CN120665897A - Zm00001eb038700 gene for regulating and controlling corn plant height and application thereof - Google Patents
Zm00001eb038700 gene for regulating and controlling corn plant height and application thereofInfo
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Abstract
The invention discloses a Zm00001eb038700 gene for regulating and controlling corn plant height and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the expression quantity of the gene is positively correlated with the height of corn plants. The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2, and the protein encodes a conserved C 2H2 type zinc finger protein Bud which possibly participates in the development regulation of internode so as to influence the plant height of corn. The 3801bp position at the downstream of the gene is SNP1-206,628,704 locus, when the locus genotype is CC, the corn shows dwarf character, when the genotype is changed into AA, the corn shows high stalk dominant character. The invention discloses a dose-response relation between the expression level of Zm00001eb038700 gene and the corn plant height for the first time, and the associated SNP can explain 3.57% of plant height phenotype variation. The invention develops a molecular marker combination combining SNP genotype and gene expression quantity, and obviously improves the accuracy of plant height prediction, so that the result of the invention provides technical support for breeding corn varieties with proper plant types and high yield.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a Zm00001eb038700 gene for regulating and controlling the plant height of corn on a corn chromosome 1 and application thereof.
Background
Corn plant height is a key agronomic trait that determines crop planting density, lodging resistance and biomass. In recent years, with the development of molecular biology techniques, a plurality of genetic loci regulating maize plant height have been identified, mainly including the following categories:
Plant hormone related genes such as auxin transporter genes (e.g., br 2), gibberellin synthesis genes, and the like. These genes affect stalk elongation primarily by regulating hormonal signaling pathways.
Cell elongation-related genes, such as genes encoding cell wall modifying enzymes, regulate internode elongation by altering cell wall plasticity.
Transcriptional regulatory factors, such as HD-ZIP-like transcriptional factors, affect stalk development by regulating downstream target gene expression.
However, existing studies have the following limitations:
(1) The identified plant height related genes are concentrated on known metabolic pathways, and the novel regulatory factors are less discovered;
(2) Most studies only focused on gene sequence variation, and the exploration of the gene expression regulation mechanism was insufficient;
(3) The traditional molecular markers are mostly based on single SNP loci, and the prediction accuracy is greatly influenced by the environment.
In addition, the prior art focuses on genetic sequence variation, and research on gene expression regulation is relatively insufficient. The invention discovers that the corn plant height can be effectively changed by regulating and controlling the expression level of the Zm00001eb038700 gene for the first time, and provides a new idea for plant height improvement.
Disclosure of Invention
In order to solve the problems, the invention provides a Zm00001eb038700 gene for regulating and controlling the plant height of corn and application thereof. The invention discloses a dose-response relation between the expression level of Zm00001eb038700 gene and the corn plant height for the first time, and the associated SNP can explain 3.57% of plant height phenotype variation. The invention develops a molecular marker combination combining SNP genotype and gene expression quantity, obviously improves plant height prediction accuracy, and provides a multi-level regulation strategy for the gene, including gene editing, expression regulation and the like.
In order to achieve the purpose, the invention provides the following technical scheme that the Zm00001eb038700 gene for regulating and controlling the plant height of corn is provided, the nucleotide sequence of the Zm00001eb038700 gene is shown as SEQ ID NO. 1, and the expression quantity of the Zm00001eb038700 gene is positively related to the plant height of corn.
The invention also provides a protein for regulating and controlling the plant height of corn, the protein is encoded by the gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The invention also provides a molecular marker for detecting the plant height property of the corn, wherein the SNP1-206,628,704 locus of the molecular marker is positioned at 3801bp of the downstream of the gene, when the genotype of the SNP1-206,628,704 locus is CC, the corn shows the dwarf property, and when the genotype is changed into AA, the corn shows the high stalk (suitable height) dominant property.
The invention also provides a product for detecting the molecular marker, and the product comprises a reagent, a kit or a gene chip. The product detects the genotype of the molecular marker or detects the expression level of the Zm00001eb038700 gene.
