CN114196685A - Corn stalk puncture strength gene ZmFLS2 and application of molecular marker thereof - Google Patents

Corn stalk puncture strength gene ZmFLS2 and application of molecular marker thereof Download PDF

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CN114196685A
CN114196685A CN202111575000.0A CN202111575000A CN114196685A CN 114196685 A CN114196685 A CN 114196685A CN 202111575000 A CN202111575000 A CN 202111575000A CN 114196685 A CN114196685 A CN 114196685A
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李鹏程
武婷婷
王后苗
吉伟东
汤潇
徐暑晖
杨泽峰
徐辰武
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Yangzhou University
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Abstract

The invention discloses a corn stalk puncture strength gene ZmFLS2 and application of a molecular marker thereof, belonging to the field of molecular biology, wherein the corn ZmFLS2 gene has the function of regulating and controlling the corn stalk puncture strength, the nucleotide sequence of the corn ZmFLS2 gene is shown as SEQ ID No.1 and is positioned at 91bp upstream of S5_210155797, the SNP molecular marker is positioned at-397 bp of the corn ZmFLS2 gene, and the basic group of the site is G or A. According to the invention, a gene ZmFLS2 related to the stalk puncture strength character is screened from a corn genome by a whole genome association analysis (GWAS) technical method, and the gene plays a very key role in regulating and controlling the stalk strength of corn, and the discovery is possibly helpful for clarifying the genetic basis of the stalk strength; in addition, the identified candidate genes and variations have potential breeding value and can be used for molecular marker-assisted selection breeding to improve the lodging resistance of the corn.

Description

Corn stalk puncture strength gene ZmFLS2 and application of molecular marker thereof
Technical Field
The invention relates to the field of molecular biology, in particular to a corn stalk puncture strength gene ZmFLS2 and application of a molecular marker thereof.
Background
Corn (Zea mays L) is an important grain, economic and feed crop in China and plays a significant role in the whole national economy. With the increase of the planting density of the corn and the frequent occurrence of natural disasters, the lodging of the corn tends to be aggravated, and the yield of the corn is reduced by about 108kg/hm every time the lodging rate of the corn is increased by 1 percent2The corn yield loss caused by lodging is 5 to 20 percent in every year in the world. Lodging not only seriously affects the yield and quality of corn, but also brings obstacles to mechanical harvesting of corn, and is one of important limiting factors for high yield, stable yield and high quality of corn. The research on the genetic mechanism of the lodging-resistant related traits of the corn has important guiding significance for breeding new lodging-resistant varieties, reducing lodging hazards and ensuring high and stable yield of the corn.
Corn lodging is divided into two types, namely root lodging and stem folding, wherein the stem folding has the greatest influence on yield. The factors influencing the lodging of corn are numerous, and the characteristics of plant type, stem quality and the like are the most important factors. A large number of researches show that the plant height and the ear height are significantly negatively related to the lodging resistance of the corn, and a breeder mainly breeds corn strains with lower plant height and ear height to improve the lodging resistance of the corn; a plurality of subject groups research the relationship between plant types and corn lodging resistance by using different corn materials, and all discover that the lodging of corn is obviously related to stem thickness, plant height, ear height, the number of nodes on the ear position and the stem thickness below the ear position, and the plant height and the stem thickness are main factors influencing the lodging of the corn stems. The quality of the stalk is an important index influencing the lodging resistance of the corn. Corn Stalk quality can be measured by properties such as Stalk vertical crush strength (SCS), Stalk crust puncture strength (RPS), and Stalk Bending Strength (SBS). Zuber et al performed high-direction and low-direction selection on stalk crushing strength by using two corn groups as materials, and research results show that the stalk crushing strength and the corn lodging resistance are in a significant positive correlation (Zuber et al, 1980); the puncture strength of the hard skin of the stems is highly related to the lodging resistance of the stems, and the measuring method is simple and convenient, so the method is widely applied to the corn lodging resistance genetic research (Li et al, 2014; Flint-Garcia et al, 2003 b; Peiffer et al, 2013). The traits have wide genetic variation in the inbred line, are main factors of corn lodging resistance and can be improved in the breeding process.
