CN116516045B - Specific molecular marker primer for methylation identification of weedy rice Os12g0622850 gene and application thereof - Google Patents
Specific molecular marker primer for methylation identification of weedy rice Os12g0622850 gene and application thereof Download PDFInfo
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Abstract
The invention discloses a specific molecular marker primer for methylation identification of a weedy rice Os12g0622850 gene and application thereof, wherein the molecular marker primer comprises forward primers OsG1-F1, osG1-F2, osG1-F3, osG1-F4 and OsG1-F5, and reverse primers OsG1-R1, osG-R2, osG1-R3, osG-R4 and OsG1-R5, and the forward primers OsG-R1, osG-R2, osG1-R3, osG-R4 and OsG-R5 respectively have nucleotide sequences shown as SEQ ID No. 2-11. According to the invention, by comparing specific methylation sites of weed rice and rice population in different regions of China, molecular marker primers based on DNA methylation and used for specific screening of weed rice in Hainan region are developed and used for specific identification of weed rice and rice population in Hainan region, and meanwhile, the primers based on sequence difference of RED pericarp gene RED4 of weed rice and cultivated rice are jointly utilized and used for rapid molecular identification of weed rice and rice, so that technical support is provided for output of weed rice in Hainan region.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and plant genetic engineering, and particularly relates to a specific molecular marker primer for methylation identification of a weedy rice Os12g0622850 gene and application thereof.
Background
Weedy rice (Oryza sativa l.f. spontanea) is a weedy rice plant that spontaneously continues to occur in paddy fields and has similar morphological and physiological characteristics to cultivated rice, and is recognized as one of three major malignant weeds in worldwide paddy fields. Rice (Oryza sativa L.) is one of the most important food crops worldwide, and breeds more than half of the population worldwide, while China is the largest rice producing country and consuming country in the world, and the safe production of rice crops is directly related to the sustainable development of food safety and agriculture in China. 10 plants/m 2 weed rice can cause yield loss of more than 20% of the rice, and further increase of density can cause sterilization and even field waste. In addition, the mixing of weedy rice also seriously affects the quality of rice, rice grains are uneven, the taste of rice is poor, the color becomes red, and the grade and commercial value of rice are reduced. The main rice planting areas in China almost have weed rice, the annual occurrence area of the weed rice is 600 ten thousand hectares, 27 hundred million kilograms of rice yield loss is caused by the weed rice each year, and about 1 ten thousand hectares of farmlands are forced to be abandoned due to the harm of the weed rice. The labor input of the weed control rice is 15000 ten thousand working days in the whole country, and the total cost of the control and loss is more than 300 hundred million yuan.
Hainan is a province of tropical islands, and is rich in biological resources but fragile in ecological environment. Hainan is used as a 'south-propagation silicon valley' of national biological breeding, is an indispensable base for rice breeding and additional generation breeding, has a plurality of breeding units, is complex in rice crop variety and source, and is extremely easy to suffer from the seed source pollution of local weed rice. The Hainan region faces dual challenges of external and internal defense output, especially as internal defense output of a south numerous base. The monitoring and detecting technology is a technical prerequisite for internal output prevention, so that the research and development of weed rice specific screening technology in Hainan area is very urgent.
At present, the conventional screening and identifying technology of plants is relatively more, and a method is established mainly based on polymorphism of DNA molecules, and reflects DNA base differences among genomes of different biological individuals or populations. Comprising the following steps: (1) Southern hybridization-based markers such as RFLP, SSCP-RFLP and DGGE-RFLP; (2) PCR-based labels, such as RAPD, AFLP, TRAP, SPAR, etc.; (3) Repeat-based markers such as SSR, SSLP, microsatellite DNA, etc.; (4) mRNA-based markers such as RT-PCR, EST, SAGE and the like; (5) single nucleotide polymorphism-based markers, such as SNPs; (6) Based on the difference of DNA sequences, CRISPR designs primers for identification; (7) Quantitative PCR and digital quantitative PCR, and designing primers for identification based on the difference of DNA sequences. However, most of these techniques can only identify species level or materials with specific genetic mutations, and it is difficult to individualize naturally occurring weedy rice from specific regional sources.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a specific molecular marker primer for methylation identification of a weed rice Os12g0622850 gene and application thereof, and the molecular marker primer based on DNA methylation specific screening of weed rice in Hainan regions is developed by comparing specific methylation sites of weed rice and rice populations from different regions in China, so that technical support is provided for the output of weed rice in Hainan regions.
