SNP Loci Related to Maize Husky Width Regulation Gene RHW1 and Their Applications

Abstract

The present invention provides an SNP locus related to the maize bract width regulatory gene RHW1 and its application, which relates to the technical field of plant molecular breeding. The SNP locus includes one or more loci among SNP1 to SNP8, and they form a haplotype marker. The haplotype marker is related to the genotype of the functional locus InDel-4337 related to the maize bract width gene RHW1 and can be used to predict or assist in predicting the maize bract width trait.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular breeding, and particularly relates to an SNP locus related to the maize husk width regulation gene RHW1 and its application. Background Art

[0002] The following statements only provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Maize is the largest food crop in China. With the adjustment of the planting structure, the mechanized harvesting of crops has become an important link in agricultural development. However, the current main maize cultivars in China have disadvantages such as high grain moisture content and slow dehydration rate in the later stage, which greatly restricts the further development of large-scale and mechanized maize planting. The maize husk is a modified leaf transformed from the leaf sheath attached to the ear stalk node. During ear development, the husk has various physiological and protective functions. In particular, a suitable husk morphological structure can directly affect the dehydration efficiency of maize grains at the harvest stage.

[0004] A haplotype marker is a set of markers screened in the region near a gene through genetic linkage analysis and associated with the gene phenotype. The haplotype marker identifies the function of a gene through a set of markers, rather than a small number of one or a few markers, and the function of the gene will not be unable to be identified due to the detection failure of a single marker. Since the detected markers are distributed inside and upstream and downstream of the gene, the problem of inaccurate identification due to recombination occurring near the gene is also avoided.

[0005] The maize genome is approximately 2.3 Gb in size, which is 18 times that of Arabidopsis thaliana and 5 times that of Oryza sativa. Based on de novo sequencing and population resequencing of multiple maize varieties, the haplotype map (Hapmap) of maize has been improved from the first generation to the third generation. Hapmap1 was constructed using the whole-genome sequences of 27 maize inbred lines and contains 3.3 million SNP and InDel data; subsequent Hapmap2 included pre-domestication varieties (Tripsacum dactyloides) and domesticated varieties of maize, and more than 50 million SNPs were identified from 103 materials; Hapmap3 covered 1,218 maize lines or varieties from all over the world. By establishing a new computational pipeline to process more than 12 trillion bp of sequencing data, more than 83 million variant sites were finally determined (Robert Bukowski et al, Construction of the third-generation Zea mays haplotype map. GigaScience, 2047-217X). On the other hand, in 2013, Wang Guoying, Yan Jianbing and others collaborated to obtain approximately 3.6 million SNP markers based on transcriptional sequences from 368 widely representative maize inbred lines using mRNA sequencing strategies (Fu et al, RNA sequencing reveals the complex regulatory network in the maize kernel. Nat Commun, 4:2832). Subsequently, the Yan Jianbing team integrated an association panel consisting of 540 inbred lines, including breeding germplasm resources representing the tropics, subtropics and temperate zones. Among them, 143 inbred lines were from widely used commercial hybrid parents in China, 95 inbred lines were from Chinese local maize varieties, 54 inbred lines were selected from the GEM (Germplasm Enhancement of Maize) project, and 235 inbred lines were from the International Maize and Wheat Improvement Center. Through RNA sequencing and GBS (Genotyping-by-Sequencing) sequencing technologies, a total of 2.65 M SNP markers were obtained (Yang et al, Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in maize. Mol Breeding 28, 511-526). Screening out haplotype markers related to the morphological and structural traits of maize bracts is helpful for maize trait evaluation and breeding work.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide an SNP locus related to the maize husk width regulation gene RHW1 and its application, for predicting or assisting in predicting the genotype of the functional locus InDel-4337 related to the maize husk width gene and measuring the maize husk width trait. RHW1 For solving the above technical problems, the present invention adopts the following technical solutions:

[0008] In this article, unless otherwise specified, all the embodiments and preferred implementation methods mentioned in this article can be combined with each other to form a new technical solution. All the technical features and preferred features mentioned in this article can be combined with each other to form a new technical solution. Each component or its preferred component involved can be combined with each other to form a new technical solution.

