CN108866231B - Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Dongfu-type rice, application and detection method - Google Patents
Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Dongfu-type rice, application and detection method Download PDFInfo
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Abstract
The invention provides a detection primer and a kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Dongmu rice, and an application and a detection method thereof, belonging to the field of crop molecular genetic breeding. The detection primer of the cytoplasmic male sterility restoring gene Rf (DW)8 of the Dongmu rice provided by the invention is designed aiming at the chromosome segment where the cytoplasmic male sterility restoring gene of the Dongmu rice is located, the exchange frequency of the gene Rf (DW)8 is low, the designed primer can very accurately judge the existence of the restoring gene, the guarantee on the molecular level is provided for the cultivation of the restoring line, and the identification goodness of fit reaches 97.81%. The detection can be conveniently and rapidly carried out; the detection primer is applied to the preparation of the detection kit of the cytoplasmic male sterility restoring gene of the Dongmu rice, so that the gene detection can be carried out more conveniently and rapidly, the specific information of a sample to be detected can be obtained, the detection primer can be specifically applied to breeding, and the detection kit has higher application value.
Description
Technical Field
The invention relates to the field of crop molecular genetic breeding, in particular to a detection primer and a kit for cytoplasmic male sterility restoring gene Rf (DW)8 of east wild type rice, and an application and a detection method thereof.
Background
The three cytoplasmic sterility types most widely used in rice production at present are: baotai type, herba Patriniae type and flos Carthami type. The Baptiji sterile line restorer gene Rf1a can shear the mitochondrial sterile gene orf79, and Rf1b can degrade atp6/orf79 bicistronic mRNA, so that the generation of toxoprotein is inhibited, and fertility is restored. The restorer genes Rf3 and Rf4 of the wild abortive sterile line are respectively positioned on No.1 and No. 10 chromosomes of rice, Tang et al in 2014 clones Rf4, the N end of the genes contains a mitochondrial localization signal, 18 PPR structural regions have high similarity with Rf1a, but the restorer mechanism of the wild abortive cytoplasmic sterility of Rf1a cannot be a post-transcriptional regulation mechanism, male sterility is inhibited by reducing the expression level of mRNA of the sterile gene WA352, allele variation analysis is carried out on loci Rf3 and Rf4 by utilizing chromosome single-segment substitution lines, Cai et al in 2013, and the result shows that the effect of Rf4 is greater than that of Rf3 under a certain genetic background and the two have an accumulation effect. However, molecular cloning of Rf3 has not been reported. The rosette type sterile line restorer genes Rf5 and Rf6 are located on rice chromosome 10 and 8 respectively, the ju uk team clones Rf5 and Rf6 in 2012 and 2015 respectively, Rf5 belongs to the PPR family of genes which cannot bind to CMS related transcripts, but its partner GRP162 protein can bind to atp6-orfH 79. GRP162 physically interacts with RF5 and binds to atp6-orfH79 through an RNA recognition motif. Both RF5 and GRP162 are components of the 400-500kD fertility restorer complex (RFC), and PPR binds directly to GRP162 to form a subunit of RFC, which cleaves CMS-related transcripts in vitro. RF6 encodes a specific PPR family protein that repeats in the 3 rd to 5 th PPR domain. The RF6 is positioned in mitochondria, is combined with OsHXK6 in the mitochondria and promotes the shearing of the protein atp6-orfH79 coded by the sterile gene at the 1238 amino acid position, thereby ensuring the normal development and fertility restoration of the pollen.
The wild rice in east and west of the Yangtze river and countryside is the ordinary wild rice in the northest latitude of the world, is praised as 'plant panda' in the rice world, and is a precious breeding material for rice because of various excellent genes. As early as the eighties of the last century, the sterile line 'international oil viscosity A' is cultivated by utilizing the cytoplasm of the Dongxiang wild rice by a breeder of our unit, and later the test recovery is carried out by utilizing a large amount of rice materials at home and abroad, but a strong restoring line cannot be screened out, so that the utilization of the male sterile line is interrupted. In recent years, in the utilization research of Dongxiang wild rice, the unit breeds Dongyu cytoplasmic sterile line DY1A by taking early-35 (DY1B) of early indica as a recurrent parent, and finds that the restoring relationship of Dongyu CMS and the existing CMS type is completely different. Most of rice materials are maintainer lines of CMS in east China and have extremely wide maintenance spectrums, so that the rice materials have wider and free resource utilization space in the cultivation of the sterile line of the three lines.