Maize lodging-resistance-related traits are controlled by multiple genetic loci and are complex quantitative traits (Radu et al, 1994). At present, researchers at home and abroad discover a large number of QTLs (quantitative trait loci) with plant height and spike height by utilizing a plurality of genetic groups, a corn genome website (www.maizegdb.org) already collects 235 QTLs with plant height and spike height, and the loci are distributed on 10 chromosomes of corn and are mostly micro-effect QTLs; to date, several maize plant height mutant genes have been cloned, such as Dwarf3, Dwarf8, Dwarf9, Brachytic2, and ZmGA3ox2, among others (Winkler and Helentjaris, 1995; Lawit et al, 2010; Multani et al, 2003; Teng et al, 2013). There has also been some progress in genetic analysis of maize stalk quality. Flint-Garcia et al utilize 4F2:3QTL analysis is carried out on the stalk puncture strength by the colony, and the total position is located to 36 QTL (Flint-Garcia et al, 2003b) which are distributed on 10 chromosomes of the corn; li and the like carry out QTL analysis on the stem puncture strength of two sets of recombinant inbred line populations (RIL), detect 7 QTL in total, and finely position a major locus qRPR3-1 within a 3.1Mb interval (Li et al, 2014); hu et al performed QTL analysis of stalk bending strength and detected 7 QTLs in total under four environmental conditions, these sites being able to account for 17.2% to 26.1% phenotypic variation (Hu et al, 2012).
Although the QTL positioning research of the lodging resistance related traits of the corn has made a certain progress, the lodging resistance of the corn is influenced by a plurality of traits, the genetic basis is complex, and the difference exists in different genetic backgrounds. Therefore, the deep research on the genetic diversity of the corn and the excavation of new genes of the corn are of great significance.
Disclosure of Invention
The invention aims to provide a corn stalk puncture strength gene ZmFLS2 and application of a molecular marker thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides application of a corn ZmFLS2 gene in regulating and controlling corn stalk puncture strength, wherein a nucleotide sequence of the corn ZmFLS2 gene is shown as SEQ ID No. 1.
Further, the maize ZmFLS2 gene is located 91bp upstream of S5_ 210155797.
The invention also provides an SNP molecular marker for selecting the corn stalk puncture strength character, the SNP molecular marker is positioned at-397 bp of the corn ZmFLS2 gene, and the basic group at the position is G or A.
The invention also provides application of the SNP molecular marker in selection of the corn stalk puncture strength character.
Further, the stalk penetration strength of the strain carrying the G allele was higher than that of the strain carrying the a allele.
The invention also provides a method for screening or identifying the puncture strength of the corn stalks, which analyzes the genotype of the site at the ZmFLS2 gene-397 bp position of the corn according to the SNP molecular marker and judges the puncture strength of the corn stalks to be detected according to the genotype.
Further, the stalk penetration strength of the strain carrying the G allele was higher than that of the strain carrying the a allele.
The invention also provides an application of the corn ZmFLS2 gene or the SNP molecular marker in corn breeding, wherein the nucleotide sequence of the corn ZmFLS2 gene is shown as SEQ ID No. 1; further, the corn is used for enhancing the lodging resistance of corn.
