CN112725519A - PARMS marker based on brown planthopper resistance gene Bph14 of rice and application - Google Patents
PARMS marker based on brown planthopper resistance gene Bph14 of rice and application Download PDFInfo
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Abstract
The invention discloses a PARMS marker and application based on a rice brown planthopper resistance gene Bph14, in particular to a functional PARMS marker and application based on nonsynonymous mutation of SNP of the 1 st exon of a rice brown planthopper resistance gene Bph 14.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of genetic engineering, relates to a PARMS marker based on a brown planthopper resistance gene Bph14 and application, and particularly relates to a functional PARMS marker based on a non-synonymous mutation of SNP of a No. 1 exon of a brown planthopper resistance gene Bph14 and application.
[ background of the invention ]
Brown planthopper belongs to the family of homoptera planthopper, takes rice as a main host, and brown planthopper is one of main pests harmful to rice production. The brown planthopper inhales the rice vascular bundle sheath juice through the oral needle, the base of a rice plant becomes black, falls down or even withers when the brown planthopper is serious, and the brown planthopper spreads the rice straw-like bushy stunt and the odontoblast when eating, and simultaneously promotes the spread of rice sheath blight and sclerotinia scleotiorum. With the change of farming modes and the large-area popularization of high-yield varieties, the harm of brown planthopper is increased year by year, and the brown planthopper becomes the first insect pest in rice production.
At present, the methods for controlling brown planthopper mainly comprise: agricultural control, biological control and chemical control, wherein breeding insect-resistant rice varieties is the most effective and economic strategy. Molecular breeding practices show that the gene structure variation of important agronomic character genes of rice is analyzed, a target gene function marker is developed, direct selection and effective polymerization of the genes can be realized, the breeding efficiency is greatly improved, and the breeding time is shortened. Related researches exist, for example, Chinese patent application CN201610631705 a molecular marker of a brown planthopper resistant gene Bph14 of rice and an application thereof, provides a molecular marker H14 of a brown planthopper resistant gene Bph14 of rice and an application method thereof, and a method for breeding brown planthopper resistant rice by using the molecular marker H14 in an auxiliary way is to perform PCR amplification by using rice genome DNA as a template and H14 as a primer, detect an amplification product through 2% agarose gel electrophoresis, and if a single band of 260bp exists, indicate that a detected sample is homozygous for the Bph14 gene; if a single band with 390bp exists, the detection sample does not contain the Bph14 gene; if the two bands of 260bp and 390bp exist, the detection sample is represented as a Bph14 gene hybrid. For another example, chinese patent application CN201910302173 is a primer pair for detecting rice brown planthopper resistant gene Bph14 and an application thereof, the primer pair for detecting rice brown planthopper resistant gene Bph14 is co-dominant marker primer B14InD, and co-dominant marker primer B14InD is: forward primer sequence: CTACTGATTGCAGATTGACG, respectively; reverse primer sequence: CACTTGGTGAACTTATTCCCTT, the primer pair for detecting the brown planthopper resistant gene Bph14 of the rice is adopted to obtain a Bph14 specificity codominant molecular marker through PCR amplification, the located position of the brown planthopper resistant gene is definite, and the primer pair can be used for detecting the genotype of rice varieties or strains through detecting the specificity codominant molecular marker of the Bph14 gene, can predict the brown planthopper resistance of rice plants, and is favorable for improving the breeding efficiency of brown planthopper resistant rice.
At present, commonly used molecular markers RFLP, RAPD, CAPS, AFLP, SSR, ISSR, STS, SRAP and IRAP can be used for linkage analysis and detection of target genes, but the markers are long in detection time, complicated in operation, low in efficiency, low in automation degree, toxic substances and the like, and are not suitable for large-scale genotype analysis.
A single nucleotide polymorphism SNP refers to a variation of a single base caused at the genomic level, which occurs at a frequency of not less than 1% in a population. The SNP can occur at any position of a genome, is used as a new generation of molecular marker, and has the advantages of large quantity, uniform distribution, rich polymorphism, high precision and the like. The PARMS technology, namely a five-primer amplification hindered mutation system, is a newly developed SNP genotyping method based on fluorescence detection, utilizes five primers (a pair of general fluorescent primers, a pair of allele specific primers and a reverse common primer) to carry out allele specific amplification on SNP or short Indel sites, carries out genotyping through fluorescence scanning, and has the advantages of simple and convenient operation, short time consumption and low cost. Based on the fluorescent molecular marker developed by the PARMS technology, a detection sample is amplified only by one-time PCR without electrophoresis detection, and amplification data is directly obtained on an original plate by using a fluorescent scanner, and a corresponding genotype result is quickly obtained through software analysis.
