Primer pair for identifying cold tolerance of rice and application of primer pair

Abstract

The invention provides a primer pair for identifying cold tolerance of rice and application thereof, and belongs to the technical field of biology. The primer pair comprises a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQ ID NO: 1 in a sequence table, and the sequence of the reverse primer is shown as SEQ ID NO: 2 in the sequence table. According to the primer pair for identifying the cold resistance of the rice and the application of the primer pair, the primer pair is used for identifying a functional variation site Indel marker site of COLD11, so that whether a to-be-detected sample carries a COLD11 excellent allele or not is accurately known, and then the cold resistance of the rice is accurately judged; and a technical support is provided for cultivating excellent rice varieties capable of resisting low-temperature cold damage.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer pair for identifying cold tolerance of rice and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and more than half of the world's population relies on rice as their staple food. Rice originated in tropical and subtropical regions, and is more sensitive to low temperatures than crops such as wheat and barley. In recent years, the annual rice planting area in the world is about 140 million hectares, of which more than 15 million hectares are threatened by low temperatures.

[0003] At present, relatively few natural variant genes that affect rice seedling cold tolerance have been cloned. Discovering more cold tolerance genes and using molecular marker-assisted selection to improve rice cold tolerance can help rice avoid the threat of low temperatures and thus cultivate excellent rice varieties that can resist low temperature damage.

[0004] Public Content

[0005] In order to solve the problems of the prior art, the present disclosure provides a primer pair for identifying cold tolerance of rice and its application. The technical solution is as follows:

[0006] In one aspect, the present disclosure provides a primer pair for identifying cold tolerance of rice, the primer pair comprising a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the reverse primer is shown in SEQ ID NO: 2 in the sequence listing.

[0007] In another aspect, the present disclosure provides an application of the above primer pair for identifying cold tolerance of rice, the application comprising:

[0008] Using the DNA of the sample to be tested as a template, using the forward primer and the reverse primer to perform PCR amplification to obtain an amplified product;

[0009] Using gel electrophoresis to detect bands in the amplified product;

[0010] If the bands in the amplified product include a target band, and the target band is 117 bp, then the sample to be tested carries the COLD11 superior allele.

[0011] Specifically, gel electrophoresis was performed using a 4% polyacrylamide gel.

[0012] Specifically, each 20 μL PCR amplification reaction system includes: 10 μL 2×Rapid Taq Plus Master Mix; 0.3 μL of the forward primer with a concentration of 10 μM; 0.3 μL of the reverse primer with a concentration of 10 μM; 3 μL of template DNA; and 6.4 μL of ddH2O.

[0013] The beneficial effects of the technical solution provided by the embodiments of the present disclosure are as follows: the embodiments of the present disclosure provide a primer pair for identifying the cold tolerance of rice and its application, the primer pair is used to identify the functional variation site Indel marker site of COLD11, so as to accurately know whether the sample to be tested carries the COLD11 superior allele, and then accurately judge the cold tolerance of rice, and provide technical support for the cultivation of excellent rice varieties that can resist low temperature damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 It is a sequence difference comparison diagram of the parent Nipponbare and the parent WD16343 at the COLD11 functional site provided in Example 2 of the present disclosure;

[0016] Figure 2 It is the amplified band diagram of the parent Nipponbare, the parent WD16343 and the F2 population provided in Example 2 of the present disclosure;

[0017] Figure 3 It is a statistical chart of the survival rate of seedlings of the parent Nipponbare, the parent WD16343 and the F2 population after cold treatment provided in Example 2 of the present disclosure. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0019] Embodiment 1

[0020] The present disclosure provides a primer pair for identifying cold tolerance of rice, the primer pair is used to identify the functional variation site Indel marker site of COLD11, the position of the Indel marker site is determined based on the sequence of the reference genome Nipponbare, and the specific sequence position is Chr11:23942756. The primer pair includes: a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID NO: 1 in the sequence list, and the sequence of the reverse primer is shown in SEQ ID NO: 2 in the sequence list. The specific sequence is shown in Table 1.

[0021] Table 1 shows the primer sequences and their corresponding Indel marker sites

[0022]

[0023] Embodiment 2

[0024] The present disclosure provides an application of the above primer pair. In this embodiment, the commercially available cold-resistant variety Nipponbare and the cold-intolerant variety WD16343 are used as samples to be tested. The application includes:

[0025] In this embodiment, DNA is first extracted from the sample to be tested: about 50 mg of fresh rice leaves to be tested are cut and placed in a 2 mL centrifuge tube, and 700 μL of 1.5×CTAB solution is added thereto. Two steel balls are added to the centrifuge tube, and the sample is placed in a proofing machine for 60 seconds, and the sample is ground until it is completely broken. Use a water bath preheated to 65°C for 60 minutes. Add chloroform:isoamyl alcohol (24:1) in an equal volume to CTAB, shake up and down to mix, and then centrifuge at 12000 r / min for 10 minutes to obtain a supernatant. Take 450 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol to the new centrifuge tube, invert and mix, and then put it in a -20°C refrigerator for precipitation for 1 hour. Centrifuge at 12000 r / min for 15 minutes, discard the supernatant, obtain a precipitate, and add 800 μL of 75% ethanol to the precipitate to wash the precipitate twice. After centrifugation again, the supernatant was discarded to obtain a precipitate (DNA). After the water in the precipitate evaporated, 50 μL of TE was used to dissolve the precipitate, and finally the precipitate was stored in a -20°C refrigerator for future use.

