CN112410458A - PCR primer for quickly and early identifying weedy rice and application thereof - Google Patents
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Abstract
The invention discloses a PCR primer for quickly and early identifying weedy rice and application thereof, wherein a double PCR primer P-Bh4 and a double PCR primer P-Bh4/P-Rc are designed in the deletion marker region of black glume gene Bh4 and red pericarp gene Rc of rice; carrying out PCR amplification on rice genome DNA by using the primers; the products are separated by agarose gel electrophoresis, and the weedy rice is identified according to an electrophoresis band. According to the invention, the weedy rice with black glumes or red pericarps can be identified by detecting the wild type genes of the black glume gene Bh4 and the red pericarp gene Rc, so that the weedy rice can be comprehensively and effectively identified; the method can rapidly identify the weedy rice in the rice seedling stage, is simple to operate, has accurate results, and is easy for batch detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a PCR primer for quickly and early identifying weedy rice and application thereof.
Background
The weedy rice is rice which continuously grows in a paddy field, is harmful to rice production and has weed characteristics, is early-maturing, easy to fall, mostly red rice grains, has strong biological competitiveness and is the third most serious weed next to barnyard grass and moleplant seed in the paddy field. In recent years, in some rice areas of Jiangsu, Hunan, Guangdong, Liaoning provinces and other provinces in China, weedy rice is more and more generated, the yield and the quality of rice are seriously influenced, and the rice production and the grain safety in China are threatened. The main reason that the weedy rice is serious is the wide popularization and application of light rice cultivation measures such as wheat-rice interplanting, direct seeding rice, no tillage or little tillage and the like; secondly, with the improvement of the level of agricultural mechanization, the mechanical harvesting of the paddy rice is more and more common, and the mechanical harvesting carries weed rice seeds, which leads to the continuous diffusion of the weed rice. The weedy rice has the same falling grain and dormancy characteristics as the wild rice, so that the weedy rice is continuously propagated and survived in the rice field and invades the cultivated rice field, is difficult to control, and is difficult to completely eradicate once the weedy rice invades the rice field. Until now, no effective weedy rice control technology, herbicide with selective killing effect specially aiming at weedy rice and matched technology exist.
The shapes, physiological backgrounds and genetic backgrounds of the weedy rice and the cultivated rice are extremely similar and are extremely difficult to distinguish from the appearance. However, the seedling emergence and the maturity of the weed rice are earlier than those of the cultivated rice, the tillering number is more, the plant is higher or lower than that of the cultivated rice, the rice husk is black or yellow, most seed coats are red, and the rice is easy to fall. The traditional weedy rice identification method is mainly based on the analysis of biological characters such as plant type, grain type, falling grain property and the like, so that the identification can be carried out only at the later growth stage of rice, and the harm caused by weedy rice is inevitable at the moment. Therefore, there is a need for rapid and accurate identification of weedy rice at the seedling stage of rice, and early removal to reduce weedy rice damage in rice fields.
At present, the reported early weedy rice identification methods are all identified by molecular techniques such as PCR (polymerase chain reaction), and PCR identification primers are mainly designed by deletion markers of red peel gene Rc, but the weedy rice with white peel cannot be detected; in addition, a method for identifying weedy rice by nested PCR and enzyme digestion detection of weedy rice SNP sites is provided, but the method has more complicated steps.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a PCR primer for quickly and early identifying weedy rice and application thereof, and can effectively identify the weedy rice in the rice seedling stage (early rice planting stage).
In order to achieve the purpose, the RCR primer for quickly and early identifying the weedy rice is designed, and the primer is a P-Bh4 primer:
the forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3', the reverse primer P-Bh4-R is: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
The invention also provides an application of the RCR primer in quickly identifying the weedy rice in a rice seedling stage (the weedy rice is identified by detecting the glume color of the rice).
The method of the above application:
1) extracting DNA, namely extracting the genomic DNA of the rice to be detected;
2) and (3) PCR amplification: performing PCR amplification by using the DNA extracted in the step 1) as a template and the RCR primer of claim 1;
3) and (3) carrying out electrophoretic observation and identification on the PCR product:
and if a 356bp band appears on the lane, determining that the rice to be detected is the weedy rice.
