CN114480705A - SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof - Google Patents
SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof Download PDFInfo
- Publication number
- CN114480705A CN114480705A CN202210036446.4A CN202210036446A CN114480705A CN 114480705 A CN114480705 A CN 114480705A CN 202210036446 A CN202210036446 A CN 202210036446A CN 114480705 A CN114480705 A CN 114480705A
- Authority
- CN
- China
- Prior art keywords
- bacterial blight
- rice bacterial
- rice
- gene
- molecular marker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an SNP molecular marker of a rice bacterial blight-resistant gene XA23 and an amplification primer and application thereof, which are characterized in that through the analysis of the sequence difference of disease-resistant and susceptible varieties in an XA23 gene region, the nucleotide sequence of the SNP molecular marker is shown in a sequence table SEQ ID NO:1, an SNP site A/C exists at the 44 th base position, the SNP site of the rice bacterial blight-resistant gene XA23 is designed and developed according to the sequence, the nucleotide sequence is shown in a sequence table SEQ ID NO:7-9, and the application of the amplification primer of the SNP molecular marker of the rice bacterial blight-resistant gene XA23in screening the rice bacterial blight-resistant varieties has the advantages of quickly, accurately and effectively detecting rice disease-resistant individuals by using the SNP marker.
Description
Technical Field
The invention belongs to a molecular biology DNA marking technology, and particularly relates to an SNP molecular marker of a rice bacterial blight resistant gene XA23, and an amplification primer and application thereof.
Background
Bacterial blight is one of three diseases of bacterial diseases and rice. In recent years, the resistance of pathogenic varieties is lost due to the variation of the bacterial blight, so that the bacterial blight is aggravated, and particularly in coastal areas, the yield of rice is reduced, and the quality is also influenced. The resistance genes such as XA4, XA7 and XA21 used before are used for a long time, so that the resistance is reduced, and a new resistance gene needs to be discovered. The identification and molecular marker location of the new gene Xa-23(t) for resisting rice bacterial blight of common wild rice (Xanthomonas oryzae pv. oryzae) and the molecular marker location [ J ] Proc. 2000, 26(005): 536-542.) found the new gene XA23 for resisting rice bacterial blight in wild rice.
XA23 is a bacterial leaf blight resistance gene having broad-spectrum resistance, and has excellent resistance to bacterial blight. The variety containing the gene has excellent disease resistance in fields, and is currently applied to bacterial blight disease resistance breeding by taking XA23 as a main resistance source. Wang Chun Lian et al (Wang, C., 2013). High-resolution genetic mapping of rice bacterial blight resistance gene Xa23 . Mol Genet Genomics289, 745-753 (2014)) finely maps the XA23 gene to chromosome 11 of rice. In order to facilitate the screening and application of XA23 gene, Zhao Kai Jun et al (2015) develop molecular marker, the 983bp band variety obtained by PCR amplification and electrophoresis contains XA23 gene, and the 869bp band variety does not contain XA23 gene, the application of the marker provides a good way to avoid a large amount of inoculation identification work in field, but the method needs electrophoresis detection and is difficult to realize high-throughput and high-efficiency detection.
Penpe et al (2020) screened for SNP markers that are closely linked to XA23, and if the closely linked markers are separated from the markers, the markers appear to be resistant to disease, but the target gene is susceptible, which has a small probability of false results. And the marked genotype directly corresponds to disease resistance or infection by designing the mark aiming at the sequence difference site of the disease resistance and infection variety of the XA23 gene region. SNP markers are developed for key disease-resistant sites in an XA23 gene region, a new generation of marker detection system is constructed by using the KASP technology, electrophoresis detection and the like are omitted, and an accurate, efficient and high-throughput detection method is constructed.
Disclosure of Invention
The invention aims to solve the technical problem of providing an SNP molecular marker for quickly, accurately and effectively detecting a rice bacterial leaf blight resistant gene XA23 of a rice disease resistant individual by using the SNP marker, and an amplification primer and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: a SNP molecular marker of a rice bacterial blight resistant gene XA23 has a nucleotide sequence shown as SEQ ID NO. 1, and the mutation type at the 44 th base is A/C (figure 1).
The SNP locus of the rice bacterial blight resistant gene XA23 is designed and developed according to a sequence, and the specific sequence is as follows:
XA23npFGC：GAAGGTGACCAAGTTCATGCTAGCTACTATAAAAGTCCCTTCCGC；
XA23npFGA：GAAGGTCGGAGTCAACGGATTAGCTACTATAAAAGTCCCTTCCGA；
XA23npR：GATGCAACAAGGAAGGGCTTTTA。
the application of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties.