[0009] In this article, unless otherwise specified, any numbering is used to distinguish one entity or behavior from another entity or behavior, rather than necessarily requiring or implying any actual such relationship, order or importance between these entities or behaviors. For example, the numbers i, ii...... iv, and SNP1, SNP2...... SNP8.

[0010] In this article, unless otherwise specified, "optionally", "optional", "optional" or "optional" means that the subsequent described event or circumstance can but does not have to occur, and this description includes the occasion where the event or circumstance occurs or does not occur.

[0011] In this article, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.

[0012] In this article, "haplotype marker" refers to a linear combination of multiple molecular markers that are tightly linked on the same chromosome.

[0013] In this article, "InDel-4337" refers to the maize husk width gene

[0014] In this article, "InDel-4337" refers to the maize husk width gene RHW1An insertion / deletion (InDel) molecular marker in the 3’UTR region, and this InDel-4337 is described in the prior art document Xia, A., Zheng, L., Wang, Z., Wang, Q., Lu, M., Cui, Z., & He, Y. (2023). The RHW1-ZCN4 regulatory pathway confers natural variation of husk leaf width in maize. New Phytologist, 239(6), 2367-2381. https: / / doi.org / 10.1111 / nph.19116. The entire content is incorporated into the application document by reference. RHW The gene encodes a MYB-like transcription factor suppressor that regulates cell proliferation and then affects the development of husk leaf width. InDel-4337 affects husk leaf traits by increasing the translation efficiency or stability of the RHW1 protein. In genome-wide association studies (GWAS), it was confirmed that InDel4337 was significantly associated with the diversity of maize husk leaf width (P = 2.97×10 -7 ). Maize lines carrying the InDel-4337Ref allele of the RHW gene showed narrower husk leaves than those carrying the InDel-4337Del allele. In this article, "InDel-4337Ref" represents the genotype of the InDel-4337 insertion, and "InDel-4337Del" represents the genotype of the InDel-4337 deletion.

[0015] In this article, "husk leaf width" refers to the width of the outer husk leaves of the maize ear, which can be obtained by known and conventional methods in the art. Usually, the width of the middle part of the third layer of husk leaves (counting from the outside to the inside) is measured. Wide husk leaves refer to husk leaves with a width higher than the average width of maize husk leaves; narrow husk leaves refer to husk leaves with a width lower than the average width of maize husk leaves. In an alternative embodiment, wide husk leaves refer to the measurement and statistical results of the representative husk leaf width of maize varieties with the InDel-4337Del RHW1 genotype; narrow husk leaves refer to those with the InDel-4337Ref RHW1Measurement and statistical results of the representative husk leaf width of 3 genotype maize varieties. The measurement and statistical results of the representative husk leaf width of the above two genotype maize varieties were significantly different through the Wilcoxon rank sum test. In an alternative embodiment, the above two genotypes are genotypes within a selected range of maize varieties, such as the maize varieties involved in the prior art literature Xia, A., Zheng, L., Wang, Z., Wang, Q., Lu, M., Cui, Z., & He, Y. (2023). The RHW1-ZCN4 regulatory pathway confers natural variation of husk leaf width in maize. New Phytologist, 239(6), 2367-2381. https: / / doi.org / 10.1111 / nph.19116, or the maize varieties shown in Tables 2 and 3.

[0016] Based on the information of 540 maize inbred lines and 2.65M SNP loci provided by the website MaizeGDB (http: / / www.maizego.org / Programs.html), the present invention distinguishes the varieties containing the target gene by comparing the published gene sequences or functional gene loci, and then develops a group of molecular markers tightly linked to the maize husk leaf width regulatory gene RHW1 The molecular marker is a haplotype marker, including one or more loci among SNP1-SNP8 located on the same chromosome. The genomic positions of each locus are as follows:

[0017] The genomic position of SNP1 is Chr5: 221649717 bp;

[0018] The genomic position of SNP2 is Chr5: 221649776 bp;

[0019] The genomic position of SNP3 is Chr5: 221649809 bp;

[0020] The genomic position of SNP4 is Chr5: 221649857 bp;

[0021] The genomic position of SNP5 is Chr5: 221649904 bp;

[0022] The genomic position of SNP6 is Chr5: 221649929 bp;

[0023] The genomic position of SNP7 is Chr5: 221649957 bp;

[0024] The genomic location of SNP8 is Chr5: 221650023 bp;

[0025] The physical positions of the SNP loci are determined based on the B73 whole-genome sequence version V4.