As a further description of the above scheme, the products include those prepared by PCR, qPCR, sanger sequencing, high throughput sequencing, fluorescent in situ hybridization method, taqMan probe method, ARMS-PCR method or KASP method for detecting the genotype of the molecular marker.
The invention also provides the use of said gene, said protein or said molecular marker, said use comprising any one of the following:
a) Corn plant height genetic diversity evaluation based on SNP1-206,628,704 locus haplotype analysis;
b) Constructing a molecular genetic map for integrating SNP1-206,628,704 sites and/or Zm00001eb038700 gene expression quantity;
c) Carrying out whole genome association analysis on the corn plant height by utilizing SNP1-206,628,704 locus and linkage markers thereof;
d) Identifying the dwarf maize variety by detecting SNP1-206,628,704 locus genotype and/or Zm00001eb038700 gene haplotype;
e) Auxiliary selective breeding based on SNP1-206,628,704 locus and/or associated haplotype molecular markers;
f) Corn plant height breeding with SNP1-206,628,704 locus as genome selection marker;
g) And (3) targeted editing of SNP1-206,628,704 locus and/or gene editing breeding for regulating and controlling Zm00001eb038700 gene expression.
The invention also provides a method for regulating and controlling the plant height of corn, which changes the gene editing target point by a gene editing technology, wherein the gene editing target point is selected from the group consisting of:
SNP1-206,628,704 site, zm00001eb038700 gene promoter region or Zm00001eb038700 gene coding region.
The invention also provides a corn plant high molecular breeding method, which comprises the following steps:
(1) Detecting genotypes of SNP1-206,628,704 sites in a corn sample to be detected;
(2) Detecting the expression level of the Zm00001eb038700 gene;
(3) And screening the corn varieties with target plant height traits by combining SNP genotype and gene expression data.
The invention also provides a corn plant height prediction model, which is used for establishing a multiple linear regression equation based on the Zm00001eb038700 gene expression quantity and SNP1-206,628,704 locus genotype and predicting the corn plant height phenotype.
The invention utilizes a temperate maize inbred line Ye107 with shorter plant height as a common male parent to hybridize with 1 temperate and 4 tropical/subtropical maize inbred lines as female parents respectively, so as to construct a maize multi-parent population with obvious plant height difference. The SNP1-206,628,704 which is located on chromosome 1 and is obviously related to the plant height is analyzed and positioned by utilizing the whole genome association analysis (GWAS), the site can explain 3.57% of plant height phenotype variation, the site is excavated to the functional gene Zm00001eb038700 for regulating and controlling the plant height of corn, and qRT-PCR results show that the gene is highly expressed in corn internodes, and the gene is proved to be highly related to the regulation and control of the corn plant height, so that the results of the invention provide technical support for molecular marker selection of proper plant types and high-yield corn varieties.
Compared with the prior art, the invention has the following beneficial effects:
1. novel plant height regulating gene
The gene function innovation is that Zm00001eb038700 (coding C 2H2 zinc finger protein) is discovered for the first time to regulate the plant height of corn, which is different from the known hormone related genes (such as auxin and gibberellin passage) or protein modification genes (such as ubiquitin hydrolase), and belongs to a new transcription regulation target.
2. Unique molecular marker design
Marker localization innovation SNP1-206,628,704 is 3801bp (non-gene internal) downstream of gene, and can affect enhancer or remote control element.
The invention develops a molecular marker system based on 5 haplotypes (Hap 1-Hap 5), wherein the Hap4 haplotype is obviously related to the high stalk character and is more accurate than a single SNP marker.
3. Specific application of tropical corn germplasm
Ecological suitability the marker has better performance in tropical/subtropical maize germplasm, and fills the blank of the prior art.
Environmental stability-verification in multiple environments such as Yunnan Jingfu and Yanshan, the association of SNP locus and phenotype is stable (PVE is 3.57%).
4. Multi-level technology integration
The composite detection system can be used for predicting plant height by combining SNP genotype, haplotype analysis and gene expression quantity.
