A caps molecular marker closely linked to the leafless pillow trait of maize and application thereof

Abstract

The application discloses a CAPS molecular marker closely linked with a leafless pillow of corn and application thereof, and belongs to the technical field of molecular genetics. The CAPS molecular marker is CAPS-M5; the molecular marker CAPS-M5 is located at 4496246-4496474 bp of corn chromosome 2; and the nucleotide sequence of the molecular marker CAPS-M5 is shown as SEQ ID NO. 1. The CAPS molecular marker can be used for detecting and identifying the leafless pillow trait of corn, and can be simply, quickly and high-throughput used in breeding practice, and can accelerate the breeding process of corn.

Description

Technical Field

[0001] This invention relates to the field of maize genetics and breeding, specifically to a CAPS molecular marker closely linked to the leafless pillow trait in maize and its application. Background Technology

[0002] Corn is an important food and cash crop, as well as a vital energy and industrial raw material. With the continuous development of animal husbandry, industry, and energy sectors, the demand for corn is increasing year by year. In recent years, corn breeding has gradually shifted from pursuing yield per plant to increasing overall yield per plant. Increasing yield per unit area by increasing planting density is an important way to address the decreasing arable land resources and the growing demand for corn. Achieving this goal requires breeding varieties with ideal plant types that tolerate high density. Crop plant type includes traits such as leaf type, root type, stem type, and ear type. The ideal corn plant type is characterized by: narrow, upward-pointing leaves above the ear, broad, flat, and thick leaves below the ear, with strong veins to maintain plant uprightness. Leaf length, width, and morphological characteristics are important agronomic traits affecting planting density. Compact plant type is the foundation for increased yield in high-density cultivation, and the leaf angle, as a key trait determining the compactness of the plant type, directly affects the light-blocking capacity of the canopy and the efficiency of light energy utilization.

[0003] Generally, varieties with smaller leaf angles have more compact plant types, higher light transmittance in the upper leaves, and better light conditions in the middle and lower leaves, which is conducive to efficient light energy utilization, thus allowing them to adapt to higher planting densities and increase yield. Studies have shown that leaf pulvinus development directly regulates leaf angle formation, with the size and number of thin-walled and thick-walled cells on the near-abaxial surface of the leaf pulvinus significantly affecting the leaf angle, and consequently influencing maize canopy structure and yield potential. Therefore, elucidating the genetic regulatory mechanism of leaf pulvinus development is the theoretical basis for optimizing leaf angles and cultivating compact, density-tolerant plant types.

[0004] CAPS (Cleaved Amplified Polymorphic Sequence) markers are functional molecular markers developed based on differences in DNA sequences caused by single nucleotide polymorphisms (SNPs) or small insertions and deletions. CAPS markers are used to amplify specific fragments using PCR primers designed at the target site, and the amplified products are then digested with restriction endonucleases. Alleles are distinguished based on differences in the length of the digested fragments. CAPS markers offer advantages such as ease of operation, high reproducibility, and insensitivity to environmental conditions, while providing genetic information that directly reflects the polymorphism of the target gene. Utilizing CAPS markers closely linked to the target gene or located at functional sites, rapid and accurate screening can be achieved in breeding work such as marker-assisted selection (MAS), gene mapping, and germplasm identification. This allows for the aggregation of superior alleles, improving breeding efficiency and accelerating the process of new variety selection.

[0005] Currently, some progress has been made in the research on the genetic basis of leaf pulvinus and leaf angle traits in maize. However, the number of reported leaf pulvinus alleles is limited, and there is a lack of efficient and precise functional molecular markers for breeding practice. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a CAPS molecular marker closely linked to the leafless bollard trait in maize and its application. The CAPS molecular marker of this invention can be used to detect and identify the leafless bollard trait in maize, and can be used simply, quickly, and with high throughput in breeding practices, thus accelerating the maize breeding process.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a CAPS molecular marker closely linked to the leafless bollicle of maize, the CAPS molecular marker being named CAPS-M5; in maize material exhibiting the leafless bollicle trait, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.1. Specifically: GAACATGGCCAGCTTCGCCGCCACGCAGCTCGGCCTCAACCTCGGCTACCGCACCTACTTCCCCCCAGAGGAGGCTACACGTACGGCCACCA CCCGC CGCGCTGCCAGGCCGAGGGCTGCAAGGCCGACCTCTCCAGCGCCAAGCGATACCACCGTCGCCACAAGGTGTGCGAGCACCACTCCAAGGCGCCCGTCGTCGTCACCGCCGGTGGACTGCATCAGAGGTTC (SEQ ID NO. 1) Note: The bolded italicized regions in the sequence are restriction enzyme sites.