The invention is realized by the following technical scheme:
specific molecular marker primers for methylation identification of rice grass Os12g0622850 gene include forward primers OsG-F1, osG1-F2, osG-F3, osG1-F4, osG1-F5, and reverse primers OsG1-R1, osG1-R2, osG1-R3, osG1-R4, osG1-R5;
the OsG-F1 has a nucleotide sequence shown as SEQ ID No. 2;
the OsG-F2 has a nucleotide sequence shown as SEQ ID No. 3;
the OsG1-F3 has a nucleotide sequence shown as SEQ ID No. 4;
the OsG-F4 has a nucleotide sequence shown as SEQ ID No. 5;
the OsG-F5 has a nucleotide sequence shown as SEQ ID No. 6;
The OsG-R1 has a nucleotide sequence shown as SEQ ID No. 7;
the OsG-R2 has a nucleotide sequence shown as SEQ ID No. 8;
The OsG R3 has a nucleotide sequence shown as SEQ ID No. 9;
the OsG-R4 have a nucleotide sequence shown as SEQ ID No. 10;
the OsG-R5 has a nucleotide sequence shown as SEQ ID No. 11.
The application of the molecular marker primer in specific identification of weedy rice.
Preferably, the weedy rice specific identification comprises the steps of:
(1) Extracting weed rice and rice DNA; bisulphite treatment, preparing template DNA; synthesizing a primer;
(2-1) result judgment: PCR amplification using OsG1-F1+ OsG1-R1/R2/R3 primer combinations or OsG1-F2+ OsG1-R1/R2/R3 primer combinations;
if single PCR band products of 85bp, 142bp and 178bp can be amplified and the gray value of the single PCR band product is the same as that of a Marker band, the single PCR band products are weed rice and paddy rice from Hainan area;
If a single band cannot be obtained, such as non-specific amplification, or if a single PCR product can be obtained, but the gray value of the product band is less than 0.8 compared with the gray value of the Marker band, other provinces of weedy rice and paddy rice are obtained.
Preferably, the method further comprises the step (2-2), specifically comprising the following steps:
(2-2) result judgment: PCR amplification was performed using OsG1-F3+ OsG1-R5 primer combinations or OsG1-F4/F5+ OsG1-R4 primer combinations;
If the single PCR band products of 352bp, 378bp and 403bp can be amplified and the gray value of the single PCR band product is the same as that of the Marker band, the single PCR band products are weed rice and paddy rice from the southern area of Yangtze river;
if a single band cannot be obtained, such as non-specific amplification, or if a single PCR product can be obtained, but the gray value of the product band is less than 0.8 compared with the gray value of the Marker band, the rice and the rice are weeds in the north of the Yangtze river.
Preferably, the method further comprises a step (3), specifically comprising the following steps:
(3) Specificity identification of weedy rice and rice: PCR amplification using RED4-for+RED4-rev primer combinations; the RED4-for has a nucleotide sequence shown as SEQ ID No. 12; the RED4-rev has a nucleotide sequence shown as SEQ ID No. 13;
If the length of the PCR amplification product is 118bp, the PCR amplification product is weedy rice;
If the length of the PCR amplification product is 104bp, the rice is obtained.
Preferably, the PCR amplification is specifically as follows:
The PCR reaction system is as follows: 100ng of sulfite modified genomic DNA, 6. Mu.L of dNTP mix, 5. Mu.L of 10X EPITAQ PCR Buffer, 2. Mu.L of 25mM MgCl, 2.0. Mu.L of 10. Mu.M forward and reverse primers, epiTaq HS 1.25U, and the Mixture was complemented to 50. Mu.L with ddH 2 O;
The PCR reaction procedure was: 95 ℃ for 5min;95 ℃ for 30 seconds, 48 ℃ to 56 ℃ delta 2 ℃,72 ℃ for 2min and 35cycles; the amplified product was scanned and recorded with a gel imager at 72℃for 10min, and the gray scale value of the single PCR product band was read using analytical software.
The technical scheme of the invention adopts the following design ideas:
Through data analysis of 64 weedy rice and rice population methylation groups, a methylation area exists between the methylation degree of the weedy rice Os12g0622850 gene in Hainan region and inland provincial weedy rice, and a methylation amplification primer is designed according to specific methylation sites in the methylation area and is used for specific identification of weedy rice and rice population in Hainan region. Meanwhile, the sequence difference primer of RED peel gene RED4 of the weedy rice and the cultivated rice is used for rapid molecular identification of the weedy rice and the rice.