Drawings
FIG. 1 is a NAM population line spectrogram with obvious plant height difference constructed by hybridizing 4 tropical/subtropical high stalk parents and 1 temperate dwarf parent Chang7-2 with excellent temperate dwarf parent Ye 107;
FIG. 2 is a plot of plant height phenotype frequency distribution and correlation matrix scatter of a multi-parent population under three environments;
FIG. 3 shows (A) SNPs marker density heat map, (B) phylogenetic tree of multiple parent population, (B) three-dimensional Principal Component Analysis (PCA), and (D) Linkage Disequilibrium (LD) attenuation map;
FIG. 4 is a Manhattan plot (left) and Q-Q plot (right) of analysis of significant SNPs of maize plant height using GWAS under different environments, (A) 21 scenic flood environment, (B) 22 Yanshan environment, and (C) 23 Yanshan environment (D) optimal linear unbiased predictive analysis (BLUP) of significant SNPs related to maize plant height;
FIG. 5 is a diagram showing 5 haplotypes of the plant height candidate gene Zm00001eb 038700. (a) represents 5 haplotypes of candidate gene Zm00001eb038700, (B) represents the distribution of 5 haplotypes of candidate gene Zm00001eb038700 in 5 RILs subgroups and the difference in regulatory maize plant height, representing p < 0.05, representing p <0.01, (C) depicts the relative positions of candidate gene Zm00001eb038700 and SNP-206,628,704;
Fig. 6 shows the relative expression levels of Zm00001eb038700 gene in different periods, a represents the 7 th internode growth development dynamic of 5 parents in four periods (V8, V14, VT, R3), B represents the relative expression level of Zm00001eb038700 gene in 5 parents, symbol represents the significance when P < 0.005, symbol represents the significance when P < 0.001, and ns represents the insignificant difference.
Detailed Description
In order to further illustrate the technical scheme of the invention, the Zm00001eb038700 gene for regulating and controlling the corn plant height and the application thereof provided by the invention are described in detail below with reference to the accompanying drawings and examples, but the invention is not to be construed as being limited by the scope of protection.
The invention provides a Zm00001eb038700 gene for regulating and controlling the plant height of corn, and the nucleotide sequence of the Zm00001eb038700 gene is shown as SEQ ID NO. 1.
Corresponding to chromosome 206614407-206626403 of maize No. 1 in genome version Zm-B73-REFERENCE-NAM-5.0. The amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 2, and the protein codes a conserved C 2H2 type zinc finger protein Bud, and can directly or indirectly regulate and control synthesis, transportation or signal transduction of auxin, thereby influencing the distribution and accumulation of the auxin in the stalk and further regulating internode elongation and plant height. SNP1-206,628,704 indicates the physical position (206,628,704 base site) of the SNP on chromosome 1 in the maize reference genome (B73 RefGen_v5).
Example 1 plant height phenotyping
Plant material, which is 917 RILs group constructed by crossing with 5 tropical/subtropical/temperate inbred lines (YML 32, CML171, TML418, NK40-1, chang 7-2) by taking temperate dwarf inbred line Ye107 as common male parent, the parent information is shown in Table 1.
TABLE 1 parental information
FIG. 1 is a constructed multi-parent population consisting of 5 subgroups of population 1 (YML 32 XYe 107), population 2 (CML 171 XYe 102), population 3 (TML 418 XYe 102), population 4 (NK 40-1 XYe 107) and population 5 (Chang 7-2 XYe 107), containing a total of 917F 8 RILs. The distribution is as follows, population 1 (173), population 2 (190), population 3 (176), population 4 (180) and population 5 (198). The parents and recombinant inbred lines of these multiple parent populations are planted.
Planting environment 2021 scenic flood (altitude 552.7 m), 2022-2023 years Yanshan (altitude 1572 m), adopting random block design, and 3 times biological repetition.
The trial used a Randomized Complete Block Design (RCBD) with three biological replicates per environment. Each test area has a row length of 3.5 meters, a row spacing of 70 cm and a plant spacing of 25 cm, and each row has 14 plants. After 20d of flowering of the maize plants, 5-10 plants in each cell were randomly selected and the plant height (PLANT HEIGHT, PH) from the ground to the top of the tassel was measured in centimeters (cm).
