Corn deep sowing tolerance gene ZmCRK10 and deep sowing tolerance molecular marker and application

Abstract

The application discloses a maize deep sowing tolerance gene ZmCRK10, a deep sowing tolerance molecular marker and application, a nucleotide sequence of the maize deep sowing tolerance gene ZmCRK10 is shown as SEQ ID No:1; a sequence of a transcript CDS of the application is shown as SEQ ID No:3; the gene ZmCRK10 of the application encodes a cysteine-rich receptor kinase, participates in regulating plant growth and development, overexpression of a T04 transcript can increase the mesocotyl length under deep sowing treatment of corn, and improves the deep sowing tolerance characteristics of corn. Subsequently, through resequencing of a related population, it is found that a large fragment structural variation exists in a maize ZmCRK10 promoter region, and when a transposon with a length of about 121.7kb exists, the expression of a T04 transcript of ZmCRK10 can be significantly improved, the maize mesocotyl elongation is promoted, and the ZmCRK10 Type A can be used as a maize deep sowing tolerance molecular marker.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a maize deep-planting tolerance gene ZmCRK10, its molecular markers, and their applications. Background Technology

[0002] Maize is my country's largest grain crop and an important forage crop, playing a vital role in ensuring national food security and economic development. However, approximately two-thirds of my country's maize planting area is located in arid and semi-arid regions. These areas experience low natural rainfall, rapid evaporation of surface soil moisture, and low soil moisture content, severely impacting early spring maize sowing. This makes maize highly susceptible to drought stress during germination and sprouting, ultimately affecting yield. Appropriate deep sowing allows seeds to fully utilize deep soil moisture, ensuring germination and sprouting in arid environments, and is a crucial way for maize seedlings to resist drought. However, to date, no clone of the QTL regulating maize deep sowing tolerance has been found. Therefore, fully utilizing maize germplasm resources, analyzing the mechanism of maize deep sowing tolerance, and screening and breeding new deep-sowing-tolerant maize varieties are of great significance for drought resistance, seedling protection, stable yield, and increased production in arid and semi-arid regions.

[0003] Under appropriate deep sowing conditions, the emergence rate of maize is significantly positively correlated with the elongation of the mesocotyl and coleoptile. Maize varieties with longer mesocotyls or coleoptiles ensure faster seed emergence from deep-sown seeds, with mesocotyl elongation playing a major role, having a greater impact than the coleoptile. The mesocotyl of maize refers to the region between the root and coleoptile of the maize seedling. Mesocotyl elongation is influenced by various internal and external factors, including light intensity, sowing depth, hormones, lignin metabolism, and varietal differences. Cysteine-rich receptor-like kinases (CRKs) are a large class of plant receptor kinases. CRKs have been reported to regulate ROS generation, calcium ion signaling, MAPK cascade reactions, ABA responses, and callose deposition, and are widely involved in plant growth and development, immune responses, and abiotic stress. Studies have found that CRK mutations in Arabidopsis thaliana affect seed germination, emergence, rosette leaf size, and root development, suggesting that CRK-mediated callose deposition may be involved in regulating cell turgor pressure-driven elongation. In conclusion, the maize CRK gene may play a role in regulating maize mesocotyl elongation development and has potential application value in the breeding of new maize varieties tolerant to deep planting.

[0004] Besides molecular breeding, the use of genetic methods to cultivate and improve maize varieties tolerant to deep planting is also of great significance. Screening for molecular markers of maize deep-planting tolerance genes can effectively improve the efficiency of breeding deep-planting tolerant maize varieties. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a maize deep-planting tolerance gene ZmCRK10, its molecular marker, and its applications. The maize deep-planting tolerance gene ZmCRK10 encodes a cysteine-rich receptor kinase, which participates in regulating plant growth and development. ZmCRK10 has two major transcripts; overexpression of the T04 transcript of ZmCRK10 can promote mesocotyl elongation under deep-planting conditions in maize. The molecular marker for maize deep-planting tolerance is ZmCRK10. Type A This includes a transposon of approximately 121.7 kb in length identified by the marker (containing multiple LTR long terminal repeat retrotransposons). The presence of this marker significantly promotes maize mesocotyl development, and its mechanism of action is to promote the expression of the downstream ZmCRK10-T04 transcript, thereby promoting mesocotyl elongation.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0007] This invention provides a maize deep-planting tolerance gene ZmCRK10, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] The present invention also provides a deep-planting tolerant transcript of the maize deep-planting tolerant gene ZmCRK10, ZmCRK10-T04, the CDS nucleotide sequence of which is shown in SEQ ID NO: 3.

