CN111100944A - Identification primer of rice grain type regulatory gene GW8 allele, identification method and application - Google Patents
Identification primer of rice grain type regulatory gene GW8 allele, identification method and application Download PDFInfo
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- CN111100944A CN111100944A CN202010047359.XA CN202010047359A CN111100944A CN 111100944 A CN111100944 A CN 111100944A CN 202010047359 A CN202010047359 A CN 202010047359A CN 111100944 A CN111100944 A CN 111100944A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to the technical field of biology, and particularly relates to an identification primer of a rice grain type regulatory gene GW8 allele, an identification method and application. Tender plant leaves are selected, total genomic DNA is extracted by adopting a CTAB method, a GW8-InDel marker of the invention is utilized to analyze specific bands of a pair of alleles of GW8, and an insertion/deletion fragment of the InDel marker is positioned on a CDS sequence of a GW8 gene. From the results of the agarose gel electrophoresis, it was observed whether specific bands were generated at 151bp and 159 bp. The method can be used for rapidly and accurately identifying the two alleles of GW8, and provides a method for identifying the GW8 allele, which has the advantages of low starting cost, high efficiency, simple operation and obvious strip specificity, for analyzing the relationship between the frequency and the contribution of the GW8 allele and various grain type traits.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an identification primer and an identification method of rice grain type regulatory gene GW8 allele.
Background
Polymerase Chain Reaction (PCR) technology is a technology for amplifying target genes in vitro firstly established by Kary Mullis of PE Cetus company in 1983, and can amplify specific DNA fragments by millions of times within a few hours.
InDel markers (Insertion/Deletion markers) are PCR primers designed to amplify Insertion Deletion sites in the genome. The InDel marker based on the PCR technology is widely applied to molecular marker-assisted selective breeding, QTL positioning and species identification due to the advantages of strong function, high efficiency, simple operation, low cost and the like, and has been successfully used for researching marker trait association and genetic variation.
The yield of cereal crops is largely determined by the grain type of the grain, and GW8(GRAIN WIDTH8) is a positive regulator of cell proliferation, and can increase the width of the grain and increase the grain weight. The insertion/deletion fragment appearing in the CDS sequence of the GW8 gene can be used for identifying the allele of the GW8, and through the research on the allele frequency, the contribution of the allele and the genetic variation, the insertion/deletion fragment can help rice breeders to formulate a breeding strategy for improving the appearance and the yield of rice and breed high-quality and high-yield rice varieties which are competitive in the international market. The invention relates to a pair of InDel markers for identifying GW8 alleles and an identification method thereof.
Disclosure of Invention
The invention aims to provide an identification primer and an identification method of a rice grain type regulatory gene GW8 allele, which are used for evaluating the relationship between the contribution of the GW8 allele and individual traits, and have the advantages of low starting cost, high efficiency, simple operation and obvious strip specificity.
In order to achieve the purpose, the invention provides the following technical scheme:
an identification primer of rice grain type regulatory gene GW8 allele, the primer sequence is as follows:
forward primer sequence "5'-TTTCAGTGTCCTCCTGTCTG-3'";
the reverse primer sequence "5'-ACCACTAAACCAGGTGCTAC-3'".
A method for identifying a rice grain type regulatory gene GW8 allele comprises the following steps:
extracting DNA of a germplasm plant to be detected;
adding the primer of claim 1 to perform a PCR reaction;
carrying out agarose gel electrophoresis on the PCR reaction product;
and (4) strip analysis.
Further, the strip analysis method comprises the following steps: observing whether specific bands appear at 151bp and 159 bp; if a specific strip appears at 159bp, the germplasm to be detected has A allele of GW8, which indicates that the germplasm to be detected is longer in grain length, narrower in grain width and thinner in grain thickness; if a specific strip appears at 151bp, the germplasm to be detected has a B allele of GW8, which indicates that the germplasm to be detected is short in grain length, wide in grain width and thick.
Further, DNA of the young leaves of the seeds to be tested is extracted by a CTAB method, and the extracted DNA is dissolved in double distilled water and stored at 4 ℃ for later use.
Further, the PCR reaction system is as follows: the volume of the reaction system is 20 mu L, which comprises: 2 XTaqMix buffer10 μ L, double distilled water 6 μ L, 10 μm primer GW8-InDel 2 μ L, and sample DNA 2 μ L.
Further, the PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 40s, and 35 cycles; extending at 72 deg.C for 10min, and storing at 4 deg.C.
Further, the PCR product was detected on agarose gel electrophoresis using 1 XTAE as electrophoresis buffer at a constant voltage of 120V for 30min, followed by observation and photographing under a gel imaging system.
Further, the agarose gel comprises: agarose 9g, 1 XTAE 300mL, EB stain 50. mu.L.
The invention also provides application of the identification primer of the rice grain type regulatory gene GW8 allele in rice trait identification.
