRP45B Gene regulating the development of maize embryo and endosperm affects the accumulation of grain storage substances, and has important application value for improving maize kernel yield and quality.

[0021] 2. ZmRRP45B The identification of the superior haplotype and the analysis of the utilization approach provide technical support and excellent germplasm resources for the utilization of the gene. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 As one embodiment of this disclosure l168 Phenotypic analysis of mutant grains; In the figure, A: Phenotypic of grains on mature segregating ears; 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: Phenotypic comparison of grain length, width, and thickness between wild-type and mutant grains; G: Difference in 100-grain weight between wild-type and mutant grains; H: Statistical differences in grain length, width, and thickness between wild-type and mutant grains; I: Difference in germination between wild-type and mutant grains; J: Representative plants at the mature stage of wild-type and mutant grains; K: Difference in germination rate between wild-type and mutant grains; L: Difference in starch content between wild-type and mutant grains.

[0023] Figure 2 This is a cytological analysis of wild-type and mutant seeds in one embodiment of this disclosure; in the figure, the first row is a comparison of paraffin sections of mutant and wild-type seeds 8-15 days after pollination; the second row is a comparison of the embryos of mutant and wild-type seeds; the third row is a comparison of the endosperm basal transfer layer of mutant and wild-type seeds.

[0024] Figure 3 As one embodiment of this disclosure ZmRRP45B Map-based cloning of genes, gene structure and function verification; in the diagram, A: ZmRRP45B Map-based cloning of genes; B: ZmRRP45B Schematic diagram of gene structure; C: ZmRRP45B Protein structure diagram; D: ZmRRP45B Functional verification and feedback.

[0025] Figure 4 This is a molecular marker map of dCAPS1-F1 / R1 and dCAPS1-F2 / R2 and their enzyme digestion, as well as a natural population validation map of the mutation sites, in one embodiment of this disclosure. A: dCAPS1-F2 / R2 PCR product in restriction endonuclease... NdeI Electrophoretic bands in a 10% nucleic acid PAGE gel after enzyme digestion, B: 178 natural populations in ZmRRP45B ( rrp45b ) Validation of the chr5.s_175415208 site on the gene.

[0026] Figure 5 This is the result of a correlation analysis of grain traits in one embodiment of this disclosure; in the figure, the upper part is... ZmRRP45B Manhattan plot of association analysis between SNPs and grain traits; the middle one is... ZmRRP45BA schematic diagram of the gene structure; below is the LD block diagram of the SNP; 100KW represents the 100-grain weight trait, KW represents the grain width trait, KNPE represents the number of grains 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.

[0027] Figure 6 In one embodiment of this disclosure, haplotypes are utilized using molecular markers; in the figure, A: agarose gel electrophoresis band of dCAPS2-F1R1 (chr5.s_175413829) PCR product; B: agarose gel electrophoresis band of dCAPS2-F2R2 PCR product; C: agarose gel electrophoresis band of dCAPS2-F2R2 PCR product. Sac After I enzyme digestion, the bands were observed on a 10% nucleic acid PAGE gel; D: agarose gel electrophoresis band of dCAPS3-F1R1 (chr5.s_175418484) PCR product; E: agarose gel electrophoresis band of dCAPS3-F2R2 PCR product; F: agarose gel electrophoresis band of dCAPS3-F2R2 PCR product. Spe After I enzyme digestion, the bands were observed in a 10% nucleic acid PAGE gel via electrophoresis. 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

[0028] Unless otherwise specified, all instruments and equipment used in the following examples are conventional instruments and equipment; all reagents and materials used are commercially available conventional products; and all experimental and detection methods used (such as genomic DNA extraction, RNA extraction and reverse transcription, RT-PCR, real-time quantitative PCR, BN-PAGE gel electrophoresis, Western blot, corn kernel paraffin sections, etc.) are conventional methods unless otherwise specified.