The application of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties is characterized in that the application is as follows: the 44 th site genotype for detecting the existence of the rice variety is AA or AC, and is selected as the rice variety resisting bacterial blight.
The application of the SNP molecular marker amplification primer of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties comprises the following specific steps:
1) cutting leaves from a variety to be detected, and extracting cDNA;
2) and (3) performing PCR amplification and fluorescence scanning on an amplification product by using the extracted DNA as a template and using the SNP molecular marker amplification primer of the rice bacterial leaf blight resistant gene XA23, genotyping each parent sample, and reserving individuals with the 44 th site genotype of AA or AC, so as to obtain the rice bacterial leaf blight resistant variety.
The PCR amplification reaction system is 0.15ul of 10mM XA23npFGC, 0.15ul of 10mM XA23npFGA, 0.4ul of 10mM XA23npR, 5ul of PARMS2X and 4.3ul of ultrapure water; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extending for 60s for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20s, annealing at 57 ℃ and extension for 60s, 30 cycles.
Compared with the prior art, the invention has the advantages that: the SNP molecular marker of the rice bacterial leaf blight resistant gene XA23 and the amplification primer and the application thereof, provided by the invention, aim at the site of sequence difference of disease-resistant and susceptible varieties in an XA23 gene region, are designed to be more accurate relative to linkage markers, the accuracy of gene identification is improved, and the SNP molecular marker can be combined with a kasp genotyping technology to identify the XA23 gene more accurately and efficiently. The marker can better promote the application of the XA23 gene in breeding and improve the resistance of rice varieties to bacterial blight. Provides SNP markers in the aspect of bacterial blight for KASP typing technology, and promotes the new generation of molecular marker technology to better serve rice breeding.
Drawings
FIG. 1 shows the alignment results of the disease-resistant and disease-susceptible varieties in the XA23 gene region in example 1;
FIG. 2 shows the results of the XA23 gene marker typing in example 2;
FIG. 3 shows the typing results of F2 individuals analyzed by XA23 gene marker in example 3.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Firstly, the sequence analysis of resistant and susceptible varieties is carried out, and primers are designed aiming at different key sites. The designed primer is used for detecting and testing materials, and finally, the tested primer is verified to ensure the accuracy of the primer.
Example 1
Primers were developed for identifying XA23 for the gene difference sites.
According to the report of Wang Chun Lian et al (Wang C,2014, High-resolution genetic mapping of rice bacterial blue resistance gene Xa23. Mol Genomics. 2014 Oct;289(5):745-53. doi: 10.1007/s00438-014 0848-y. Epub 2014 Apr 9. PMID: 24715026.), 15 pairs of sequencing primers were designed at the XA23 gene position, and because the sequence variation of the sequence is large, part of the sequencing primers can only have amplification products in part of varieties, and part of the primers have no amplification products in the existing varieties, and only the pair of primers of Xa23 index 3F and Xa23 index 3R2 have amplification products in the existing materials. The sequencing primer sequence is as follows: xa23ind 3F: CTTCCGCGTCACTAACATCAGCTACT, respectively; xa23indc3R 2: TTAAACAGGGAGAATAACCATCTTG is added.
The sequencing primer is used for sequencing 6 parts of the existing breeding materials, namely 3 parts of XA23 gene-containing disease-resistant materials and 3 parts of XA23 gene-free disease-sensitive materials, namely A17, A17-1, A25, 20F163, F7562 and F7540. Wherein 20F163, F7562, and F7540 are susceptible, and A17, A17-1, and A25 are resistant to diseases. The sequencing sequences of A17, A17-1, 20F163, A25, F7562 and F7540 are respectively SEQ ID NO:1-6, the sequencing sequence is the interval 22203769 to 22204190 on 11 chromosome of Nipponbare of a variety, specifically shown in a sequence table, each sequence is subjected to sequence alignment analysis by utilizing MegAlign software, and the result is shown in a figure 1, and the disease-resistant material and the disease-sensitive material are different at the base 44 of the sequence SEQ ID NO: 1. According to the sequence 44, the base of the susceptible variety is C, and the base of the disease-resistant variety is A, and a primer is developed and designed aiming at the sequence difference site. Designing upstream primers with slightly different sequence fragment lengths, selecting several different conserved regions and designing downstream primers, and designing 8 groups of primers. When the 8 groups of primers are used for material detection, the XA23np group of primers are found to have typing on the material detection result and the typing is clear, so that the XA23np group of primers are determined to be used for the identification of the XA23 gene. Primer XA23np group primer sequences were as follows, and the primer sequences were synthesized by Shanghai.