[0008] In wild-type maize material with leaf sheaths, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.2. Specifically: GAACATGGCCAGCTTCGCCGCCACGCAGCTCGGCCTCAACCTCGGCTACCGCACCTACTT CCCGC CCAGAGGAGGCTACACGTACGGCCACCA CCCGCCGCGCTGCCAGGCCGAGGGCTGCAAGGCCGACCTCTCCAGCGCCAAGAGATACCACCGTCGCCACAAGGTGTGCGAGCACCACTCCAAGGCGCCCGTCGTCGTCACCGCCGGTGGACTGCATCAGAGGTTC (SEQ ID NO. 2) Note: The bolded italicized regions in the sequence are restriction enzyme sites.

[0009] The CAPS-M5 of this invention is located at 4496246-4496474 bp on maize chromosome 2, with the B73RefGen_v5 genome as the reference genome. Compared with the wild type, the CAPS-M5 in maize materials with the leafless pillow trait has a nucleotide deletion at one site, resulting in a reduction of one FauⅠ (CCCGC) restriction site.

[0010] In maize materials with leafless pillow characteristics, CAPS-M5, after being digested with FauⅠ, showed two bands at 101bp and 127bp on electrophoresis.

[0011] In wild-type maize material with leaf cushions, CAPS-M5, after being digested with FauⅠ, theoretically produces three fragments of 127 bp, 69 bp, and 33 bp by electrophoresis. Among them, the 33 bp fragment is too short and migrates beyond the dye front in conventional gel electrophoresis and cannot be detected. Therefore, the actual distinguishable characteristic bands are 127 bp and 69 bp.

[0012] Therefore, the leaf pulvinus characteristics of maize can be identified based on the size of the electrophoretic bands after enzyme digestion.

[0013] A second aspect of the present invention provides the application of the above-described CAPS molecular marker in either (1) or (2) below: (1) Screening or identifying maize with the leafless pillow trait; (2) Marker-assisted breeding of maize.

[0014] Furthermore, the aforementioned marker-assisted breeding of maize specifically involves the innovation of maize germplasm that is tolerant to dense planting without leaf pillows or the selection of hybrid varieties.

[0015] A third aspect of the present invention provides primer pairs for amplifying the above-mentioned CAPS molecular marker, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; specifically as follows: Upstream primer: 5'-GAACATGGCCAGCTTCGCCGCC-3'; (SEQ ID NO.3) Downstream primer: 5'-GAACCTCTGATGCAGTCCACCGGC-3'. (SEQ ID NO.4) In a fourth aspect, a kit containing the primer pairs described above is provided.

[0016] Furthermore, the kit also includes: DNA template, Taq Master Mix, and ddH2O.

[0017] A fifth aspect of the invention provides the use of the above primer pairs or kits in the following (1) or (2): (1) Screening or identifying maize with the leafless pillow trait; (2) Marker-assisted breeding of maize.

[0018] In the above applications, the method for screening or identifying maize with the leafless pillow trait is as follows: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR products were then subjected to... Fau I. Gel electrophoresis analysis was performed after enzyme digestion; If two bands, 101bp and 127bp, are present, the maize is identified as having the leafless pillow trait.

[0019] Furthermore, in the above applications, the aforementioned marker-assisted breeding of maize specifically refers to the innovation of maize leafless pillow germplasm or the selection of hybrid varieties.

[0020] A sixth aspect of the present invention provides a method for detecting the leaf sheath trait of maize, comprising the following steps: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain the amplification products; Sequencing analysis of the amplified products, or sequencing the amplified products... Fau I. Gel electrophoresis analysis was performed after enzyme digestion; The maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.1 was identified as maize with the leafless pillow trait, and its ear leaf angle was smaller than that of maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.2. Or, the amplification product is... Fau Ⅰ. After enzyme digestion and electrophoresis, if two bands of 101bp and 127bp appear, it is identified as maize with the leafless pillow trait. If two bands, 127bp and 69bp, are present, the maize is identified as having a leaf pillow. If three bands of 127bp, 101bp, and 69bp are present, it is identified as heterozygous.