Therefore, it is necessary to research and establish a PARMS marker based on the brown planthopper resistance gene Bph14 and application thereof.
[ summary of the invention ]
Aiming at the defects that the marker detection for preventing and controlling brown planthoppers in the prior art is long in time consumption, tedious in operation, low in efficiency, low in automation degree, toxic substances are used, and the marker is not suitable for large-scale genotype analysis, the invention provides a PARMS marker and application based on a rice brown planthopper resistance gene Bph14, in particular to a functional PARMS marker and application based on SNP nonsynonymous mutation of exon 1 of a rice brown planthopper resistance gene Bph14, the invention utilizes first-generation sequencing to detect the difference of Bph14 in high brown planthopper resistance material Y009 and Nipponbare of a susceptible brown planthopper variety, finds that T → C mutation occurs at bp site of exon 1 of the gene to cause the change from phenylalanine to leucine to cause the reactive function of Bph14, introduces the difference of the two bases into a designed forward primer, adds two different fluorescence labeling universal primers on the basis, develops a rice brown planthopper resistance gene Bph14 fluorescence function marker based on the PARMS technology, the Bph14 gene can be conveniently applied to rice resistance molecule breeding.
The invention relates to a PARMS marker and application based on a brown planthopper resistance gene Bph14, in particular to a functional PARMS marker and application based on nonsynonymous mutation of SNP of exon 1 of a brown planthopper resistance gene Bph14, comprising the following steps:
1) identifying the resistance of the rice material brown planthopper: taking Nipponbare as a reference, artificially inoculating and identifying the brown planthopper resistance of a plurality of rice materials in the seedling stage, and screening brown planthopper resistant materials Y009;
2) structural analysis of the Bph14 gene: extracting DNA of Y009 and Japanese Qingxin fresh leaves, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Bph14 gene in two rice materials by using a software Vector NTI 11, and finding that T → C mutation occurs at 448bp site of the No. 1 exon, so that phenylalanine is changed into leucine, and the non-function of Bph14 is caused;
3) design and synthesis of functional markers of Bph14 gene: according to the T → C mutation of the No. 1 exon 488 site of Bph14, 1 PARMS molecular marker PARMS-Bph14 is designed, the marker is composed of 3 specific primers of Bph14 genes, and the difference of two bases is introduced into the designed forward primer:
forward primer BPH 14-Ra: TGGCTCTGGTCGGAACTTAAAGAAGGTGACCAAGTTCATGCT；
Forward primer BPH 14-Rg: TGGCTCTGGTCGGAACTTAAGGAAGGTCGGAGTCAACGGATT；
Reverse primer BPH 14-F: AACCGTATTCTGTTTCGTTATAGGA, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Bph 14: the marked 3 primers designed according to the Bph14 gene sequence and two general fluorescent primers are added into a PCR reaction system at the same time for amplification, the Bph14 allele sequence is matched with a forward primer Bph14-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Bph14-Fa to obtain an HEX fluorescent signal value through amplification, and if the rice sample is in a heterozygous state at the site, the two forward primers are amplified at the same time;
5) PCR amplification and genotyping of Bph14 gene: after identifying the resistance of brown planthopper of multiple rice materials for polymerizing multiple resistance genes, parting the brown planthopper gene Bph14 by using PARMS-Bph14, and simultaneously adding 3 designed primers Bph14-Fg, Bph14-Fa, BPH14-R and two universal primers of #1 and #2 into a PCR reaction system, wherein the PCR reaction system is 10 mu L: 5 μ L of 2 XPAMS master mix, 0.15 μ L of 10 mM Bph14-Fg labeled primer, 0.15 μ L of 10 mM Bph14-Fa labeled primer, 0.4 μ L of 10 mM Bph14-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O；
The PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are susceptible brown planthopper Bph14 allele (T) type materials, HEX fluorescence signals (green) obtained are anti-brown planthopper Bph14(C) type materials, and gray is negative control;
6) PARMS-Bph14 efficacy assessment: rice material with multiple polymerized resistance genes is analyzed to determine phenotype and genotype.
In the invention:
the multiple parts in the step 1) are 200 parts and 300 parts.