The invention discloses the following technical effects:
according to the invention, 3 characters (stalk puncture strength RPR, stalk crushing strength SCS and stalk bending strength SBS) related to stalk strength and 4 characters (plant height PH, ear height EH, stalk thickness SD and stalk length SL) related to plant type are analyzed through 3 field tests, and the characters are detected to have great phenotypic variation. Genome-wide association analysis (GWAS) was performed using 372331 Single Nucleotide Polymorphisms (SNPs) using a general linear model and a mixed linear model. A total of 94 Quantitative Traits (QTL) were detected, including 241 SNPs. Furthermore, combining GWAS data and gene expression profiles, 60 candidate genes were identified within a 50kb range of significant SNPs, including some genes encoding flavonol synthase (GRMZM2G069298, ZmFLS2), nitrate reductase (GRMZM5G878558, ZmNR 2). The GRMZM2G069298 is subjected to resequencing in all test materials to find 2 variants which are obviously related to RPR, so that the gene plays a very key role in regulating and controlling the stalk strength of the corn, and the finding is possibly helpful for explaining the genetic basis of the stalk strength; in addition, the identified candidate genes and variations may be associated with genetic improvement in lodging resistance in maize.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the relevant loci and candidate genes of RPR on chromosome 5; wherein, (A) a Manhattan plot of the RPR on chromosome 5; (B) genes around the peak signal (± 50 kb); (C) gene-associated localization of GRMZM2G069298(ZmFLS 2); (D) phenotypic differences of RPR between different alleles of GRMZM2G069298(ZmFLS 2);
FIG. 2 shows the expression profiles of candidate genes at different developmental stages.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The sequence of the maize ZmFLS2 gene is shown in SEQ ID No. 1.
SEQ ID No.1:
>GRMZM2G069298
ATGGGGGGCGAGACGCACCTGAGCGTGCAGGAGCTGGCGGCGTCGCTGGGCGCGCTACCGCCGGAGTTCGTGCGGTCTGAGCAGGACCAGCCGGGCGCGACCACGTACCGCGGGGCCGCCGTGCCGGACGCGCCGGTGATCGACATGTCGGAGCCCGGGTTCGGCGCGCGCATGGCCGCAGCCGCCAGGGAGTGGGGGCTGTTCCAGGTGGTGAACCACGGCGTGCCCTCAGCGGCGGTGGCGGAGCTCCAGCGCGTCGGGCGGGCCTTCTTCGCGCTTCCGACGGAGGAGAAGGAGCGCTACGCCATGGACCCGGCGTCCGGCAAGATCGAGGGCTACGGCACCAAGCTGCAGAGGGACCTCGAGGGCAAGAAGACGTGGAACGACTTCTTCTTCCACGTCGTCGCGCCGCCGGAGAAGGTGGACCACGCCGTCTGGCCCCGGAGCCTCGCCGGGTACAGGGAGGCCAACGAGGAGTACTGCCGCCACATGCAGCGCCTGACGCGCGAGCTGTTCGAGCACCTCTCGCTGGGGCTGGGGCTCCACGGGAGCGCCATGGCGGAGGCGTTCGGCGGAGACGGCCTGGTGTTCCTGCAGAAGATCAACTTCTACCCGCCGTGCCCGCAGCCGGAGCTCACGCTCGGCGTCGCGCCGCACACCGACATGAGCACGCTCACCGTCCTCGTGCCCAACGAGGTGCAGGGGCTCCAGGTCTTCAAAGATGGTCAATGGTACGAGGCCAAGTACGTGCCCGACGCACTCATCGTCCATATCGGCGATCAGATCGAGGCAAGCGACCTACGTAGCATCTTTTTTTTTCTTTCTATTGTGTTGTGATGATCTTGCTCCCCTCAAGTATTTCTTTGATGTCCCGATCGAGTAGATTTTCAGCAACGGGGCATACAAGGCGGTGCTGCACCGTACGACGGTGAACAAGGAGAAGACGCGGATGTCATGGCCGATGTTCGTGGAGCCGCCGGGGGAGCTCGTCGTCGGGCCGCACCCCAAGCTGGTCACGGAGGAGAGCCCGGCCAAGTACAAGGCCAAGAAGTACAAGGACTACCAGCACTGCAAGATCAACAAGCTCCCCATGTAA
Example 1
1. Materials and methods
1.1 plant Material and phenotypes
The related population included 345 maize inbred lines from different regions of china. These inbred lines were field tested three times in three in 2015 to 2017. Each field experiment adopts a completely random block design, a single row block design and twice repetition. Each row comprises 13 plants, 3.5 meters long and 0.5 meter wide. At the flowering stage, 9 strains per line were randomly selected from each replicate for phenotypic identification. The average value of 7 characters of stem thickness, stem length, plant height, spike height, straw bending strength, straw crushing strength and straw puncture strength is calculated. Except for