CN110878375A - Specific molecular marker for detecting rice cold-resistant gene CTB4a and application thereof - Google Patents
Specific molecular marker for detecting rice cold-resistant gene CTB4a and application thereof Download PDFInfo
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- 235000009566 rice Nutrition 0.000 title claims abstract description 72
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- 239000003147 molecular marker Substances 0.000 title claims abstract description 40
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- 238000009395 breeding Methods 0.000 claims abstract description 16
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- 101100274508 Oryza sativa subsp. japonica CKI1 gene Proteins 0.000 description 3
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Abstract
The invention discloses a specific molecular marker for detecting a rice cold-resistant gene CTB4a, which is named as SNP2063, has the molecular marker polymorphism of T/C, and is detected by using a group of primer combinations, wherein the primer combinations comprise primers CTB4a-Ft, CTB4a-Fc and CTB4 a-R. The molecular marker for detecting the cold-resistant gene CTB4a of the rice has high specificity, the provided application method of the molecular marker is convenient to operate, the detection result is accurate and reliable, the molecular marker can be used for identification and auxiliary breeding of the cold-resistant gene CTB4a of the rice, and the problems of low efficiency and long period of traditional breeding can be solved.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a specific molecular marker for detecting a rice cold-resistant gene CTB4a and application thereof.
Background
Rice (Oryza sativa L.) is an important food crop. The population of rice as staple food in the world is about 50%, and the demand for rice in asian areas increases year by year. Due to rapid development of global economy, increased population pressure, drastic increase of industrial land and reduction of cultivated land area, rice production faces huge pressure, so that the continuous improvement and excavation of the high-yield potential of rice are the main research directions of scientists. The cold resistance of the rice at the bud stage is closely related to the quality and yield of the rice.
The low temperature is an important limiting factor influencing the rice yield, the rice is sensitive to the low temperature in the bud stage, and the cold injury of japonica rice and indica rice occurs below 15 ℃ and 18 ℃ respectively. The cold damage of rice in the bud stage can cause the rice seeds to be prevented from sprouting and stop growing at growing points, and the death of the seeds can be caused by long-term low temperature. In high latitude and high altitude areas, the temperature in the rice seeding period is low, so that the popularization of a plurality of rice varieties is limited.
Wild rice grows in a complex natural environment and contains a large number of excellent genes, and wild rice growing in the north of Guangxi Guilin can normally overwinter, germinate and grow at subzero temperature and contains a large number of cold-resistant genes. In recent years, the cloning of cold-resistant genes in rice sprout stage by using a molecular biological method becomes a hotspot of research, and the cloned wild rice sprout stage cold-resistant genes have important theoretical and practical guiding significance for improving the stress resistance of rice.
The COLD-resistant mechanism of rice is not deeply and systematically researched, the former obtains several genes, namely qLTG3-1, COLD1, LTG1, LTT7, CTB1 and CTB4a, by forward cloning, and several COLD-resistant genes are widely involved in the process of signal transduction.Fujino clones one of QTL qLTG3-1, the gene is expressed in embryo and encodes an unknown protein.Liu and the like hybridize towards No.2 by using a strong COLD-resistant material IL112 and a COLD-sensitive material Guigui, 7 COLD-resistant QTLs are positioned in the later generation population, and combined with expression spectrum analysis, a COLD-resistant gene LTT7 gene encoding an extension protein is obtained and is related to fresh plant weight at low temperature after sowing, the COLD-resistant gene is induced to improve the COLD resistance of rice, Lu and the like clone a COLD-resistant gene LTG1, an LTG1 encoding protein kinase, which is involved in the synthesis and regulation of COLD-resistant gene, and regulation of a COLD-resistant gene, the COLD-resistant gene encoding protein is involved in the COLD-resistant gene encoding protein kinase, the COLD-resistant gene is very low temperature, the COLD-resistant gene encoding protein of COLD-resistant gene of a COLD-resistant gene encoding protein, the COLD-resistant gene of a COLD-resistant gene encoding COLD-resistant gene of a COLD-resistant gene encoding COLD-resistant gene of japonica rice, the COLD-resistant gene of a COLD-resistant gene encoding COLD-resistant gene of a COLD-resistant gene of a COLD-resistant.