[0029] Example 1: l168 Phenotypic analysis of mutants

[0030] l168 The mutant (collected by Tang Jihua in June 2020 from the Yuanyang Science and Education Park of Henan Agricultural University) was a grain mutant discovered during field breeding and selection. Observation of mature wild-type and mutant grains on the same ear revealed that, compared to the wild-type, l168The mutant grains are smaller, the endosperm is not fully filled, and the top is slightly collapsed, resulting in a significant reduction in the 100-grain weight of the mutant (75.75% lower than the wild type) (see...). Figure 1 AH); most mutant grains failed to germinate normally, and mutant seedlings showed weak growth after emergence (see...). Figure 1 IJ), its germination rate (24.8%) was lower than that of the wild type (91.7%) (see...). Figure 1 K), the starch content of the mutant was reduced by 8.98% at the same dry weight (see K). Figure 1 L).

[0031] For 8–15 days after pollination (8–15 DAP) l168 Paraffin sections of mutant and wild-type seeds were observed, revealing delayed embryonic development and reduced intracellular proliferation in the basal transfer layer of the endosperm in the mutant (see...). Figure 2 ).

[0032] Example 2: ZmRRP45B Map-based cloning and functional verification of genes

[0033] use l168 Using the F2 segregating population derived from the inbred line Zheng 58 as material, the target gene was progressively mapped to a physical region of approximately 500 kb on chromosome 5 of maize through genetic analysis and map-based cloning (see [link to original text]). Figure 3 A).

[0034] 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... Zm00001d016770 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 9 exons, and in the mutant... l168 middle Zm00001d016770 The gene has a G deletion at position 85bp in exon 7, causing the 302nd amino acid it encodes to change from valine to a stop codon (see...). Figure 3 B).

[0035] Table 1 ZmRRP45B Primers and sequences used for cloning

[0036]

[0037] Zm00001d016770The gene cloning method is as follows: ① Using a plant polysaccharide-polyphenol RNA extraction kit, RNA was extracted from the endosperm of mutants and wild-type individuals 10 days after pollination, according to the kit instructions. Total RNA was thoroughly dissolved in RNase-free double-distilled water, and possible DNA residues were removed using DNase I; ② RNA quality was initially assessed using agarose gel electrophoresis, and RNA concentration and absorbance at 260 nm and 280 nm were detected using a NanoDrop instrument. Based on OD... 260 / 280 ① Determine the integrity and purity of RNA by checking if the saturation is between 1.8 and 2.2 (28S:18S>1.0); ② Use the obtained RNA as a template for reverse transcription, and aliquot the resulting cDNA and store it at -20℃ for later use; ③ Based on the maize B73 reference genome... Zm00001d016770 Transcript sequence, design amplification primers:

[0038] F: 5'-ATGGAGCACCTCCGGTG-3' (SEQ ID No. 3);

[0039] R: 5'-CTAACTCCTGTCACTTTTCTCTT-3' (SEQ ID No. 4).

[0040] To validate this candidate gene and further clarify its biological function, a [system / mechanism] was constructed. Zm00001d016770 The overexpression vector was used to transform maize immature embryos and transgenic contemporary seeds were obtained. Overexpression-positive plants were then compared with + / rrp45b Hybridization was performed, and the F1 ears obtained from the hybridization were genotyped to obtain those that were overexpressing positive and + / rrp45b The grains were used for self-pollination to obtain F2, and the phenotypic characteristics and genotypic identification of the segregating ears of F2 were performed. l168 The mutation site is rrp45b / rrp45b At that time, transgenic positive plants exhibited the normal grain phenotype, while transgenic negative plants exhibited the mutant grain phenotype. This indicates that... Zm00001d016770 Can be replenished l168 The grain mutant phenotype is the result of l168 The functional genes of the grain mutant phenotype were identified and named. ZmRRP45B .

[0041] ZmRRP45B The primers for detecting genetically modified organisms are as follows:

[0042] F: 5'- GCCCTGCCTTCATACGCTA-3' (SEQ ID No. 9);

[0043] R: 5'-TAGGGCTTTCTCGGTGGTGTAT-3' (SEQ ID No. 10).

