CN111763760A - Fluorescent molecular marker for identifying maize folate genotype and primer thereof - Google Patents

Abstract

The invention discloses a fluorescent molecular marker for identifying a maize folate genotype and a primer thereof, belonging to the technical field of gene detection. The invention discloses a fluorescent molecular marker and a primer thereof for identifying the genotype of maize folic acid, the molecular marker detection means has simple and convenient process, the DNA extraction is rapid and stable, the PCR reaction is simple, the fluorescent data acquisition is accurate and environment-friendly, and compared with the result obtained by a similar method, the invention has the advantages of accurate and efficient identification of the target genotype and lower cost.

Description

Fluorescent molecular marker for identifying maize folate genotype and primer thereof

Technical Field

The invention relates to the technical field of gene detection, in particular to a fluorescent molecular marker for identifying a maize folate genotype and a primer thereof.

Background

Folic acid is also called pteroylglutamic acid, which plays an important role in the processes of human protein synthesis, cell division and growth, folic acid deficiency of pregnant women can cause fetal malformation, fresh corn is rich in natural folic acid, and high folic acid becomes a high-value label of fresh corn products. At present, China enters a nutritional agricultural new era with a crop nutrition strengthening technical system as a core, so that the development of the high-folic acid fresh-eating corn type is bound to cater to the development direction of the future fresh-eating corn industry. The gene ZmGFT1 is proved to be a main effective gene which is obviously related to the metabolism of maize folic acid, and the 1060-position 1062 base of the gene has three types: the gene type detection method comprises the following steps of GAA, GGG and CAA, wherein GGG is a high-folic acid type, different genotypes of the variation site in different maize germplasms can be effectively identified through a genotype detection technology, and the gene type detection method is used for high-folic acid molecular marker assisted selection.

The current molecular marker technical method for detecting genotype mainly comprises the following steps: detecting the amplified fragments by polyacrylamide gel by designing a mismatch primer; sequencing the product amplified based on PCR, and analyzing according to the base comparison result; KASP-based genotyping assays, and the like. However, the prior art has the defects of complex operation, high toxicity of used test reagents, high detection cost, expensive instruments and equipment and the like.

Therefore, it is an urgent problem to provide a fluorescent molecular marker and a primer thereof for identifying the maize folate genotype for solving the urgent problem of the technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention provides a fluorescent molecular marker for identifying maize folate genotypes and a primer thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a fluorescent molecular marker for identifying a maize folate genotype and a primer thereof have the following sequences:

haplotype primer Fol-A1: 5'-GAAGGTGACCAAGTTCATGCTAGCACGGGGACATTGTAGTTC-3', respectively; SEQ ID No. 1;

haplotype primer Fol-A2: 5'-GAAGGTGACCAAGTTCATGCTAGCACGGGGACATTGTAGTTG-3', respectively; SEQ ID NO. 2;

haplotype primer Fol-A3: 5'-GAAGGTCGGAGTCAACGGATTCACGGGGACATTGTAGCCC-3', respectively; SEQ ID NO. 3;

common primer Fol-C2: 5'-AAGAGAGGCGCTACGACGG-3', respectively; SEQ ID No. 4;

the 5' ends of the primers Fol-A1, Fol-A2 and Fol-A3 are labeled with fluorescent molecules, the fluorescent molecules labeled by Fol-A1 and Fol-A2 are the same, and the fluorescent molecules labeled by Fol-A1, Fol-A2 and Fol-A3 are different.

Further, the fluorescent molecule is FAM, HEX, ROX, not limited to the above fluorescent molecule.

Further, a detection method for identifying the genotype of the maize folic acid comprises the following specific steps:

(1) extracting corn leaf genome DNA by an alkaline cooking method;

(2) the haplotype primer Fol-A1, Fol-A2, Fol-A3 and the common primer Fol-C2 of the primer of claim 1 are simultaneously involved in PCR reaction;

(3) and detecting the PCR product by using a microplate reader under the condition of containing a fluorescence signal, and classifying materials and genotyping according to the fluorescence intensity signal value.

Further, the specific steps of the alkaline cooking method for extracting the corn leaf genome DNA in the step (1) are as follows:

① is 1cm in length²Grinding the leaves with 100. mu.l of 0.3M sodium hydroxide;

② centrifuging at 3000rpm for 1min, then boiling water bath for 1min, adding 200 μ l of 0.2M Tris-HCl with pH of 7.0 to neutralize and reduce pH;

③ boiling water bath for 1min, centrifuging at 3000rpm for 1min, diluting the supernatant by 10-30 times, and using the diluted supernatant for PARMS reagent detection.

According to the technical scheme, compared with the prior art, the fluorescent molecular marker and the primers thereof for identifying the maize folate genotype are disclosed and provided, the PARMS technology is adopted, the molecular marker detection method is simple and convenient in process, DNA extraction is rapid and stable, PCR reaction is simple, fluorescence data is accurate and environment-friendly, and compared with results obtained by similar methods, the fluorescent molecular marker and the primers are accurate and efficient in identification of the target genotype and lower in cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of the amplification of the invention to identify ZmGFT1 allelic forms GAA, CAA and GGG;

FIG. 2 is a graph showing the results of measuring the folate content of three genotype samples according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

2x PARMS mastermix was purchased from peptide Biotech, Inc. of the city of Wuhan.