Successful application of the rice three-line hybrid system, cultivation of an undisclosed restorer line and positioning research of a restorer gene thereof. Along with the cloning of wild abortive type restoring genes, the revealing of the sterile essence and the regulation mechanism becomes possible, and meanwhile, the breeding process of the restoring line is accelerated through the auxiliary selection of molecular markers.
However, the cloning of the "east wild type" three-line sterility restorer gene has not been reported.
Disclosure of Invention
The first purpose of the invention is to provide a detection primer of the cytoplasmic male sterility restoring gene Rf (DW)8 of the east wild type rice, which can conveniently and quickly detect the cytoplasmic male sterility restoring gene Rf (DW)8 of the east wild type rice.
The second purpose of the invention is to provide the application of the detection primer of the east wild type rice cytoplasm male sterility restoring gene Rf (DW)8 in the detection of the east wild type rice cytoplasm male sterility restoring gene Rf (DW) 8.
The third purpose of the invention is to provide the application of the detection primer of the east wild type rice cytoplasm male sterility restoring gene Rf (DW)8 in the preparation of the detection kit of the east wild type rice cytoplasm male sterility restoring gene Rf (DW) 8.
The fourth purpose of the invention is to provide a detection kit of the Dongmu rice cytoplasmic male sterile restoring gene Rf (DW)8, which is convenient for detecting the gene.
The fifth purpose of the invention is to provide the application of the detection kit for the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice in the detection of the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice.
The sixth purpose of the present invention is to provide a method for detecting cytoplasmic male sterility restoring gene Rf (DW)8 of rice of the Shandong wild type, which is easy to carry out and can rapidly obtain the detection result.
In order to achieve the above purpose of the invention, the following technical scheme is adopted:
the detection primers of the Dongmu rice cytoplasm male sterility restoring gene Rf (DW)8 comprise a first primer pair and a second primer pair; the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4.
The application of the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 in the detection of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The application of the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 in preparing the detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 comprises the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 is applied to the detection of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The detection method of the Dongfu rice cytoplasm male sterility restoring gene Rf (DW)8 comprises the following steps:
extracting genome DNA of a sample to be detected as a template, respectively carrying out PCR amplification reaction by using a first primer pair and a second primer pair to obtain amplification products, carrying out gel electrophoresis on the amplification products, and analyzing the sample to be detected according to the gel electrophoresis result.
Compared with the prior art, the invention has the beneficial effects that: the detection primer of the cytoplasmic male sterility restoring gene Rf (DW)8 of the Dongmu rice provided by the invention is designed aiming at the cytoplasmic male sterility restoring gene of the Dongmu rice, the exchange frequency of the gene Rf (DW)8 is low, the designed primer can very accurately judge the existence of the restoring gene, the guarantee on the molecular level is provided for the cultivation of a restoring line, and the identification goodness of fit reaches 97.81%. The detection can be conveniently and rapidly carried out; the detection primer is applied to the preparation of the detection kit of the cytoplasmic male sterility restoring gene of the Dongmu rice, so that the gene detection can be carried out more conveniently and rapidly, the specific information of a sample to be detected can be obtained, the detection primer can be specifically applied to breeding, and the detection kit has higher application value.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it should be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes specific detection primers, kits, and applications and detection methods for cytoplasmic male sterile restorer gene Rf (DW)8 of Oriental wild type rice in the embodiments of the present invention.
The detection primers of the Dongmu rice cytoplasm male sterility restoring gene Rf (DW)8 comprise a first primer pair and a second primer pair; the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4.
Two groups of primer pairs with stronger specificity are designed in a targeted manner, and the detection primer pairs can specifically detect the Dongmu rice cytoplasm male sterility restoring gene Rf (DW)8 in a sample.
A first primer pair:
the upstream primer SEQ ID NO.1 is IdZ8-39F: 5'-TGTGGTTCAGGGGGTAATTC-3';
the downstream primer SEQ ID NO.2 is IdZ8-39R 5'-GCGATAGCGTTATGTTGAGC-3';
the product length is 115 bp.