Genetic force analysis
After systematic sorting and quality control of the phenotype data collected three consecutive years, statistical analysis of the data was performed using SPSS (SPSS STATISTICS). Including calculating mean, minimum, maximum, standard Deviation (SD), coefficient of Variation (CV), skewness, and kurtosis, and using kurtosis and skewness to evaluate the normal distribution of the phenotypic data. The pearson correlation coefficient analysis and the fabrication of correlation graphs were performed using Origin (Origin 2022) software. Generalized genetic forces are calculated with reference to the method of Knapp et al.
The experimental results show that the plant height phenotype data of the 5 RILs (Recombinant Inbred Lines, recombinant inbred line) group are subjected to preliminary statistical analysis, and the obvious difference exists between the plant heights (156.0-216.0 cm) of all female parents and the plant height (155.0 cm) of the male parent Ye 107. The strain heights of RILs populations also exhibited a broad range of phenotypic variations (fig. 1). The strain height phenotype data for the 5 RILs populations were descriptive statistically shown in table 2. Frequency distribution analysis of plant height phenotype data shows that the skewness and kurtosis absolute values of all populations in different environments are smaller than 1, normal distribution characteristics (table 2 and figure 2) are met, and the precondition of quantitative trait genetic analysis is met. Population 1-population 5 had a broad range of plant heights, 97.9%, 96.9%, 98.3%, 97.5% and 96.2%, respectively, and Pearson correlation analysis (p < 0.05 significance level) showed that there was a very significant positive correlation (0.94-0.97) between plant heights of RILs in the same population in three different environments (fig. 2), indicating that maize plant heights were predominantly regulated by genetic factors, with less environmental impact.
TABLE 2 descriptive statistical analysis of the plant heights of the multiple parent populations
Note that 21 scenic floods, 22 Yanshan and 23 Yanshan represent trials performed in 2021 at scenic floods and 2022 and 2023 at Yanshan, respectively. H 2 generalized genetic transmission.
Example 2 SNPs Density distribution, phylogenetic Tree analysis, principal Component Analysis (PCA) and linkage disequilibrium analysis
DNA extraction and genome sequencing
Genomic DNA was first extracted from young leaves of F 8 RILs maize using the cetyl trimethylammonium bromide (CTAB) method. DNA purity was assessed using a nanophotometer (IMPLEN, CA, USA) and DNA concentration was measured using a QubitTMDNA Assay Kit detection kit and a Qubit 2.0 fluorometer (Life Technologies, USA, grand Island, NY, USA). In library preparation, 1.5 μg of high quality DNA was processed per sample using TruSeq nano DNA HT sample preparation kit (Illumina, USA). DNA was fragmented to an average 350bp size by sonication, followed by end repair, 3' adenylation and Illumina adaptor ligation. The adaptor-ligated DNA was amplified by Polymerase Chain Reaction (PCR) and the resulting library was purified using the AMPure XP system. Library fragment size distribution was verified using an Agilent 2100 bioanalyzer and quantified by qT-PCR. Sequencing was performed on Illumina NovaSeq 6000 platforms, yielding 150bp double ended sequencing data with an insertion length of 350bp. Raw sequencing data was subjected to Quality Control (QC) filtering, and CLEAN READS was aligned with reference genome B73 (refgen_v5) using BWA (Burrows-WHEELER ALIGNER) software to identify all genetic variations across the genome. And SNPs were called using Genome Analysis Toolkit software, PLINK v1.9 filter SNP, parameters were set to-geno 0.2 and-MAF 0.05, SNPs with deletion rates greater than 10% and Minimal Allele Frequencies (MAF) less than 5% were removed. And finally, carrying out functional annotation on the SNP obtained by screening by using ANNOVAR software.
Phylogenetic tree, PCA and linkage disequilibrium analysis
And calculating a distance matrix by adopting a maximum reduction method (Maximum parsimony, MP) to construct a phylogenetic tree. The present invention uses GCTA software for Principal Component Analysis (PCA) and GAPIT software package in R software for cluster analysis, followed by visualizing the PCA results through a scatter plot. And calculating linkage disequilibrium degree (r 2) between every two SNP markers within the whole genome range by using PopLDdecay software, and visualizing LD attenuation analysis results by using a software self-contained script plot_OnePop.pl. Based on the LD decay pattern, the number of markers required for GWAS and their detection efficiency were further evaluated.