XA23npFGC：GAAGGTGACCAAGTTCATGCTAGCTACTATAAAAGTCCCTTCCGC；
XA23npFGA：GAAGGTCGGAGTCAACGGATTAGCTACTATAAAAGTCCCTTCCGA；
XA23npR：GATGCAACAAGGAAGGGCTTTTA。
Example 2
Detection of marker XA23np
Using the markers developed above, 21 parts of the material (see Table 1) were tested, 5 of which were susceptible (no XA23 gene) and 16 of which were resistant (containing XA23 gene).
1. Extracting DNA of the material, and extracting genome DNA from rice leaves by adopting an SDS method.
Sampling and placing the sample into a 2.0ml tube, adding two steel balls and 500 mu L of SDS solution in advance, and oscillating and homogenizing the sample for 1.5 min; heating at 65 deg.C for 0.5-1 h; cooling to room temperature, adding 500 μ L chloroform/isoamyl alcohol (24: 1) solution in fume hood, and mixing; centrifuging at 12000rmp for 10min, taking about 400 μ L of supernatant, and transferring to a new 1.5ml centrifuge tube; adding isopropanol solution of the same volume, shaking, precipitating at-20 deg.C for more than 1 hr, centrifuging at 12000rmp for 10min, and removing supernatant; adding 800 μ L70% ethanol, gently flicking to precipitate, centrifuging at 1000rmp for 3min, and removing supernatant; adding 300 mu L H2O is dissolved overnight for use.
2. KASP reaction test
The KASP reaction test was performed on a PCR instrument. The 10ul reaction system was XA23npFGC (10 mM) 0.15ul, XA23npFGA (10 mM)) 0.15ul, XA23npR (10 mM)) 0.4ul, PARMS2X (reagent purchased from Wuhan dynasty peptide Biotech Co., Ltd.) 5ul, and ultrapure water 4.3 ul. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extending for 60s for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20s, annealing at 57 ℃ and extension for 60s, 30 cycles. And (4) after the reaction is finished, reading fluorescence data by using a microplate reader, and converting the result of fluorescence scanning into a scatter diagram.
The molecular marker amplification primer XA23np is used for KASP primary screening reaction verification of 21 rice varieties including Xa23 gene-containing materials, other gene donors and susceptible materials, the results are shown in Table 1 and FIG. 2, the detection results of 15 rice varieties containing Xa23 gene at XA23np test site are all basic group A, and the detection results of 5 rice varieties without Xa23 gene at test site are basic group C.
TABLE 1 disease resistance of the cultivars and XA23 genotype
Example 3
Application and validation of the marker XA23np
Using infection without XA23 gene20F171 against disease-resistant XA 23-containing gene20F3792 was crossed and analyzed at F2 generation for a total of 168 strains. The 168 individuals were inoculated with the bacterial suspension of the bacterial blight of the leaf blight fungus by leaf cutting, and then observed and counted for their onset of disease, the specific onset of disease is shown in table 2. Further, the individual plants were subjected to leaf extraction to extract DNA. They were genotyped with XA23np marker.
TABLE 2 detailed disease conditions
By analysis, 20 of the strains were homozygous genotype AA, indicating that they contain the XA23 gene. The 49 strains were genotypic AC, indicating heterozygous XA23 gene individuals. In addition, 99 strains were homozygous genotype CC, and indicated as homozygous individuals without XA23 gene (see FIG. 3).