In the traditional rice resistance breeding, phenotype selection is carried out on plants through resistance identification, the time consumption is long, the limitation of environmental conditions is easy, errors are easily caused by identification results, and the selection efficiency is low. The Molecular Marker-assisted breeding (MAS) is simple and effective, the breeding cost can be reduced, and the breeding period can be shortened. At present, the main problems of cold-resistant breeding of rice are that the applicable cold-resistant genes are too few, the cold-resistant genes of rice are detected, the cold-resistant genetic background of different rice varieties is known, the blindness of cold-resistant breeding work can be reduced, and the breeding efficiency is improved, so that the identification of the stress-resistant genes of rice variety resources by using gene-specific molecular markers is very important. The CTB4a gene is a stress tolerance gene with a cold tolerance function of rice, the cold tolerance function still maintains the yield of the rice under a complex low-temperature environment background, and the CTB4a specific molecular marker is suitable for being introduced into varieties expected to improve the popularization and planting area in high latitude areas to develop, so that a theoretical basis is provided for molecular design, breeding and improvement of rice stress resistance in the breeding process of rice breeders, efficient breeding is finally realized, and huge social and economic benefits are brought.
Disclosure of Invention
The invention aims to provide a specific molecular marker for detecting a cold-resistant gene CTB4a and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows: the invention provides a specific molecular marker for detecting a rice cold-resistant gene CTB4a, which is named as SNP2063 and has a molecular marker polymorphism of T/C.
The specific molecular marker can be detected by using a group of primer combinations, wherein the primer combinations comprise primers ctb4a-Ft, ctb4a-Fc and ctb4a-R, the sequence of ctb4a-Ft is shown in SEQ ID.NO.1, the sequence of ctb4a-Fc is shown in SEQ ID.NO.2, and the sequence of ctb4a-R is shown in SEQ ID.NO. 3.
The reagent or the kit containing the primer combination belongs to the protection scope of the invention.
The invention provides application of the primer combination in detecting a rice cold-resistant gene CTB4 a.
The application further comprises the following steps:
(1) extracting the genomic DNA of the rice to be detected;
(2) performing PCR reaction detection by using the primer combination with the rice genome DNA to be detected as a template;
(3) if only the base T is detected at the specific molecular marker site, judging that the rice sample to be detected carries the CTB4a gene; if only the base C is detected, judging that the rice sample to be detected is an allele with CTB4 a; if bases T and C are detected simultaneously, determining that the rice sample to be detected is a heterozygous genotype; if neither the base T nor the base C is detected, the rice sample to be detected is judged to have neither the CTB4a gene nor the CTB4a allele.
The PCR detection reaction conditions in the step (2) are as follows: pre-denaturation at 95 ℃ for 5 min; then, circulating amplification reaction is carried out for 35 cycles of 95 ℃ for 40sec, 55 ℃ for 30sec and 72 ℃ for 50 sec; finally, extending for 10min at 72 ℃; after the reaction is finished, detecting the fluorescence signal intensity of the PCR amplification product by utilizing a HEX, FAM and ROX three-channel fluorescence scanning microplate reader, and converting the read result into a graph.
The invention provides application of the specific molecular marker in auxiliary identification of a rice cold-resistant gene CTB4 a.
The invention provides application of the specific molecular marker or the primer combination or the kit containing the primer combination in rice germplasm resource improvement.
The invention provides application of the specific molecular marker or the primer combination or the kit containing the primer combination in cultivating rice with cold resistance.
The invention has the advantages of
The invention provides a specific molecular marker for detecting a rice cold-resistant gene CTB4a, and provides a primer group for detecting the molecular marker, which have high specificity for detecting CTB4a gene sites, can quickly identify the CTB4a gene in rice varieties by utilizing a PCR detection technology, and can overcome the defects of long detection period and abundant experience requirement of the traditional breeding identification method. The application method of the specific molecular marker provided by the invention is convenient to operate, has accurate and reliable detection results, can be used for identification and assisted breeding of the CTB4a gene of rice, and can solve the problems of low efficiency and long period of traditional breeding.
Drawings
FIG. 1.41 part of rice variety cold-resistant gene CTB4a test result chart
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 development of specific molecular marker and primer design of Rice Cold-resistant Gene CTB4a
According to a difference fragment found by comparing a cold-resistant gene CTB4a sequence of a cold-resistant parent Kunming small white grain rice with a CTB4a gene sequence of a corresponding genome region of a rice variety 9311, a molecular marker with good specificity is screened, SNP2063 is selected from the molecular marker, and the SNP2063 has a T/C difference in a CTB4a coding region.
Designing a specific Primer combination for detecting the molecular marker by using Primer 6.0, and converting the SNP into the molecular marker which can be detected by a PCR technology, wherein the Primer combination comprises primers ctb4a-Ft, ctb4a-Fc and ctb4a-R, the sequence of ctb4a-Ft is shown as SEQ ID.NO.1, the sequence of ctb4a-Fc is shown as SEQ ID.NO.2, and the sequence of ctb4a-R is shown as SEQ ID.NO. 3; ctb4a-Ft can be matched and combined with HEX fluorescent primers in a PARMS master mix, ctb4a-Fc can be matched and combined with FAM fluorescent primers in the PARMS master mix, and therefore different genotypes are obtained according to different fluorescent signals; the primers were synthesized by Beijing Hua big Biotechnology services GmbH.
