CN116814841B - Primer group for identifying rice black brown glume gene HK4, and method and application thereof - Google Patents
Primer group for identifying rice black brown glume gene HK4, and method and application thereof Download PDFInfo
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- CN116814841B CN116814841B CN202311028389.6A CN202311028389A CN116814841B CN 116814841 B CN116814841 B CN 116814841B CN 202311028389 A CN202311028389 A CN 202311028389A CN 116814841 B CN116814841 B CN 116814841B
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 53
- 235000009566 rice Nutrition 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 20
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 52
- 230000003321 amplification Effects 0.000 claims description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 15
- 229910000831 Steel Inorganic materials 0.000 claims description 6
- 239000010959 steel Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims 1
- 238000012408 PCR amplification Methods 0.000 abstract description 3
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 101150105245 HK4 gene Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Abstract
The present disclosure provides a primer set for identifying rice black brown glume gene HK4, a method and an application thereof, and belongs to the field of molecular genetics. The primer set includes: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each primer pair comprising: forward primer and reverse primer. The embodiment of the invention provides a primer group for identifying rice black brown glume gene HK4, a method and application thereof, wherein a rice variety AC-1613 has the black brown glume gene HK4, the primer group can accurately identify the rice black brown glume gene HK4 by utilizing PCR amplification reaction, and meanwhile, the identification method is convenient and rapid and is not influenced by environment.
Description
Technical Field
The present disclosure relates to the field of molecular genetics, and in particular, to a primer set for identifying rice black brown glume gene HK4, a method and an application thereof.
Background
The rice has obvious heterosis phenomenon, two varieties with certain difference in characters and complementary dominant characters are utilized for hybridization to produce hybrid seeds with heterosis, and the hybrid seeds are used for producing hybrid rice with greatly improved yield, namely hybrid rice. The average yield of the hybrid rice planted in China is increased by 10 to 15 percent.
The glume color of the rice seeds is the character shown after the rice is heading and maturing, and the rice seeds with large glume color contrast can be separated by a color selector. Therefore, sterile lines and restorer lines with different glume colors are selected to be mixed and sowed according to a certain proportion, mixed harvesting is carried out, and the mechanized level of seed production can be improved through sorting by the difference of the glume colors of the hybrid seeds and the male parent seeds. The glume color of the rice variety AC-1613 provided by the International Rice institute (IRRI) is black brown after the rice seeds are mature, and the glume color is greatly different from most of the current cultivated rice seeds which are yellow when mature, so that the rice seeds have great potential for improving the mechanization degree of hybrid rice seed production. However, when the rice encounters excessive rainwater in the heading and flowering period, the glume is easy to generate wounds, and the rice glume is easy to turn into black brown due to the fact that the glume is infected by some saprophytes, therefore, when the rice glume is black brown, whether the rice variety contains the black brown glume gene HK4 or not or whether other rice varieties turn into black brown after being infected by saprophytes can not be determined.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problems of the prior art, the embodiment of the disclosure provides a primer group for identifying rice black brown glume gene HK4, a method and application thereof, which can accurately screen rice varieties with black brown glume color when seeds are mature by identifying the rice black brown glume gene HK4. The technical scheme is as follows:
in one aspect, the present disclosure provides a primer set for identifying rice black brown glume gene HK4, the primer set comprising: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each primer pair comprising: forward primer and reverse primer;
the forward primer of the first primer pair is shown as SEQ ID NO: as shown in figure 1, the number of the components,
the reverse primer of the first primer pair is shown as SEQ ID NO: as shown in figure 2, the number of the parts is two,
the forward primer of the second primer pair is shown as SEQ ID NO: as shown in figure 3, the number of the holes in the steel plate is,
the reverse primer of the second primer pair is shown as SEQ ID NO: as shown in figure 4, the number of the parts is,
the forward primer of the third primer pair is shown as SEQ ID NO: as shown in figure 5,
the reverse primer of the third primer pair is shown as SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,
the forward primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 7 of the drawings,
the reverse primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 8,
the forward primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 9,
the reverse primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 10,
the forward primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 11,
the reverse primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 12,
the forward primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 13,
the reverse primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 14,
the forward primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 15,
the reverse primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 16,
the forward primer of the ninth primer pair is shown as SEQ ID NO: as indicated by the reference numeral 17,
the reverse primer of the ninth primer pair is shown as SEQ ID NO: as indicated at 18, the number of the cells,
the forward primer of the tenth primer pair is shown as SEQ ID NO: as indicated by the numeral 19,
the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
In another aspect, the present disclosure provides a method for identifying rice black brown glume gene HK4 using the primer set described above, the method comprising:
obtaining genome DNA of rice to be detected;
amplifying the genome DNA by adopting the primer group to obtain an amplified product;
when the amplification product comprises amplification fragments of 227bp, 398bp, 210bp, 253bp, 364bp, 151bp, 140bp, 215bp, 187bp and 162bp, the rice black brown glume gene HK4 exists in the rice to be detected.
