Positive regulatory factors associated with ear height of maize, SNP molecular markers and application thereof

Abstract

The present application belongs to the field of crop genetic breeding, and relates to a corn ear height regulating factor, in particular to a positive regulating factor related to corn ear height, a SNP molecular marker and application thereof. The regulating factor relates to a gene Zm00001d012763 ZmKRP16 ) composed of 872 amino acids and containing a kinesin-related family conservative functional domain, and a molecular biology function thereof has not been reported in corn. The present application studies the molecular biology function of the gene in regulating corn ear height, and provides a potential new gene resource for breeding a new excellent high-yield corn variety with suitable ear height.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and relates to the regulatory factors of maize ear height, especially the positive regulatory factors, SNP molecular markers and their applications related to maize ear height. Background Technology

[0002] Corn is one of the most important crops in my country. Currently, corn has wide applications in food, feed, beverages, and industry. Due to its versatility, high yield, and resistance to drought, cold, and pests, it has become one of the most widely cultivated crops in the world. Recent research indicates that increasing corn planting density is currently one of the most effective ways to increase yield. Modern research shows that ear height is a crucial indicator directly affecting corn planting density. Excessively high ear height can easily cause lodging, while excessively low ear height hinders the transport of photosynthetic products to the ear. Simultaneously, ear height significantly impacts the growth of corn stems and roots, forcing them to bear greater pressure. Therefore, developing precise functional molecular markers for ear height traits in corn breeding, rationally improving ear height traits, and formulating targeted breeding programs will help breeders develop new corn varieties tolerant to high planting densities, thereby increasing overall corn yield.

[0003] Ear height is a typical quantitative trait controlled by multiple genes. Its genetic mechanism is significantly influenced by dominant, additive, and environmental interactions, and it exhibits relatively high narrow-sense heritability, indicating that major genes contributing significantly to ear height are at play. Currently, QTL mapping has identified many QTL regions and candidate genes related to ear height. However, QTL regions typically span a large area, usually several centimorgans (cm), and include numerous candidate genes, making fine gene mapping difficult and the development of precise functional molecular markers challenging. Therefore, developing precise functional molecular markers for ear height in maize breeding is crucial for improving selection accuracy and accelerating variety development. This is of great significance for marker-assisted breeding and genetic improvement of related traits in maize. Patent CN202110064297.8 discloses three SNP molecular markers of the ZmRzf gene located on chromosome 8 of maize that are significantly associated with ear height, which can be used as auxiliary selection markers for ear height in maize breeding. Our research group previously discovered a negative regulatory factor associated with ear height, which is a C2C2-CO-like transcription factor. In order to further explore the molecular markers and regulatory factors associated with maize ear height, our research group has conducted in-depth research. Summary of the Invention

[0004] To further investigate key genes related to ear height, this invention proposes a SNP molecular marker, a positive regulator, and its application related to ear height in maize.

[0005] The technical solution of this invention is implemented as follows: Positive regulatory factors associated with ear height in maize, the positive regulatory factors comprising kinesin-related functional proteins.

[0006] Furthermore, the positive regulatory factor is a nucleotide sequence that is more than 90% similar to the coding region of SEQ ID No. 1 from 334 to 2952 bp.

[0007] Furthermore, the coding region sequence of the positive regulatory factor is shown as 334-2952bp in SEQ ID No. 1, and is named... ZmKRP16 Gene.

[0008] The aforementioned SNP molecular marker is located at the 2054th base C upstream of the cleavage site in the 9th intron region of the ZmKRP16 gene; the coding region sequence of the ZmKRP16 gene is the bases from 334 to 2952 bp in SEQ ID No. 1; the promoter region is the bases from 1 to 333 bp in SEQ ID No. 1.

[0009] Furthermore, the promoter region of the gene ZmKRP16 contains a base mutation (SNP), such as... Figure 1 As shown.

[0010] The SNP molecular marker of the above-mentioned positive regulatory factor is located in the 9th intron region of ZmKRP16. There is one SNP (C / T) in the low-ear position DH521 and the high-ear position inbred line 953. The SNP molecular marker is located in the 9th intron region of the gene ZmKRP16 at the 2054th base C upstream of the 10th exon.

[0011] The primer pair used to identify the above-mentioned SNP molecular markers is KRP16-F1 and KRP16-R1, wherein the KRP16-F1 sequence is shown in SEQ ID No. 2 and the KRP16-R1 sequence is shown in SEQ ID No. 3.

[0012] The above primer pairs are used to identify the position of maize ears. The steps are as follows: (1) Using the DNA genome of the maize plant to be tested as a template, and the above primer pairs as primers, PCR amplification was performed; (2) Compare the sequence of the PCR amplification product of step (1) with the nucleotide sequence of the reference sequence of YuDH521 (as shown in SEQ ID No.7) to determine the height of the ear.