[0026] In a first aspect, there is provided the use of a substance for detecting the above-mentioned maize haplotype markers in any one of (i) to (iv):

[0027] (i) Predicting the genotype of the functional locus InDel-4337 related to the maize husk width gene RHW1 ;

[0028] (ii) Preparing a product for predicting the genotype of the functional locus InDel-4337 related to the maize husk width gene RHW1 ;

[0029] (iii) For predicting the maize husk width trait:

[0030] (iv) Preparing a product for predicting the maize husk width trait.

[0031] In an optional embodiment, the maize haplotype markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 located on the same chromosome.

[0032] In an optional embodiment, the substance for detecting the maize haplotype markers includes one or more of a probe for SNP detection, an SNP chip, a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a reagent for constructing a sequencing library, and a reagent for sequencing. More specifically, the substance for detecting the maize haplotype markers includes, but is not limited to, primers, probes, enzymes for nucleic acid amplification reactions, fluorescent labels, buffer reagents, dNTPs, salts, etc. According to different specific detection methods, those skilled in the art can select the above-mentioned reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing a sequencing library, and reagents for sequencing according to the methods described in general and more specific textbooks, reference documents, process manuals, product descriptions, and standard documents. The present invention does not limit this.

[0033] In an alternative embodiment, the substance for detecting maize haplotype markers includes an SNP chip. The SNP chip technology mainly forms a dense oligonucleotide probe array by immobilizing SNP markers on a carrier, and then performs allele-specific reactions with the target DNA. The polymorphism of SNP sites is determined based on the presence, absence, and intensity of signals after the reaction. This technology can rapidly and densely scan the entire genome of crops. Especially when performing throughput genotyping on population samples in breeding work, the cost of a single detection site is very low. It is a highly integrated, high-throughput, miniaturized, and automated means for detecting SNPs. In an alternative embodiment, the SNP chip includes a Maize 10K low-density gene chip.

[0034] In an alternative embodiment, the substance for detecting maize haplotype markers includes reagents for KASP (Kompetitive Allele Specific PCR) reaction.

[0035] In a second aspect, a kit is provided, which contains the substance for detecting maize haplotype markers described in the first aspect.

[0036] In an alternative embodiment, the kit contains one or more of the probes for SNP detection, SNP chips, reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. More specifically, the substance for detecting maize haplotype markers includes, but is not limited to, primers, probes, enzymes for nucleic acid amplification reactions, fluorescent labels, buffer reagents, dNTPs, salts, etc. According to different specific detection methods, those skilled in the art can select the composition of the kit according to the methods described in general and more specific textbooks, reference documents, process manuals, product descriptions, and standard documents. The present invention does not limit this.

[0037] In an alternative embodiment, the kit contains an SNP chip, preferably including a Maize 10K low-density gene chip.

[0038] In an alternative embodiment, the kit contains reagents for KASP (Kompetitive Allele Specific PCR) reaction.

[0039] In a third aspect, a method for predicting the genotype of the functional locus InDel-4337 related to the maize husk width gene RHW1 is provided, and the method includes detecting the maize haplotype markers described in the first aspect.

[0040] If the test sample meets one or more of the following conditions: SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, then it is predicted that the maize husk width gene RHW1 has an InDel-4337 insertion in its 3' UTR region.

[0041] In an alternative embodiment, the maize haplotype markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 located on the same chromosome; if the test sample meets the conditions that SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, then it is predicted that the maize husk width gene RHW1 has an InDel-4337 insertion in its 3' UTR region.

[0042] In a fourth aspect, a method for predicting the maize husk width trait is provided, which includes detecting the maize haplotype markers described in the first aspect.

