CN115820895A - Molecular marker closely linked with chlorophyll content of corn and application thereof - Google Patents
Molecular marker closely linked with chlorophyll content of corn and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with the chlorophyll content of corn and application thereof. The molecular marker closely linked with the chlorophyll content of the corn disclosed by the invention is a DNA fragment shown in SEQ ID No.3 or a DNA fragment shown in SEQ ID No.4, and can be detected by using a primer pair consisting of two single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2 in a sequence table. Experiments prove that the molecular marker closely linked with the chlorophyll content of the corn is related to the chlorophyll content of the corn, the chlorophyll content of the corn can be successfully identified by utilizing the molecular marker, and the molecular marker has the advantages of simplicity, convenience, rapidness, high efficiency, accuracy, good repeatability and high specificity, can be used for the auxiliary breeding of the molecular marker of the corn, and can be used for breeding new corn varieties with excellent comprehensive properties, thereby greatly saving the breeding cost and improving the breeding efficiency.
Description
Technical Field
The invention relates to a molecular marker closely linked with the chlorophyll content of corn and application thereof in the field of biotechnology.
Background
Corn (Zea mays L.) is a crop which is used for both food, feed and economy, and is the only crop which is steadily increased in sowing area and yield in China. In recent years, global economy has been rapidly developed, and the demand for corn is expected to increase in future industrial production and life. In 2021, the corn planting area in China is 4332 million hectares, 206 million hectares are increased compared with the last year, the yield is 27255 million tons, and the yield accounts for about 39.91 percent of the total grain yield in China. Therefore, the corn production has an important strategic position in guaranteeing the food safety in China.
Chlorophyll is an important pigment involved in photosynthesis in green plant chloroplasts, is usually combined with proteins to form a stable complex, synthesizes organic substances through absorption, transmission and conversion of light energy in photosynthesis, and is the basis of crop yield formation. The chlorophyll content is an effective index for evaluating the photosynthesis rate, the photosynthesis rate is enhanced along with the increase of the chlorophyll content in a certain range, but excessive chlorophyll is easy to catalyze and generate oxygen free radicals under illumination, so that the normal physiological metabolic process of plants is influenced. The chlorophyll content is also one of important target characters of high-photosynthetic-efficiency breeding of crops, and the higher chlorophyll content can effectively improve the photosynthesis rate, synthesize more photosynthetic products and finally realize the yield potential of the crops. Therefore, further analyzing the genetic basis of the chlorophyll content of the corn plants and developing molecular markers closely linked with Quantitative Trait Loci (QTL) of the chlorophyll content of the corn plants have important significance for breeding new corn varieties with strong photosynthetic capacity, good greenness and excellent comprehensive traits.
The molecular markers have the advantages of large quantity, no influence of environmental conditions, development period, expression regulation and other factors on detection, capability of providing complete and rich genetic information and the like, and are widely applied to the aspects of germplasm resource identification, QTL positioning, molecular marker-assisted selection and the like. An Insertion-Deletion (InDel) marker is one of the commonly used molecular markers based on the difference of DNA levels, and specifically refers to the difference between two materials, wherein, relative to one material, a certain number of nucleotide insertions or deletions exist in certain sites of the genome of the other material, and PCR primers for amplifying the Insertion-Deletion sites are designed according to the Insertion-Deletion sites. By utilizing the InDel marker which is tightly linked with the target gene, linkage drag is reduced, favorable genes are aggregated, the breeding process is accelerated, and the selection efficiency and effect can be effectively improved through auxiliary backcross selection, auxiliary pedigree selection and even whole genome selection.
Currently, although there are QTLs reported to control chlorophyll content of maize plants on all chromosomes of maize, there are few reports of developing molecular markers closely linked to the target QTL and applying for patent applications, and there are no patent reports related to chlorophyll content of maize plants in the qCHL4-1 segment.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect the chlorophyll content character of corn.
In order to solve the technical problems, the invention firstly provides application of a substance for detecting a corn chlorophyll content molecular marker in detection or auxiliary detection of corn chlorophyll content characters, wherein the corn chlorophyll content molecular marker is a DNA fragment shown as SEQ ID No.3 and a DNA fragment shown as SEQ ID No.4.