[0009] The present invention also provides a deep-seated protein ZmCRK10-T04 encoded by the deep-seated transcript ZmCRK10-T04, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0010] The primer pairs used to obtain the above-mentioned deep-seated transcript ZmCRK10-T04 sequence are as follows:

[0011] ZmCRK10-T04-F0: 5'-GTCGACAAACGCACTAGTATCCCGGGA

[0012] TGGATTTCGACCCTAAGCTC-3',

[0013] ZmCRK10-T04-R0: 5'-GGGTACATTTGGCGCGCCTTCCCGGG

[0014] TCTAGGATACAGTTCACTGAGTGTA-3'.

[0015] The present invention also provides an application of the deep-planting tolerance transcript ZmCRK10-T04 or the deep-planting tolerance protein ZmCRK10-T04 in improving the deep-planting tolerance of maize.

[0016] The present invention also provides the application of the deep-planting tolerant transcript ZmCRK10-T04 or the deep-planting tolerant protein ZmCRK10-T04 in the breeding of new deep-planting tolerant maize varieties.

[0017] This invention also provides a molecular marker for maize deep-planting tolerance, ZmCRK10. Type A The maize deep-planting tolerant molecular marker ZmCRK10 Type A The nucleotide sequence is shown in SEQ ID NO: 4.

[0018] The present invention also provides the aforementioned molecular marker ZmCRK10 for maize deep-planting tolerance. Type A Applications in improving maize's tolerance to deep planting and in assisted breeding.

[0019] The present invention also provides a method for obtaining the maize deep-planting tolerant molecular marker ZmCRK10. Type A The primer pairs are as follows:

[0020] ZmCRK10 Type A -F1:5'-ACCTCATCTCCTTCATGTGTGGTC-3',ZmCRK10 Type A -R1: 5'-GCATAGGTGGAATCGTCGCAG-3'.

[0021] The present invention also provides an application of a kit for identifying maize varieties with excellent deep-planting tolerance, the kit comprising the primer pairs described above.

[0022] This invention also provides a method for identifying maize varieties with excellent deep-planting tolerance using the above-mentioned kit, comprising the following steps:

[0023] (1) Extract DNA from the maize variety to be tested;

[0024] (2) Design the primer pairs as follows:

[0025] ZmCRK10 Type A -F1:5'-ACCTCATCTCCTTCATGTGTGGTC-3', as shown in SEQ ID NO:5

[0026] ZmCRK10 Type A -R1: 5'-GCATAGGTGGAATCGTCGCAG-3', as shown in SEQ ID NO: 6;

[0027] (3) Perform PCR amplification using the above DNA as a template;

[0028] (4) Electrophoresis: The amplification showed a target band of 403bp, indicating that the variety has a strong tolerance to deep sowing.

[0029] The principle of this invention:

[0030] This invention utilizes maize transformation technology to obtain maize ZmCRK10-T04 overexpression material. The overexpression material, isolated negative control material, and the transformed background material KN5585 were seeded at a depth of 15 cm. The results showed that the mesocotyl length of the overexpression material was significantly higher than that of the wild type. Further gel section analysis of the mesocotyls revealed that the mesocotyl cell length of the overexpression material was significantly higher than that of the isolated negative control and background materials, indicating that ZmCRK10-T04 may positively regulate maize mesocotyl elongation under deep seeding conditions by promoting cell elongation. Related population resequencing revealed a large structural variation in the maize ZmCRK10 promoter region, approximately 121.7 kb in length (ZmCRK10...). Type A ) and 63.3kb (ZmCRK10 Type B The transposable of ZmCRK10 Type A The presence of this substance significantly enhances the expression of the T04 transcript of ZmCRK10, promotes the development of the maize mesocotyl, and can serve as a molecular marker for maize tolerance to deep sowing.

[0031] The beneficial effects of this invention are:

[0032] This invention utilizes the mesocotyl length under 15cm deep-sown treatment in maize populations and 550,000 SNP markers for GWAS analysis, identifying a gene related to deep-sown tolerance, ZmCRK10. This gene encodes a cysteine-rich receptor kinase involved in regulating plant growth and development. Overexpression of the transcript ZmCRK10-T04 promotes mesocotyl elongation under deep-sown conditions, enhancing maize's tolerance to deep sowing. The presence of a 121.7kb transposon structural variation in its promoter region significantly promotes maize mesocotyl development, and as a molecular marker for maize deep-sown tolerance, it can effectively improve the efficiency of breeding deep-sown maize varieties. Attached Figure Description

[0033] Figure 1 A schematic diagram illustrating the construction and identification of maize overexpression materials;

[0034] In the figure, a is a schematic diagram of the construction of the overexpression vector pZZ0153-UBIp-ZmCRK10-T04-3HA.