[0043] If the test sample meets one or more of the following conditions: SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, then it is predicted that the maize husk width trait is narrow husk.

[0044] In an alternative embodiment, the maize haplotype markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 located on the same chromosome; if the test sample meets the conditions that SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, then it is predicted that the maize husk width trait is narrow husk.

[0045] In an alternative embodiment, the method for assisting in predicting that maize has narrow husk further includes detecting other molecular markers related to the husk width trait, such as but not limited to one or more of other SNP molecular markers, SSR molecular markers, STR molecular markers, and InDel molecular markers, or combining other known prediction methods in the art to predict the husk width trait.

[0046] In a fifth aspect, provided is the use of the maize haplotype markers described in the first aspect, or the kit described in the second aspect, or the method for determining the genotype of the functional locus InDel-4337 related to the maize husk width gene described in the third aspect, or the method for predicting the maize husk width trait described in the fourth aspect in predicting the maize grain dehydration efficiency at the harvest stage or in maize breeding. RHW1 ​

[0047] In alternative embodiments, the maize breeding includes, but is not limited to, marker-assisted selection breeding at the maize flowering stage, gene pyramiding breeding, or genome-wide selection breeding.

[0048] In a sixth aspect, a method for regulating the width of maize husks is provided, the method including regulating the genotype of the maize haplotype markers described in the first aspect.

[0049] In alternative embodiments, the method includes making at least one of the SNP1-SNP8 loci in the maize genome conform to the following: SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8, so that the maize obtains the trait of narrow husks.

[0050] In alternative embodiments, the method includes making SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 in the maize genome conform to the following: SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, so that the maize obtains the trait of narrow husks.

[0051] The specific means for making the maize genome obtain a specific genotype can be achieved by known and conventional methods in the art, and the present invention does not limit this. For example, maize with a target genotype can be obtained by gene editing methods.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] Based on the information of 540 maize inbred lines and 2.65M single nucleotide polymorphism (SNP) loci, the present invention obtains the genotype information of a total of 1,612 SNPs in the regions 100 Kb upstream and downstream of the functional locus InDel-4337 of the maize husk width gene RHW1. Through haplotype analysis, 8 SNP loci associated with the RHW1 functional genotype are found. These 8 haplotype markers can be used for the functional identification of the maize flowering stage gene RHW1, effectively avoiding the problem of identification accuracy caused by the failure of a few marker detections, as well as the problem of gene function loss caused by recombination near the gene, and improving the accuracy of functional gene identification.

[0054] This haplotype marker can be used to be transformed into a single SNP marker (such as a KASP marker) based on basic PCR and applied to SNP chip design, and applied to maize breeding, gene pyramiding breeding, and genome-wide selection breeding, further improving the efficiency and accuracy of molecular breeding and accelerating maize genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0056] Figure 1 For the linkage disequilibrium analysis results of the 200kb interval of haplotype analysis generated by Haploview in Example 1 (including 100kb upstream and downstream of the functional site InDel-4337);

[0057] Figure 2 For the 3 haplotypes and frequency results of the adjacent sequences of the functional site InDel-4337 of haplotype analysis generated by Haploview provided in Example 1. Specific Embodiments

[0058] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0059] Example 1

[0060] According to the website MaizeGDB (http: / / www.maizego.org / Programs.html), 2.65M SNP locus information of 540 maize inbred lines was obtained, and the haplotype markers associated with the functional site InDel-4337 of the maize husk leaf width regulatory gene were screened. RHW1 The functional site InDel-4337 is associated with haplotype markers.