A second primer pair:
the upstream primer SEQ ID NO.3 is IdZ8-52F 5'-TTTGAATTTAAGGAGGTTACCG-3';
the downstream primer SEQ ID NO.4 is IdZ8-52R 5'-ATGCTCACCAGCAGGGTTTAT-3';
the length of the product is 103 bp.
The application of the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 in the detection of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The application of the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 in preparing the detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 comprises the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
Further, in a preferred embodiment of the present invention, at least one of a genome extraction reagent, a 10 × PCR reaction buffer, and a DNA polymerase is further included.
The kit comprises an integrated detection primer and related matched chemical reagents, so that the gene can be conveniently detected; meanwhile, the corresponding detection reagent is provided by the kit, so that the stability, reliability and accuracy of detection can be improved.
Further, in a preferred embodiment of the invention, the genome extraction reagent CTAB reagent and chloroform.
The detection kit of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 is applied to the detection of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
The detection method of the Dongfu rice cytoplasm male sterility restoring gene Rf (DW)8 comprises the following steps:
extracting genome DNA of a sample to be detected as a template, respectively carrying out PCR amplification reaction by using a first primer pair and a second primer pair to obtain amplification products, carrying out gel electrophoresis on the amplification products, and analyzing the sample to be detected according to the gel electrophoresis result.
Further, in a preferred embodiment of the present invention, the reaction system of the PCR amplification reaction is: 10 XPCR reaction buffer 1 uL; 0.1. mu.L of DNA polymerase of 1. mu.L, 5U/. mu.L each of the primer pairs; genomic DNA1 μ L at a concentration of 50 ng/. mu.L, made up to 10 μ L with deionized water.
Further, in the preferred embodiment of the present invention, the annealing temperature in the PCR reaction is 53-57 ℃ and the annealing time is 17-23 s.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a detection primer of an east wild type rice cytoplasm male sterility restoring gene Rf (DW)8, wherein the detection primer comprises a first primer pair and a second primer pair; the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4.
A first primer pair of detection primers for detecting the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice:
the upstream primer SEQ ID NO.1 is IdZ8-39F: 5'-TGTGGTTCAGGGGGTAATTC-3';
the downstream primer SEQ ID NO.2 is IdZ8-39R 5'-GCGATAGCGTTATGTTGAGC-3';
the product length is 115 bp.
A second primer pair:
the upstream primer SEQ ID NO.3 is IdZ8-52F 5'-TTTGAATTTAAGGAGGTTACCG-3';
the downstream primer SEQ ID NO.4 is IdZ8-52R 5'-ATGCTCACCAGCAGGGTTTAT-3';
the length of the product is 103 bp.
The detection primers for detecting the cytoplasmic male sterile restoring gene Rf (DW)8 of the east wild type rice have strong pertinence to the cytoplasmic male sterile restoring gene Rf (DW)8 of the east wild type rice, so that the detection primers have better specificity, and the detection result is more reliable and accurate through two groups of detection primers, so that the detection primers for detecting the cytoplasmic male sterile restoring gene Rf (DW)8 of the east wild type rice can be applied to the detection of the cytoplasmic male sterile restoring gene Rf (DW)8 of the east wild type rice.
Meanwhile, a detection primer of the Dongmu rice cytoplasm male sterility restoring gene Rf (DW)8 can be applied to the preparation of a detection kit of the Dongmu rice cytoplasm male sterility restoring gene Rf (DW) 8.
Example 2
The embodiment provides a detection kit of an east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8, which comprises a detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8, wherein the detection primer comprises a first primer pair and a second primer pair; the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4.
A first primer pair of detection primers for detecting the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice:
the upstream primer SEQ ID NO.1 is IdZ8-39F: 5'-TGTGGTTCAGGGGGTAATTC-3';
the downstream primer SEQ ID NO.2 is IdZ8-39R 5'-GCGATAGCGTTATGTTGAGC-3';
the product length is 115 bp.
A second primer pair:
the upstream primer SEQ ID NO.3 is IdZ8-52F 5'-TTTGAATTTAAGGAGGTTACCG-3';
the downstream primer SEQ ID NO.4 is IdZ8-52R 5'-ATGCTCACCAGCAGGGTTTAT-3';
the length of the product is 103 bp.
The detection kit at least comprises one of genome extraction reagent, 10 XPCR reaction buffer solution and DNA polymerase. Wherein, the genome extraction reagent CTAB reagent and chloroform. The detection kit for the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice provided by the embodiment can also be applied to the detection of the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice.