The DNA extraction of the Y009 and the Nippon fresh leaves in the step 2) is the DNA extraction of the Y009 and the Nippon fresh leaves by adopting a CTAB method.
The step 5) of identifying and polymerizing the multiple parts of rice materials of the multiple resistance genes is to identify and polymerize 40-50 parts of rice materials of the multiple resistance genes.
The PCR reaction program in the PCR reaction system in the step 5) is as follows: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1 min.
The phenotype in the step 6) is that brown planthopper is sensed or felt, and brown planthopper is resisted or highly resisted; the genotype identification result is TT type and CC type.
Compared with the prior art, the invention has the following advantages:
1. the PARMS marker based on the brown planthopper resistance gene Bph14 and the application only need one-time PCR amplification, the operation process is simple and easy, the control is convenient, the DNA fragments do not need to be separated by gel electrophoresis, and the toxic chemical substances are prevented from being contacted.
2. The PARMS marker based on the rice brown planthopper resistance gene Bph14 and the application thereof have the advantages of excellent polymorphism, clear gene typing, convenient and fast data reading of software, difficult error and reliable preparation of PARMS-Bph14 detection results.
[ description of the drawings ]
FIG. 1 is a diagram showing the sequence differences of the Bph14 gene in two rice materials, Y009 and Nipponbare, in example 1 of the present invention, and showing that a T → C mutation occurs at the 448bp site in exon 1, resulting in a change from phenylalanine to leucine, resulting in a non-functionality of Bph 14.
FIG. 2 is a fluorescence image of the Bph14 gene in Y009 and Nipponbare rice material in example 1 of the present invention.
[ detailed description ] embodiments
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1:
the PARMS marker and the application based on the brown planthopper resistance gene Bph14 are functional PARMS markers and the application based on nonsynonymous mutation of SNP of exon 1 of the brown planthopper resistance gene Bph14, and comprise the following steps:
1) identifying the resistance of the rice material brown planthopper: taking Nipponbare as a control, artificially inoculating and identifying the brown planthopper resistance of 226 rice materials in a seedling stage, and screening the brown planthopper resistant material Y009;
2) structural analysis of the Bph14 gene: extracting DNA of Y009 and Japanese Qingxin fresh leaves by a CTAB method, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Bph14 gene in two rice materials by using a software Vector NTI 11, and finding that T → C mutation occurs at 448bp site of the No. 1 exon, so that phenylalanine is changed into leucine (see attached figure 1) to cause the non-function of Bph 14;
3) design and synthesis of functional markers of Bph14 gene: according to the T → C mutation of the No. 1 exon 488 site of Bph14, 1 PARMS molecular marker PARMS-Bph14 is designed, the marker is composed of 3 specific primers of Bph14 genes, and the difference of two bases is introduced into the designed forward primer:
forward primer BPH 14-Ra: TGGCTCTGGTCGGAACTTAAAGAAGGTGACCAAGTTCATGCT；
Forward primer BPH 14-Rg: TGGCTCTGGTCGGAACTTAAGGAAGGTCGGAGTCAACGGATT；
Reverse primer BPH 14-F: AACCGTATTCTGTTTCGTTATAGGA, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Bph 14: the marked 3 primers designed according to the Bph14 gene sequence and two general fluorescent primers are added into a PCR reaction system at the same time for amplification, the Bph14 allele sequence is matched with a forward primer Bph14-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Bph14-Fa to obtain an HEX fluorescent signal value through amplification, and if the rice sample is in a heterozygous state at the site, the two forward primers are amplified at the same time;
5) PCR amplification and genotyping of Bph14 gene: after identifying the resistance of the brown planthopper of 44 rice materials for polymerizing a plurality of resistance genes, carrying out typing on the brown planthopper gene Bph14 by using PARMS-Bph14, and simultaneously adding 3 designed primers Bph14-Fg, Bph14-Fa, BPH14-R and two general primers of #1 and #2 into a PCR reaction system, wherein the PCR reaction system is 10 mu L: 5 μ L of 2 XPAMS master mix, 0.15 μ L of 10 mM Bph14-Fg labeled primer, 0.15 μ L of 10 mM Bph14-Fa labeled primer, 0.4 μ L of 10 mM Bph14-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O; the PCR reaction procedure was: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1min
The PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are susceptible brown planthopper Bph14 allele (T) type materials, HEX fluorescence signals (green) obtained are anti-brown planthopper Bph14(C) type materials, and gray is negative control;
6) PARMS-Bph14 efficacy assessment: 40 of 44 materials have phenotype of feeling the brown planthopper or sensing the brown planthopper, and the genotype identification result is TT type; 4 parts of the material is resistant or highly resistant to brown planthopper, the genotype is CC (see figure 2), the effectiveness of the marker is 100 percent, and the marker can be used for molecular breeding of brown planthopper resistance of rice.