In the application, the substance for detecting the molecular marker of the chlorophyll content in the corn can be a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table.
The invention also provides a method for detecting the chlorophyll content character of corn, which comprises the following steps: taking genome DNA of corn to be detected as a template, and carrying out PCR amplification by using a primer pair consisting of two single-chain DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, wherein the chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.3 is higher or more than candidate chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.4, the chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.3 is higher or more than candidate chlorophyll content of the heterozygous corn with the PCR product sequences of SEQ ID No.3 and SEQ ID No.4, and the chlorophyll content of the heterozygous corn with the PCR product sequences of SEQ ID No.3 and SEQ ID No.4 is higher or more than candidate chlorophyll content of the homozygous corn with the PCR product sequences of SEQ ID No.4.
The invention also provides a method for detecting the chlorophyll content character of corn, which comprises the following steps: taking the genome DNA of the corn to be detected as a template, and carrying out PCR amplification by using a primer pair consisting of two single-chain DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, wherein the chlorophyll content of the homozygous corn with the size of 185bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the homozygous corn with the size of 286bp of the obtained PCR product in a candidate mode, the chlorophyll content of the homozygous corn with the size of 185bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the heterozygous corn with the sizes of 185bp and 286bp of the obtained PCR product in a candidate mode, and the chlorophyll content of the heterozygous corn with the sizes of 185bp and 286bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the homozygous corn with the size of 286bp of the obtained PCR product in a candidate mode.
The invention also provides a method for breeding corn, which comprises the following steps: taking the genome DNA of the corn to be tested as a template, carrying out PCR amplification by using a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, and selecting the corn to be tested with a PCR product of SEQ ID No.3 as a parent to finish breeding.
In the above, the reaction system for performing PCR amplification using the primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in the sequence listing may be: 1 mu L of single-stranded DNA shown in SEQ ID No.1 with the concentration of 10 mu mo 1/L; 1 mu L of single-stranded DNA shown in SEQ ID No.2 with the concentration of 10 mu mo 1/L; 1 μ L of genomic DNA at a concentration of 100 ng/. Mu.L; 5 μ L of 2 XTAQQ PCR StarMix with Loading Dye (Biotech Co., ltd., product number: A012-01, kyoto Kang Runcheng); 2 μ L ddH 2 O。
The reaction conditions for PCR amplification by using a primer pair consisting of two single-stranded DNAs shown by SEQ ID No.1 and SEQ ID No.2 in the sequence table may be: pre-denaturation at 95 ℃ for 10min; denaturation at 95 ℃ for 45s, annealing at 58 ℃ for 45s, extension at 72 ℃ for 40s,35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
The application of the molecular marker for detecting the chlorophyll content of the corn in the preparation of products for detecting the chlorophyll content character of the corn also belongs to the protection scope of the invention.
The application of the molecular marker for the content of the corn chlorophyll in detecting or assisting in detecting the content and the property of the corn chlorophyll also belongs to the protection scope of the invention.
The application of the molecular marker for the chlorophyll content of the corn in corn breeding also belongs to the protection scope of the invention.
In the invention, the chlorophyll content is the chlorophyll content of the leaf (such as the ear position leaf).
The molecular marker for the chlorophyll content of the corn is related to the chlorophyll content of the corn, can be used for successfully identifying the chlorophyll content of the corn, has the advantages of simplicity, convenience, rapidness, high efficiency, accuracy, good repeatability and high specificity, can be used for the molecular marker-assisted breeding of the corn, can be used for breeding new corn varieties with excellent comprehensive properties, greatly saves the breeding cost and improves the breeding efficiency.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
Drawings
FIG. 1 shows the alignment of the amplified sequence of the molecular marker qCHL4-1 of the present invention in parents.
FIG. 2 is an electropherogram of the PCR amplification product of the molecular marker qCHL4-1 of the invention in parents. Wherein, W is the amplification banding pattern of a maize inbred line W22, C is the amplification banding pattern of a maize wild relative species CIMMYT 8759, and the sizes of bands of a Marker from bottom to top are 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp and 2000bp in sequence.