[0035] Figure b shows the identification results of DNA insertion into the overexpression vector of the transgenic material.

[0036] c is a graph identifying the expression level of ZmCRK10 in transgenic materials (the expression level in KN5585 is set to 1).

[0037] Figure 2 Phenotypic analysis of ZmCRK10-T04 overexpressing materials and controls at a seeding depth of 15cm;

[0038] In the figure, a shows the mesocotyl phenotype images of the overexpressing material and the control under a 15cm deep seeding treatment, where Bar = 1cm.

[0039] b is a statistical analysis of the mesocotyl length of the overexpressing material and the control under 15cm deep seeding treatment.

[0040] c shows mesocotyl sections of the overexpression material and control after seeding at a depth of 15 cm. The sections were stained with Calcofluor; blue indicates the cell wall location. Bar = 50 μm.

[0041] d is a schematic diagram of the mesodermal cell length of the overexpression material and the control under 15cm deep seeding treatment. Each point represents the average length of 10 mesodermal cells.

[0042] Figure 3 Allelic variation analysis diagram for ZmCRK10;

[0043] In the figure, 'a' represents the structural variation of the ZmCRK10 promoter and the ZmCRK10 in the associated population. Type A Genotyping gel electrophoresis

[0044] b is ZmCRK10 Type A Association analysis diagram between genotype and hypocotyl length in associated populations.

[0045] c is ZmCRK10 Type A Association analysis diagram between genotype and ZmCRK10-T04 transcript abundance.

[0046] Figure 4 For F 2:3 ZmCRK10 in the population, BC3F3 population and RIL F8 population Type A A diagram illustrating how natural variations affect maize's tolerance to deep sowing;

[0047] a is a seeding depth of 15cm, with LIAO5263×CML423 as the parent F. 2:3 A schematic diagram of the mesocotyl length in the population.

[0048] b represents a seeding depth of 15cm, with LIAO5114×CIMBL83 as the parent F. 2:3 A schematic diagram of the mesocotyl length in the population.

[0049] c is a schematic diagram of the mesocotyl length of the BC3F3 population with LY042×CML423 as the parent at a sowing depth of 15cm.

[0050] A schematic diagram of the mesocotyl length of the RIL F8 population with BY815×KUI3 as the parent at a sowing depth of 15cm.

[0051] Figure 5 This diagram illustrates the use of primers and kits to detect the genotype of different maize varieties and the length of the mesocotyl in deeply sown maize.

[0052] a is ZmCRK10 Type A Gel images for identifying maize varieties by genotype.

[0053] b is ZmCRK10 Type B Gel images for identifying maize varieties by genotype.

[0054] c is ZmCRK10 Type A Mesocot length of maize varieties with different genotypes at a sowing depth of 15cm.

[0055] d is ZmCRK10 Type B Mesocotyl length of maize varieties with different genotypes at a sowing depth of 15 cm. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0057] Example 1: Localization of the deep-planting tolerance gene ZmCRK10 in maize

[0058] Genome-wide association analysis (GWAS) was performed using genotypic data from 380 natural maize populations (data source: http: / / maizego.org / Resources.html) combined with mesocotyl length data under a 15cm deep sowing treatment. Referring to the maize genome annotation file, the target gene ZmCRK10 was located. The expression of this gene in the mesocotyl increases with increasing sowing depth. Its nucleotide sequence is shown in SEQ ID No: 1. The amino acid sequence of the cysteine-rich receptor kinase encoded by this gene is shown in SEQ ID No: 2.