plant height and ear height, other characters enter the middle part of the third internode flat edge by analyzing the third internode at 2 weeks after floweringAnd (4) carrying out measurement. Specifically, the bending strength of the straw, the crushing strength of the straw and the puncture strength of the straw were measured using different pressure sensors of a YYD-1 instrument (topun agriculture science and technology ltd, zhejiang, china). Data in N/mm2(stalk puncture strength) and N (stalk crush strength and stalk puncture strength) are recorded. Each phenotypic analysis was performed according to a linear model: y isijk=μ+Gi+Ej+GEij+Bk(Ej)+eilkWhere μ represents the population mean, GiRepresenting a genotype effect, EjRepresenting an environmental effect, GEijRepresenting genotype-environment interactions, Bk(Ej) Representing the block effect, eilkRepresenting a random error. The BLUP value for each trait was calculated using MLM in R-package lme 4. Use of LME4 software package for estimating genotype
Figure BDA0003424529180000051
Genotype and environment interactions
Figure BDA0003424529180000052
And error of
Figure BDA0003424529180000053
The variance. The generalized heritability is calculated using the following formula:
Figure BDA0003424529180000054
where e and r represent the number of contexts and the number of groups in each context, respectively.
1.2 genotype analysis and Whole genome Association analysis
345 inbred lines were genotyped using a genotyping-sequencing strategy. 372,331 SNPs were retained for GWAS after quality control (deletion rate 20%; MAF 0.05%). Principal Component Analysis (PCA) was performed using TASSEL, and a population structure matrix was built using the first 5 principal components to control the population structure. And (4) calculating a genetic relationship matrix according to a TASSEL center IBS method, and estimating the genetic relationship between individuals. GWAS was performed in TASSEL using GLM (with PC) and MLM (with PC and kinship). The P value threshold is 2.69 multiplied by 10-6(1/n, n isNumber of SNPs). Linkage Disequilibrium (LD) decay was determined using PopLDdecay software, with the average LD for all chromosomes decaying to r within about 50kb20.20. If the physical distance between two adjacent SNPs is less than 50kb, a meaningful SNP is classified as a region. Furthermore, the SNPs with the smallest P-value in QTL were considered to be significant SNPs.
1.3 candidate Gene analysis and Gene-based Association mapping
We identified all potential candidate genes within 50kb of the detection site. Gene annotation information was obtained from the MaizeGDB database (http:// www.maizegdb.org). The physical location of these genes and SNPs was determined using the maize B73 RefGen _ V3 genome (version 5 b.60). Compared transcriptome analysis is carried out on the high stalk puncture strength and low stalk puncture strength strains in different periods by using published gene expression data, and the result shows that 8327 genes have differential expression in one or more periods.
In the case of genetic mapping, genomic DNA was extracted from fresh young leaves of 345 inbred lines. Candidate genes from the test inbred were sequenced by watma gene life science ltd using the target sequence capture technique of the NimbleGen platform. Multiple sequence alignment analysis was performed using MAFFT software (v 7.313). Gene-based polymorphisms were identified using TASSEL (MAF. gtoreq.0.05). And analyzing the correlation significance of the maize SNPs and the target traits according to a tassel MLM model (MLM + PCA + genetic relationship). The threshold P value for controlling the whole genome type 1 error rate is 1/n (where n is the number of markers of the candidate gene).