The invention also provides a double PCR primer combination for quickly and early identifying weedy rice and cultivated rice, wherein the combined primer consists of two pairs of primers of a P-Bh4 primer and a P-Rc primer, and the P-Bh4 primer is as follows:
the forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3', the reverse primer P-Bh4-R is: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
The P-Rc primer is:
the forward primer P-Rc-F is: 5'-CATTCTCCAGATGGACTACTCC-3', the reverse primer P-Rc-R is: 5'-GATGGCACCGACTTTTCGCGT-3' are provided.
(the P-Bh4 primer is designed according to 22bp deletion marker of Bh4 gene, a 356bp band can be amplified by Bh4 wild-type gene, and a 191bp band can not be amplified by Bh4 deletion type; the P-Rc primer is designed according to 14bp deletion marker of Rc gene, and a 191bp band can be amplified by Rc wild-type gene, and the Rc deletion type can not be amplified).
The invention also provides a detection kit for rapidly and early identifying the weedy rice and the cultivated rice, and the kit comprises the dual PCR primer combination.
The invention also provides an application of the double PCR primer combination or the kit in quickly identifying the weedy rice and the cultivated rice in a rice seedling stage (the weedy rice and the cultivated rice are identified by detecting the color of glumes and pericarps of the rice).
The invention also provides a method for rapidly identifying weedy rice and cultivated rice in the seedling stage of rice, which comprises the following steps:
1) extracting DNA, namely extracting the genomic DNA of the rice to be detected;
2) and (3) PCR amplification: performing double PCR amplification by using the DNA extracted in the step 1) as a template and adopting the double PCR primer combination of claim 4;
3) and (3) carrying out electrophoretic observation and identification on the PCR product:
if two or one target strip is arranged on the lane, the paddy rice to be detected is judged to be weedy rice,
if there is no specific band, it is cultivated rice.
Preferably, in the step 3),
if one strip of 356bp and one strip of 191bp simultaneously appear on the lane, the rice to be detected is judged to be the weedy rice with black glume red peel;
if a 356bp strip appears on the lane, the rice to be detected is judged to be the weedy rice with black glume white pericarp;
if a stripe of 191bp appears on the lane, the rice to be detected is judged to be the weedy rice with yellow glume red peel;
and if no specific strip exists on the lane, judging that the rice to be detected is the cultivated rice.
Preferably, in the step 2), the dual PCR reaction system is:
the double PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 64 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 7 min.
The principle of the invention is as follows:
the black glume and red peel are the basic characteristics of common wild rice, and the cultivated rice is mainly yellow glume and white peel. Glume color and red pericarp are one of the important phenotypic identification indexes for the differentiation of wild rice and cultivated rice. Bh4 is a gene for controlling black glumes of rice, and most of Bh4 genes of yellow glume cultivated rice contain a 22bp deletion, so that the function of the protein is lost, and the glumes are changed into yellow. Rc is the most important gene for regulating red pericarp of rice, and the Rc gene in most rice varieties contains 14bp deletion, so that anthocyanin cannot be synthesized, and the rice varieties are expressed as white pericarp. Wild-type Bh4 and Rc genes exhibited black glume and red pericarp traits, while the deletion-type Bh4 and Rc genes exhibited yellow glume and white pericarp traits. Many traits of weedy rice are intermediate between cultivated and wild rice. Early-stage field investigation researches show that the phenotypes of the weedy rice in China are rich and diverse, some weedy rice is similar to wild rice and has the characteristics of black glumes or red pericarps, other weedy rice has the characteristics of yellow glumes or white pericarps of cultivated rice, and most weedy rice is black-shelled red skin, yellow-shelled red skin and black-shelled white skin.
The invention has the beneficial effects that:
1. the wild-type gene Bh4 is applied to identification of weedy rice, and the weedy rice can be identified as the weedy rice as long as the wild-type gene Bh4 can amplify a target strip.
2. The invention combines the detection of the Bh4 wild-type gene and the Rc wild-type gene, is applied to the identification of the weedy rice, and can be identified as the weedy rice as long as the Bh4 wild-type gene and/or the Rc wild-type gene can amplify a target strip, namely the weedy rice has black glumes and/or red pericarps, so that the weedy rice can be comprehensively and effectively identified, and the weedy rice with the black glumes and the white pericarps cannot be missed.