Genotype AA contains the XA23 gene and resistance appears to be resistance. The genotype is CC, and does not contain XA23 gene, and the resistance is expressed as the sense. The genotype was AC, the hybrid XA23 gene, and resistance was expressed as resistance. The instruction marker XA23np can be used for the identification of XA23 gene.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Sequence listing
<110> Ningbo city institute of agricultural science
<120> SNP molecular marker of rice bacterial blight resistant gene XA23, and amplification primer and application thereof
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 480
<212> DNA
<213> A17 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCG [ A/C ] AACATCTTCCTCCCGCATCACTAACATCAGCTTCTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACCGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACATCGCGACAGGGGCGCGG)
<400> 1
<210> 2
<211> 475
<212> DNA
<213> A17-1 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCGAAACATCTTCCTCCCGCATCACTAACATCAGCTTCTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACCGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACTCGCTACATAGCGGCGC)
<400> 2
<210> 3
<211> 481
<212> DNA
<213> 20F163 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCGCGTCACTAACATCTTCCTCCCGCATCACTAACATCAGCTACTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACTGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACTCGCTAAATGGCACAGG)
<400> 3
<210> 4
<211> 476
<212> DNA
<213> A25 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCGAAACATCTTCCTCCCGCATCACTAACATCAGCTTCTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACCGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACATCGCTAAATGCGCCCCG)
<400> 4
<210> 5
<211> 479
<212> DNA
<213> F7562 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCGCGTCACTAACATCTTCCTCCCGCGTCACTAACATCAGCTACTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACTGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACTCGCTACCGCCACGC)
<400> 5
<210> 6
<211> 482
<212> DNA
<213> F7540 Gene sequence (TCTTCCGCGTCACTAACATCAGCTACTATAAAAGTCCCTTCCGCGTCACTAACATCTTCCTCCCGCGTCACTAACATCAGCTACTATAAAAGCCCTTCCTTGTTGCATCATCTCAAGGAGCTGCAAGCACTTCCTCTCTGGCAGCACTTCCTCATCTCAAGGAGTTGCAAATGTTGCATCATCTCAAGGAGCTGGCAGCCGTAGCCGGTATACACATGATCCTCATCTACCTCTGCCGCTTTCTCCTCCGCCGCAGCCGCAACGTATTATTCACTGTTTCCAACAGCCTCCGTTTTCGCCTCAAGGTATTAACTGTATTGTTGTACATATGTCTCTCGGTCATGCTGTTCTACCTGTTTGGCTCCATCATGCCGCTGCCGCCGTGGGGCCTCGTGGTCGGTTGGGTCATGGCCCTCATCGCCGTCGAGCTCGCCTACGCCTTCATCTTCCATATAGCTCGCTACTCCGCAAATCGCGCGGGA)
<400> 6
<210> 7
<211> 45
<212> DNA
<213> XA23npFGC amplification primer (GAAGGTGACCAAGTTCATGCTAGCTACTATAAAAGTCCCTTCCGC)
<400> 7
<210> 8
<211> 45
<212> DNA
<213> XA23npFGA（GAAGGTCGGAGTCAACGGATTAGCTACTATAAAAGTCCCTTCCGA）
<400> 8
<210> 9
<211> 23
<212> DNA
<213> XA23npR （GATGCAACAAGGAAGGGCTTTTA）
<400> 9
<210> 10
<211> 26
<212> DNA
<213> xa23indc3F sequencing primer (CTTCCGCGTCACTAACATCAGCTACT)
<400> 10
<210> 11
<211> 25
<212> DNA
<213> xa23indc3R2 sequencing primer (TTAAACAGGGAGAATAACCATCTTG)
<400> 11
Claims (6)
1. An SNP molecular marker of a rice bacterial blight resistant gene XA23, which is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1, and the mutation type at the 44 th base is A/C.
2. The amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23 as claimed in claim 1, which is characterized in that the specific sequence is as follows:
XA23npFGC：GAAGGTGACCAAGTTCATGCTAGCTACTATAAAAGTCCCTTCCGC；
XA23npFGA：GAAGGTCGGAGTCAACGGATTAGCTACTATAAAAGTCCCTTCCGA；
XA23npR：GATGCAACAAGGAAGGGCTTTTA。
3. the use of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23 as claimed in claim 2 in screening rice bacterial blight resistant varieties.
4. The application of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties according to claim 3, is characterized in that the application is as follows: the 44 th site genotype for detecting the existence of the rice variety is AA or AC, and is selected as the rice variety resisting bacterial blight.
5. The application of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties according to claim 4 is characterized by comprising the following specific steps:
1) cutting leaves from a variety to be detected, and extracting cDNA;
2) and (3) performing PCR amplification and fluorescence scanning on an amplification product by using the extracted DNA as a template and using the amplification primer marked by the SNP molecule of the rice bacterial leaf blight resistant gene XA23, genotyping each variety sample, and reserving an individual with the 44 th site genotype of AA or AC, so as to obtain the rice bacterial leaf blight resistant variety.