[0044] Example Three: Maize ZmRRP45B Molecular marker development for mutation site

[0045] For ZmRRP45B A PCR molecular marker dCAPS1 was developed for the single base mutation, as follows:

[0046] l168 The actual position of the mutation site in the fourth version of the maize B73 reference genome is 175,415,208 of chromosome 5, and the following dCAPS primers are designed:

[0047] dCAPS1-F1: 5'-AGGAAGCCACTATCACTATGGTTG-3' (SEQ ID No. 5);

[0048] dCAPS1-R1: 5'-AACAGTACCCGGCAAATCAG-3' (SEQ ID No. 6);

[0049] dCAPS1-F2: 5'-CCTTTTTATACTTTGCTGGTTCT-3' (SEQ ID No. 7);

[0050] dCAPS1-R2: 5'-CCTCAGAAAATTAATGTCACATAT-3' (SEQ ID No. 8).

[0051] Among them, dCAPS1-F1R1 is an outer primer pair, which can specifically amplify ZmRRP45B the sequence of the gene; dCAPS1-F2R2 is an inner primer pair, and the second round of amplification is carried out with the PCR product diluted by 10 times of dCAPS1-F1R1 as a template.

[0052] For l168The molecular marker detection of mutation sites includes the following steps: (1) The reaction system is: DNA template (30-50 ng / μL) 1μL; Left primer (10μM) 0.5μL; Right primer (10μM) 0.5μL; ddH2O 2 μL; 2×PCR Mix 5 μL; (2) The PCR amplification program of dCAPS1-F1R1 is: 1 cycle 95℃ pre-denaturation 3 min; 35 cycles 95℃ denaturation 15s, 58℃ annealing 20s, 72℃ extension 30s; Delay 72℃ 5min; Soak 25℃; (3) The PCR amplification program of dCAPS1-F2R2 is: 1 cycle 95℃ pre-denaturation 3 min; 35 cycles 95℃ denaturation 15 s, 58℃ annealing 20 s, 72℃ extension 15s; Delay 72℃ 5 min; Soak 25℃.

[0053] The PCR product of dCAPS1-F2R2 was diluted 10-fold and subjected to restriction endonuclease. Nde I. Enzyme digestion, followed by detection with a 10% nucleic acid PAGE gel. Wild-type material showed smaller bands after enzyme digestion (see...). Figure 4 ).

[0054] Example 4: ZmRRP45B Candidate gene association analysis

[0055] To clarify ZmRRP45B The gene utilization pathway involved using 129 SNPs (single nucleotide polymorphism sites) (B73V4:Chr5:175412532-175422968) of this gene in 471 inbred lines as genotypes, and conducting candidate gene association analysis based on the grain phenotype, population structure (Q), and phylogenetic relationships (K) of the population. p With a threshold of <7.75E-03 (1 / 129), a total of 45 SNPs were detected that were significantly associated with grain width, grain thickness, 100-grain weight, number of grains per row, grain starch, grain protein, and grain oil content (see Table 2).

[0056] Table 2 ZmRRP45B Candidate gene association analysis results

[0057]