Example 3
This example provides a method for detecting cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice, which uses the detection kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice provided in example 2 to perform an assay, comprising the following steps:
1.1 cutting rice leaves with the length of 4cm into pieces, placing the pieces into a 2mL centrifuge tube, and adding liquid nitrogen to carry out quick freezing;
1.2 crushing a rice sample by using a tissue crusher;
1.3 adding 500 μ L of preheated 2% CTAB reagent solution, incubating at 65 deg.C for 30min, and shaking upside down every 5 min;
1.4 adding 500 mu L chloroform, violently overturning and shaking for 20 times, and centrifuging at 12000rpm for 10 Min;
1.5 sucking 400 μ L of supernatant, adding 1mL of absolute ethanol into a new 1.5mL centrifuge tube, mixing uniformly, and freezing at-20 deg.C for 30 min;
1.6 taking the frozen stock solution in the step 1.5, centrifuging at 12000rpm for 10min, and precipitating the genome DNA at the bottom of the tube;
1.7 discarding the supernatant, air-drying at normal temperature, adding 300 mu L of deionized water, fully dissolving, and freezing and storing at-20 ℃ for later use;
1.8 performing PCR amplification by using a first primer pair and a second primer pair respectively, wherein the reaction system is as follows: 10 XPCR reaction buffer 1 uL; 0.1. mu.L of DNA polymerase of 1. mu.L, 5U/. mu.L each of the primer pairs; 1 μ L of genomic DNA at a concentration of 50 ng/. mu.L, made up to 10 μ L with deionized water; the reaction program is pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 20s, annealing at 53 ℃ for 23s, extension at 72 ℃ for 20s, circulation for 30 times, and final extension at 72 ℃ for 5 min;
1.9 carrying out gel electrophoresis on the amplification product obtained by PCR amplification, and analyzing the sample to be detected according to the electrophoresis result.
If the first primer pair and the second primer pair have bands in the gel electrophoresis, the fact that the east wild type rice cytoplasm male sterility restoring gene Rf (DW)8 of the target gene is contained in the sample to be detected is shown.
Example 4
This example provides a method for detecting cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice, which uses the detection kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice provided in example 2 to perform an assay, comprising the following steps:
1.1 cutting rice leaves with the length of 4cm into pieces, placing the pieces into a 2mL centrifuge tube, and adding liquid nitrogen to carry out quick freezing;
1.2 crushing a rice sample by using a tissue crusher;
1.3 adding 500 μ L of preheated 2% CTAB reagent solution, incubating at 65 deg.C for 30min, and shaking upside down every 5 min;
1.4 adding 500 mu L chloroform, violently overturning and shaking for 20 times, and centrifuging at 12000rpm for 10 Min;
1.5 sucking 400 μ L of supernatant, adding 1mL of absolute ethanol into a new 1.5mL centrifuge tube, mixing uniformly, and freezing at-20 deg.C for 30 min;
1.6 taking the frozen stock solution in the step 1.5, centrifuging at 12000rpm for 10min, and precipitating the genome DNA at the bottom of the tube;
1.7 discarding the supernatant, air-drying at normal temperature, adding 300 mu L of deionized water, fully dissolving, and freezing and storing at-20 ℃ for later use;
1.8 performing PCR amplification by using a first primer pair and a second primer pair respectively, wherein the reaction system is as follows: 10 XPCR reaction buffer 1 uL; 0.1. mu.L of DNA polymerase of 1. mu.L, 5U/. mu.L each of the primer pairs; 1 μ L of genomic DNA at a concentration of 50 ng/. mu.L, made up to 10 μ L with deionized water; the reaction program is pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 20s, fire at 55 ℃ for 20s, extension at 72 ℃ for 20s, circulation for 30 times, and finally extension at 72 ℃ for 5 min;
1.9 carrying out gel electrophoresis on the amplification product obtained by PCR amplification, and analyzing the sample to be detected according to the electrophoresis result.
If the first primer pair and the second primer pair have bands in the gel electrophoresis, the fact that the east wild type rice cytoplasm male sterility restoring gene Rf (DW)8 of the target gene is contained in the sample to be detected is shown.