FIG. 3 shows the molecular marker qCHL4-1 at F of the present invention 2 Electropherograms of PCR amplification products in the population. Wherein, W is the amplification band type of the homozygous W22 genotype, C is the amplification band type of the homozygous CIMMYT 8759 genotype, H is the amplification band type of the heterozygous genotype, and the size of the Marker bands from bottom to top is 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp and 2000bp in sequence.
FIG. 4 shows the molecular marker qCHL4-1 of the present invention at F 2 Single mark of plant chlorophyll content character in colonyAnd (5) analyzing the results. NIL _ W22 indicates homozygous W22 genotype, het indicates heterozygous genotype, NIL _ CIMMYT 8759 indicates homozygous CIMMYT 8759 genotype, and indicates significant difference (P _ CIMMYT 3242 genotype)<0.05 Denotes that the difference is extremely significant (P)<0.01)。
Detailed Description
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, the 1 st position of each nucleotide sequence in the sequence Listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA, unless otherwise specified.
The maize inbred line W22 and the maize wild relative species CIMMYT 8759 in the following examples are both described in the literature (Identification and fine mapping of qualitative trail loci logic for the number of mutant crop in mail step, JIntegr plant biol.2016 Jan;58 (1): 81-90. Doi.
MR0429 in the following examples is a product of the American corn germplasm storage Center (Maize Genetics Cooperation storage Center) under the website: www.maizecoop.cropsci.uiuc.edu, which is also available to the public from the applicant, is only useful for repeating the experiments relevant to the present invention and is not useful for other applications. MR0429 is introgression line material derived from maize inbred line W22 and maize wild relative species CIMMYT 8759 by crossing, backcrossing and inbreeding.
Example 1: molecular marker closely linked with chlorophyll content of corn plant
The invention provides a molecular marker qCHL4-1 (marked as a molecular marker of chlorophyll content of a corn plant) for identifying or assisting in identifying the chlorophyll content of the corn plant, wherein the molecular marker is a DNA fragment obtained by taking corn genome DNA as a template and performing PCR amplification on A1 by using a primer, and the sequence of the obtained DNA fragment is SEQ ID No.3 or SEQ ID No.4. The primer pair A1 has the following sequences:
forward amplification primer CHL4-F:5'-TGTAGTTGTCAAATGGGCTCC-3' as shown in SEQ ID No. 1;
reverse amplification primer CHL4-R:5'-TGCCCACCTGTTTATTTCCT-3' as shown in SEQ ID No. 2;
taking genome DNA of a maize inbred line W22 with higher plant chlorophyll content and a maize wild relative species CIMMYT 8759 with lower plant chlorophyll content as templates, carrying out PCR amplification by using a forward amplification primer shown in SEQ ID No.1 and a reverse amplification primer shown in SEQ ID No.2, and detecting the sequence of an obtained PCR product.
Wherein, the reaction system for 10 μ L PCR amplification is as follows:
(1) 1 mu L of forward amplification primer shown in SEQ ID No.1 with the concentration of 10 mu mo 1/L;
(2) 1 mu L of reverse amplification primer shown in SEQ ID No.2 with the concentration of 10 mu mo 1/L;
(3) 1 μ L of DNA template at a concentration of 100 ng/. Mu.L;
(4) 5 μ L of 2 XTAQQ PCR StarMix with Loading Dye (Biotech Co., ltd., product number: A012-01, kyoto Kang Runcheng);
(5)2μL ddH 2 O。
the procedure for PCR amplification was as follows:
(1) Pre-denaturation at 95 ℃ for 10min;
(2) Denaturation at 95 ℃, annealing at 58 ℃, extension at 72 ℃ for 40s,35 cycles;
(3) Extending for 10min at 72 ℃;
(4) Storing at 4 ℃.
The PCR instrument model: eppendorf Mastercycler nexus.
Carrying out electrophoretic separation on the PCR amplification product in 4.0% agarose gel (each 100mL of gel solution contains 4.0g of agarose), and carrying out sequencing analysis, wherein the result shows that the PCR amplification is carried out by taking the genome DNA of the maize inbred line W22 as a template, the molecular weight of the PCR amplification product is 185bp, and the nucleotide sequence of the PCR amplification product is shown as SEQ ID No. 3; the genome DNA of a corn wild relative species CIMMYT 8759 is used as a template for PCR amplification, the molecular weight of a PCR amplification product is 286bp, and the nucleotide sequence of the PCR amplification product is shown as SEQ ID No.4.