Calculating the average resistance grade of the brown planthopper of each strain, wherein the formula is as follows:
average resistance ═ Σ (number of each resistant strain × corresponding resistance level)/total number of strains.
The average resistance rating was:
1.0-1.9 is High Resistance (HR),
2.0-3.9 is anti (R),
4.0-5.9 is medium-resistant (MR),
(iii) a neutral feeling (MS) of 6.0-7.9,
high Sensitivity (HS) is 8.0-9.0.
Example 2:
the PARMS marker and the application based on the brown planthopper resistance gene Bph14 are functional PARMS markers and the application based on nonsynonymous mutation of SNP of exon 1 of the brown planthopper resistance gene Bph14, and comprise the following steps:
1) identifying the resistance of the rice material brown planthopper: taking Nipponbare as a control, artificially inoculating 200 parts of rice materials in a seedling stage to identify the resistance of brown planthopper, and screening brown planthopper resistant materials Y009;
2) structural analysis of the Bph14 gene: extracting DNA of Y009 and Japanese Qingxin fresh leaves by a CTAB method, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Bph14 gene in two rice materials by using a software Vector NTI 11, and finding that T → C mutation occurs at 448bp site of the No. 1 exon, so that phenylalanine is changed into leucine (see attached figure 1) to cause the non-function of Bph 14;
3) design and synthesis of functional markers of Bph14 gene: according to the T → C mutation of the No. 1 exon 488 site of Bph14, 1 PARMS molecular marker PARMS-Bph14 is designed, the marker is composed of 3 specific primers of Bph14 genes, and the difference of two bases is introduced into the designed forward primer:
forward primer BPH 14-Ra: TGGCTCTGGTCGGAACTTAAAGAAGGTGACCAAGTTCATGCT；
Forward primer BPH 14-Rg: TGGCTCTGGTCGGAACTTAAGGAAGGTCGGAGTCAACGGATT；
Reverse primer BPH 14-F: AACCGTATTCTGTTTCGTTATAGGA, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Bph 14: the marked 3 primers designed according to the Bph14 gene sequence and two general fluorescent primers are added into a PCR reaction system at the same time for amplification, the Bph14 allele sequence is matched with a forward primer Bph14-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Bph14-Fa to obtain an HEX fluorescent signal value through amplification, and if the rice sample is in a heterozygous state at the site, the two forward primers are amplified at the same time;
5) PCR amplification and genotyping of Bph14 gene: after identifying the resistance of the brown planthopper of 40 parts of rice materials for polymerizing a plurality of resistance genes, parting the brown planthopper gene Bph14 by using PARMS-Bph14, and simultaneously adding 3 designed primers Bph14-Fg, Bph14-Fa, BPH14-R and two general primers of #1 and #2 into a PCR reaction system, wherein the PCR reaction system is 10 mu L: 5 μ L of 2 XPAMS master mix, 0.15 μ L of 10 mM Bph14-Fg labeled primer, 0.15 μ L of 10 mM Bph14-Fa labeled primer, 0.4 μ L of 10 mM Bph14-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O; the PCR reaction procedure was: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1min
The PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are susceptible brown planthopper Bph14 allele (T) type materials, HEX fluorescence signals (green) obtained are anti-brown planthopper Bph14(C) type materials, and gray is negative control;
6) PARMS-Bph14 efficacy assessment: 40 of 44 materials have phenotype of feeling the brown planthopper or sensing the brown planthopper, and the genotype identification result is TT type; 4 parts of the material is resistant or highly resistant to brown planthopper, the genotype is CC, the effectiveness of the marker is 100 percent, and the marker can be used for molecular breeding of brown planthopper resistance of rice.