Illustratively, the amplification procedure for amplifying the genomic DNA using the primer set is: 98 ℃ for 2min; each cycle includes: 15sec at 98 ℃, 20sec at 55-65 ℃ and 20sec at 72 ℃ for 35 cycles in total; and at 72℃for 5min.
Illustratively, the amplification system for amplifying the genomic DNA using the primer set is: 10 XPCR Buffer 1. Mu.L; 25mMMg 2+ 0.6. Mu.L; 10mM dNTP 0.2. Mu.L; 0.3. Mu.L of each of the forward primer and the reverse primer in the 10. Mu.M primer set; 5U/. Mu.LTaqDNApolymerase 0.2. Mu.L; 0.3 μl of the genomic DNA; adding ddH 2 O to the amplification system was 10. Mu.L.
In yet another aspect, the present disclosure provides an application of the above primer set, the application comprising: the primer group is used for identifying rice black brown glume gene HK4.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the invention provides a primer group for identifying rice black brown glume genes HK4, a method and application thereof, wherein a rice variety AC-1613 is provided with the black brown glume genes HK4, the primer group can identify the rice black brown glume genes HK4 by detecting a molecular marker linked with a gene locus by utilizing Indel molecular markers of PCR amplification reaction, so that the rice variety with black brown glume is accurately screened, and meanwhile, the identification method is convenient and rapid and is not influenced by the environment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a diagram showing electrophoresis results of a first primer pair to a fifth primer pair in a primer set according to a second embodiment of the present disclosure;
fig. 2 is a diagram showing electrophoresis results of a sixth primer pair to a tenth primer pair in the primer set provided in the second embodiment of the present disclosure.
Detailed Description
For the purposes of clarity, technical solutions and advantages of the present disclosure, the following further details the embodiments of the present disclosure with reference to the accompanying drawings.
Example 1
The present example provides a primer set for identifying rice black brown glume gene HK4, comprising: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each primer pair comprising: forward primer and reverse primer;
the forward primer of the first primer pair is shown as SEQ ID NO: as shown in figure 1, the number of the components,
the reverse primer of the first primer pair is shown as SEQ ID NO: as shown in figure 2, the number of the parts is two,
the forward primer of the second primer pair is shown as SEQ ID NO: as shown in figure 3, the number of the holes in the steel plate is,
the reverse primer of the second primer pair is shown as SEQ ID NO: as shown in figure 4, the number of the parts is,
the forward primer of the third primer pair is shown as SEQ ID NO: as shown in figure 5,
the reverse primer of the third primer pair is shown as SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,
the forward primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 7 of the drawings,
the reverse primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 8,
the forward primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 9,
the reverse primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 10,
the forward primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 11,
the reverse primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 12,
the forward primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 13,
the reverse primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 14,
the forward primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 15,
the reverse primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 16,
the forward primer of the ninth primer pair is shown as SEQ ID NO: as indicated by the reference numeral 17,
the reverse primer of the ninth primer pair is shown as SEQ ID NO: as indicated at 18, the number of the cells,
the forward primer of the tenth primer pair is shown as SEQ ID NO: as indicated by the numeral 19,
the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
Example two
The present embodiment provides a method for identifying rice black brown glume gene HK4 using the primer set provided in the first embodiment, the method comprising:
obtaining genome DNA of rice to be detected;
amplifying the genome DNA by using the primer group to obtain an amplified product;
referring to Table 1, when the amplified products include amplified fragments of 227bp, 398bp, 210bp, 253bp, 364bp, 151bp, 140bp, 215bp, 187bp and 162bp, rice black brown glume gene HK4 exists in the rice to be detected. The black brown glume gene HK4 of the rice is physically located in a 1.75Mb interval between 21,849,914 bp-23,602,163 bp of the long arm of chromosome 4.
Table 1 shows the primer sequences corresponding to the molecular markers
The embodiment of the invention provides application of the primer group, which comprises the following steps: the primer group is used for identifying the rice black brown glume gene HK4, so that rice varieties containing the HK4 gene can be accurately screened.
The specificity of the primer pair provided in the first embodiment of the present invention was detected. Specifically, samples to be tested are black brown glume material AC-1613 and yellow glume material Japanese sunny, 9311, hui No. 5, zhonghua No. 11 and Hui No. 1 respectively, and seedling leaf genomic DNA of the samples to be tested is taken for identification.
Genomic DNA of the sample to be tested is extracted by using a genomic DNA extraction kit (RTG 2404-01) provided by Zhongkeshitai (Beijing) biotechnology limited company, and the specific method is referred to the specification of the kit. The concentration of 1. Mu.L of the genomic DNA of the sample to be measured was measured by a nucleic acid concentration meter (Thermo), and the concentrations of the genomic DNA of the sample to be measured were adjusted to 50 ng/. Mu.L.