The primer design principle is as follows:
When the rice DNA is treated with a reagent such as bisulfite, cytosine which undergoes DNA methylation remains unchanged, while cytosine which does not undergo DNA methylation undergoes deamination to uracil, which is replaced with thymine during polymerase chain reaction. Therefore, by sequencing and sequence alignment of the PCR products, the methylation occurrence of the oryza sativa gene can be clarified. Methylation analysis of the genes of Hainan, jiangsu, heilongjiang and other provincial weed rice Os12g0622850 shows that the methylation degree of the genes of Hainan weed rice Os12g0622850 is very high, and the methylation degree of the genes of other provincial weed rice is remarkably lower than that of Hainan weed rice. Therefore, a molecular marker primer for specifically screening weed rice in Hainan area is designed based on methylation difference of Os12g0622850 genes among different weed rice populations.
The beneficial effects of the invention are as follows:
(1) The detection technology is advanced, and the purpose of specific screening of weed rice in Hainan area can be met by utilizing the specific molecular marker primer.
(2) The detection is rapid, the identification and screening work of weed rice in Hainan area can be completed within a few hours, and the detection period is greatly shortened.
(3) The identification work of a plurality of plants can be simultaneously carried out, so that the production cost is saved, and the screening efficiency is greatly improved.
Drawings
FIG. 1 shows amplification results of Os12g0622850 gene-differential methylation region specific amplification primers for different weedy rice in example 2: 1 is a Marker, GZ is a Guizhou weed population, HU is a Hunan weed rice population, HA is a Hainan weed rice population, GX is a Guangxi Zhuang autonomous region weed rice population, GD is a Guangdong weed rice population, YN is a Yunnan weed rice population, ZJ is a Zhejiang weed rice population, and JX is a Jiangxi weed rice population;
FIG. 2 shows polyacrylamide gel electrophoresis of RED4 primer amplified fragments of weedy rice and rice in example 3: 1 is Marker,2 and 6 are rice, and the rest is weed rice.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific embodiments.
Example 1 screening of different weedy Rice and Rice differential methylation regions
The methylation group data of the 64 weedy rice (191 single plants) and the rice population (77 single plants) are used for screening methylation areas with significant differences between weedy rice and rice in Hainan area and other provinces. The results show that the Os12g0622850 gene (shown as SEQ ID No. 1) undergoes high-level methylation in rice and paddy rice in Hainan region, but does not undergo methylation in rice and paddy rice in other provinces such as Heilongjiang, jiangsu and the like. Therefore, a molecular marker primer for specifically screening the weed rice in the Hainan area is designed by utilizing the differential methylation region of the Os12g0622850 gene and is used for identifying the weed rice and the rice in the Hainan area.
Specific molecular marker primers for methylation identification of rice grass Os12g0622850 gene include forward primers OsG1-F1, osG1-F2, osG1-F3, osG1-F4, osG1-F5, and reverse primers OsG1-R1, osG1-R2, osG1-R3, osG1-R4, osG1-R5, as shown in Table 1.
TABLE 1 molecular marker primers designed based on Os12g0622850 gene
Example 2 rapid identification of weedy Rice/Rice in Hainan region and other provinces and North and south of the Yangtze river
114 Weed rice seed groups of 19 provinces such as Hainan province, guangdong province, jiangxi province and Heilongjiang province are selected for rapid identification and research of weed rice and other province weed rice in Hainan region, and each group has 3 single plants. The weed rice and the mature and full seeds of the rice are washed 3 times by deionized water and sterilized by NaClO with the volume fraction of 10 percent for 10 minutes. The sterilized seeds were washed 5 times with running water. Spreading filter paper in a disposable plastic culture dish, adding 8mL of sterilized water, uniformly spreading the cleaned seeds on the filter paper, and culturing in a plant incubator at 28 ℃ for 12h/d until the seeds are exposed. The weed rice and the rice seeds after white exposure are planted in sterilized nutrient soil and vermiculite (2:5), the plant is cultivated for about 5 weeks under the conditions of 28 ℃ and 12h/d illumination time, when the second complete leaf grows to 10cm, the whole plant of the plant material is taken down, quick frozen by liquid nitrogen and then placed in a refrigerator at the temperature of minus 80 ℃ for preservation. DNA extraction was performed using a DNA extraction kit (Tiangen), and the extracted DNA sample was subjected to concentration and quality detection using micro spectrophotometry. DNA methylation was performed using the EZ DNA Methylation-GoldTM KIT methylation kit of ZYMO RESEARCH.
PCR amplification was performed using the sulfite-treated DNA as a template, the PCR amplification primers are shown in Table 1, and the amplification system and the reaction procedure are shown in tables 2 and 3.