[0058] Example 5: ZmRRP45B Excellent haplotype identification

[0059] Of the 45 significantly associated SNPs, located in ZmRRP45BThe chr5.s_175413975 of 5' UTR and the chr5.s_175413829 and chr5.s_175418484 of 3' UTR of the gene 5 are the three SNPs most significantly associated with the grain traits (p < 4.41E-03, see Figure 5 ). Based on the three SNPs, haplotype analysis was carried out, and there were seven combinations (Hap1 AAA, Hap2 TAA, Hap3 ACA, Hap4 TCA, Hap5 AAG, Hap6 TAG, and Hap7 ACG) in 471 inbred lines, and the differences in the associated significant traits reached a significant level. Taking the starch content of the grain as an example, the average starch content of the grains of Hap2 and Hap3 and Hap5 and Hap6 was close, and each was calculated according to one type of haplotype p = 1.49E-08) (see Table 2): Hap1 (4), Hap2 / 3 (195), Hap4 (2), Hap5 / 6 (69), and Hap7 (146). Among them, the average starch content of the excellent haplotype Hap1 was 66.567%, and the distribution frequency in the inbred lines was low (2 / 471), which belonged to a rare haplotype and had potential for use in breeding improvement; the average starch content of the adverse haplotype Hap4 was 62.338%, and the distribution frequency in the inbred lines was extremely low (2 / 471), which belonged to a rare haplotype; most of the inbred lines were the haplotype with a higher distribution frequency selected, and the average starch content thereof was between 65 and 66% (see Table 3).

[0060] Table 3 Haplotype analysis

[0061]

[0062] Example Six: ZmRRP45B Development of molecular markers of excellent haplotype

[0063] Based on the excellent haplotype of starch content, simple and easy-to-operate PCR markers dCAPS2 and dCAPS3 were developed for the two SNPs of chr5.s_175413829 and chr5.s_175418484.

[0064] dCAPS primers:

[0065] dCAPS2-F1: 5' -CCGTGTCGTAAATGGGCCTT-3' (SEQ ID No. 11);

[0066] dCAPS2-R1: 5' -TTTGTATGCCCCAGGTGAGT-3' (SEQ ID No. 12);

[0067] dCAPS2-F2: 5'-ATGTATGGCACCCCGGATGAGAGCT-3' (SEQ ID No. 13);

[0068] dCAPS2-R2: 5'-GATTGCGCATGCTACAACTGTATG-3' (SEQ ID No. 14);

[0069] dCAPS3-F1: 5'-AACTCGGTGAATATGGCCAGC-3' (SEQ ID No. 15);

[0070] dCAPS3-R1: 5'-GCCCAAAAGGTAGACGCAAT-3' (SEQ ID No. 16);

[0071] dCAPS3-F2: 5'-TTTCACATCCAAACCGGAAAAACTA-3' (SEQ ID No. 17);

[0072] dCAPS3-R2: 5'-GCTTCACTCTTGCATATTTCGAAC-3' (SEQ ID No. 18).

[0073] wherein, dCAPS2-F1R1 and dCAPS3-F1R1 are outer primer pairs, which can specifically amplify the genomic sequences containing two SNPs (see Figure 6 A and Figure 6 D); dCAPS2-F2R2 and dCAPS3-F2R2 are inner primer pairs of chr5.s_175413829 and chr5.s_175418484, respectively, and the PCR product of dCAPS2-F1R1 and dCAPS3-F1R1 is diluted 10 times as a template for the second round of amplification.

[0074] For the molecular marker detection of the excellent haplotype, the following steps are included:

[0075] (1) The reaction system is: DNA template (30-50 ng / μL) 1 μL; Left primer (10 μM) 0.5 μL; Right primer (10 μM) 0.5 μL; ddH2O 2 μL; 2x PCR Mix 5 μL.

[0076] (2) The PCR amplification procedure of dCAPS2-F1R1 and dCAPS3-F1R1 is as follows: 1 cycle 95°C pre-denaturation 3 min; 35 cycles 95°C denaturation 15 s, 58°C annealing 20 s, 72°C extension 30 s; Delay 72°C 5 min; Soak 25°C.

[0077] (3) The PCR amplification procedure of dCAPS2-F2R2 is as follows: 1 cycle 95°C pre-denaturation 3 min; 35 cycles 95°C denaturation 15 s, 58°C annealing 20 s, 72°C extension 15 s; Delay 72°C 5 min; Soak 25°C.

[0078] (4) The PCR amplification procedure of dCAPS3-F2R2 is as follows:

[0079] 1 cycle 95°C pre-denaturation 3 min; 35 cycles 95°C denaturation 15 s, 58°C annealing 20 s, 72°C extension 15 s; Delay 72°C 5 min; Soak 25°C.