[0021] Preferably, the PCR amplification reaction system consists of: 0.5 μL of the upstream primer shown in SEQ ID NO.3 at a concentration of 10 μM; 0.5 μL of the downstream primer shown in SEQ ID NO.4 at a concentration of 10 μM; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; and 3 μL of ddH2O.

[0022] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; and storage at 10℃.

[0023] Fau The reaction system for enzyme digestion was as follows: 5 μL PCR product; 1 μL Fau I. Restriction endonuclease; 2 μL 10 × NEB buffer; 2 μL H2O.

[0024] The beneficial effects of this invention are: This invention screens and identifies a CAPS molecular marker closely linked to the leafless bollicle trait in maize from a specific locus on chromosome 2. Using this marker, the leafless bollicle trait can be identified at the molecular level during the early growth stages of maize plants, avoiding the problems of long cycles, susceptibility to environmental influences, and low identification efficiency associated with relying solely on field phenotypic observation. This marker detection method is simple to operate, provides intuitive results, and is accurate. It has advantages such as good repeatability, high specificity, low detection cost, and suitability for batch screening, effectively improving the early selection efficiency of leafless bollicle maize materials and providing a reliable technical means for maize plant architecture improvement and marker-assisted breeding. Attached Figure Description

[0025] Figure 1 Electrophoretic patterns of PCR amplification products of the molecular marker CAPS-M5 in the F2 population of this invention. Wherein, W represents the amplification band pattern of maize with leaf pillow, N represents the amplification band pattern of maize with leafless trait, H represents the amplification band pattern of heterozygous genotype, and M represents the marker. The band sizes from bottom to top are 69 bp, 101 bp, and 127 bp, respectively.

[0026] Figure 2 The results of the analysis of the leaf angle at the ear position in maize (W) with a leaf pillow and maize (N) with a leafless trait, identified using the molecular marker CAPS-M5 of this invention, are presented. Data are expressed as mean ± standard error. P <0.0001 (two-tailed t-test). Detailed Implementation

[0027] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0028] Terminology Explanation: Liguleless trait in maize: refers to a morphological phenotype in which the pulvinus structure (including ligule and auricle) at the junction of the leaf and leaf sheath of the maize plant is absent or completely undeveloped.

[0029] The direct basis for judging this trait is the presence or absence of a leaf pulvinus structure; as a result, the leaf angle at the ear position of plants without a leaf pulvinus will be significantly smaller than that of normal plants. Therefore, in breeding screening, the leaf angle at the ear position can also be used for auxiliary judgment and quantitative analysis.

[0030] As mentioned earlier, breeding leafless varieties suitable for high-density planting is an important way to improve plant population effects and increase maize yield. Since the leafless trait in maize may be regulated by one or more genes, and may exhibit polymorphism and interactions, this increases the difficulty of identifying CAPS molecular markers closely linked to the leafless trait. Currently, research on maize leafless genes is still limited.

[0031] In view of this, this invention has conducted in-depth research on the genes controlling the leafless bollard trait in maize. This invention uses genetic methods to determine the inheritance pattern of the leafless bollard trait and identifies the locations of genes closely associated with it through association analysis and map-based cloning techniques. Primer design and optimization are also among the key technical challenges in developing CAPS molecular markers closely linked to the leafless bollard trait. Due to the variability of the target DNA sequence, designing specific primers can be challenging. Primer design needs to consider the length of the target DNA sequence, GC content, specificity, and site coverage between primers. Furthermore, primer temperature and amplification procedures also need to be optimized to ensure reliable amplification results. The presence of polymorphism in the leafless bollard trait may also require the design of multiple sets of primers to cover various polymorphic sites.