[0061] 1. The article "The regulatory pathway confers natural variation of husk leaf width in maize" published by Professor He Yan of China Agricultural University in "New Phytologist" RHW1 - ZCN4 clarifies that the sequence variation of the functional site InDel-4337 of the gene (Regulator of Husk leaf Width) regulates the diversity of husk leaf width at the population level by affecting its protein abundance. RHW1 The sequence variation of the functional site InDel-4337 of the gene (Regulator of Husk leaf Width) regulates the diversity of husk leaf width at the population level by affecting its protein abundance. RHW1Encoded is a MYB-like transcriptional repressor, which regulates cell proliferation and then affects the development of bract width by positively regulating ZCN4 expression. The gene RHW1 was found to be located on chromosome (Chr5, 221642735 bp - 221651646 bp) in the reference genome B73, and its gene ID is Zm00001d018482; InDel-4337 is an 18-bp insertion / deletion polymorphism in the RHW1 3’UTR region, which is significantly associated with bract width diversity. Maize lines carrying the InDel-4337Ref allele showed narrower bracts than those carrying the InDel-4337Del allele. The above physical positions are based on the V4 version of the B73 whole-genome sequence.

[0062] 2. Uploaded the information of 2.65M SNP sites of 540 maize inbred lines to the server, and extracted the SNP information in the regions 100 kb upstream and downstream of the InDel-4337 functional locus of maize bract width gene, and a total of 1612 SNP variant site information was extracted.

[0063] 3. Saved the above 1612 SNP site information from 540 maize inbred lines in hapmap format, converted it to plink format using Tassel5, and then imported the plink-format SNP site information into Haploview software for haplotype analysis. According to the physical position of the functional locus, 183 LD BLOCKs ( Figure 1 ) were formed, and 3 groups of haplotypes Hap1 - 3 were obtained, which consisted of 8 SNP molecular markers. Among them, Hap1 and 2 were tightly linked to InDel-4337Ref (narrow bracts) ( Figure 2 and Tables 1 and 2), and the physical positions of the SNP sites were determined based on the V4 version of the B73 whole-genome sequence.

[0064] The chromosomal positions corresponding to the 8 SNP markers are:

[0065] The genomic position of SNP1 is Chr5: 221649717 bp;

[0066] The genomic position of SNP2 is Chr5: 221649776 bp;

[0067] The genomic position of SNP3 is Chr5: 221649809 bp;

[0068] The genomic position of SNP4 is Chr5: 221649857 bp;

[0069] The genomic location of SNP5 is Chr5: 221649904 bp;

[0070] The genomic location of SNP6 is Chr5: 221649929 bp;

[0071] The genomic location of SNP7 is Chr5: 221649957 bp;

[0072] The genomic location of SNP8 is Chr5: 221650023 bp;

[0073] When the haplotypes composed of the above SNP markers in ascending order of position are AACTCGAA or AACCCGAA, it shows the functional genotype with narrow bract leaves.

[0074] 4. Further comparative analysis of the haplotypes of narrow bract leaves and wide bract leaves found that the genotypes of 7 SNP loci of the two haplotypes Hap1 and Hap2 with narrow bract leaves are exactly the same (Table 1).

[0075] Table 1 Haplotype markers tightly linked to the maize bract width gene RHW1 composed of 7 SNP loci

[0076]

[0077] In summary, in this example, a set of haplotype molecular markers composed of 8 SNPs was developed. When the haplotypes composed of these 8 SNP markers in ascending order of position are AACTCGAA or AACCCGAA, RHW1 the genotype of the functional locus InDel - 4337 of the maize bract width gene is InDel - 4337Ref, showing narrow bract leaves. The physical positions of the SNP loci are determined based on the V4 version of the whole - genome sequence of B73.

[0078] Example 2

[0079] 1. According to the article "The RHW1 - ZCN4"regulatory pathway confers natural variation of husk leaf width in maize", the article announced the polymorphisms of 220 maize inbred lines at the InDel-4337 locus. Among them, 193 maize inbred lines contained the narrow leaf allele InDel-4337Ref at the InDel-4337 locus. Querying the SNP sequences of these 193 maize inbred lines near the InDel-4337 functional locus, it was found that the genotypes of 69 varieties such as 04K5686, 04K5702, 05W002, 07KS4, 1323, 150, 238, 384-2, 81162 at the 8 SNP loci in Example 1 were exactly the same as the genotype shown by Hap1 in Example 1 (Table 1, only showing the results of some inbred lines). The genotypes of 114 varieties such as BGY, BS16, C8605, CF3, CHANG3, CHANG7-2, CIMBL101, CIMBL105, CIMBL106, CIMBL111, CIMBL124, CIMBL129 at the 8 SNP loci in Example 1 were exactly the same as the genotype shown by Hap2 in Example 1 (Table 2, only showing the results of some inbred lines).