[0013] If the sequence of the PCR amplification product is consistent with the nucleotide sequence of the reference sequence of YuDH521 (as shown in SEQ ID No. 7), then the maize plant to be tested is a maize plant with a low ear position.

[0014] The present invention has the following beneficial effects: 1. The regulatory factor involved in this application is gene Zm00001d012763 ( ZmKRP16 ) is a protein composed of 872 amino acids (as shown in SEQ ID No. 6) containing conserved functional domains of the kinesin-related family, and its molecular biological function has not yet been reported in maize. This invention investigates the molecular biological function of this gene in regulating ear height in maize and provides a potential new gene resource for breeding suitable high-yielding maize varieties with high ear height.

[0015] 2. The purpose of this invention is to provide a gene, ZmKRP16, that controls the ear height of maize, and to accurately isolate the candidate gene using genome-wide association analysis. This gene has the DNA fragment shown in SEQ ID No. 1. Increased accumulation of this gene mRNA leads to increased ear height in maize; therefore, cloning this gene contributes to understanding the molecular mechanism of maize ear height.

[0016] 3. This invention will be based on Figure 1 Primers designed based on the SNPs of the sequences shown, such as SEQ ID No. 2 and SEQ ID No. 3, can be applied to the identification of ear height in maize. This can predict the ear height of different germplasm materials and has important practical application significance for maize breeding. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1Evidence for ZmKRP16 regulation of ear height in maize. (A) Population structure analysis of an associated population of 381 inbred lines; (B) Genome-wide association analysis demonstrating a key region on chromosome 8 associated with ear height; (C) Association analysis of candidate genes demonstrating a highly significant SNP within the intron region of ZmKRP16 associated with ear height; (D) Haplotype analysis of ZmKRP16, with the top chart showing a significantly associated SNP within the intron region of ZmKRP16. The bottom left chart compares ear height in maize varieties with different haplotypes. The bottom right chart compares ZmKRP16 expression levels between haplotype 1 and haplotype 2 inbred lines. (E) Sequence analysis of DH521, a representative inbred line with low ear height, and 953, a representative inbred line with high ear height.

[0019] Figure 2 for ZmKRP16 Expression analysis of haplotype I representative inbred line DH521 and haplotype II representative inbred line 953. (A) Zm KRP16 expression levels in haplotype I representative inbred line DH521 and haplotype II representative inbred line 953. (B) Ear height of haplotype I representative inbred line DH521 and haplotype II representative inbred line 953.

[0020] Figure 3 To utilize overexpression transgenic technology, the transgene of ZmKRP16 significantly reduced the ear height phenotype of inbred line B104. (A) qRT-PCR analysis of ZmKRP16 demonstrated that the expression level of the overexpressing transgenic line was significantly higher than that of the wild-type B104. (B) Phenotypes of the control plant B104 and the positive transgenic plant overexpressing ZmKRP16 at maturity. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0023] Positive regulatory factors associated with ear height in maize, wherein the positive regulatory factors contain conserved functional domains of the kinesin-related family.

[0024] Furthermore, the positive regulatory factor is a nucleotide sequence that is more than 90% similar to the coding region of SEQ ID No. 1 from 334 to 2952 bp.

[0025] Furthermore, the coding region sequence of the positive regulatory factor is shown as 334-2952bp in SEQ ID No. 1, and is named... ZmKRP16 Gene.

[0026] Furthermore, the gene ZmKRP16 There are base mutations (SNPs) in the upstream region, such as Figure 1 As shown.

[0027] The SNP molecular markers of the aforementioned positive regulatory factors are located in ZmKRP16 The intron region contains one SNP (C / T) in the inbred line DH591 with a lower ear position and the inbred line 953 with a higher ear position. The SNP molecular marker is located at ZmKRP16 The 9th intron region of the gene is located at the 2054th base C upstream of the 10th exon.

[0028] The primer pair used to identify the above-mentioned SNP molecular markers is KRP16-F1 and KRP16-R1, wherein the KRP16-F1 sequence is shown in SEQ ID No. 2 and the KRP16-R1 sequence is shown in SEQ ID No. 3.

