CN110628932A - CAPS marker for identifying maize germplasm folate genotype and application thereof - Google Patents
CAPS marker for identifying maize germplasm folate genotype and application thereof Download PDFInfo
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Abstract
The invention provides a CAPS marker for identifying the folate genotype of a maize germplasm and application thereof, belonging to the technical field of molecular assisted breeding, wherein primers for amplifying the CAPS marker comprise a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2. The invention utilizes the forward primer and the reverse primer to amplify the corn genome, and after the obtained product is enzyme-digested, the genotype of the maize germplasm folic acid to be detected is judged according to an electrophoresis strip of the enzyme-digested product. The molecular marker provided by the invention can quickly and effectively identify the maize germplasm folic acid genotype, has low cost, can assist in breeding, improves the efficiency of selecting the high folic acid genotype in the maize breeding process, and can accelerate the maize breeding process.
Description
Technical Field
The invention belongs to the technical field of molecular assisted breeding, and particularly relates to a CAPS marker for identifying the folate genotype of a maize germplasm and application thereof.
Background
Folic acid is a water-soluble B vitamin, also known as VB9, and comprises tetrahydrofolic acid and derivatives thereof. They are involved in the one-carbon unit transfer reaction in animals and plants, play an important role in the biosynthesis of purine, thymidylate, DNA, amino acid and protein and the methyl cycle, and are micronutrients essential for the growth and development of animals and plants. Since humans do not have the ability to synthesize folic acid themselves, they rely primarily on dietary intake to meet their needs, and adults need to take at least 400 μ g of folic acid per day to meet the needs of their life activities, increasing to 600 μ g during pregnancy. Inadequate levels of folate intake in adults can lead to megaloblastic anemia and hyperhomocysteinemia, and also increase the risk of Alzheimer's disease, coronary sclerosis, stroke, and cancer; the lack of folic acid in pregnant women may lead to conditions such as low fetal weight, cleft lip and palate, heart disease, and neural tube defects. Although almost all human diets contain folic acid, folic acid is ingested in amounts of less than 400 μ g per day in many parts of the world, and folate deficiency is a global health problem. Folate deficiency is more likely to worsen in pregnant and lactating women. The corn, which is the main cultivation crop in the world and the first large food crop in China, has the folic acid content (40-60 mug/100 g) which is only one tenth of the daily folic acid diet requirement of human beings on average. Therefore, the cultivation of fresh sweet and waxy corn varieties with high folic acid content is an important way for folic acid nutrition enhancement of human beings.
The enzyme-Cleaved Amplified Polymorphic Sequence (CAPS) labeling technology is a simple, economic, reliable and co-dominant technology, is a molecular labeling technology combining a PCR technology based on SNP and an RFLP technology, and is suitable for many aspects of biological research. The CAPS marking technology is widely applied to aspects such as plant genotyping, molecular marker-assisted selective breeding, variety identification and the like.
At present, a CAPS marker for quickly and accurately identifying the folate genotype of maize germplasm does not exist.
Disclosure of Invention
In view of the above, the invention aims to provide a CAPS marker capable of identifying the folate genotype of a maize germplasm quickly, accurately and at low cost and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a CAPS marker for identifying the folate genotype of maize germplasm, wherein primers for amplifying the CAPS marker comprise a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2.
Preferably, the nucleotide sequence obtained by PCR amplification by using the forward primer and the reverse primer is shown as SEQ ID No.3 by using a corn genome as a template.
Preferably, the maize germplasm comprises sweet maize germplasm and waxy maize germplasm.
The invention provides application of the CAPS marker in identification of maize germplasm folate genotypes.
Preferably, the method comprises the following steps:
1) extracting the corn genome DNA to be identified, and performing PCR amplification by using the forward primer and the reverse primer to obtain an amplification product;
2) carrying out enzyme digestion on the amplification product by using a restriction enzyme MnlI to obtain an enzyme digestion product;
3) electrophoresing the enzyme digestion product, and judging the folic acid genotype of the corn to be identified according to the band type obtained by electrophoresis; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 82bp, the maize germplasm to be detected is the maize germplasm with a low folate genotype; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 47bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid homozygous genotype; if the electrophoresis band of the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid heterozygous genotype.