[0059] Example 2 Construction of Maize ZmCRK10-T04 Overexpression Vector

[0060] Based on the expression vector pZZ0153-UBIp-3HA, a cDNA fragment of the deep-seeding transcript ZmCRK10-T04 was inserted downstream of the UBI promoter and between the 3HA tag via homologous recombination, and expressed by the UBI promoter. Figure 1 Electrophoresis and sequencing analysis showed that the maize ZmCRK10-T04 overexpression vector, named pZZ0153-UBIp-ZmCRK10-T04-3HA, was successfully obtained. The specific experimental steps are as follows:

[0061] 1. Amplification of the deep-seated transcript ZmCRK10-T04:

[0062] A pair of homologous recombination primers was designed based on the cDNA sequence of ZmCRK10-T04 for PCR amplification. The primer sequences are as follows:

[0063] ZmCRK10-T04-F0: 5'-GTCGACAAACGCACTAGTATCCCGGGA

[0064] TGGATTTCGACCCTAAGCTC-3',

[0065] ZmCRK10-T04-R0: 5'-GGGTACATTTGGCGCGCCTTCCCGGGTC

[0066] TAGGATACAGTTCACTGAGTGTA-3';

[0067] Gene amplification was performed using Vazyme Phanta Max Super-Fidelity DNA polymerase to obtain PCR products. Sequencing yielded the deep-seated transcript ZmCRK10-T04, whose CDS nucleotide sequence is shown in SEQ ID NO: 3. The reaction system was as follows (total volume 20 μL):

[0068] 2×Phanta Buffer 10μL dNTP 0.4μL ZmCRK10-T04-F0 0.8μL ZmCRK10-T04-R0 0.8μL Phanta max 0.4μL cDNA (maize B73) 1.5μL <![CDATA[ddH2O]]> 6.9μL

[0069] The reaction program was as follows: 95℃ for 3 min; 95℃ for 15 s; 58℃ for 30 s; 72℃ for 60 s; 72℃ for 5 min; 35 cycles.

[0070] 2. Linearization of vector preparation: The pZZ0153-UBIp-3HA vector was linearized by digesting the restriction endonuclease XmaI.

[0071] 3. Detection and recovery of PCR products and vector enzyme digestion products: Agarose gel electrophoresis was used to detect PCR products and vector enzyme digestion products. The target fragment was recovered using the Omega Bio-tek Gel Extraction Kit;

[0072] 4. Recombination of Gel Recycling Products: Homologous recombination was performed using the Vazyme ClonExpress II One Step Cloning Kit; the reaction system is as follows:

[0073] Linearized carrier 200ng Insert fragment 120ng 5×CE II buffer 4μL Exnase II 2μL <![CDATA[ddH2O]]> to 20μL

[0074] Gently pipette and mix well. After a short centrifugation, collect the reaction solution at the bottom of the centrifuge tube. Incubate in a 37°C water bath for 30 minutes, then place on ice to cool to room temperature.

[0075] 5. Transformation of recombinant products: Transform the recombinant products into E. coli DH5α competent cells, following the guidelines for molecular cloning experiments.

[0076] 6. Sequencing Identification: Positive plasmids were selected and sequenced correctly using colony PCR and plasmid PCR, indicating successful acquisition of the maize ZmCRK10 overexpression vector pZZ0153-UBIp-ZmCRK10-T04-3HA. PCR and sequencing primers are as follows:

[0077] Primer F: 5'-CACATACTCAGACAGGCAGAGTTG-3',

[0078] Primer R: 5'-TCTGGAACGTCGTATGGG-3'.

[0079] Example 3: Obtaining Transgenic Maize Lines

[0080] The maize ZmCRK10 overexpression vector pZZ0153-UBIp-ZmCRK10-T04-3HA obtained in Example 2 was sent to Jiangsu Weimi Biotechnology Co., Ltd. for transformation. The transformation background was maize inbred line KN5585. T0 generation transgenic seeds obtained from the company were used to cultivate T1 generation plants. T1 positive plants were identified by DNA detection of the overexpression vector insertion, and seeds were harvested and cultivated to obtain T2 generation plants. PCR detection was performed on the T2 generation plants to confirm that the target gene had not undergone genetic segregation or loss. Finally, two positive homozygous transgenic maize families containing stable ZmCRK10-T04 inheritance and corresponding negative segregation materials were obtained. The PCR primers were the same as in Example 2.6.

[0081] Example 4: Detection of expression level of ZmCRK10-T04 transgenic maize plants

[0082] Both ZmCRK10-T04 overexpressing maize and wild-type maize were planted. Maize leaves from three-week-old plants were harvested, and RNA was extracted using the Trizol method. The extracted RNA was digested with DNase I and then reverse transcribed using Promega MLV reverse transcriptase. The extracted cDNA was then analyzed by RT-PCR. The maize Actin gene was used as a control.