[0029] Example 1 This application relates to a positive regulator of maize ear height. ZmKRP16 Polymorphism was observed between the low-ear-position maize inbred line DH591 and the high-ear-position inbred line 953, showing differences in water level. To further verify this, the following experiments were conducted: An associated population was constructed using 381 maize inbred lines with significant differences in ear height. These 381 inbred lines were then sequenced using Illumina high-throughput resequencing, achieving an average genome coverage of 7.4-fold. By comparing the sequencing data with the B73 reference genome (V4), a total of 13,749,539 SNPs were identified. Based on these SNP data, genetic structure analysis was performed on the associated population. The 381 inbred lines were divided into four groups: the Lérid group, the Lancaster group, the Tangsipingtou group, and a germplasm group derived from American hybrids. Figure 1 A). Ear height was identified from 381 inbred lines in Zhengzhou over two seasons. Genome-wide association analysis (rMVP) was performed on ear height using a mixed linear model. Strong associations were found in candidate regions located on chromosome 8. Figure 1B), and further association analysis of candidate genes showed that the strongest SNP significantly associated with ear height was identified within the gene region of Zm00001d012763 ( Figure 1 C). The candidate gene within this interval was initially identified as Zm00001d012763. The full-length gene can be searched on MaizeGDB using the gene ID Zm00001d012763. This gene encodes a kinesin-related family protein located on chromosome 8 and is named ZmKRP16. This gene consists of 23 exons and 22 introns. One SNP in the 9th intron region significantly affects maize ear height. Using these two SNPs, 381 inbred lines were divided into two haplotypes, and the difference in ear height between the two haplotypes was statistically significant. Ten inbred lines from each haplotype were selected to analyze the expression level of ZmKRP16, showing that the expression level of haplotype I was significantly lower than that of haplotype II (…). Figure 1 D). Using the low-ear-position inbred line DH521 (haplotype I) and the high-ear-position inbred line 953 (haplotype II) as materials, the gene was cloned using cDNA reverse transcription of mRNA from DH521 and 953 as templates. Simultaneously, using the DNA from these two materials as templates, the upstream SNP sequences of this gene were isolated using primer pairs KRP16-F1 and KRP16-R1. Sequence difference analysis showed that there were significantly different SNPs in the upstream sequence of this gene between DH521 and 953. Figure 1 E).

[0030] The sequences of primer pairs KRP16-F1 and KRP16-R1 are as follows: KRP16-F1 (SEQ ID No. 2): 5'-TCCAAACCGAATCCCGCTAAA-3'; KRP16-R1 (SEQ ID No. 3): 5'-TGGCTCGCTCTCTAGGAACA-3'.

[0031] Therefore, the steps for applying the above primer pairs in identifying the position of maize ears are as follows: (1) Using the DNA genome of the maize plant to be tested as a template, and the above primer pairs as primers, PCR amplification was performed; (2) Compare the sequence of the PCR amplification product of step (1) with the nucleotide sequence of the reference sequence of YuDH521 (as shown in SEQ ID No.7) to determine the height of the ear.

[0032] If the sequence of the PCR amplification product is consistent with the nucleotide sequence of the reference sequence of YuDH521 (as shown in SEQ ID No. 7), then the maize plant to be tested is a maize plant with a low ear position.

[0033] Example 2 ZmKRP16 Positive regulation of corn ear height: To further clarify ZmKRP16 The expression levels of different types of representative inbred lines were measured. DH521, an inbred line with a lower ear position selected from haplotype I, and 953, an inbred line with a higher ear position selected from haplotype II. ZmKRP16 Gene expression status.

[0034] qRT-PCR technology was used (referencing Liu Huafeng, Identification of Ds insertion sites in maize Ac / Ds mutant library and study on drought resistance function of Ds mutant ZmC2H2-149, Doctoral dissertation, Henan Agricultural University, 2024) for analysis. ZmKRP16 Expression level analysis.

[0035] First, using Primer Premier 5.0 software, a pair of specific primers, qA3A1-F1 and qA3A10-R1, were designed based on the CDS sequence of the gene: qA3A1-F1: 5'-TGACAGTCGCCAACAGATCC-3'; qA3A10-R1: 5'-CTGCTACTGAGCAGCCATCA-3'; Using cDNA as a template and the housekeeping gene Tubulin as a control, PCR was performed: Tubulin-qF:CTACCTCACGGCATCTGCTATGT; Tubulin-qR: GTCACACACACTCGACTTCACG; The reaction system is as follows: qPCR reaction procedure (three-step method): The results showed that the leaves at the 9-leaf stage of maize growth and development... ZmKRP16 The abundance of haplotype I representative inbred line DH521 was significantly lower than that of haplotype II representative inbred line 953. Figure 2 Therefore, it is proposed that... ZmKRP16 It acts as a positive regulatory factor to regulate the ear height of maize.

[0036] Example 3 ZmCOL6 The overexpression of transgenic genes was used to verify their gene function. 1. Construction of overexpression vectors according to ZmKRP16A pair of primers, KRP16-F2 and KRP16-R2, were designed based on the coding region sequence of the gene, with BglII and SpeI restriction sites added to the 5' ends of the primers, respectively. These primers were used to amplify the DH521 material. ZmKRP16 The cDNA sequence was obtained, and the PCR product was detected by agarose gel electrophoresis. The recovered target PCR product was ligated into the pMD18-T vector, transformed, and single clones were selected. PCR detection and sequencing were performed, and plasmids were extracted from the correctly sequenced bacterial cultures. The plasmid and the pCAMBIA1304 vector plasmid were double-digested using Bgl II and SpeI restriction enzymes, respectively. The digested target fragments were recovered, and the vector fragment and the target fragment were ligated using T4 ligase. The ligation product was transformed into E. coli, positive clones were identified, and the recombinant plasmid was extracted and named pCAMBIA1304- ZmKRP16 The recombinant plasmid was transformed into Agrobacterium competent cells and used to transform the maize inbred line B104.