Preferably, the PCR amplification system in the step 1) comprises the following components in 25 μ L: 2 XPCR M Six 12.5. mu.L, ddH2O11 uL, forward primer 0.5 uL, reverse primer 0.5 uL, DNA template 0.5 uL; the forward primer andthe concentration of the reverse primer was 10. mu.M.
Preferably, the procedure of PCR amplification described in step 1) is as follows: denaturation at 95 deg.C for 3 min; denaturation at 95 deg.C for 30s, annealing at 62.5 deg.C for 30s, and extension at 72 deg.C for 1min, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃.
Preferably, the enzyme-digested system in the step 2) comprises the following components in 25 μ L: 17.3-17.4 mu LddH2O, 10 XBuffer 2.5. mu.L, amplification product 5. mu.L, 1UMnl I restriction endonuclease 0.1-0.2. mu.L.
Preferably, the temperature of the enzyme digestion in the step 2) is 37 ℃, and the time of the enzyme digestion is 15-30 min.
The invention provides application of the CAPS marker in corn breeding assistance.
The invention has the beneficial effects that: the invention provides a CAPS marker for identifying the folate genotype of maize germplasm, wherein primers for amplifying the CAPS marker comprise a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2. The invention utilizes the forward primer and the reverse primer to amplify the corn genome, and after the obtained product is enzyme-digested, the genotype of the maize germplasm folic acid to be detected is judged according to an electrophoresis strip of the enzyme-digested product. The molecular marker provided by the invention can quickly and effectively identify the maize germplasm folic acid genotype, and has low cost; the CAPS marker for identifying the maize germplasm folate genotype can screen sweet and waxy maize germplasm materials with high and low folate genotypes, assist breeding, improve the efficiency of selecting the high folate genotype in the maize breeding process and accelerate the maize breeding process.
Compared with the prior art (detecting gene locus variation by utilizing first-generation DNA sequencing), the method is more convenient, effective and accurate, has obvious price advantage and shorter time for obtaining results. The price of one reaction of DNA sequencing is 12 yuan, and the time is two days; one reaction cost for using the CAPS marker of the present invention is 1.4 yuan, which takes one hour.
Drawings
FIG. 1 is a diagram showing the electrophoresis result of the cleaved products after amplification by the CAPS marker of the present invention.
Detailed Description
The invention provides a CAPS marker for identifying the folate genotype of maize germplasm, wherein primers for amplifying the CAPS marker comprise a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2; the method comprises the following specific steps:
F:5’-ACGCCGCTTCTCTGGCTAGG-3’(SEQ ID No.1)
R:5’-ACGGGTGATCCTCCTGACGG-3’(SEQ ID No.2)。
in the invention, the nucleotide sequence obtained by PCR amplification by using the forward primer and the reverse primer is shown as SEQ ID No.3 by using a corn genome as a template, and specifically comprises the following steps:
ACGCCGCTTCTCTGGCTAGGCAGGTAGCGTCTGACATTGCCTGCATCGCAGCAGTCCCAGTGTTCCTGTACGCAGCAGCCCACCCGGCGGGGAAGTCGGTGGGCGCGGTCCGGCGTGAGCTCGGCTACTACCGCCCAAACTACAGGGGCAACCAGTGGTCAGGCAGCGTGCTCCCCAACGTCCTGCCGGTGAAGCCTGACGTCGGTCCGGCCCACGTCGTCTCGCACAAGAGAGGCGCTACGACGGTGGGTGTCACGCCTTGGATCGAGAACTACAATGTCCCCGTGCTGTGTAAGGACGTCGCCACCGTCAGGAGGATCACCCGT。
in the present invention, the maize germplasm preferably includes sweet maize germplasm and waxy maize germplasm; the maize genome is preferably a sweet maize genome or a waxy maize genome.