[0032] This invention ultimately located the leafless pillow gene between two molecular markers, InDel-Y13 and InDel-Y17, on maize chromosome 2, at a physical distance of 123 kb. Sequencing within this region revealed that the maize leafless pillow mutant, compared to the wild type, exhibits a one-nucleotide deletion, resulting in a change in the number of FauⅠ (CCCGC) restriction sites. Therefore, this region can serve as a CAPS functional marker tightly linked to the maize leafless pillow, and is named CAPS-M5.

[0033] Based on this CAPS functional marker, the present invention further designed specific primers for specifically amplifying the above-mentioned CAPS functional marker, as follows: Upstream primer: 5'-GAACATGGCCAGCTTCGCCGCC-3'; Downstream primer: 5'-GAACCTCTGATGCAGTCCACCGGC-3'.

[0034] BLAST analysis of the above-mentioned specific primers, based on the existing maize genome sequence, revealed that the molecular marker CAPS-M5 is located at 4496246-4496474 bp on maize chromosome 2, with the physical location referenced from the B73 RefGen_v5 genome version.

[0035] The molecular marker CAPS-M5 of this invention can accurately identify the leafless pillow trait in maize at an early stage, which is beneficial for breeding leafless pillow varieties suitable for high-density planting. This invention was proposed based on this.

[0036] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0037] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0038] Example 1: Obtaining CAPS molecular markers tightly linked to leafless pillows in maize Maize leafless pillow mutant jd30 Derived from the male parent JD30 of the hybrid Shannong 206. A leafless, pillow-less mutant. jd30 Using maize inbred lines B73 and Lx9801 as the female parent, an F2 segregating population was constructed to finely map the gene controlling the leafless bollicle in maize. The constructed F2 segregating population was planted in the field, and phenotypic identification was performed at the 5-leaf stage. Total DNA was extracted from leaves without a leafless bollicle, and polymorphic screening of the leafless bollicle mutant and parents was conducted using existing markers from the synthetic maize IBM map and a self-developed SSR marker. Primers were used for encryption based on the published maize genome, ultimately locating the leafless bollicle gene between two molecular markers, Indel-Y13 and Indel-Y17, with a physical distance of 123 kb.

[0039] 1. Obtaining the leafless pillow mutant jd30This is a naturally occurring leafless ecliptic mutant discovered in the field. This mutant exhibits the leafless ecliptic phenotype from stage V2 until maturity. As growth and development progress, the mutant... jd30 The plant has a compact shape, and the phenotype of narrow, upright leaves with a small leaf angle is becoming more pronounced.

[0040] Table 1: jd30 Mutant phenotypic analysis 2. Genetic analysis Using leafless pillow mutant jd30 Genetic analysis of the F2 generation obtained by crossing maize inbred lines Lx9801 and B73, which have complete leaf pillow structures, revealed that the ratio of normal phenotype to mutant phenotype in the F2 population was 3:1 (χ2c<χ20.05), indicating that the leaf pillow trait is controlled by a pair of recessive nuclear genes.

[0041] Table 2: jd30 Genetic analysis of leafless pillow mutant 3. Preliminary localization of the gene for the absence of leaf cushions The F2 segregating population constructed from the inbred line B73 and the leafless pillow mutant was planted in the field. Phenotypic identification of the segregating population was performed at the 5-leaf stage, and total DNA was extracted from leaves with the leafless pillow phenotype.

[0042] by jd30 Using the inbred line Lx9801 as the male parent and the inbred line Lx9801 as the female parent, an F2 localization population was constructed. Simultaneously, 208 pairs of SSR primers evenly distributed across the 10 maize chromosomes were used to screen the parents for polymorphism, identifying 32 pairs of polymorphic SSR molecular markers. DNA from 20 Lx9801 lines and 20 other lines were randomly selected. jd30 Equal amounts of DNA were mixed to construct normal DNA and mutant DNA pools. Preliminary linkage analysis was performed on molecular markers exhibiting polymorphism, revealing that the molecular marker p-ucl552 located on chromosome 2 may be tightly linked to the target trait. Furthermore, mutant DNA was used... jd30 Validation was performed on 235 leafless boll-forming plants paired with Lx9801, revealing that the SSR marker was linked to the target gene. Subsequently, using B73 as a reference genome sequence, 48 pairs of SSR markers were developed on chromosome 2 using SSR hunter and Primer Premier 5.0 software. Polymorphism screening was performed on these 48 pairs of markers among the parents, revealing that one pair of markers showed polymorphism differences among the parents. Further validation was performed on 237 leafless boll-forming individuals in the F2 population, preliminarily locating the target gene between the p-umcl552 and K1 markers on the short arm of maize chromosome 2, at a physical distance of 3.3 Mb.