[0037] Primer KRP16-F2 (SEQ ID No. 4): '5- AGATCT ATGGGGAAGGTGGAAGACGA-3', the underlined part is the Bgl II restriction site; KRP-R2 (SEQ ID No. 5): 5'-AAGTTCTTCTCCTTT ACTAGT CTACCGCCTCTTAGGGGGTT-3', the underlined part is the SpeI restriction enzyme site. 2. Genetic transformation The method of Agrobacterium-mediated maize genetic transformation was adopted (refer to Wang Ping'an, Creation of transgenic maize materials resistant to maize rough dwarf disease based on RNA interference, Master's thesis of Henan Agricultural University, 2011).

[0038] Using 1.2-1.8 mm immature embryos as recipient material, maize immature embryos were infected with Agrobacterium LBA4404 under conditions of OD600 value of 0.5 and infection time of 10 minutes. After co-culture, resting culture, callus induction, low-pressure screening of callus (PPT concentration 3 mg / L), and high-pressure screening (PPT concentration 6 mg / L), regenerated plants were obtained. The regenerated plants were phenotypically screened with a herbicide concentration of 200 mg / L (PPT), and seedlings sensitive to herbicide were removed. Total DNA was extracted from the leaves of herbicide-insensitive seedlings, and PCR detection of plant marker genes (Bar) and target genes was performed. Positive plants were transplanted to the field and self-pollinated to obtain T1 generation seeds, thus completing the identification process of transgenic T1 generation.

[0039] In this embodiment, a total of 7 independent transgenic positive plants were obtained. Genetically stable transgenic positive plants were obtained through multiple generations of continuous self-pollination. Phenotypic identification in the field showed that the ear height of the positive transgenic lines was significantly lower than that of the wild type. Figure 3 A).

[0040] Example 4 ZmKRP16 Positive regulation of corn ear height: ZmKRP16 We investigated the expression of the ZmKRP16 gene because it reduced ear height in transgenic lines and increased ear height in wild-type B104.

[0041] qRT-PCR technology was used (referencing Liu Huafeng, Identification of Ds insertion sites in maize Ac / Ds mutant library and study on drought resistance function of Ds mutant ZmC2H2-149, Doctoral dissertation, Henan Agricultural University, 2024) for analysis. ZmKRP16 Expression level analysis.

[0042] Amplification was performed using specific primers qA3A1-F1 and qA3A10-R1, with cDNA as a template and the housekeeping gene Tubulin as a control. qA3A1-F1: 5'-TGACAGTCGCCAACAGATCC-3'; qA3A10-R1: 5'-CTGCTACTGAGCAGCCATCA-3'.

[0043] The primers for the housekeeping gene Tubulin are as follows: Tubulin-qF:CTACCTCACGGCATCTGCTATGT; Tubulin-qR: GTCACACACACTCGACTTCACG.

[0044] The reaction system is as follows: qPCR reaction procedure (three-step method): The results showed that the leaves at the 10-leaf stage of maize growth and development... ZmKRP16 The expression level in the overexpressing transgenic lines was significantly higher than that in wild-type B104. Figure 3 B), indicating ZmKRP16 The expression level of this substance is high, and the ear position in maize increases, thus suggesting... ZmKRP16 It acts as a positive regulator of maize ear height.

[0045] This demonstrates that gene overexpression technology can stimulate endogenous genes in maize. ZmKRP16 Increased gene expression leads to increased ear height in maize, proving that it controls... ZmKRP1 Gene expression can be used to cultivate inbred lines with suitable ear height as the basic material for breeding, and to cultivate suitable hybrids with high ear height for production and application.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. ZmKRP16 The application of genes in regulating ear height in maize is characterized by, The steps are as follows: overexpression in the plants to be cultivated ZmKRP16 Genes were used to obtain maize plants with increased ear position; ZmKRP16 The amino acid sequence encoded by the gene is shown in SEQ ID No.

6.

2. The method according to claim 1 ZmKRP16 The application of genes in regulating ear height in maize is characterized by: The ZmKRP16 The nucleotide sequence of the gene is more than 90% similar to the coding region of SEQ ID No. 1, from 334 to 2952 bp.

3. The method according to claim 2 ZmKRP16 The application of genes in regulating ear height in maize is characterized by: The ZmKRP16 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

Images