The experimental result is that the WGS technology is adopted to scan the whole genome of the corn, 6,389,682 high-quality SNPs are totally identified on 10 chromosomes, and are distributed on 10 chromosomes of the corn. The marker densitometry of SNPs showed that the number of SNPs on chromosome 1-10 was 891,814, 699,810, 717,023, 880,707, 669,104, 485,237, 564,060, 545,295, 473,708, 462,924, respectively. Wherein the number of SNPs on chromosome 1 is the largest and the number of SNPs on chromosome 10 is the smallest (FIG. 3A). Phylogenetic analysis showed that phylogenetic trees constructed using the adjacency method can be divided into 5 significantly different subspecies (fig. 3B). The results of PCA are highly consistent with phylogenetic classification, with five different subgroups (fig. 3C). 6,389,682 high quality SNPs were used for LD decay analysis, and it was found that when r 2 was reduced to a plateau threshold of 0.3, the physical distance of LD decay was approximately 20 kb (FIG. 3D). Based on this feature, the present invention screens candidate genes within 20 kb upstream and downstream of significant SNPs. The above results indicate that whole genome association analysis can be further performed.
EXAMPLE 3 Whole genome correlation analysis
After Illumina NovaSeq-6000 sequencing, BAM files were processed and then subjected to a whole genome association study (GWAS) of PH using Mixed Linear Model (MLM) in GEMMA software. In the GWAS analysis process, individual relationships and population stratification are the major contributors to false positives. Therefore, the study adopts MLM to carry out marker-character association analysis, takes the genetic structure of the population as a fixed effect and the genetic relationship of the individuals as a random effect so as to correct the influence of the genetic structure of the population and the genetic relationship of the individuals. Minor allele frequencies (minor allele frequency, MAF) >5% and r 2 <0.2 were used to screen for PH-related SNPs, set to plink-indep-pariewise 5050.2. Statistical significance criteria for a SNP significance threshold of-log 10 (p) > 6 were calculated by the formula-log 10 (1/SNP total number), and SNP sites that were significantly correlated to PH were identified based on the threshold. The quantile-quantile (Q-Q) diagram and manhattan diagram were then plotted using R software (v4.3.3). And significant SNP sites were screened by Bed Tools v 1.7. Based further on the B73 v5 reference genome and annotation information, candidate genes associated with PH were identified within 20kb upstream and downstream of the significant SNPs.
Experimental results GWAS analysis was performed using a mixed linear model (Mixed Linear Model, MLM) based on the phenotype data of 917F 8 RILs and 6,389,682 high quality SNPs in the multiple parental populations. A number of SNPs significantly associated with PH were co-detected at a significance threshold of-log 10 (p) >6, with 1-206,628,704 being detected in all environments (21 scenic floods, 22 Yanshan, 23 Yanshan and blu), with a higher p-value for SNPs 1-206,628,704 of 6.67-8.07 and pves (phenotypic variation interpretation) of 2.31% -3.57%. The SNP locus provides an important clue for further excavating related candidate genes for regulating and controlling the PH of the corn. SNP1-206,628,704 indicates that the SNP is at the 206,628,704 th base site on chromosome 1 in the maize reference genome (B73 RefGen_v5).
Example 4 identification and functional annotation of candidate genes
Identifying SNPs which are obviously related to PH based on GWAS analysis and LD attenuation results, locating SNP physical positions by using BLAST functions in the Maize GDB, mining candidate genes within 20kb range on the upstream and downstream of the obvious SNPs, and functionally annotating the screened candidate genes by using NCBI, maize GDB, interPro and Unit databases, thereby screening SNP loci and candidate genes which are obviously related to PH of corn.