[0043] 4. Fine mapping of the gene without leaf cushion To further narrow the positioning interval, the F2 segregating population was expanded, and inbred lines Lx9801 and B73 were used with... jd30 F1 was obtained through hybridization, and F2 segregating populations were obtained by self-crossing F1. We developed 17 pairs of Indel markers between the two molecular markers p-umncl552 and K1. Through parental polymorphism screening and linkage analysis, 6 pairs of molecular markers closely linked to the trait were screened out. These markers were then used to develop mutants. jd30 The target gene was validated in 2047 leafless phenotypes obtained by crossbreeding with the inbred line Lx9801, narrowing the gap to the two markers Y4 and Y7, with a physical distance of 247 kb. Because... jd30 Molecular markers showing no polymorphic differences between the two parents, Lx9801 and Lx9801, were used to identify mutants. jd30 A new F2 segregating population was created by pairing the inbred line B73 with the inbred line B73, and five new Indel markers were developed in the localization region, of which three pairs of markers were found in the localization region. jd30 There are polymorphic differences between B73 and B73. These three pairs of markers can be used to... jd30 The target gene was validated in 1865 leafless phenotypes paired with inbred line B73, and was finally located within a physical distance of approximately 123 kb between InDel-Y13 and InDel-Y17; the nucleotide sequence of the target gene associated with the leafless phenotype is shown in SEQ ID NO.5.

[0044] InDel-Y13-F: 5'-AGTCGCTGGGATGCATTTTC-3'; (SEQ ID NO.6) InDel-Y13-R: 5'-AACTGATTCAACTAAGGGTGCT-3'. (SEQ ID NO.7) InDel-Y17-F: 5'-GTCCCGTCCATCCATTCCTC-3'; (SEQ ID NO.8) InDel-Y17-R: 5'- CCCTATACACGTGAACATGCA-3'. (SEQ ID NO.9) Finally, a CAPS molecular marker closely linked to the absence of a leaf pillow in maize was screened at 4496246-4496474 bp on chromosome 2, and named molecular marker CAPS-M5. The physical location is referenced from the B73 RefGen_v5 genome version. In maize materials with the leaf pillow trait, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.1. In wild-type maize materials with a leaf pillow, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.2.

[0045] Example 2: Development and application of CAPS molecular markers based on maize leafless pillow gene Based on the molecular marker CAPS-M5 screened in Example 1, primer pairs for amplifying the molecular marker were designed. The designed primer pair sequences are as follows: Upstream primer: 5'-GAACATGGCCAGCTTCGCCGCC-3'; (SEQ ID NO.3) Downstream primer: 5'-GAACCTCTGATGCAGTCCACCGGC-3'. (SEQ ID NO.4) The method for detecting the leaf pulvinus trait of maize using the above molecular marker CAPS-M5 and primer pairs is as follows: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain the amplification products; Sequencing analysis of the amplified products, or sequencing the amplified products... Fau I. After enzyme digestion, gel electrophoresis analysis was performed.

[0046] For sequencing, the method for determining the leaf sheath trait in maize is as follows: The maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.1 was identified as maize with the leafless pillow trait, and its ear leaf angle was smaller than that of maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.2.

[0047] For amplified products Fau For gel electrophoresis analysis following enzyme digestion, the method for determining the leaf sheath trait in maize is as follows: amplification products Fau Ⅰ After enzyme digestion and electrophoresis, if two bands of 101bp and 127bp appear, it is identified as maize (N) with the leafless pillow trait; If two bands, 127bp and 69bp, are present, it is identified as a leaf-pillowed maize (W); If three bands of 127bp, 101bp, and 69bp are present, it is identified as heterozygous (H).

[0048] The PCR amplification reaction system consisted of: 0.5 μL of the 10 μM upstream primer shown in SEQ ID NO.3; 0.5 μL of the 10 μM downstream primer shown in SEQ ID NO.4; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; and 3 μL of ddH2O.