In the invention, by utilizing the high and low folic acid related differential sites in the sweet corn germplasm and the waxy corn germplasm, the 269-bit 271 in the SEQ ID No.3 sequence is GGG in the high folic acid sweet corn and waxy corn germplasm, the GAA or CAA in the low folic acid genotype, the 267-bit 270 in the SEQ ID No.3 sequence is found to be the restriction enzyme site CTCC which can obviously distinguish the high and low folic acid from the heterozygous site, the corresponding restriction endonuclease is MnlI, and meanwhile, the 232-bit 236-bit restriction enzyme site CTCC in the SEQ ID No.3 sequence is utilized to monitor the quality of the enzyme digestion product, if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 82bp, the corn germplasm to be detected is the low folic acid genotype corn germplasm; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 47bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid homozygous genotype; if the electrophoresis band of the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid heterozygous genotype. The 235bp enzyme digestion product is a monitoring band, and the accuracy of the PCR experimental process and the enzyme digestion process can be proved only by cutting out the band from all detected germplasm.
The invention also provides application of the CAPS marker in identification of maize germplasm folate genotypes.
In the present invention, the application comprises the following steps: 1) extracting the corn genome DNA to be identified, and performing PCR amplification by using the forward primer and the reverse primer to obtain an amplification product; 2) carrying out enzyme digestion on the amplification product by using a restriction enzyme MnlI to obtain an enzyme digestion product; 3) electrophoresing the enzyme digestion product, and judging the folic acid genotype of the corn to be identified according to the band type obtained by electrophoresis; if the electrophoresis strip has two fragments of 235bp and 82bp, the maize germplasm to be detected is the maize germplasm with a low folate genotype; if the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the maize germplasm to be detected is the maize germplasm with a high folic acid genotype.
In the invention, corn genome DNA to be identified is extracted, and PCR amplification is carried out by using the forward primer and the reverse primer to obtain an amplification product. In the present invention, the maize to be identified is preferably sweet maize or waxy maize; the invention has no special requirement on the extraction method of the maize genome to be identified, and the conventional plant genome extraction method in the field can be adopted. In the specific implementation process of the invention, the TPS method is adopted to extract the genome, and the extracted genome is preferably stored at-20 ℃ for later use.
After the corn genomic DNA to be identified is obtained, the forward primer and the reverse primer are utilized to carry out PCR amplification, and an amplification product is obtained. In the present invention, the PCR amplification system preferably comprises the following components in 25. mu.L: 2 XPCRMix 12.5 uL, ddH2O11 muL, forward primer 0.5 muL, reverse primer 0.5 muL, DNA template 0.5 muL; the concentration of the forward primer and the reverse primer is 10 mu M; the above-mentionedThe procedure for PCR amplification of (a) is preferably as follows: denaturation at 95 deg.C for 3 min; denaturation at 95 deg.C for 30s, annealing at 62.5 deg.C for 30s, and extension at 72 deg.C for 1min, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃. The invention obtains an amplification product after the PCR amplification.
After the amplification product is obtained, the amplification product is subjected to enzyme digestion by using restriction enzyme Mnl I to obtain an enzyme digestion product. In the present invention, the enzyme-digested system is 25 μ L, and preferably comprises the following components: ddH217.3-17.4 mu L of O, 2.5 mu L of 10 multiplied by Buffer, 5 mu L of amplification product and 0.1-0.2 mu L of 1U MnlI restriction endonuclease; the enzyme cutting temperature is preferably 37 ℃, and the enzyme cutting time is preferably 15-30 min. In the present invention, the restriction enzyme MnlI is preferably available from NEB or Thermo Fisher; when the restriction enzyme MnlI is purchased from NEB, the system for enzyme cleavage is preferably as follows: ddH2O17.3 mu L, 10 Xbuffer 2.5 mu L, amplification product 5 mu L, 1U MnlI restriction endonuclease, wherein the temperature of enzyme digestion is preferably 37 ℃, and the time of enzyme digestion is preferably 15 min. When the restriction enzyme MnlI is available from Thermo Fisher, the system for the enzyme cleavage is preferably as follows: ddH2O17.4. mu.L, 10 Xbuffer 2.5. mu.L, amplification product 5. mu.L, 1U MnlI restriction endonuclease, the temperature of the enzyme digestion is preferably 37 ℃, and the time of the enzyme digestion is preferably 30 min.