Example 5
This example provides a method for detecting cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice, which uses the detection kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice provided in example 2 to perform an assay, comprising the following steps:
1.1 cutting rice leaves with the length of 4cm into pieces, placing the pieces into a 2mL centrifuge tube, and adding liquid nitrogen to carry out quick freezing;
1.2 crushing a rice sample by using a tissue crusher;
1.3 adding 500 μ L of preheated 2% CTAB reagent solution, incubating at 65 deg.C for 30min, and shaking upside down every 5 min;
1.4 adding 500 mu L chloroform, violently overturning and shaking for 20 times, and centrifuging at 12000rpm for 10 Min;
1.5 sucking 400 μ L of supernatant, adding 1mL of absolute ethanol into a new 1.5mL centrifuge tube, mixing uniformly, and freezing at-20 deg.C for 30 min;
1.6 taking the frozen stock solution in the step 1.5, centrifuging at 12000rpm for 10min, and precipitating the genome DNA at the bottom of the tube;
1.7 discarding the supernatant, air-drying at normal temperature, adding 300 mu L of deionized water, fully dissolving, and freezing and storing at-20 ℃ for later use;
1.8 performing PCR amplification by using a first primer pair and a second primer pair respectively, wherein the reaction system is as follows: 10 XPCR reaction buffer 1 uL; 0.1. mu.L of DNA polymerase of 1. mu.L, 5U/. mu.L each of the primer pairs; 1 μ L of genomic DNA at a concentration of 50 ng/. mu.L, made up to 10 μ L with deionized water; the reaction program comprises pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 20sec, annealing at 57 ℃ for 17sec, extension at 72 ℃ for 20sec, circulation for 30 times, and final extension at 72 ℃ for 5 min;
1.9 carrying out gel electrophoresis on the amplification product obtained by PCR amplification, and analyzing the sample to be detected according to the electrophoresis result.
If the first primer pair and the second primer pair have bands in the gel electrophoresis, the fact that the east wild type rice cytoplasm male sterility restoring gene Rf (DW)8 of the target gene is contained in the sample to be detected is shown.
Experimental example 1
The experimental example identifies the detection accuracy of the detection primers provided by the invention, and tests are carried out by using the experimental method of the example 4.
Firstly, Dongxiang wild rice is used as female parent, Zhongzao 35 is used as male parent, and 8 generations of backcross are carried out to cultivate a stable Dongxiang cytoplasmic male sterile line DY 1A. Selecting complete fertile rice in backcross generations by using Dongxiang wild rice as male parent and DY1A as recurrent parentThe plant (full anther and smooth dusting) is used as a male parent to be hybridized with DY 1A. After 5 backcrosses, a total of fully fertile individuals were obtained, including 2 BC2F11, BC3F11, BC4F118 BC5F1Planting the 18 BC5F2And (4) a group. First, 61 completely fertile individuals including 2 BC were selected from the group2F11, BC3F11, BC4F14 BC5F1And 50 from BC5F2The samples were subjected to DNA chip scanning using the 6K chip technology developed by the Chinese seed group for individual plants of the population (Huihuiyu et al, 2014). Statistical analysis shows that three QTLs positioned at 8 th, 10 th and 8 th chromosomes exist in all the completely fertile individuals. The restoring genes contained in the three sites are respectively named as: rf (DW)8, Rf (DW)10, and Rf (DW) 8.
Using the map-based cloning method, Rf (DW)8 was located between the markers IdZ8-39 and IdZ8-52 at a physical position within about 207kb relative to the Nipponbare reference genome: 26,830,517 bp-27,637,741 bp. From BC5F2In the population, 183 completely fertile individuals were randomly selected and further used as a verification population for the accuracy of Rf (DW)8 marker identification. The recombination frequency between the marker and the gene is analyzed, and the selection efficiency of the marker on the restored gene is calculated.
183 BC5F2The complete fertile individual plants, the banding pattern statistics are shown in table 1; in the table, 1 represents the molecular marker fragment derived from Dongxiang wild rice alone, 2 represents the molecular marker fragment derived from Miyao 35 alone, and 3 represents the heterozygous banding pattern of both parents.