3. The invention provides a double PCR/double PCR method, which can effectively identify weedy rice in the rice seedling stage (early rice planting stage), and has simple and quick operation and accurate result.
Drawings
FIG. 1: test result chart of example 1 of the present invention;
in the figure, M is DL 2000Marker in bp, wherein 1, 3, 6, 7, 9 and 10 are black glume weedy rice;
FIG. 2: test result chart of embodiment 2 of the present invention
In the figure, M is DL 2000Marker, the unit is bp, 1-5 are black glume red pericarp weedy rice, 6-10 are black glume white pericarp weedy rice, 11-15 are yellow glume red pericarp weedy rice, and 16-20 are cultivated rice.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1
The method for quickly and early identifying the weedy rice specifically comprises the following steps:
1) P-Bh4 primer design
The forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3' the flow of the air in the air conditioner,
the reverse primer P-Bh4-R is as follows: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
2) DNA extraction
Selecting 10 parts of sample in a paddy field flooded by weedy rice, collecting leaves, grinding by liquid nitrogen, and extracting the genomic DNA of the sample by using a plant genomic DNA extraction kit;
3) PCR amplification of sample genomic DNA
The reaction system of PCR amplification is as follows: 10.7. mu.L of sterile double distilled water, 12.5. mu.L of 2 XPCR Mix, 0.4. mu.L of each of primers P-Bh4-F and P-Bh4-R (10. mu. mol/L), and 1. mu.L of template DNA (10 ng/. mu.L); the amplification conditions were: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 64 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 7 min.
4) PCR product electrophoresis detection and result judgment
After the PCR was completed, 7. mu.L of the product was electrophoretically detected on a 2% agarose gel. If a 356bp band appears in the lane, it is identified as weedy rice. The electrophoresis results of 10 samples are shown in FIG. 1, in which 1, 3, 6, 7, 9 and 10 are black glume weedy rice.
Example 2
The method for rapidly identifying the weedy rice and the cultivated rice specifically comprises the following steps:
1) primer design
a.P-Bh4 primer design:
the forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3' the flow of the air in the air conditioner,
the reverse primer P-Bh4-R is as follows: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
The amplification product size was 356 bp.
b.P-Rc primer design:
the forward primer P-Rc-F is: 5'-CATTCTCCAGATGGACTACTCC-3' the flow of the air in the air conditioner,
the reverse primer P-Rc-R is: 5'-GATGGCACCGACTTTTCGCGT-3' are provided.
The size of the amplified product is 191 bp;
2) DNA extraction
Selecting 120 parts of weedy rice sample and 20 parts of cultivated rice sample, collecting young leaves in seedling stage, grinding by liquid nitrogen, and extracting genome DNA of the samples by using a plant genome DNA extraction kit;
3) performing double PCR amplification on sample genomic DNA
The reaction system of the double PCR amplification is as follows: 9.5. mu.L of sterile double distilled water, 2 XPCR Mix12.5. mu.L, 0.4. mu.L of each of the primers P-Bh4-F and P-Bh4-R (10. mu. mol/L), 0.6. mu.L of each of the primers P-Rc-F and P-Rc-R (10. mu. mol/L), and 1. mu.L of template DNA (10 ng/. mu.L). The amplification conditions were: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 64 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 7 min.
4) PCR product electrophoresis detection and result judgment
After the PCR was completed, 7. mu.L of the product was electrophoretically detected on a 2% agarose gel. If one strip of 356bp and one strip of 191bp appear on the lane, the weedy rice is black glume red peel; if a 356bp band appears on the lane, the weed rice is black glume white pericarp weed rice; if a stripe of 191bp appears on the lane, the weedy rice is yellow glume red peel; if no specific band is found in the lane, the cultivated rice is used. The results of electrophoresis of a portion of the sample are shown in FIG. 2. Wherein 1-5 are black glume red pericarp weedy rice, 6-10 are black glume white pericarp weedy rice, 11-15 are yellow glume red pericarp weedy rice, and 16-20 are cultivated rice.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. An RCR primer for quickly and early identifying weedy rice, which is characterized by comprising the following components in percentage by weight: the primer is a P-Bh4 primer:
the forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3' the flow of the air in the air conditioner,
the reverse primer P-Bh4-R is as follows: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