Example 3:
the PARMS marker and the application based on the brown planthopper resistance gene Bph14 are functional PARMS markers and the application based on nonsynonymous mutation of SNP of exon 1 of the brown planthopper resistance gene Bph14, and comprise the following steps:
1) identifying the resistance of the rice material brown planthopper: taking Nipponbare as a control, artificially inoculating and identifying the resistance of brown planthopper of 300 parts of rice material in seedling stage, and screening brown planthopper resistant material Y009;
2) structural analysis of the Bph14 gene: extracting DNA of Y009 and Japanese Qingxin fresh leaves by a CTAB method, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Bph14 gene in two rice materials by using a software Vector NTI 11, and finding that T → C mutation occurs at 448bp site of the No. 1 exon, so that phenylalanine is changed into leucine (see attached figure 1) to cause the non-function of Bph 14;
3) design and synthesis of functional markers of Bph14 gene: according to the T → C mutation of the No. 1 exon 488 site of Bph14, 1 PARMS molecular marker PARMS-Bph14 is designed, the marker is composed of 3 specific primers of Bph14 genes, and the difference of two bases is introduced into the designed forward primer:
forward primer BPH 14-Ra: TGGCTCTGGTCGGAACTTAAAGAAGGTGACCAAGTTCATGCT；
Forward primer BPH 14-Rg: TGGCTCTGGTCGGAACTTAAGGAAGGTCGGAGTCAACGGATT；
Reverse primer BPH 14-F: AACCGTATTCTGTTTCGTTATAGGA, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Bph 14: the marked 3 primers designed according to the Bph14 gene sequence and two general fluorescent primers are added into a PCR reaction system at the same time for amplification, the Bph14 allele sequence is matched with a forward primer Bph14-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Bph14-Fa to obtain an HEX fluorescent signal value through amplification, and if the rice sample is in a heterozygous state at the site, the two forward primers are amplified at the same time;
5) PCR amplification and genotyping of Bph14 gene: after identifying the resistance of 50 parts of rice materials for polymerizing a plurality of resistance genes to brown planthopper, parting the brown planthopper gene Bph14 by using PARMS-Bph14, and simultaneously adding 3 designed primers Bph14-Fg, Bph14-Fa, BPH14-R and two general primers of #1 and #2 into a PCR reaction system, wherein the PCR reaction system is 10 mu L: 5 μ L of 2 XPAMS master mix, 0.15 μ L of 10 mM Bph14-Fg labeled primer, 0.15 μ L of 10 mM Bph14-Fa labeled primer, 0.4 μ L of 10 mM Bph14-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O; the PCR reaction procedure was: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1min
The PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are susceptible brown planthopper Bph14 allele (T) type materials, HEX fluorescence signals (green) obtained are anti-brown planthopper Bph14(C) type materials, and gray is negative control;
6) PARMS-Bph14 efficacy assessment: 40 of 44 materials have phenotype of feeling the brown planthopper or sensing the brown planthopper, and the genotype identification result is TT type; 4 parts of the material is resistant or highly resistant to brown planthopper, the genotype is CC, the effectiveness of the marker is 100 percent, and the marker can be used for molecular breeding of brown planthopper resistance of rice.
To summarize:
1. in the prior art, the conventional DNA marker detection methods mainly comprise polyacrylamide electrophoresis and agarose gel electrophoresis, and the detection methods have the defects of long time consumption, complex operation, low efficiency, low automation degree, toxic substance use and the like; capillary electrophoresis has poor preparation capability due to small sample injection amount; the small diameter of the capillary tube leads to too short light path, and the sensitivity is lower when some detection methods (such as ultraviolet absorption spectroscopy) are used; electroosmosis varies depending on the sample composition, and further affects the reproducibility of separation. Through the examples 1-3, the PARMS marker based on the brown planthopper resistance gene Bph14 and the application of the marker are shown, only one-time PCR amplification is needed, the operation process is simple and easy, the control is convenient, the DNA fragment is separated without gel electrophoresis, and the toxic chemical substances are prevented from being contacted.
2. The PARMS marker based on the rice brown planthopper resistance gene Bph14 and the application thereof have the advantages of excellent polymorphism, clear gene typing, convenient and fast data reading of software, difficult error and reliable preparation of PARMS-Bph14 detection results.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention.