[0080] The PCR products of dCAPS2-F2R2 and dCAPS2-F3R3 were digested by restriction enzymes Sac I and Spe I respectively, and then detected by 10% acrylamide gel electrophoresis. Hap1 and Hap2 are two higher bands; Hap7 is two lower bands; Hap3 and Hap4 are lower bands after Sac I digestion, Spe I digestion are higher bands, Hap5 and Hap6 are higher bands after Sac I digestion, Spe I digestion are lower bands (see Figure 6 ).

[0081] Although some preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0082] Obviously, various modifications and changes can be made to the present disclosure by those skilled in the art without departing from the spirit and scope of the inventive concept. Thus, if these modifications and changes fall within the scope of the claims and their equivalents, they are intended to be included in the present application.

Claims

1. Use of a dCAPS primer in identifying a gene ZmRRP45B application of a superior haplotype or a variety / line selected for the grain starch content trait, characterized in that, The dCAPS primers include: the outer primer pair shown as SEQ ID NO. 11 and SEQ ID NO. 12, the inner primer pair shown as SEQ ID NO. 13 and SEQ ID NO. 14, the outer primer pair shown as SEQ ID NO. 15 and SEQ ID NO. 16, the inner primer pair shown as SEQ ID NO. 17 and SEQ ID NO. 18; The identified gene ZmRRP45B The superior haplotype comprises the following steps: taking the extracted DNA of the material to be identified as a template, respectively taking the outer primer pair specific PCR amplification product containing the SNP sites of chr5.s_175413829 and chr5.s_175418484; then respectively taking the inner primer pair and the PCR product after dilution as a template to carry out the second round of amplification, and respectively using restriction enzymes Sac I and Spe I to cut, and carrying out gel electrophoresis detection, the AAA and TAA haplotypes are two higher bands; the two markers of the ACG and TCG haplotypes are lower bands; the ACA and TCA haplotypes are Sac I lower bands after cutting, Spe I higher bands after cutting, the AAG and TAG haplotypes are Sac I higher bands after cutting, Spe I lower bands after cutting; The breeding of the corn kernel starch content trait variety / line includes the following steps: identifying the genes of the material to be identified based on the method ZmRRP45B Haplotype, selecting AAA and TAA haplotype as starch content breeding improvement material.

2. A method of identifying a gene ZmRRP45B A method of haplotyping comprising the steps of: PCR product containing SNP site of chr5.s_175413829 and chr5.s_175418484 is amplified by PCR with outer primer pairs specific to the SNP site, respectively, using DNA of the extracted material to be identified as template; the PCR product is diluted and used as template for the second round of amplification with inner primer pairs specific to the SNP site, respectively; the second round of PCR product is digested with restriction enzyme Sac I and Spe I, and gel electrophoresis is performed; the AAA and TAA haplotypes are two higher bands; the ACG and TCG haplotypes are two lower bands; the ACA and TCA haplotypes are Sac I, and gel electrophoresis is performed; the AAA and TAA haplotypes are two higher bands; the ACG and TCG haplotypes are two lower bands; the ACA and TCA haplotypes are Spe I, and gel electrophoresis is performed; the AAA and TAA haplotypes are two higher bands; the ACG and TCG haplotypes are two lower bands; the ACA and TCA haplotypes are Sac I, and gel electrophoresis is performed; the AAA and TAA haplotypes are two higher bands; the ACG and TCG haplotypes are two lower bands; the ACA and TCA haplotypes are Spe I, and gel electrophoresis is performed; the AAA and TAA haplotypes are two higher bands; the ACG and TCG haplotypes are two lower bands; the ACA and TCA haplotypes are 3. A method of selecting a maize genotype ZmRRP45B The method of selecting an elite haplotype variety / line is characterized in that, The method of claim 2 identifies a gene of the material to be identified ZmRRP45B Haplotypes, AAA and TAA haplotypes are selected as breeding materials for improving starch content.

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