SEQ ID No.3：
TGTAGTTGTCAAATGGGCTCCACGTGGAAGTATGCCAAAATCCAGACACAATTGTATGGTATACATATCTACATAATTCATTATAACTGGTACTATCGTAGTATGATTTGCAATTTAAGTAAAACTTCTTTCTCAGATTTAGTTCAATATATAGCTAACTCAGAAAGGAAATAAACAGGTGGGCA。
SEQ ID No.4：
TGTAGTTGTCAAATGGGCTCCACGTGGAAGTATGCCAAAATCCAGACACAATTGTATGGTAATTTACCTCAGAATGCTGTATTGGCAACAAGGGTACCTTGTTGCCAATACAGCATTCTGAGGTAAATACCCTTGTTGCCAATACAGCATTCTGAGGTAAATTACATATCTACATAATTCATTATAACTGGTACTATCTTAGTATGATTTGCAATTTAAGTAAAATTTCTTTCTCAGATTTAGTTTAATATATAGCTAACTCAGAAAGGAAATAAACAGGTGGGCA。
Wherein, the amplified banding pattern of the maize inbred line W22 is an excellent allele for increasing the chlorophyll content of the plants. Therefore, if the molecular weight of the PCR amplification product of the corn sample to be detected is 185bp, the corn sample to be detected contains the allele for increasing the chlorophyll content of the corn plant; if the molecular weight of the PCR amplification product of the corn sample to be detected is 286bp, the corn sample to be detected contains the allele for reducing the chlorophyll content of the corn plant.
The result of alignment of SEQ ID No.3 and SEQ ID No.4 is shown in FIG. 1. The electrophoresis results of the PCR amplification products of the maize inbred line W22 and the maize wild relative species CIMMYT 8759 are shown in FIG. 2.
Example 2: method for obtaining molecular marker CHL4
The method for obtaining the molecular marker CHL4 specifically comprises the following steps:
step 1: construction of BC containing 866 families 2 S 3 Introgression line population
Taking a maize inbred line W22 as a receptor parent and a maize wild relative species CIMMYT 8759 as a donor parent, and obtaining the maize inbred line by hybridizing 1 generation, backcrossing 2 generation and inbreeding 3 generationBC containing 866 families 2 S 3 A population of introgression lines.
Step 2: field planting and phenotypic determination of introgression line populations
BC was planted in the spring of 2019 at the national crop variety regional test station of Liuyang city (28.2 degree N,113.6 degree E) in Hunan province 2 S 3 A population of introgression lines. The field experiment adopts an amplification type incomplete random block design. 2 rows are planted in each plot, 15 plants are planted in each row, and the planting distance is 25cm. 2 families are planted on each ridge. The ridge height is 15cm, the ridge width is 70cm, and the furrow width is 30cm.
The SPAD value of the ear position leaves of the plants was measured at the flowering stage of maize using a portable chlorophyll measuring instrument (SPAD-502plus, konica Minolta, japan). 5 single plants with consistent growth vigor are selected for each family, the SPAD value of 1/2 position of each ear position leaf of each single plant is determined, each single plant is repeatedly determined for 3 times, the mean value of 3 times of determination is used as the chlorophyll content of the ear position leaf of the plant, and the mean value of 5 single plant SPAD values is used as the chlorophyll content of the ear position leaf of the family.
And step 3: performing QTL localization analysis
QTL localization analysis was performed using the multiple QTL model of R/QTL. Firstly, carrying out QTL simple interval positioning analysis by using Haley-Knott regression, and determining the LOD threshold (alpha = 0.05) of the plant chlorophyll content QTL by adopting a method of 10000 times of replacement test. And (3) carrying out multi-QTL model fitting on the QTL model obtained by simple interval positioning, and optimizing the position of each QTL by utilizing a refieqtl command of R/QTL. And further detecting whether other QTL (quantitative trait loci) of the significant improvement model exist in the genome by using an addqtl command, if a new QTL is detected, re-fitting the multi-QTL model and optimizing the QTL position, and repeating the process until the new QTL is not detected. And finally, calculating the total phenotypic variation of all QTL interpretations and the additive effect and phenotypic contribution rate of the single QTL by using a fitqtl command.