[0083] The test results showed that the ZmCRK10-T04 gene was successfully transferred into maize. Figure 1 c).

[0084] The primers used in the detection process are as follows:

[0085] qZmCRK10-F: 5'-GGGACCTGAAGGCAAATAAC-3',

[0086] qZmCRK10-R: 5'-GTCCCAACAACTCTGCCTGT-3';

[0087] qZmActin-F: 5'-GCTGGATCTTGCTGGCCGTG-3',

[0088] qZmActin-R: 5'-AGGCGCCACGACCTTGATCT-3';

[0089] Example 5: Detection of deep-sowing tolerance phenotype in ZmCRK10-T04 transgenic maize

[0090] Currently, a sowing depth of 15 cm better reflects the deep-sowing tolerance of different varieties. Furthermore, using 27 randomly selected core population materials, different sowing depth treatments (10, 15, and 18 cm) were conducted. The results showed that under the 15 cm sowing depth treatment, the mesocotyl length had the highest correlation with the emergence rate, and the linear relationship showed the best fit (R0).2 =0.62), which can better reflect the emergence ability of seedlings sown at deep soil.

[0091] Therefore, ZmCRK10-T04 positive transgenic material, negative isolate, and background material KN5585 were simultaneously planted in pots (length × width × height = 50cm × 35cm × 25cm, with drainage holes at the bottom) with a 5cm layer of soil at the bottom, and covered with a 15cm layer of soil. Water was drawn from the bottom of the pot. After culturing the materials in a dark room at 28℃ for 10 days, the seedlings were carefully removed, the soil near the mesocotyl was removed to preserve the integrity of the mesocotyl as much as possible, and the mesocotyl length of the seedlings was photographed and measured. The results showed that overexpression of ZmCRK10-T04 significantly promoted the elongation of the maize mesocotyl under deep sowing treatment. Figure 2 a, b). Mesocotyl samples were taken 1 cm below the coleoptile and gel-sectioned to a thickness of 60 μm. The mesocotyl sections were stained with Calcofluor, and cell wall signals were observed under 405 nm excitation light. Figure 2 c) It was found that the length of mesocotyl cells in the overexpression material was significantly higher than that in the isolated negative control and background material. Figure 2 d).

[0092] In conclusion, ZmCRK10-T04 may positively regulate mesocotyl elongation in maize under deep-sowing conditions by promoting cell elongation. ZmCRK10-T04 transgenic maize exhibits stronger tolerance to deep sowing.

[0093] Example 6: Molecular marker for maize deep-planting tolerance, ZmCRK10 Type A Filtering and association analysis

[0094] 1. ZmCRK10 in the associated population Type A Genotyping and association analysis with mesocotyl length

[0095] Analysis of the ZmCRK10 sequences of maize materials B73 and SK, whose sequencing sequences have been published, revealed a transposon of approximately 121.7 kb in the promoter region of B73, while another transposon of approximately 63.3 kb with significant sequence differences was found in the promoter region of SK. Primers were designed based on the sequences to identify the transposon insertion status in the ZmCRK10 promoter region of the associated population. Figure 3 a). Used to identify 121.7kb (ZmCRK10) Type A The primer pair for the transposon is ZmCRK10. Type A -F1 / R1 is as follows (amplified partial sequence):

[0096] ZmCRK10Type A -F1:5'-ACCTCATCTCCTTCATGTGTGGTC-3',ZmCRK10 Type A -R1: 5'-GCATAGGTGGAATCGTCGCAG-3'.

[0097] The PCR amplification system is as follows (total volume 20 μL):

[0098] DNA 100ng 2×Taq mix 10μL <![CDATA[ZmCRK10 Type A -F1]]> 0.4μL <![CDATA[ZmCRK10 Type A -R1]]> 0.4μL <![CDATA[ddH2O]]> to 20μL

[0099] The reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s; 58℃ for 30 s; 72℃ for 45 s; 72℃ for 5 min; 35 cycles.

[0100] 2.ZmCRK10 Type A Association analysis between genotype and mesocotyl length and ZmCRK10-T04 transcript abundance in associated populations

[0101] Using the above primers, ZmCRK10 Type A -F1 / R1 was used for PCR identification of ZmCRK10 Type A Genotype, used to distinguish it from another genotype.