TABLE 2 amplification system (10. Mu.L)
| 2×Rapid PCR Mix | 5μL |
| Forward primer | 0.3μL |
| Reverse primer | 0.3μL |
| DNA template | 1.3μL |
| ddH2O | 3.1μL |
Table 3 reaction procedure
And (3) performing 1.0% agarose gel electrophoresis on the PCR product, performing photographing detection by using a gel imager, and reading the gray value of the PCR product band by using Image J analysis software on the single PCR product obtained by amplification.
After PCR amplification using OsG1-F1+ OsG1-R1/R2/R3 or OsG1-F2+ OsG1-R1/R2/R3 primer combinations, as shown in FIG. 1 and Table 4, PCR products of Hainan Area (HA) weedy rice were single bright bands of 85bp, 142bp and 178bp, and the brightness was equivalent to that of a Marker, and the gray value ratio was 1. The PCR amplification produces non-characteristic amplification of Guizhou (GZ), guangxi (GX), guangdong (GD), yunnan (YN), zhejiang (ZJ) and other provinces of partial weed rice and rice populations, so that the PCR product can be distinguished from weed rice and rice in Hainan area. Although single PCR products with the same size as Hainan weed rice can be amplified from the weed rice and rice populations in the provinces of Henan, hunan (HU), jiangxi (JX), liaoning and the like, the strip brightness is very low, and the contrast gray value with a Marker is below 0.8, so that the weed rice and rice in the Hainan area can be distinguished from the provinces according to the gray value.
TABLE 4 differentiation of Hainan from other provincial weedy rice
When carrying out PCR amplification by using OsG1-F3+ OsG1-R5 or OsG1-F4/F5+ OsG1-R4 primer combinations, as shown in Table 5, the single PCR products of 352bp, 378bp and 403bp can be obtained by amplifying weed rice and paddy rice in the south of Yangtze river such as Hainan, guangdong, guangxi and Hubei, and the like, the brightness is equivalent to that of a Marker, and the gray value ratio is 1. The rice or paddy rice in the north of the Yangtze river such as Heilongjiang, liaoning and the like can generate nonspecific amplification, or can obtain single PCR products of 352bp, 378bp and 403bp by amplification, but has very low strip brightness and a contrast gray value with a Marker of less than 0.8, so that the rice or paddy rice in the south of the Yangtze river and the north of the Yangtze river can be identified according to the strip or gray value.
TABLE 5 differentiation of weed Rice in the regions south of the Yangtze river and in the regions north of the Yangtze river
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Example 3 specific screening and identification of weedy Rice and Rice populations
After DNA extraction from the planted rice and paddy material described in example 2, PCR amplification was performed using the primers RED4-for and RED4-rev (Table 6), the amplification system and conditions were the same as above, and the PCR product was run on a 2% polyacrylamide gel.
TABLE 6 primers designed based on Red4 Gene
As shown in FIG. 2, the PCR amplified product band size of the rice population was 118bp, while the PCR amplified product band size of the rice population was 104bp. Therefore, the weedy rice and the rice can be directly identified according to the result of polyacrylamide gel electrophoresis.
In conclusion, the specificity screening and identification of the weed rice in the Hainan area can be realized by utilizing the weed rice and rice specificity detection primers designed based on the Os12g0622850 gene differential methylation region and combining the weed rice and rice specificity identification primers designed based on RED4 gene sequence differential.
Claims (4)
1. Specific molecular marker primers for methylation identification of rice grass 12g0622850 genes are characterized by comprising forward primers OsG1-F1, osG1-F2, osG1-F3, osG1-F4 and OsG1-F5 and reverse primers OsG1-R1, osG1-R2, osG1-R3, osG1-R4 and OsG1-R5;
the nucleotide sequence of OsG-F1 is shown as SEQ ID No. 2;
the nucleotide sequence of OsG-F2 is shown as SEQ ID No. 3;
the nucleotide sequence of OsG1-F3 is shown as SEQ ID No. 4;
the nucleotide sequence of OsG-F4 is shown in SEQ ID No. 5;
the nucleotide sequence of OsG to F5 is shown as SEQ ID No. 6;
the nucleotide sequence of OsG-R1 is shown as SEQ ID No. 7;
the nucleotide sequence of OsG-R2 is shown as SEQ ID No. 8;
The nucleotide sequence of OsG-R3 is shown as SEQ ID No. 9;
the nucleotide sequence of OsG-R4 is shown as SEQ ID No. 10;
the nucleotide sequence of OsG to R5 is shown as SEQ ID No. 11.