Example 1

The design of PARMS primers for the target genotype was carried out by the technique of five-primer amplification hindered mutation system (PARMS) based on the sequence information of ZmGFT1 gene (GRMZM2G124863) obtained at NCBI, and the design results are shown in Table 1.

TABLE 1

3 primers (Fol-A1, Fol-A2 and Fol-A3) designed according to the gene type of a ZmGFT1 gene target sequence and a common primer (Fol-C2) are simultaneously added into a PCR reaction system for amplification, and the GAA gene type can be matched with a haplotype primer Fol-A1 according to the SNP difference to amplify to obtain a FAM fluorescence signal value; the CAA genotype can be matched with a haplotype primer Fol-A2 according to the SNP difference to obtain an FAM fluorescence signal value through amplification; the GGG genotype can be amplified to obtain a HEX fluorescence signal value by matching the SNP difference with a haplotype primer Fol-A3; Fol-C2 is a common primer; heterozygous sites combine FAM and HEX signals. Amplification schemes for the identification of ZmGFT1 allelic forms GAA, CAA and GGG are shown in FIG. 1.

In FIG. 1, green spots represent HEX signals (GGG), red spots represent heterozygous signals (GGG and GAA or CAA), blue spots represent FAM signals (GAA or CAA), and gray spots represent indeterminate samples.

Example 2

The method for identifying the maize folate genotype by using the fluorescent molecular marker and the primer thereof comprises the following specific steps:

(1) the method for extracting the corn leaf genome DNA rapidly at low cost by an alkaline cooking method comprises the following specific steps:

① is 1cm in length²Grinding the leaves with 100. mu.l of 0.3M sodium hydroxide, centrifuging at ② 3000rpm for 1min, then boiling in a water bath for 1min, adding 200. mu.l of 0.2M Tris-HCl (pH7.0) to neutralize and reduce the pH, boiling in a water bath for 1min at ③, centrifuging at 3000rpm for 1min, diluting the supernatant by 10-30 times, and using the diluted supernatant for PARMS reagent detection.

(2) Haplotype primers Fol-A1, Fol-A2, Fol-A3 and a common primer Fol-C2 simultaneously participate in PCR reaction, and the reaction system is as follows:

the reaction procedure was as follows:

(3) the PCR product was detected by a microplate reader under a fluorescent signal, and material classification and genotyping were performed based on the fluorescence intensity signal values (http:// www.snpway.com), where FAM signals represent GAA and CAA genotypes, HEX represents GGG genotype, and the presence of HEX represents heterozygous genotype.

Example 3 accuracy verification

Carrying out folic acid determination by using fluorescent molecular markers for identifying the maize folic acid genotypes and three genotype samples obtained by primer detection of the fluorescent molecular markers, wherein the three genotype samples comprise 16 parts of GGG, GAA and CAA respectively, and the result is shown in figure 2; the comparison of the differences of the mean values shows that the mean value of the folic acid content of the sample with the GGG genotype is obviously higher than that of the other two genotypes, and the accuracy and the practicability of the invention are further verified.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims (4)

1. A fluorescent molecular marker for identifying a maize folate genotype and a primer thereof are characterized in that the primer sequence is as follows:

haplotype primer Fol-A1: 5'-GAAGGTGACCAAGTTCATGCTAGCACGGGG ACATTGTAGTTC-3', respectively; SEQ ID No. 1;

haplotype primer Fol-A2: 5'-GAAGGTGACCAAGTTCATGCTAGCACGGGGA CATTGTAGTTG-3', respectively; SEQ ID No. 2;

haplotype primer Fol-A3: 5'-GAAGGTCGGAGTCAACGGATTCACGGGGAC ATTGTAGCCC-3', respectively; SEQ ID No. 3;

common primer Fol-C2: 5'-AAGAGAGGCGCTACGACGG-3', respectively; SEQ ID No. 4;

the 5' ends of the primers Fol-A1, Fol-A2 and Fol-A3 are labeled with fluorescent molecules, the fluorescent molecules labeled by Fol-A1 and Fol-A2 are the same, and the fluorescent molecules labeled by Fol-A1, Fol-A2 and Fol-A3 are different.

2. The fluorescent molecular marker and the primer for identifying the maize folate genotype as claimed in claim 1, wherein the fluorescent molecule is FAM, HEX or ROX.

3. A detection method for identifying the genotype of maize folic acid is characterized by comprising the following specific steps:

(1) extracting corn leaf genome DNA by an alkaline cooking method;

(2) the haplotype primer Fol-A1, Fol-A2, Fol-A3 and the common primer Fol-C2 of the primer of claim 1 are simultaneously involved in PCR reaction;

(3) and detecting the PCR product by using a microplate reader under the condition of containing a fluorescence signal, and classifying materials and genotyping according to the fluorescence intensity signal value.

4. The detection method for identifying the maize folate genotype as claimed in claim 3, wherein the alkaline cooking method for extracting the maize leaf genome DNA in the step (1) comprises the following specific steps:

① is 1cm in length²Adding 100 μ l of 0.3M hydrogen and oxygenSodium dissolving and grinding;

② centrifuging at 3000rpm for 1min, then boiling water bath for 1min, adding 200 μ l of 0.2M Tris-HCl with pH of 7.0 to neutralize and reduce pH;

③ boiling water bath for 1min, centrifuging at 3000rpm for 1min, diluting the supernatant by 10-30 times, and using the diluted supernatant for PARMS reagent detection.

Images