2. Results and analysis
2.1 analysis of phenotypic variation
In order to discuss the genetic basis of lodging resistance of corn stalks, 345 parts of corn inbred lines are subjected to field tests in the mature period under 3 environments. The evaluation of 7 traits was carried out, which included 3 traits related to Stalk strength (rod penetration strength (RPR), Stalk Crushing Strength (SCS) and Stalk Bending Strength (SBS)) and 4 traits related to plant type (plant height, PH), Ear Height (EH), Stem Diameter (SD) and Stem Length (SL)). Analysis of the trait distribution showed a somewhat left continuous distribution. Furthermore, all examined traits were quite different. The coefficient of variation for the three environments is between 8.02% (SD) and 22.81% (SBS). The difference among all the traits is more than 1.5 times, and the difference among strains is 1.59-3.07. Two-way anova showed that genotype, environment and genotype-environment interactions had significant effects on most traits (table 1).
TABLE 1 descriptive statistics of straw Strength traits
Figure BDA0003424529180000061
Figure BDA0003424529180000071
The phenotype correlation analysis shows that the 3 traits related to the stalk strength have extremely obvious positive correlation (r is 0.422 to 0.764). Plant configuration-related traits were also highly positively correlated (r ═ 0.326-0.570), except for the relationship between SD and SL. The correlation between the stalk strength related traits and the plant configuration related traits is researched, and the results show that the 4 plant configuration related traits are in extremely obvious positive correlation with the relative growth rate (r is 0.134-0.368). Among the four properties, the plant height, the ear height and the stem thickness are in positive correlation with the crushing strength and the bending strength of the straws.
2.2 genome-wide Association analysis (GWAS)
Correlation analysis was performed on 372331 SNP markers according to the General Linear Model (GLM) and the Mixed Linear Model (MLM). The GLM and MLM methods identified 907 and 3 markers and trait associations, respectively (P < 1/372331). Among them, GLM shows many false positives; in contrast, the MLM method is too rigorous. After relaxing the MLM significance threshold (P <1/10000), a total of 241 tokens and their associations were detected for 7 personalities. Multiple SNPs for a particular genomic region are closely related to the traits of the manhattan cell. And (3) carrying out cluster analysis on the 7 traits, detecting 94 QTLs (table 2) in total, and detecting 11-20 QTLs for each trait. On chromosome 1, Q14 contained 33 significant SNPs associated with SD, with the most significant association with SNPs being S1 — 288149975, accounting for 6.9% of phenotypic variation. On chromosome 5, Q56 contained 14 significant SNPs associated with RPR (fig. 1A), with the most significant SNP associated with S5_210155797 accounting for 9.7% of phenotypic variation. A total of 5 pleiotropic QTLs involving 4 traits were detected. More specifically, Q15 is associated with SCS and RPR, Q35, Q37 and Q49 are associated with SCS and SBS, and Q66 is associated with SCS and SD.
TABLE 2 Whole genome Association analysis results (parts)
Figure BDA0003424529180000072
Figure BDA0003424529180000081
2.3 identification of candidate genes
Based on the results of GWAS and the set of predicted genes screened from the annotated B73 maize reference genome (version 5b.60), a total of 265 potential candidate genes were identified within 50kb of 94 major SNPs (fig. 1B). To examine candidate gene expression profiles, genes differentially expressed at different developmental stages were analyzed for high RPR and low RPR lines. Among the differentially expressed genes detected, 60 were within the sites recognized by GWAS (fig. 1C). GRMZM2G079768, which encodes the LOB domain protein, was detected in Q35 and is associated with SCS and SBS. This gene was differentially expressed (DAS) between high and low RPR lines at 6, 12, 18 and 27 days post-sowing (fig. 2).