QTL positioning result analysis: 10 QTLs controlling the chlorophyll content of the corn plants are detected in total, wherein a QTL qCHL4-1 with larger phenotypic effect is detected on the 4 th chromosome. The LOD value of qCHL4-1 is 10.04, the additive effect size is-3.03, the dominant effect size is 2.99, the phenotype contribution rate is 3.75%, and the LOD is located in the range of 231902829bp to 234112186bp of the maize 4 th chromosome.
And 4, step 4: development and synthesis of molecular marker CHL4
qCHL4-1 is searched in a physical interval of 231902829bp to 234112186bp of a 4 th chromosome by using online primer design software primer3 (https:// primer3.Ut. Ee /), a forward amplification primer CHL4-F and a reverse amplification primer CHL4-R are designed, the primers are synthesized by Beijing optimaceae biotechnology, inc., and the nucleotide sequences are as follows:
forward amplification primer CHL4-F:5'-TGTAGTTGTCAAATGGGCTCC-3' as shown in SEQ ID No. 1;
reverse amplification primer CHL4-R:5'-TGCCCACCTGTTTATTTCCT-3' as shown in SEQ ID No. 2.
Example 3: application of molecular marker CHL4
Introgression line MR0429 heterozygous only for the qCHL4-1 segment and homozygous at other sites in the genome was used as starting material and self-pollinated to produce an F segregating only for the qCHL4-1 segment 2 And (4) a group. To contain F of 612 individuals 2 The population is used as a material to verify the molecular marker CHL4 obtained by the invention so as to determine the accuracy of the molecular marker applied to molecular marker-assisted selective breeding. The method specifically comprises the following steps:
step 1: f 2 Determination of chlorophyll content of population plants
F was determined according to the method of example 2 2 The chlorophyll content of the plants of the population.
Step 2: and (3) extracting the DNA of the corn leaves by adopting a CTAB method.
And 3, step 3: PCR amplification
The reaction system for PCR amplification is 10. Mu.L, and comprises:
(1) 1 mu L of forward amplification primer shown in SEQ ID No.1 with the concentration of 10 mu mo 1/L;
(2) 1 mu L of reverse amplification primer shown in SEQ ID No.2 with the concentration of 10 mu mo 1/L;
(3) 1 μ L of DNA template at a concentration of 100 ng/. Mu.L;
(4) 5 μ L of 2 XTAQQ PCR StarMix with Loading Dye (Biotech Co., ltd., product number: A012-01, kyoto Kang Runcheng);
(5)2μL ddH 2 O。
the procedure for PCR amplification was as follows:
(1) Pre-denaturation at 95 ℃ for 10min;
(2) Denaturation at 95 ℃ for 45s, annealing at 58 ℃ for 45s, extension at 72 ℃ for 40s,35 cycles;
(3) Extending for 10min at 72 ℃;
(4) Storing at 4 ℃.
The PCR instrument model: eppendorf Mastercycler nexus.
And 4, step 4: electrophoresis
Molecular marker CHL4 in part F 2 The electrophoretogram of the PCR amplified product of the individual strain is shown in FIG. 3.
And 5: analysis of results
Determining the genotype of the corn sample to be detected according to the molecular weight of the PCR amplification product: if the PCR amplification product of the corn sample to be detected only has a 185bp strip, the corn to be detected is homozygous W22 genotype (namely the genotype is the same as the genotype of the corn inbred line W22); if the PCR amplification product of the corn sample to be detected only has a 286bp strip, the corn to be detected is homozygous CIMMYT 8759 genotype (namely the same genotype as the wild relative species CIMMYT 8759 of the corn); if the PCR amplification product of the corn sample to be detected has not only a 185bp band but also a 286bp band, the corn to be detected is a heterozygous genotype.