After the enzyme digestion is finished, electrophoresing the enzyme digestion product, and judging the folic acid genotype of the corn to be identified according to the type of a band obtained by electrophoresis; electrophoresing the enzyme digestion product, and judging the folic acid genotype of the corn to be identified according to the band type obtained by electrophoresis; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 82bp, the maize germplasm to be detected is the maize germplasm with a low folate genotype; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 47bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid homozygous genotype; if the electrophoresis band of the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid heterozygous genotype. In the present invention, the electrophoresis is preferably 2% agarose gel electrophoresis, and the electrophoresis result is preferably imaged using a gel imager. The invention has no special requirements on the specific operation steps and parameter setting of the electrophoresis, and the conventional electrophoresis operation steps and parameter setting in the field can be adopted.
The invention also provides application of the CAPS marker in auxiliary corn breeding, sweet and waxy corn germplasm materials with high and low folic acid content and genotypes can be screened by using the CAPS marker for identifying the maize germplasm folic acid genotypes, the breeding is assisted, the efficiency of selecting the high folic acid genotypes in the corn breeding process is improved, and the breeding process of the corn can be accelerated.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The nucleotide sequence of the primer marked by CAPS is as follows:
F:5’-ACGCCGCTTCTCTGGCTAGG-3’
R:5’-ACGGGTGATCCTCCTGACGG-3’
the method for identifying the maize germplasm folate genotype by using the CAPS marked primer comprises the following steps:
1. extracting corn genome DNA (including sweet corn and waxy corn) to be detected by using a TPS method;
the primers are used for carrying out PCR amplification on the extracted sweet corn and waxy corn genome DNA to obtain PCR products.
The amplification system of PCR amplification is: 2 XPCRMix 12.5 uL, ddH2mu.L of O11, 0.5. mu.L of each of the forward primer and the reverse primer (stock solution concentration 10. mu.M), and 0.5. mu.L of the maize genomic DNA.
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 30s, annealing at 62.5 deg.C for 30s, and extension at 72 deg.C for 1min, and circulating for 35 times; finally, extending for 5min at 72 ℃; obtaining PCR amplification products, and storing at 4 ℃ for later use.
2. The amplification product was digested with MnlI restriction endonuclease from NEB, 17.3. mu.L ddH2O, 10 XBuffer 2.5 uL, PCR product 5 uL, 1U MnlI restriction endonuclease, the reaction conditions are as follows:the enzyme digestion temperature is 37 ℃, the enzyme digestion reaction time is 15min, and an enzyme digestion product is obtained.
3. The resulting cleavage products were detected by 2% agarose gel electrophoresis and imaged by a gel imager. If the enzyme digestion product has two fragments of 235bp and 47bp, the germplasm of the sweet corn and the waxy corn to be detected is the corn with the high folic acid genotype; if the enzyme digestion product has two fragments of 235bp and 82bp, the germplasm of the sweet and waxy corns to be detected is or is candidate to be the corn with low folate genotype; if the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the germplasm of the sweet and waxy corns to be detected is or is selected as the corn with high folic acid heterozygous genotype; the electrophoresis result is shown in figure 1, wherein the electrophoresis lane is a marker band, an amplification product band and an enzyme digestion band of materials No. 1-15 from left to right in sequence.