And (3) performing PCR (polymerase chain reaction) amplification reaction on the obtained genome DNA of the rice to be detected by using the primer set provided by the embodiment of the invention to obtain an amplified fragment.
Illustratively, an amplification system for amplifying genomic DNA using a primer set is: 10 XPCRBuffer 1. Mu.L; 25mMMg 2+ 0.6. Mu.L; 10mM dNTP 0.2. Mu.L; 0.3. Mu.L each of the forward primer and the reverse primer in the 10. Mu.M primer set; 5U/. Mu.LTaqDNApolymerase 0.2. Mu.L; genomic DNA 0.3. Mu.L; adding ddH 2 O was made up to 10. Mu.L.
Illustratively, the amplification procedure for amplifying genomic DNA using the primer set is: 98 ℃ for 2min; each cycle includes: 15sec at 98 ℃, 20sec at 55-65 ℃ and 20sec at 72 ℃ for 35 cycles in total; and at 72℃for 5min.
After the PCR amplification reaction is finished, an amplification product is obtained, the amplification product is subjected to electrophoresis analysis on agarose gel with the concentration of 3.5%, the electrophoresis results are shown in figures 1 and 2, and as can be seen from figures 1 and 2, the black brown glume material AC-1613 (lane L1) and the yellow glume material Japanese sunny (lane L2), 9311 (lane L3), ezhong No. 5 (lane L4), zhonghua No. 11 (lane L5) and Huaqihui No. 1 (lane L6) of the sample to be detected have the same electrophoresis results as the actual glume color of the sample to be detected, and the primer group provided by the embodiment of the invention has accurate identification.
Meanwhile, the five samples to be tested are respectively subjected to 3 repeated tests, and the results of the 3 repeated tests are consistent, so that the primer set provided by the embodiment has good reproducibility and accuracy.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (5)
1. A primer set for identifying rice black brown glume gene HK4, the primer set comprising: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each primer pair comprising: forward primer and reverse primer;
the forward primer of the first primer pair is shown as SEQ ID NO: as shown in figure 1, the number of the components,
the reverse primer of the first primer pair is shown as SEQ ID NO: as shown in figure 2, the number of the parts is two,
the forward primer of the second primer pair is shown as SEQ ID NO: as shown in figure 3, the number of the holes in the steel plate is,
the reverse primer of the second primer pair is shown as SEQ ID NO: as shown in figure 4, the number of the parts is,
the forward primer of the third primer pair is shown as SEQ ID NO: as shown in figure 5,
the reverse primer of the third primer pair is shown as SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,
the forward primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 7 of the drawings,
the reverse primer of the fourth primer pair is shown as SEQ ID NO: as shown in figure 8,
the forward primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 9,
the reverse primer of the fifth primer pair is shown as SEQ ID NO: as shown in the drawing 10,
the forward primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 11,
the reverse primer of the sixth primer pair is shown as SEQ ID NO: as shown in the drawing 12,
the forward primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 13,
the reverse primer of the seventh primer pair is shown as SEQ ID NO: as shown in the drawing 14,
the forward primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 15,
the reverse primer of the eighth primer pair is shown as SEQ ID NO: as indicated by the numeral 16,
the forward primer of the ninth primer pair is shown as SEQ ID NO: as indicated by the reference numeral 17,
the reverse primer of the ninth primer pair is shown as SEQ ID NO: as indicated at 18, the number of the cells,
the forward primer of the tenth primer pair is shown as SEQ ID NO: as indicated by the numeral 19,
the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
2. A method for identifying rice black brown glume gene HK4 using the primer set of claim 1, comprising:
obtaining genome DNA of rice to be detected;
amplifying the genome DNA by adopting the primer group to obtain an amplified product;
when the amplification product comprises amplification fragments of 227bp, 398bp, 210bp, 253bp, 364bp, 151bp, 140bp, 215bp, 187bp and 162bp, the rice black brown glume gene HK4 exists in the rice to be detected.
3. The method of claim 2, wherein the amplification procedure for amplifying the genomic DNA using the primer set is: 98 ℃ for 2min; each cycle includes: 15sec at 98 ℃, 20sec at 55-65 ℃ and 20sec at 72 ℃ for 35 cycles in total; and at 72℃for 5min.
4. The method of claim 2, wherein the amplification system for amplifying the genomic DNA using the primer set is: 10 XPCR Buffer 1. Mu.L; 25mM Mg 2+ 0.6. Mu.L; 10mM dNTP 0.2. Mu.L; 0.3. Mu.L of each of the forward primer and the reverse primer in the 10. Mu.M primer set; 5U/. Mu. LTaq DNApolymerase 0.2.2. Mu.L; 0.3 μl of the genomic DNA; adding ddH 2 O to the amplification system was 10. Mu.L.
5. The use of the primer set of claim 1, wherein the use comprises: the primer group is used for identifying rice black brown glume gene HK4.
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