[0080] Table 2 Genotypes of some maize inbred lines consistent with Hap1

[0081]

[0082] Table 3 Genotypes of some maize inbred lines consistent with Hap2

[0083]

[0084] 2. In practical applications, compare the haplotype of the sample to be tested with Figure 2 the genotype. If the haplotype is consistent with Hap1 or Hap2, it is considered to contain RHW1 the narrow bract genotype InDel-4337Ref of the gene, showing narrower bracts.

[0085] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting maize haplotype markers in any one of (i) to (iv): (ⅰ)Genotype of the functional locus InDel-4337 related to the gene predicting the width of maize husk RHW1 ; Genotype of the functional locus InDel-4337 (ii) A product for preparing a genotype of the functional locus InDel-4337 related to genes for predicting the husk width of maize RHW1 related to the genotype of the functional locus InDel-4337; (iii) for predicting the maize husk width trait; (iv) preparing a product for predicting the maize husk width trait; The maize haplotype markers are composed of SNP1 to SNP8 located on the same chromosome; The genomic position of SNP1 is Chr5: 221649717 bp; The genomic position of SNP2 is Chr5: 221649776 bp; The genomic position of SNP3 is Chr5: 221649809 bp; The genomic position of SNP4 is Chr5: 221649857 bp; The genomic position of SNP5 is Chr5: 221649904 bp; The genomic position of SNP6 is Chr5: 221649929 bp; The genomic position of SNP7 is Chr5: 221649957 bp; The genomic position of SNP8 is Chr5: 221650023 bp; The haplotype composed of SNP1 to SNP8 in ascending order of position is AACTCGAA or AACCCGAA; The physical positions of the SNP loci are determined based on the V4 version of the whole-genome sequence of B73.

2. The application according to claim 1, characterized in that, The substance for detecting maize haplotype markers includes one or more of a probe for SNP detection, a SNP chip, a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a reagent for constructing a sequencing library, and a reagent for sequencing.

3. The application according to claim 2, wherein The substance for detecting maize haplotype markers includes a reagent for KASP reaction.

4. Kit, characterized in that, Comprising the substance for detecting maize haplotype markers according to any one of claims 1 to 3.

5. Method for predicting the genotype of the functional site InDel-4337 related to the maize husk width gene RHW1 which is characterized in that Including detecting the maize haplotype markers according to any one of claims 1 to 3; If the sample to be tested meets one or more of the following conditions: SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A, and SNP8 is A, then it is predicted that there is an insertion of InDel-4337 in the 3' UTR region of the maize husk width gene RHW1 ​ 6. The method according to claim 5, wherein The maize haplotype markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 located on the same chromosome; if the sample to be tested meets the conditions that SNP1 is A, SNP2 is A, SNP3 is C, SNP4 is T or C, SNP5 is C, SNP6 is G, SNP7 is A and SNP8 is A, then it is predicted that there is an insertion of InDel-4337 in the 3'UTR region of the maize husk width gene RHW1 .

7. A method for predicting the trait of the husk width of corn, characterized in that, Including detecting the maize haplotype markers according to any one of claims 1 to 3; If the test sample meets one or more of SNP1 being A, SNP2 being A, SNP3 being C, SNP4 being T or C, SNP5 being C, SNP6 being G, SNP7 being A, and SNP8 being A, then the maize husk width trait is predicted to be narrow husk.

8. The method according to claim 7, characterized in that, The maize haplotype markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 located on the same chromosome; if the test sample meets SNP1 being A, SNP2 being A, SNP3 being C, SNP4 being T or C, SNP5 being C, SNP6 being G, SNP7 being A, and SNP8 being A, then the maize husk width trait is predicted to be narrow husk.

9. A method for regulating the width of maize husks, characterized in that, Including regulating the genotype of the maize haplotype markers according to any one of claims 1 to 3.

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