TABLE 1 identification of maize germplasm folate genotype by CAPS markers and relationship to germplasm folate content
| Numbering | Name of Material | Water content of fresh ear | Folic acid content | Band pattern after enzyme digestion | Type (B) |
| 1 | SKT1 | 58.2 | 208.8±11.6 | High folate genotype | Sweet corn |
| 2 | SKT2 | 73.5 | 36.5±3.3 | Low folate genotype | Sweet corn |
| 3 | SKT3 | 78.6 | 43.4±3.4 | Low folate genotype | Sweet corn |
| 4 | SKT4 | 77.6 | 122.8±10.3 | Heterozygous genotype | Sweet corn |
| 5 | SKT5 | 57 | 139.8±8.4 | Heterozygous genotype | Sweet corn |
| 6 | SKT6 | 84.3 | 20.5±0.3 | Low folate genotype | Sweet corn |
| 7 | SKN1 | 61.6 | 102.8±10.9 | Heterozygous genotype | Waxy corn |
| 8 | SKN2 | 62.2 | 73.2±4.2 | Low folate genotype | Waxy corn |
| 9 | SKN3 | 51.9 | 110.6±5.0 | Heterozygous genotype | Waxy corn |
| 10 | SKN4 | 71.1 | 190.1±6.7 | High folate genotype | Waxy corn |
| 11 | SKN5 | 61.7 | 68.1±4.3 | Low folate genotype | Waxy corn |
| 12 | SKN6 | 62 | 60.1±4.7 | Low folate genotype | Waxy corn |
| 13 | SKN7 | 68.9 | 42.5±1.8 | Low folate genotype | Waxy corn |
| 14 | SKN8 | 60.2 | 34.8±2.5 | Low folate genotype | Waxy corn |
| 15 | SKN9 | 62.2 | 73.2±4.2 | Low folate genotype | Waxy corn |
According to the embodiments, the molecular marker provided by the invention can be used for rapidly and effectively identifying the maize germplasm folate genotype, the identification result is all consistent with the biochemical detection result, and the identification efficiency and the accuracy are high.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shanghai city academy of agricultural sciences
<120> CAPS marker for identifying maize germplasm folate genotype and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
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<213> Artificial Sequence
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acgggtgatc ctcctgacgg 20
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tcggctacta ccgcccaaac tacaggggca accagtggtc aggcagcgtg ctccccaacg 180
tcctgccggt gaagcctgac gtcggtccgg cccacgtcgt ctcgcacaag agaggcgcta 240
cgacggtggg tgtcacgcct tggatcgaga actacaatgt ccccgtgctg tgtaaggacg 300
tcgccaccgt caggaggatc acccgt 326
Claims (10)
1. A CAPS marker for identifying the folate genotype of a maize germplasm is characterized in that primers for amplifying the CAPS marker comprise a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 2.
2. The CAPS marker of claim 1, wherein the nucleotide sequence obtained by PCR amplification using the forward primer and the reverse primer is shown as SEQ ID No.3 using a maize genome as a template.
3. The CAPS marker of claim 1 or 2, wherein the maize germplasm comprises sweet maize germplasm and waxy maize germplasm.
4. Use of a CAPS marker according to any of claims 1 to 3 for identifying the folate genotype of maize germplasm.
5. Use according to claim 4, characterized in that it comprises the following steps:
1) extracting corn genome DNA to be identified, and carrying out PCR amplification by using the forward primer and the reverse primer in claim 1 to obtain an amplification product;
2) carrying out enzyme digestion on the amplification product by using a restriction enzyme MnlI to obtain an enzyme digestion product;
3) electrophoresing the enzyme digestion product, and judging the folic acid genotype of the corn to be identified according to the band type obtained by electrophoresis; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 82bp, the maize germplasm to be detected is the maize germplasm with a low folate genotype; if the electrophoresis strip of the enzyme digestion product has two fragments of 235bp and 47bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid homozygous genotype; if the electrophoresis band of the enzyme digestion product has three fragments of 235bp, 47bp and 82bp, the maize germplasm to be detected is the maize germplasm of the high-folic acid heterozygous genotype.
6. The use of claim 5, wherein the PCR amplification system of step 1) comprises the following components in 25 μ L: 2 XPCRMix 12.5 uL, ddH2O11 muL, forward primer 0.5 muL, reverse primer 0.5 muL, DNA template 0.5 muL; the concentration of the forward primer and the reverse primer was 10. mu.M.
7. The use according to claim 6, wherein the PCR amplification procedure in step 1) is as follows: denaturation at 95 deg.C for 3 min; denaturation at 95 deg.C for 30s, annealing at 62.5 deg.C for 30s, and extension at 72 deg.C for 1min, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃.
8. The use of claim 5, wherein the enzyme-digested system in step 2) comprises the following components in 25 μ L: ddH2O17.3-17.4. mu.L, 10 Xbuffer 2.5. mu.L, amplification product 5. mu.L, 1U MnlI restriction endonuclease 0.1-0.2. mu.L.
9. The application of claim 8, wherein the temperature of the enzyme digestion in the step 2) is 37 ℃, and the time of the enzyme digestion is 15-30 min.
10. Use of the CAPS marker of claim 1 to aid in maize breeding.
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