[0049] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; and storage at 10℃.

[0050] Fau The reaction system for enzyme digestion was as follows: 5 μL PCR product; 1 μL Fau I. Restriction endonuclease; 2 μL 10 × NEB buffer; 2 μL H2O.

[0051] The enzyme digestion reaction was carried out at a temperature of 55℃ for 2 hours.

[0052] Using maize inbred line B73 and jd30 The mutant was used as the experimental material. PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4, and the amplification products were sequenced and analyzed. The results showed that: jd30 The mutant's molecular marker CAPS-M5 has a nucleotide deletion at one site compared to wild-type maize B73.

[0053] maize inbred line B73 and jd30 F1 was obtained through hybridization, and F1 plants were self-crossed to obtain a new F2 segregating population. The plants in the F2 segregating population were subjected to PCR amplification as described above, and the amplification products were then processed... Fau I. Gel electrophoresis analysis was performed after enzyme digestion; the gel electrophoresis analysis results of some plants are as follows: Figure 1 As shown.

[0054] choose Figure 1 The ear-leaf angle was measured in maize identified as having a leaf pillow (W) and maize identified as lacking a leaf pillow (N). The results are as follows: Figure 2 As shown in the figure. The results indicate that the ear-leaf angle of maize identified by molecular markers as having a leaf pillow (W) is significantly larger than that of maize identified by molecular markers as lacking a leaf pillow (N). This demonstrates that genotyping this population using CAPS-M5 is accurate and feasible, and can be easily, rapidly, and with high throughput applied to breeding practices, which will greatly accelerate the process of molecular breeding for maize lacking a leaf pillow.

[0055] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A CAPS molecular marker closely linked to the leafless pillow of maize, characterized in that, The CAPS molecular marker is named CAPS-M5; in maize materials with no leaf pillow trait, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.1; in wild-type maize materials with leaf pillow, the nucleotide sequence of CAPS-M5 is shown in SEQ ID NO.

2.

2. The application of the CAPS molecular marker according to claim 1 in either (1) or (2): (1) Screening or identifying maize with the leafless pillow trait; (2) Marker-assisted breeding of maize.

3. The application according to claim 2, characterized in that, The specific purpose of the molecular marker-assisted breeding of maize is the innovation of maize germplasm that is tolerant to dense growth without leaf pillows or the selection of hybrid varieties.

4. A primer pair for amplifying the CAPS molecular marker of claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

5. A kit containing the primer pair of claim 4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes: DNA template, TaqMaster Mix and ddH2O.

7. The use of the primer pair of claim 4 or the kit of claim 5 in (1) or (2) below: (1) Screening or identifying maize with the leafless pillow trait; (2) Marker-assisted breeding of maize.

8. The application according to claim 7, characterized in that, The method for screening or identifying maize with the leafless pillow trait is as follows: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.

4. The PCR products were then subjected to... Fau I. Gel electrophoresis analysis was performed after enzyme digestion; If two bands, 101bp and 127bp, are present, the maize is identified as having the leafless pillow trait.

9. A method for detecting the leaf sheath trait of maize, characterized in that, Includes the following steps: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain the amplification products; Sequencing analysis of the amplified products, or sequencing the amplified products... Fau I. Gel electrophoresis analysis was performed after enzyme digestion; The maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.1 was identified as maize with the leafless pillow trait, and its ear leaf angle was smaller than that of maize with the sequencing result of the amplified product being the sequence shown in SEQ ID NO.

2. Or, the amplification product is... Fau I. After enzyme digestion and electrophoresis, if two bands of 101bp and 127bp appear, it is identified as maize with the leafless pillow trait.

10. The method according to claim 9, characterized in that, The PCR amplification reaction system consisted of 0.5 μL of the upstream primer shown in SEQ ID NO.3 at a concentration of 10 μM; 0.5 μL of the downstream primer shown in SEQ ID NO.4 at a concentration of 10 μM; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; 3 μL of ddH2O. The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; and storage at 10℃. Fau The reaction system for enzyme digestion was as follows: 5 μL PCR product; 1 μL Fau I. Restriction endonuclease; 2 μL 10 × NEBbuffer; 2 μL H2O.

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