2. The RCR primer of claim 1 is applied to rapid identification of weedy rice in rice seedling stage.
3. Use according to claim 2, characterized in that: the application method comprises the following steps:
1) extracting DNA, namely extracting the genomic DNA of the rice to be detected;
2) and (3) PCR amplification: performing PCR amplification by using the DNA extracted in the step 1) as a template and the RCR primer of claim 1;
3) and (3) carrying out electrophoretic observation and identification on the PCR product:
and if a 356bp band appears on the lane, determining that the rice to be detected is the weedy rice.
4. A dual PCR primer combination for quickly and early identifying weedy rice and cultivated rice is characterized in that: the combined primer consists of two pairs of primers of a P-Bh4 primer and a P-Rc primer, wherein the P-Bh4 primer is as follows:
the forward primer P-Bh4-F is: 5'-TGATATGCTTCCTGCTGTGCAC-3' the flow of the air in the air conditioner,
the reverse primer P-Bh4-R is as follows: 5'-GACGGCCACGTTGAGCATCGAT-3' are provided.
The P-Rc primer is:
the forward primer P-Rc-F is: 5'-CATTCTCCAGATGGACTACTCC-3' the flow of the air in the air conditioner,
the reverse primer P-Rc-R is: 5'-GATGGCACCGACTTTTCGCGT-3' are provided.
5. A detection kit for rapidly and early identifying weedy rice and cultivated rice is characterized in that: the kit comprises the dual PCR primer combination of claim 4.
6. Use of the dual PCR primer combination of claim 4 or the kit of claim 5 for rapid identification of both weedy and cultivated rice.
7. A method for rapidly identifying weedy rice and cultivated rice in a rice seedling stage is characterized by comprising the following steps: the method comprises the following steps:
1) extracting DNA, namely extracting the genomic DNA of the rice to be detected;
2) and (3) PCR amplification: performing double PCR amplification by using the DNA extracted in the step 1) as a template and adopting the double PCR primer combination of claim 4;
3) and (3) carrying out electrophoretic observation and identification on the PCR product:
if two or one target strip is arranged on the lane, the paddy rice to be detected is judged to be weedy rice,
if there is no specific band, it is cultivated rice.
8. The method for rapidly identifying weedy rice and cultivated rice at the rice seedling stage as claimed in claim 7, wherein: in the step 3), the step of the method comprises the following steps,
if one strip of 356bp and one strip of 191bp simultaneously appear on the lane, the rice to be detected is judged to be the weedy rice with black glume red peel;
if a 356bp strip appears on the lane, the rice to be detected is judged to be the weedy rice with black glume white pericarp;
if a stripe of 191bp appears on the lane, the rice to be detected is judged to be the weedy rice with yellow glume red peel;
and if no specific strip exists on the lane, judging that the rice to be detected is the cultivated rice.
9. The method for rapidly identifying weedy rice and cultivated rice at the rice seedling stage as claimed in claim 7, wherein: in the step 2), the dual PCR reaction system is as follows:
9.5 mu L of sterilized double distilled water
2×PCR Mix 12.5μL
0.4. mu.L of each primer P-Bh4
0.6. mu.L each of the primers P to Rc
Template DNA 1. mu.L
The double PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 64 ℃ for 30s and 72 ℃ for 30 s; extension at 72 ℃ for 7 min.
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| CN114214447A (en) * | 2021-07-20 | 2022-03-22 | 南京农业大学 | PCR (polymerase chain reaction) combined primer for detecting weed rice mixing in large-batch cultivated rice seeds and application |
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| 刘丹 等: "InDel分子标记及其在水稻研究中的应用", 种子, vol. 36, no. 9, pages 49 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214447A (en) * | 2021-07-20 | 2022-03-22 | 南京农业大学 | PCR (polymerase chain reaction) combined primer for detecting weed rice mixing in large-batch cultivated rice seeds and application |
| CN114214447B (en) * | 2021-07-20 | 2023-08-25 | 南京农业大学 | PCR (polymerase chain reaction) combined primer for detecting hybridization of weed rice in large-scale cultivated rice seeds and application |
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