TABLE 1 sample test results
As can be seen from Table 1, 4 crossover-type gametes appeared between the insertion deletion marker IdZ8-39 and the restorer gene Rf (DW)8, and the recombination rate was 2.19%. 3 crossover gametes appear between the insertion deletion marker IdZ8-52 and the restorer gene Rf (DW)8, and the recombination rate is 1.64%. The efficiency of simultaneous selection of the Rf (DW)8 gene by two pairs of markers was 97.81%. The existence of the restorer gene Rf (DW)8 can be identified efficiently by using the two molecular markers of the invention. The two markers can be used for molecular marker-assisted selection, effectively quicken the cultivation process of the east wild type cytoplasmic male sterility restorer line, control the scale of breeding populations and save the cost.
In summary, the detection primer of the cytoplasmic male sterility restoring gene Rf (DW)8 of the Oriental wild type rice and the detection kit prepared by using the detection primer have the advantages of good specificity, high sensitivity, accurate and reliable detection result, and can be applied to rice breeding.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110> Rice research institute of agricultural science institute of Jiangxi province
<120> detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Dongmu rice, application and detection method
<160> 4
<170> PatentIn version 3.5
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<211> 20
<212> DNA
<213> Artificial Synthesis
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tgtggttcag ggggtaattc 20
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<213> Artificial Synthesis
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gcgatagcgt tatgttgagc 20
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tttgaattta aggaggttac cg 22
<210> 4
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<212> DNA
<213> Artificial Synthesis
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Claims (10)
1. The detection primer of the Dongmu rice cytoplasm male sterility restoring gene Rf (DW)8 is characterized by comprising a first primer pair and a second primer pair; the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4.
2. The application of the detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 in the detection of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 according to claim 1, wherein the size of the product fragment of the Dongxiang wild rice genome amplified by the first primer pair is only 115bp, and the size of the product fragment of the Dongxiang wild rice genome amplified by the second primer pair is only 103bp, so that the sample to be detected has the homozygous target gene of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW) 8.
3. The use of the detection primer of the cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice as claimed in claim 1 in the preparation of a detection kit for the cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild type rice.
4. A detection kit of an east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8, which is characterized by comprising a detection primer of the east wild type rice cytoplasmic male sterility restoring gene Rf (DW)8 of claim 1.
5. The detection kit for cytoplasmic male sterility restorer gene Rf (DW)8 of claim 4, further comprising at least one of a genome extraction reagent, a 10 XPCR reaction buffer and a DNA polymerase.
6. The detection kit for cytoplasmic male sterility restorer gene Rf (DW)8 of claim 5, wherein the genome extraction reagent comprises CTAB reagent and chloroform.
7. The use of the detection kit for cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild rice as claimed in any one of claims 4 to 6 in the detection of cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild rice, wherein the size of the product fragment of the genome of Oriental wild rice amplified by the first primer pair is only 115bp, and the size of the product fragment of the genome of Oriental wild rice amplified by the second primer pair is only 103bp, so that the sample to be detected has the homozygous target gene of cytoplasmic male sterility restoring gene Rf (DW)8 of Oriental wild rice.
8. The detection method of the Dongfu rice cytoplasm male sterility restoring gene Rf (DW)8 is characterized by comprising the following steps:
extracting genome DNA of a sample to be detected as a template, respectively carrying out PCR amplification reaction by using a first primer pair and a second primer pair to obtain amplification products, carrying out gel electrophoresis on the amplification products, and analyzing the sample to be detected according to the result of the gel electrophoresis, wherein the base sequences of the first primer pair are respectively shown as SEQ ID No.1-2, and the base sequences of the second primer pair are respectively shown as SEQ ID No. 3-4; the size of the product fragment of the Dongxiang wild rice genome amplified by the first primer pair is only 115bp, and the size of the product fragment of the Dongxiang wild rice genome amplified by the second primer pair is only 103bp, so that the sample to be detected has the homozygous target gene Dongxiang wild rice cytoplasmic male sterility restoring gene Rf (DW) 8.
9. The method for detecting cytoplasmic male sterility restorer gene Rf (DW)8 of claim 8, wherein the reaction system of the PCR amplification reaction is: 10 XPCR reaction buffer 1 uL; 0.1. mu.L of DNA polymerase of 1. mu.L, 5U/. mu.L each of the primer pairs; genomic DNA1 μ L at a concentration of 50 ng/. mu.L, made up to 10 μ L with deionized water.
10. The method for detecting cytoplasmic male sterility restorer gene Rf (DW)8 of claim 9, wherein the annealing temperature in the PCR reaction is 53-57 ℃ and the annealing time is 17-23 s.
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