Sequence listing
<110> Guangxi Zhuang nationality college of autonomous region agro-sciences
<120> PARMS marker based on brown planthopper resistance gene Bph14 and application
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tggctctggt cggaacttaa ggaaggtcgg agtcaacgga tt 42
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Claims (6)
1. PARMS mark and application based on brown planthopper resistant gene Bph14 of rice, which is characterized in that: is a functional PARMS marker based on nonsynonymous mutation of SNP of exon 1 of a brown planthopper resistance gene Bph14 and application thereof, and comprises the following steps:
1) identifying the resistance of the rice material brown planthopper: taking Nipponbare as a reference, artificially inoculating and identifying the brown planthopper resistance of a plurality of rice materials in the seedling stage, and screening brown planthopper resistant materials Y009;
2) structural analysis of the Bph14 gene: extracting DNA of Y009 and Japanese Qingxin fresh leaves, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Bph14 gene in two rice materials by using a software Vector NTI 11, and finding that T → C mutation occurs at 448bp site of the No. 1 exon, so that phenylalanine is changed into leucine, and the non-function of Bph14 is caused;
3) design and synthesis of functional markers of Bph14 gene: according to the T → C mutation of the No. 1 exon 488 site of Bph14, 1 PARMS molecular marker PARMS-Bph14 is designed, the marker is composed of 3 specific primers of Bph14 genes, and the difference of two bases is introduced into the designed forward primer:
forward primer BPH 14-Ra: TGGCTCTGGTCGGAACTTAAAGAAGGTGACCAAGTTCATGCT；
Forward primer BPH 14-Rg: TGGCTCTGGTCGGAACTTAAGGAAGGTCGGAGTCAACGGATT；
Reverse primer BPH 14-F: AACCGTATTCTGTTTCGTTATAGGA, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Bph 14: the marked 3 primers designed according to the Bph14 gene sequence and two general fluorescent primers are added into a PCR reaction system at the same time for amplification, the Bph14 allele sequence is matched with a forward primer Bph14-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Bph14-Fa to obtain an HEX fluorescent signal value through amplification, and if the rice sample is in a heterozygous state at the site, the two forward primers are amplified at the same time;
5) PCR amplification and genotyping of Bph14 gene: after identifying the resistance of brown planthopper of multiple rice materials for polymerizing multiple resistance genes, parting the brown planthopper gene Bph14 by using PARMS-Bph14, and simultaneously adding 3 designed primers Bph14-Fg, Bph14-Fa, BPH14-R and two universal primers of #1 and #2 into a PCR reaction system, wherein the PCR reaction system is 10 mu L: 5 μ L of 2 XPAMS master mix, 0.15 μ L of 10 mM Bph14-Fg labeled primer, 0.15 μ L of 10 mM Bph14-Fa labeled primer, 0.4 μ L of 10 mM Bph14-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O；
The PCR product is rapidly detected in an enzyme-labeling instrument containing three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, and then the fluorescence signal value file passes through an SNP decoderhttp://www.snpway.com/ snpdecoder01/Analyzing by software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, outputting each signal point in a graphic mode, and finally automatically carrying out genotyping according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, obtaining an FAM fluorescence signal by fluorescence scanning, obtaining an HEX fluorescence signal by blue susceptible brown planthopper Bph14 allele T type materials, obtaining an anti-brown planthopper Bph 14C type materials by green, and obtaining a negative control by gray;
6) PARMS-Bph14 efficacy assessment: rice material with multiple polymerized resistance genes is analyzed to determine phenotype and genotype.
2. The PARMS marker based on the brown planthopper resistance gene Bph14 as claimed in claim 1 and the application thereof, wherein the marker comprises: the multiple parts in the step 1) are 200 parts and 300 parts.
3. The PARMS marker based on the brown planthopper resistance gene Bph14 as claimed in claim 1 and the application thereof, wherein the marker comprises: the DNA extraction of the Y009 and the Nippon fresh leaves in the step 2) is the DNA extraction of the Y009 and the Nippon fresh leaves by adopting a CTAB method.
4. The PARMS marker based on the brown planthopper resistance gene Bph14 as claimed in claim 1 and the application thereof, wherein the marker comprises: the step 5) of identifying and polymerizing the multiple parts of rice materials of the multiple resistance genes is to identify and polymerize 40-50 parts of rice materials of the multiple resistance genes.
5. The PARMS marker based on the brown planthopper resistance gene Bph14 as claimed in claim 1 and the application thereof, wherein the marker comprises: the PCR reaction program in the PCR reaction system in the step 5) is as follows: 95 ℃ for 5 min; then 10 cycles of 95 ℃, 20s, 65 ℃, minus 0.8 ℃/cycle for 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1 min.
6. The PARMS marker based on the brown planthopper resistance gene Bph14 as claimed in claim 1 and the application thereof, wherein the marker comprises: the phenotype in the step 6) is that brown planthopper is sensed or felt, and brown planthopper is resisted or highly resisted; the genotype identification result is TT type and CC type.
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