2. Use of the molecular marker primer according to claim 1 for specific identification of weedy rice, comprising the steps of:
(1) Extracting weed rice and rice DNA; bisulphite treatment, preparing template DNA; synthesizing a primer;
(2-1) result judgment: PCR amplification using OsG1-F1+ OsG1-R1/R2/R3 primer combinations or OsG1-F2+ OsG1-R1/R2/R3 primer combinations;
if single PCR band products of 85bp, 142bp and 178bp can be amplified and the gray value of the single PCR band product is the same as that of a Marker band, the single PCR band products are weed rice and paddy rice from Hainan area;
If a single band cannot be obtained, such as non-specific amplification is generated, or if a single PCR product is obtained, but the gray value of the product band is less than 0.8 compared with the gray value of the Marker band, the product band is selected from Anhui, fujian, guangdong, gansu, guangxi, guizhou, hubei, hebei, helong river, henan, hunan, jilin, jiangsu, jiangxi, liaoning, ningxia, shanxi, sichuan, shandong, shanghai, xinjiang, yunnan or Zhejiang weed rice and paddy rice;
(3) Specificity identification of weedy rice and rice: PCR amplification using RED4-for+RED4-rev primer combinations; the nucleotide sequence of RED4-for is shown as SEQ ID No. 12; the nucleotide sequence of RED4-rev is shown as SEQ ID No. 13;
If the length of the PCR amplification product is 118bp, the PCR amplification product is weedy rice;
If the length of the PCR amplification product is 104bp, the rice is obtained.
3. The use according to claim 2, further comprising step (2-2), in particular:
(2-2) result judgment: PCR amplification was performed using OsG1-F3+ OsG1-R5 primer combinations or OsG1-F4/F5+ OsG1-R4 primer combinations;
If the single PCR band products of 352bp, 378bp and 403bp can be amplified and the gray value of the single PCR band product is the same as that of the Marker band, the single PCR band products are weed rice and paddy rice from Hainan, anhui, fujian, guangdong, guangxi, guizhou, hubei, hunan, jiangsu, jiangxi, sichuan, shanghai, yunnan or Zhejiang;
If a single band cannot be obtained, such as non-specific amplification, or if a single PCR product is obtained, but the gray value of the product band is less than 0.8 compared with the gray value of the Marker band, the product band is weed rice and paddy rice from Gansu, hebei, heilongjiang, henan, jilin, liaoning, ningxia, shanxi, shandong or Xinjiang.
4. Use according to claim 2 or 3, characterized in that the PCR amplification is in particular as follows:
The PCR reaction system is as follows: the genome DNA 100 ng, dNTP mix 6 [ mu ] L, 10× EPITAQ PCR Buffer 5 [ mu ] L, 25mM MgCl 2 [ mu ] L, 10 [ mu ] M forward and reverse primers after sulfite modification are respectively 2.0 [ mu ] L and EpiTaq HS 1.25.25U, and ddH 2 O is used for complementing the primers to 50 [ mu ] L;
The PCR reaction procedure was: 95 ℃ 5 min;95 ℃ for 30 s, 48-56 ℃ delta 2 ℃,72 ℃ for 2min and 35 cycles; the amplified products were recorded by scanning with a gel imager at 72℃10 min and the grey scale values of the individual PCR product bands were read using analytical software.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101338338A (en) * | 2008-08-15 | 2009-01-07 | 江苏省农业科学院 | Molecule marker of red peel gene of weedy rice |
| CN103074443A (en) * | 2013-02-22 | 2013-05-01 | 南京农业大学 | Primer for weedy rice detection and application of primer |
| CN108949925A (en) * | 2018-07-06 | 2018-12-07 | 中国水稻研究所 | A kind of molecular detecting method for quick and precisely identifying Weedy Rice and cultivated rice |
| CN110904257A (en) * | 2019-11-08 | 2020-03-24 | 南京农业大学 | Specific molecular marker primer for methylation determination of OsICE1 gene promoter of rice and weedy rice and application thereof |
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| WO2007033370A2 (en) * | 2005-09-14 | 2007-03-22 | Cornell Research Foundation, Inc. | Rc, brown pericarp and seed coat |
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| CN101338338A (en) * | 2008-08-15 | 2009-01-07 | 江苏省农业科学院 | Molecule marker of red peel gene of weedy rice |
| CN103074443A (en) * | 2013-02-22 | 2013-05-01 | 南京农业大学 | Primer for weedy rice detection and application of primer |
| CN108949925A (en) * | 2018-07-06 | 2018-12-07 | 中国水稻研究所 | A kind of molecular detecting method for quick and precisely identifying Weedy Rice and cultivated rice |
| CN110904257A (en) * | 2019-11-08 | 2020-03-24 | 南京农业大学 | Specific molecular marker primer for methylation determination of OsICE1 gene promoter of rice and weedy rice and application thereof |
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