2.4 validation of candidate Gene ZmFLS2
ZmFLS2 was selected for resequencing to study the association between allelic and phenotypic variation. GWAS results determined S5 — 210155797 to be an important SNP associated with Q56 RPR. The GRMZM2G069298 gene is located at 91bp upstream of S5_210155797, encodes flavonol synthetase, a GRMZM2G069298 genome region is analyzed, the total length is 3641bp, the GRMZM2G069298 genome region comprises an upstream region of 1799bp, a coding region of 1003bp and a downstream region of 503bp (after a translation termination point), 234 sequence variations are detected in total, the secondary allele frequency (MAF) is more than or equal to 0.05, and the GRMZM2G069298 gene comprises 181 SNPs and 53 Indels. The MLM-based marker and trait association analysis detected 47 variants that were significantly associated with RPR (table 3), with the most significant SNP (SNP-397) located in the upstream region. The RPR of the line carrying the G allele was higher than the line carrying the a allele (fig. 1D).
TABLE 3 correlation analysis results of ZmFLS2 and stalk penetration strength
Figure BDA0003424529180000091
Figure BDA0003424529180000101
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> corn stalk puncture strength gene ZmFLS2 and application of molecular marker thereof
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atggggggcg agacgcacct gagcgtgcag gagctggcgg cgtcgctggg cgcgctaccg 60
ccggagttcg tgcggtctga gcaggaccag ccgggcgcga ccacgtaccg cggggccgcc 120
gtgccggacg cgccggtgat cgacatgtcg gagcccgggt tcggcgcgcg catggccgca 180
gccgccaggg agtgggggct gttccaggtg gtgaaccacg gcgtgccctc agcggcggtg 240
gcggagctcc agcgcgtcgg gcgggccttc ttcgcgcttc cgacggagga gaaggagcgc 300
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atcaacttct acccgccgtg cccgcagccg gagctcacgc tcggcgtcgc gccgcacacc 660
gacatgagca cgctcaccgt cctcgtgccc aacgaggtgc aggggctcca ggtcttcaaa 720
gatggtcaat ggtacgaggc caagtacgtg cccgacgcac tcatcgtcca tatcggcgat 780
cagatcgagg caagcgacct acgtagcatc tttttttttc tttctattgt gttgtgatga 840
tcttgctccc ctcaagtatt tctttgatgt cccgatcgag tagattttca gcaacggggc 900
atacaaggcg gtgctgcacc gtacgacggt gaacaaggag aagacgcgga tgtcatggcc 960
gatgttcgtg gagccgccgg gggagctcgt cgtcgggccg caccccaagc tggtcacgga 1020
ggagagcccg gccaagtaca aggccaagaa gtacaaggac taccagcact gcaagatcaa 1080
caagctcccc atgtaa 1096

Claims (9)

1. The application of the corn ZmFLS2 gene in regulating and controlling the corn stalk puncture strength is characterized in that the nucleotide sequence of the corn ZmFLS2 gene is shown as SEQ ID No. 1.
2. The use of claim 1, wherein the maize ZmFLS2 gene is located 91bp upstream of S5_ 210155797.
3. The SNP molecular marker for selecting the corn stalk puncture strength character is characterized in that the SNP molecular marker is positioned at a-397 bp position of a corn ZmFLS2 gene, and the basic group of the position is G or A.
4. The use of the SNP molecular marker of claim 3 for selecting corn stalk puncture strength traits.
5. The use of claim 4, wherein the strain carrying the G allele has a higher stalk-piercing strength than the strain carrying the A allele.
6. A method for screening or identifying the puncture strength of corn stalks is characterized in that the genotype of the site at bp-397 of ZmFLS2 gene of corn is analyzed according to the SNP molecular marker of claim 3, and the puncture strength of the corn stalks to be detected is judged according to the genotype.
7. The method of claim 6, wherein the strain carrying the G allele has a higher stalk penetration strength than the strain carrying the A allele.
8. The application of the corn ZmFLS2 gene or the SNP molecular marker of claim 3 in corn breeding, wherein the nucleotide sequence of the corn ZmFLS2 gene is shown as SEQ ID No. 1.
9. The use according to claim 8 for enhancing the lodging resistance of maize.
CN202111575000.0A 2021-12-21 2021-12-21 Corn stalk puncture strength gene ZmFLS2 and application of molecular marker thereof Pending CN114196685A (en)

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