[0026] Using the DNA of the sample to be tested as a template, PCR amplification is performed using forward primers and reverse primers to obtain an amplified product;

[0027] Specifically, each 20 μL PCR amplification reaction system includes: 10 μL 2× Rapid Taq Plus Master Mix (provided by Novazonics); 0.3 μL forward primer with a concentration of 10 μM; 0.3 μL reverse primer with a concentration of 10 μM; 3 μL template DNA; and 6.4 μL ddH2O.

[0028] Further, the PCR amplification procedure is:

[0029]

[0030]

[0031] Gel electrophoresis is used to detect the target band in the amplified product; specifically, a 4% polyacrylamide gel is used for gel electrophoresis. In this embodiment, the preparation method of the 4% polyacrylamide gel includes: adding 45mL of 10×TBE to 360mL of a 30% mixed solution of acrylamide / bisacrylamide (29:1) (provided by Biosharp), and then adding ddH2O to make the volume to 900mL to prepare a PAGE mother solution with a concentration of 12%. Take 35mL of the 12% PAGE mother solution, 250μL of ammonium persulfate AP and 25μL of tetramethylethylenediamine (TEMED) and mix them to prepare a 4% polyacrylamide gel (working gel), and add the working gel to a glass plate and solidify it before use.

[0032] 1.5 μL of DNA template was spotted on each spotting well, the electrophoresis voltage was 150 V, and the electrophoresis time was 2 h.

[0033] Add 1.5 g of AgNO3 to 900 mL of ddH2O to prepare a silver staining solution.

[0034] Add 15 g of NaOH and 1.5 mL of formaldehyde to 1 L of ddH2O to prepare a developing solution.

[0035] First, place the polyacrylamide gel after electrophoresis in the silver stain solution for about 10 minutes; then place it in ddH2O to wash away the silver stain solution, then place it in the developer until the bands appear, and observe it on the gel imaging instrument.

[0036] The cold-tolerant variety Nipponbare and the cold-intolerant variety WD16343 were sequenced and analyzed. Figure 1 As shown. Figure 1 It can be seen that there are differences in the functional site sequences of the COLD11 gene between the cold-tolerant variety Nipponbare and the cold-intolerant variety WD16343. Specifically, the functional site sequence of the COLD11 gene of the cold-tolerant variety Nipponbare is (TCG+3GCG)×2, and the functional site sequence of the COLD11 gene of the cold-intolerant variety WD16343 is (TCG+3GCG).

[0037] If the bands in the amplified product include the target band, and the target band is 117 bp, then the sample to be tested carries the COLD11 superior allele.

[0038] The cold-tolerant variety Nipponbare and the cold-intolerant variety WD16343 were used as parents to construct the F2 population. The primer pair was used to detect the F2 population to obtain the genotype of the Indel marker site of each sample to be tested, as shown in the following figure: Figure 2 As shown, Figure 2 The middle band 1 shows the 100bp size marker, band 2 shows the genotype of the parent WD16343, band 3 shows the genotype of the parent Nipponbare, and bands 4 to 25 show the genotypes of the F2 population. Figure 2 It can be seen that band 3 should be the 117bp target gene, and the target gene was partially amplified in bands 4 to 25. The seedlings of the parent cold-tolerant variety Nipponbare, the parent cold-intolerant variety WD16343, and the F2 population were cold-treated at 4℃ for 36h, then restored at 25℃ for 7d, and the survival rate of the seedlings after cold treatment was calculated. Figure 3 As shown. Figure 3 It can be seen that A is the COLD11 genotype from the cold-tolerant Nipponbare, B is the COLD11 genotype from the cold-intolerant WD16343, and H is the heterozygous genotype. By comparison, rice carrying the Nipponbare COLD11 superior genotype is more cold-tolerant than rice carrying the WD16343 COLD11 genotype. The COLD11 genotype from Nipponbare can improve the cold tolerance of rice seedlings, and the heterozygous genotype will also improve the cold tolerance of rice to a certain extent.

[0039] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer pair for identifying cold tolerance of rice, characterized in that: The primer pair includes a forward primer and a reverse primer. The sequence of the forward primer is shown as SEQ ID NO: 1 in the sequence listing, and the sequence of the reverse primer is shown as SEQ ID NO: 2 in the sequence listing.

2. A use of the primer pair for identifying cold tolerance of rice as claimed in claim 1, characterized in that: The applications include: Using the DNA of the sample to be tested as a template, using the forward primer and the reverse primer to perform PCR amplification to obtain an amplified product; Using gel electrophoresis to detect bands in the amplified product; If the bands in the amplified product include a target band, and the target band is 117 bp, then the sample to be tested carries the COLD11 superior allele.

3. The use according to claim 2, characterized in that: Gel electrophoresis was performed using 4% polyacrylamide gel.

4. The use according to claim 2, characterized in that: Each 20 μL PCR amplification reaction system includes: 10 μL 2× Rapid Taq Plus Master Mix; 0.3 μL of the forward primer with a concentration of 10 μM; 0.3 μL of the reverse primer with a concentration of 10 μM; 3 μL of template DNA; and 6.4 μL of ddH2O.

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