Example 2 application of specific molecular marker of Rice Cold-resistant Gene CTB4a
(1) Extracting the genomic DNA of rice to be detected
The total 41 parts of the rice sample to be detected comprises 4 parts of restorer line, 12 parts of maintainer line and 26 parts of other main cultivated varieties and local species, and the CTAB method is used for extracting the genome DNA of the rice sample.
(2) Detection of PCR reaction
Carrying out PCR reaction detection on the genome DNA of the rice material to be detected,
the reaction system and conditions for PCR detection are as follows:
the PCR reaction system is 10 μ L, and comprises 2 XPCR MS master mix 5 μ L, 10mmol/L forward marker primer (ctb4a-Ft)0.15 μ L, 10mmol/L forward marker primer (ctb4a-Fc)0.15 μ L, 10mmol/L universal reverse marker primer (ctb4a-R)0.15 μ L, template DNA (10-30 ng)1 μ L, and ddH2O 3.55.55 μ L.
After the reaction is finished, detecting the fluorescence signal intensity of the PCR amplification product by utilizing a HEX, FAM and ROX three-channel fluorescence scanning microplate reader, and converting the read result into a graph.
(3) The result of the detection
The detection results are shown in a figure 1 and a table 1, and the detection results show that after PCR amplification, scanning is carried out by an enzyme-labeling instrument, and the analysis results show that only FAM fluorescent signals are detected in Kunming small white valley samples, which indicates that only base T is detected, and other 41 rice parents detect HEX fluorescent signals, which indicates that only base C is detected. By combining with the principle analysis of primer design, the CTB4a gene exists in Kunming white valley, and only HEX fluorescence signals are detected in the other 41 rice parent materials, which indicates that the 41 rice samples have CTB4a allele. In conclusion, the marker of the invention can amplify the cold-resistant gene CTB4a specific fragment and clearly distinguish the cold-resistant gene CTB4a from the CTB4a allele on the fluorescence signal.
TABLE 1 phenotype and genotype of reference Rice Material
Sequence listing
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Claims (10)
1. A specific molecular marker for detecting a rice cold-resistant gene CTB4a is characterized in that the specific molecular marker is named as SNP2063, and the polymorphism of the molecular marker is T/C.
2. A specific molecular marker as claimed in claim 1 wherein said specific molecular marker is detected using a set of primer combinations comprising primers ctb4a-Ft, ctb4a-Fc and ctb4a-R, the sequence of ctb4a-Ft is shown in SEQ ID No.1, the sequence of ctb4a-Fc is shown in SEQ ID No.2 and the sequence of ctb4a-R is shown in SEQ ID No. 3.
3. Primer combination for detecting specific molecular markers according to claim 1, wherein the primer combination comprises primers ctb4a-Ft, ctb4a-Fc and ctb4a-R, wherein the sequence of ctb4a-Ft is shown in SEQ ID No.1, the sequence of ctb4a-Fc is shown in SEQ ID No.2, and the sequence of ctb4a-R is shown in SEQ ID No. 3.
4. A reagent or a kit comprising the primer set according to claim 3.
5. The primer combination of claim 3 is used for detecting rice cold-resistant gene CTB4 a.
6. Use according to claim 5, characterized in that it comprises the following steps:
(1) extracting the genomic DNA of the rice to be detected;
(2) performing PCR reaction detection by using the primer combination with the rice genome DNA to be detected as a template;
(3) if only the base T is detected at the specific molecular marker site, judging that the rice sample to be detected carries the CTB4a gene; if only the base C is detected, judging that the rice sample to be detected is an allele with CTB4 a; if bases T and C are detected simultaneously, determining that the rice sample to be detected is a heterozygous genotype; if neither the base T nor the base C is detected, the rice sample to be detected is judged to have neither the CTB4a gene nor the CTB4a allele.
7. The use according to claim 6, wherein the PCR detection reaction conditions in step (2) are: pre-denaturation at 95 ℃ for 5 min; then, circulating amplification reaction is carried out for 35 cycles of 95 ℃ for 40sec, 55 ℃ for 30sec and 72 ℃ for 50 sec; finally, extending for 10min at 72 ℃; after the reaction is finished, detecting the fluorescence signal intensity of the PCR amplification product by utilizing a HEX, FAM and ROX three-channel fluorescence scanning microplate reader, and converting the read result into a graph.
8. The application of the specific molecular marker of claim 1 in the auxiliary identification of the rice cold-resistant gene CTB4 a.
9. The molecular marker of claim 1, the primer combination of claim 3 or the kit of claim 4, for use in rice germplasm resource improvement.
10. Use of the molecular marker of claim 1 or the primer combination of claim 3 or the kit of claim 4 for breeding rice with cold tolerance.
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Cited By (2)
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Application publication date: 20200313 |