Experimental results this study screened candidate genes that regulated Maize PH within 20kb upstream and downstream of the significant SNP1-206,628,704 using the B73 -RefGen- v5 reference genome and functionally annotated the candidate genes using the maze GDB, interPro, uniProt, NCBI, and other databases. The invention identifies candidate functional genes Zm00001eb038700 on chromosome 1. Wherein the gene Zm00001eb038700 is 3801bp (FIG. 5C) located upstream of SNP1-206,628,704, and the sequence of the gene Zm00001eb038700 is shown as SEQ ID NO: 1. The Zm00001eb038700 gene codes a conserved C 2H2 type zinc finger protein Bud, the zinc finger protein is a protein superfamily, has a typical zinc ion binding domain, can specifically identify and bind nucleic acid, and plays an important role in regulating and controlling plant growth and development and environmental adaptation.
Example 5 haplotype analysis of candidate Gene Zm00001eb038700
And carrying out haplotype analysis on the screened candidate genes by using software Haploview V4.2. Significant haplotype blocks are identified based on LD analysis, and group comparison is performed according to plant height phenotype data corresponding to each haplotype of the candidate genes. Haplotype-phenotype association box plots were drawn using the ggplot package in R software (V4.3.3) and the significance of phenotype differences between different haplotypes was assessed.
As a result of the experiment, haplotype analysis was performed on the candidate gene Zm00001eb038700 determined by analysis of GWAS under various environments (FIG. 5). The bases at positions 206615938, 206615941, 206616123, 206616156, 206616181, 206616210, 206617611, 206617660, 206617686 and 206617726 of the Zm00001eb038700 gene show five haplotypes (fig. 5A):
Hap1: ACTGGCTTGG;
Hap2: ACCGGCCCTG;
Hap3:GCCGATCCTG;
Hap4:ATCTACCCGG;
Hap5: ATCTACCCGA. Of these, the line with Hap4 was significantly higher in height than the lines with other haplotypes (fig. 5B), and was thus considered to be an advantageous haplotype of the Zm00001eb038700 gene.
Example 6 analysis of relative expression of candidate Gene Zm00001eb038700 in parents
The present invention samples the middle of the 7 th internode of the 5 parental lines at four developmental stages of maize 8 leaf stage (V8), rapid growth stage (V14), male withdrawal stage (VT) and milk maturation stage (R3) (fig. 6A). Total RNA was extracted using the Tiangen RNAprep Pure plant Kit kit and genomic DNA removal and cDNA synthesis were performed using the FASTKING RT KIT (WITH GDNASE) kit. The relative expression levels of 4 candidate genes in 5 parental lines were determined by real-time fluorescent quantitative PCR (qRT-PCR) technique based on the root SuperReal PreMix Plus (SYBR Green) kit (root, beijing). The maize Actin 1 gene (action) was used as an internal reference gene. qRT-PCR was performed using procedures and systems strictly referring to the standardized procedure used by Bi et al. Each sample was set up with 3 technique replicates and usedThe method calculates the relative expression amount of the genes.
Experimental results in order to study the regulation and control effect of candidate gene Zm00001eb038700 on corn PH, qRT-PCR was used to analyze the expression patterns of 5 parents in 4 different development stages of corn. Internode length measurements indicated that the internode length of the parent NK40-1 was longest at all times, while Ye107 remained the shortest (fig. 6A). Therefore, the invention takes the dwarf parent Ye107 as a reference system. The analysis result shows that the expression level of the gene Zm00001eb038700 in the parents YML32, CML171, TML418 and NK40-1 is obviously increased from V8 to V14, and V14 is the most critical period for stem growth and development. And the relative expression level of gene Zm00001eb038700 in NK40-1 and CML171 was significantly higher than YML32 and TML418 (fig. 6B). This pattern of expression is consistent with the trend of the plant height phenotype size distribution of NK40-1 and CML171, indicating that the gene Zm00001eb038700 may have a positive regulatory effect on maize plant height.
The amino acid sequence of the protein coded by the Zm00001eb038700 gene is shown as SEQ ID NO. 2, the protein codes a conserved C 2H2 type zinc finger protein Bud, the zinc finger protein is a protein superfamily, has a typical zinc ion binding domain, can specifically identify and bind nucleic acid, and can also directly or indirectly regulate and control synthesis, transportation or signal transduction of auxin, thereby influencing the distribution and accumulation of the auxin in the stalks and further regulating the internode elongation and plant height.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
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