[0102] The results showed that ZmCRK10 Type A The mesocotyl length of the material with genotype 1 under deep seeding treatment was significantly higher than that of the other genotype 2. Figure 3 b). Structural variations in promoter regions often have a significant impact on gene expression levels. Analysis of existing leaf transcriptome data from associated populations revealed that ZmCRK10... Type A In the genotype materials, the abundance of the T04 transcript in ZmCRK10 was significantly higher than that in another genotype ( Figure 3 c). Explanation of ZmCRK10 Type A Genotype may promote maize mesocotyl elongation by increasing the expression of the T04 transcript of ZmCRK10; therefore, the molecular marker ZmCRK10 for maize deep-planting tolerance was obtained. Type A The nucleotide sequence is shown in SEQ ID NO: 4.

[0103] Example 7F 2:3 ZmCRK10 in populations, BC3F3 populations and NIL populations Type A Natural variations affect corn's tolerance to deep planting.

[0104] Utilizing ZmCRK10 Type A Molecular markers of genotype ( Figure 3 a) and mesocotyl length phenotype, selecting parents to construct F1 strains 2:3 Phenotypic analysis was performed on the F8 segregating populations of the BC3F3, NIL, and BC3F3 populations (same as in Example 5) at a depth of 15 cm to verify the variant site.

[0105] The results showed that, under a seeding depth of 15cm, ZmCRK10 Type A Homozygous plants of the genotype have a longer mesocotyl ( Figure 4 This indicates that ZmCRK10 Type A Allelic variation is a superior allelic variation that promotes mesocotyl elongation and is suitable for deep-planting tolerance. It can be used as a molecular marker for deep-planting tolerance in maize to improve the efficiency of breeding deep-planting tolerant maize varieties.

[0106] Example 8: Identification of maize varieties with different mesocotyl lengths using kit primers

[0107] 1. Kit and instructions for identifying maize varieties with excellent deep-planting tolerance

[0108] The kit includes the molecular marker ZmCRK10 for obtaining maize deep-planting tolerance. Type A Primer pair ZmCRK10 Type A -F1 / R1, where primer pair ZmCRK10 Type A -F1 / R1 is:

[0109] ZmCRK10 Type A -F1:5'-ACCTCATCTCCTTCATGTGTGGTC-3',ZmCRK10 Type A -R1:5'-GCATAGGTGGAATCGTCGCAG-3'

[0110] The method for identifying maize varieties with excellent deep-planting tolerance using the above-mentioned kit includes the following steps:

[0111] (1) Extract DNA from the maize variety to be tested;

[0112] (2) Design the primer pairs as follows:

[0113] ZmCRK10 Type A-F1:5'-ACCTCATCTCCTTCATGTGTGGTC-3',ZmCRK10 Type A -R1:5'-GCATAGGTGGAATCGTCGCAG-3'

[0114] (3) Perform PCR amplification using the above DNA as a template;

[0115] (4) Electrophoresis: The amplification showed a target band of 403bp, indicating that the variety has a strong tolerance to deep sowing.

[0116] 2. Identification of maize varieties with different mesocotyl lengths using a kit.

[0117] Verify ZmCRK10 Type A Allelic variations can be used as molecular markers for maize deep-planting tolerance. Then, ZmCRK10 was selected using a kit. Type A and ZmCRK10 Type B Different genotypes of maize varieties were treated with a 15cm deep sowing depth. Figure 5 a) and b) include commonly used maize inbred lines Chang7-2, B73, BY804, and 18-599. Results show that ZmCRK10 Type A In maize varieties with deep planting treatment, the length of the mesocotyl was significantly higher than that of ZmCRK10. Type B Genotype maize varieties ( Figure 5 (c, d) This provides an effective reagent kit for the preliminary identification of maize varieties tolerant to deep planting.

[0118] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A deep-seated transcript ZmCRK10-T04 The application of the deep-planting tolerance protein ZmCRK10-T04 in improving the deep-planting tolerance of maize is characterized by: The deep-seed-resistant transcript ZmCRK10-T04 The CDS nucleotide sequence is shown in SEQ ID NO: 3; The amino acid sequence of the deep-seeding protein ZmCRK10-T04 is shown in SEQ ID NO:

2.

2. A deep-seated transcript ZmCRK10-T04 The application of the deep-planting tolerant protein ZmCRK10-T04 in the breeding of deep-planting tolerant maize varieties is characterized by: The deep-seed-resistant transcript ZmCRK10-T04 The CDS nucleotide sequence is shown in SEQ ID NO: 3; The amino acid sequence of the deep-seeding protein ZmCRK10-T04 is shown in SEQ ID NO: 2.

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