Further, the rice traits include rice seed grain length, grain width and grain thickness.
The technical scheme provided by the invention realizes the following beneficial effects:
there have been few reports on identifying alleles using InDel marker and investigating the frequency, contribution and genetic variation, and there has been no report on identifying GW8 allele or on guiding breeding using the allele of the gene. The invention relates to an InDel marker for identifying GW8 allele and an identification method thereof, and evaluates the relationship between the contribution of GW8 allele and individual traits. The method has the advantages of low starting cost, high efficiency, simple operation and obvious specificity of the strip.
Drawings
FIG. 1 shows the labeling results of the GW8-InDel primer of the A, B allele;
FIG. 2 is a 199 germplasm strip pattern (numbers 1-24);
FIG. 3 is a 199 germplasm strip pattern (numbers 25-48);
FIG. 4 is a 199 germplasm strip pattern (numbers 49-72);
FIG. 5 is a 199 germplasm strip pattern (numbers 73-96);
FIG. 6 is a 199 germplasm strip pattern (numbers 97-120);
FIG. 7 is 199 germplasm strip patterns (No. 121-144);
FIG. 8 is a 199 germplasm strip map (accession number 145-168);
FIG. 9 is 199 germplasm strip patterns (No. 169-192);
FIG. 10 is a 199 germplasm strip map (No. 193-199).
Detailed Description
Example 1
Few reports have been made to date on the use of InDel markers to identify alleles and study their frequency, contribution and genetic variation. The invention relates to an InDel marker for identifying GW8 allele and an identification method thereof, and evaluates the relationship between the contribution of GW8 allele and individual traits. A specific band of GW8 allele can be amplified by using a primer GW 8-InDel; moreover, the invention discloses application of a primer GW8-InDel in identifying GW8 allele. A method for identifying GW8 alleles using the primers, comprising:
1) extraction of genomic DNA
Extracting DNA from 199 germplasm plants to be detected;
2) PCR reaction
Adding a primer GW8-InDel to perform PCR reaction, wherein the primer sequence is as follows:
forward primer sequence "5'-TTTCAGTGTCCTCCTGTCTG-3'"
Reverse primer sequence "5'-ACCACTAAACCAGGTGCTAC-3'";
3) agarose gel electrophoresis
Carrying out agarose gel electrophoresis on the PCR reaction product;
4) and (4) strip analysis.
2. The specific process of the step 1) is as follows: when 199 germplasm plants to be detected are subjected to fast heading, extracting DNA of leaves by adopting a CTAB method, dissolving the extracted DNA in double distilled water, and storing at 4 ℃ for later use.
3. The PCR reaction system of the step 2) is as follows: the reaction volume was 20. mu.L, which included: 2 XTaqMix buffer10 μ L, double distilled water 6 μ L, 10 μm primer GW8-InDel 2 μ L, sample DNA 2 μ L to be tested; the PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 40s, 35 cycles: extending at 72 deg.C for 10min, and storing at 4 deg.C.
4. The specific process of the step 3) is as follows: the PCR product was detected by agarose gel electrophoresis (agarose gel; 9g agarose +300mL 1 XTAE) using 1 XTAE as the electrophoresis buffer at a constant voltage of 120V.
5. The specific process of the step 4) is as follows:
shooting: observation and shooting under gel imaging system
And (3) analysis: specific bands appear at 151bp and 159bp respectively; two alleles of GW8 were identified.
Example 2
1. Extraction of genomic DNA
Selecting 199 different germplasms (see table 1 specifically), taking young leaves, extracting genome DNA by a conventional CTAB method, dissolving with double distilled water, and storing at 4 ℃ for later use.
TABLE 1.199 germplasm and its grain length, grain width, grain thickness and thousand grain weight data
2. PCR-based InDel molecular marker analysis
The PCR reaction volume was 20. mu.L, which included: 2 XTaqMix buffer10 μ L, double distilled water 6 μ L, 10 μm primer GW8-InDel 2 μ L, sample DNA 2 μ L to be tested; the PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 40s, 35 cycles: extending at 72 deg.C for 10min, and storing at 4 deg.C. The reaction was performed in an eppendorf PCR instrument.
The primer sequences are as follows;
forward primer sequence "5'-TTTCAGTGTCCTCCTGTCTG-3'"
Reverse primer sequence "5'-ACCACTAAACCAGGTGCTAC-3'";
3. agarose gel electrophoresis
The PCR product was detected by electrophoresis on an agarose gel (formulation shown in Table 2), and 10. mu.L of the sample was pipetted into a well using 1 XTAE as an electrophoresis buffer at a constant electrophoresis voltage of 120V for 30min, followed by observation and photographing under a gel imaging system.