F 2 A total of 148 strains in a single plant are homozygous W22 genotype, and the sequencing of PCR products shows that the sequences of the W22 genotype are all SEQ ID No.3 and the W22 genotype F homozygous for 148 strains 2 The chlorophyll content of the individual plant is 42.9 +/-5.4; the total 157 strains are homozygous CIMMYT 8759 genotypes, and the sequencing of PCR products shows that the sequences of the CIMMYT 8759 genotypes are all SEQ ID No.4 and 157 strains homozygous CIMMYT 8759 genotype F 2 The chlorophyll content of the individual plant is 35.9 +/-5.6; the total 307 strains are heterozygous genotypes, the sequencing of PCR products shows that the sequences are SEQ ID No.3 and SEQ ID No.4, the 307 strains are heterozygous genotypes F 2 The chlorophyll content of the individual plant is 40.1 +/-5.0.
Further analysis of variance was performed on the phenotypic values of chlorophyll content of plants of each group (fig. 4). The results show that: homozygous W22 genotype F 2 The chlorophyll content of the single plant is remarkably higher than that of homozygous CIMMYT 8759 genotype F 2 Individual, homozygous W22 genotype F 2 The chlorophyll content of the single plant is obviously higher than that of the heterozygous genotype F 2 Individual, heterozygous genotype F 2 The chlorophyll content of the single plant is remarkably higher than that of homozygous CIMMYT 8759 genotype F 2 The single plant shows that the molecular marker CHL4 is related to the chlorophyll content of the corn plant, and has important breeding application value.
In conclusion, the molecular marker CHL4 provided by the invention is closely linked with qCHL4-1, so that the chlorophyll content of a corn plant can be quickly and accurately identified, the application of the locus in breeding of a new corn variety can be promoted, and the molecular polymerization breeding of the locus and other excellent character loci is facilitated. The method provided by the invention can identify and screen the plant chlorophyll content of the corn germplasm resources at any stage of the corn, has the advantages of simplicity, convenience, rapidness, high efficiency and accuracy, and is suitable for large-scale popularization and application.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. The application of a substance for detecting a corn chlorophyll content molecular marker in detecting or assisting in detecting the corn chlorophyll content character, wherein the corn chlorophyll content molecular marker is a DNA fragment shown as SEQ ID No.3 and a DNA fragment shown as SEQ ID No.4.
2. Use according to claim 1, characterized in that: the substance for detecting the molecular marker of the chlorophyll content of the corn is a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table.
3. The method for detecting the chlorophyll content character of the corn comprises the following steps: taking genome DNA of corn to be detected as a template, and carrying out PCR amplification by using a primer pair consisting of two single-chain DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, wherein the chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.3 is higher or more than candidate chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.4, the chlorophyll content of the homozygous corn with the PCR product sequence of SEQ ID No.3 is higher or more than candidate chlorophyll content of the heterozygous corn with the PCR product sequences of SEQ ID No.3 and SEQ ID No.4, and the chlorophyll content of the heterozygous corn with the PCR product sequences of SEQ ID No.3 and SEQ ID No.4 is higher or more than candidate chlorophyll content of the homozygous corn with the PCR product sequences of SEQ ID No.4.
4. The method for detecting the chlorophyll content character of the corn comprises the following steps: taking the genome DNA of the corn to be detected as a template, and carrying out PCR amplification by using a primer pair consisting of two single-chain DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, wherein the chlorophyll content of the homozygous corn with the size of 185bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the homozygous corn with the size of 286bp of the obtained PCR product in a candidate mode, the chlorophyll content of the homozygous corn with the size of 185bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the heterozygous corn with the sizes of 185bp and 286bp of the obtained PCR product in a candidate mode, and the chlorophyll content of the heterozygous corn with the sizes of 185bp and 286bp of the obtained PCR product is higher than or is higher than the chlorophyll content of the homozygous corn with the size of 286bp of the obtained PCR product in a candidate mode.
5. A method of maize breeding comprising: taking the genome DNA of the corn to be tested as a template, carrying out PCR amplification by using a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, and selecting the corn to be tested with a PCR product of SEQ ID No.3 as a parent to finish breeding.
6. The use of the substance for detecting the molecular marker of the chlorophyll content in the corn as claimed in claim 1 or 2, in the preparation of products for detecting the chlorophyll content character of the corn.
7. The use of the molecular marker for maize chlorophyll content according to claim 1 or 2 for detecting or assisting in detecting the trait of maize chlorophyll content.
8. The use of the molecular marker for maize chlorophyll content according to claim 1 or 2 in maize breeding.
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