TABLE 2 agarose gel formulation
| Reagent | Amount of one plate |
| IO WEST regular agarose G-10 | 9g |
| 1×TAE | 300mL |
| EB staining agent | 50μL |
4. The screened InDel primers are used for carrying out PCR reaction on the DNA of 199 germplasms, the result of the PCR reaction is observed through agarose gel electrophoresis, and a pair of specific primers are screened out, wherein the specific primers have special bands (151 bp and 159bp respectively) in different germplasms.
5. In 199 germplasms, the grain length, grain width and grain thickness of five grains at the top of the main stem ear of rice were measured for each germplasm, the average value was taken, and the thousand kernel weight of each germplasm was measured.
6. The data of Grain Length (GL), Grain Width (GW), Grain Thickness (GT), and Thousand Grain Weight (TGW) were analyzed together with the tape type data, respectively. Alleles were divided into A, B alleles based on banding patterns and data from germplasm containing the A allele was ranked and compared to that of germplasm containing the B allele.
TABLE 3 analysis of the data for Grain Length (GL), Grain Width (GW), Grain Thickness (GT), and Thousand Grain Weight (TGW)
The primer of the invention is designed by utilizing insertion/deletion fragments in CDS sequences of GW8 genes, whether the insertion/deletion fragments are contained or not is the key for distinguishing two alleles, and the strip analysis method comprises the following steps: observing whether specific bands appear at 151bp and 159 bp; if a specific strip appears at 159bp, the germplasm to be detected has A allele of GW8, which indicates that the germplasm to be detected is longer in grain length, narrower in grain width and thinner in grain thickness; if a specific strip appears at 151bp, the germplasm to be detected has a B allele of GW8, which indicates that the germplasm to be detected is short in grain length, wide in grain width and thick.
As shown in the table, the average grain length of the a allele was significantly reduced compared to the average grain length of the B allele, and the grain width and grain thickness were significantly reduced. Therefore, in long grain breeding, the A allele should be considered, and in the case of increasing grain width and grain thickness, the B allele should be considered.
Claims (10)
1. An identification primer of rice grain type regulatory gene GW8 allele is characterized in that the primer sequence is as follows:
forward primer sequence "5'-TTTCAGTGTCCTCCTGTCTG-3'";
the reverse primer sequence "5'-ACCACTAAACCAGGTGCTAC-3'".
2. A method for identifying a rice grain type regulatory gene GW8 allele is characterized by comprising the following steps:
extracting DNA of a germplasm plant to be detected;
adding the primer of claim 1 to perform a PCR reaction;
carrying out agarose gel electrophoresis on the PCR reaction product;
and (4) strip analysis.
3. The method for identifying the rice grain type regulatory gene GW8 allele according to claim 2, wherein the strip analysis method comprises: observing whether specific bands appear at 151bp and 159 bp; if a specific strip appears at 159bp, the germplasm to be detected has A allele of GW8, which indicates that the germplasm to be detected is longer in grain length, narrower in grain width and thinner in grain thickness; if a specific strip appears at 151bp, the germplasm to be detected has a B allele of GW8, which indicates that the germplasm to be detected is short in grain length, wide in grain width and thick.
4. The method for identifying the allele of the regulatory gene GW8 of rice grain type as claimed in claim 2, wherein the DNA of young leaves of the seed to be tested is extracted by CTAB method, and the extracted DNA is dissolved in double distilled water and stored at 4 ℃ for further use.
5. The method for identifying the rice grain type regulatory gene GW8 allele according to claim 2, wherein the PCR reaction system is: the volume of the reaction system is 20 mu L, which comprises: 2 XTaqMix buffer10 μ L, double distilled water 6 μ L, 10 μm primer GW8-InDel 2 μ L, and sample DNA 2 μ L.
6. The method for identifying the alleles of the rice grain type regulatory gene GW8 as claimed in claim 2, wherein the PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 40s, and 35 cycles; extending at 72 deg.C for 10min, and storing at 4 deg.C.
7. The method for identifying the alleles of GW8 of the rice grain type regulatory gene, as claimed in claim 2, wherein the PCR products are detected by agarose gel electrophoresis using 1 XTAE as electrophoresis buffer with electrophoresis constant voltage of 120V for 30min, followed by observation and photographing under a gel imaging system.
8. The method for identifying the GW8 allele according to claim 2, wherein the agarose gel comprises: agarose 9g, 1 XTAE 300mL, EB stain 50. mu.L.
9. The use of the primers for identifying the alleles of the rice grain type regulatory gene GW8 as claimed in claim 1 in the identification of rice traits.
10. The use of primers for identifying the alleles of the rice grain type regulatory gene GW8 as claimed in claim 9, wherein the rice traits include rice seed grain length, grain width and grain thickness.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308563A (en) * | 2021-05-28 | 2021-08-27 | 扬州大学 | Method for rapidly developing Australian wild rice specific molecular marker |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308563A (en) * | 2021-05-28 | 2021-08-27 | 扬州大学 | Method for rapidly developing Australian wild rice specific molecular marker |
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Application publication date: 20200505 |