6. The application of the amplification primer of the SNP molecular marker of the rice bacterial blight resistant gene XA23in screening rice bacterial blight resistant varieties according to claim 5, wherein the amplification primer comprises the following components in percentage by weight: the PCR amplification reaction system is 0.15ul of 10mM XA23npFGC, 0.15ul of 10mM XA23npFGA, 0.4ul of 10mM XA23npR, 5ul of PARMS2X and 4.3ul of ultrapure water; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extending for 60s for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20s, annealing at 57 ℃ and extension for 60s, 30 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210036446.4A CN114480705A (en) | 2022-01-13 | 2022-01-13 | SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210036446.4A CN114480705A (en) | 2022-01-13 | 2022-01-13 | SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114480705A true CN114480705A (en) | 2022-05-13 |
Family
ID=81512199
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210036446.4A Pending CN114480705A (en) | 2022-01-13 | 2022-01-13 | SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480705A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334290A (en) * | 2023-04-12 | 2023-06-27 | 湖北省农业科学院粮食作物研究所 | Primer group and kit for identifying rice functional genes and application of primer group and kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087578A (en) * | 2014-07-07 | 2014-10-08 | 合肥丰乐种业股份有限公司 | Molecular marker closely linked with rice bacterial blight resistance gene and primers and application thereof |
| CN107236811A (en) * | 2017-07-04 | 2017-10-10 | 华智水稻生物技术有限公司 | Bacterial leaf spot resistance gene Xa21 assistant breedings molecular labeling and its application |
| WO2019047074A1 (en) * | 2017-09-06 | 2019-03-14 | 中国农业科学院作物科学研究所 | Snp molecular marker combination for rice genotyping, and application thereof |
| CN109628629A (en) * | 2018-12-11 | 2019-04-16 | 华智水稻生物技术有限公司 | The SNP marker development and application of rice bacterial leaf spot resistant gene xa5 |
| CN112501344A (en) * | 2020-12-22 | 2021-03-16 | 华智生物技术有限公司 | SNP molecular marker for detecting bacterial leaf blight resistance gene Xa23 and application thereof |
-
2022
- 2022-01-13 CN CN202210036446.4A patent/CN114480705A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087578A (en) * | 2014-07-07 | 2014-10-08 | 合肥丰乐种业股份有限公司 | Molecular marker closely linked with rice bacterial blight resistance gene and primers and application thereof |
| CN107236811A (en) * | 2017-07-04 | 2017-10-10 | 华智水稻生物技术有限公司 | Bacterial leaf spot resistance gene Xa21 assistant breedings molecular labeling and its application |
| WO2019047074A1 (en) * | 2017-09-06 | 2019-03-14 | 中国农业科学院作物科学研究所 | Snp molecular marker combination for rice genotyping, and application thereof |
| CN109628629A (en) * | 2018-12-11 | 2019-04-16 | 华智水稻生物技术有限公司 | The SNP marker development and application of rice bacterial leaf spot resistant gene xa5 |
| CN112501344A (en) * | 2020-12-22 | 2021-03-16 | 华智生物技术有限公司 | SNP molecular marker for detecting bacterial leaf blight resistance gene Xa23 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| WANG, C., ZHANG, X., FAN, Y., GAO, Y., ZHU, Q., ZHENG, C., QIN, T., LI, Y., CHE, J., ZHANG, M., YANG, B., LIU, Y. AND ZHAO, K.: "XA23 is an executor R protein and confers broad-spectrum disease resistance in rice", MOL PLANT, vol. 8, no. 2, pages 293 * |
| 周雷;陈志军;马银花;刘凯;李三和;杨国才;胡刚;万丽云;游艾青;: "水稻白叶枯病抗性基因Xa23的分子标记优化及应用", 湖北农业科学, vol. 50, no. 24, pages 5243 - 5246 * |
| 高利军;高汉亮;李容柏;李道远;周萌;颜群;周维永;张晋;邓国富;: "水稻抗白叶枯病基因Xa23分子标记的优化和验证", 分子植物育种, vol. 8, no. 04, pages 660 - 664 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334290A (en) * | 2023-04-12 | 2023-06-27 | 湖北省农业科学院粮食作物研究所 | Primer group and kit for identifying rice functional genes and application of primer group and kit |
| CN116334290B (en) * | 2023-04-12 | 2024-04-05 | 湖北省农业科学院粮食作物研究所 | Primer group and kit for identifying rice functional genes and application of primer group and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868131B (en) | SNP molecular marker of upland cotton No. 6 chromosome related to fiber strength | |
| CN113584216B (en) | Development and application of KASP marker of wheat grain weight gene TaCYP78A16 | |
| CN111575400B (en) | Wheat stripe rust resistance QTL molecular marker IWB12253 and application thereof | |
| CN111850161B (en) | Sand-melt aegilops 7SshChromosome specific molecular marker and application thereof | |
| CN113584215B (en) | Development and application of KASP marker of wheat powdery mildew resistance gene pmCH7015 | |
| CN112695114B (en) | SNP molecular marker for detecting rice blast resistance Pik gene and application thereof | |
| CN114480705A (en) | SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof | |
| CN111560464B (en) | Molecular marker IWB59718 and application thereof in detection of wheat stripe rust resistance | |
| CN117683927A (en) | Functional KASP molecular marker of rice blast resistance gene and application thereof | |
| CN106755465B (en) | Molecular marker closely linked with wheat flag leaf length QTL QFLL | |
| CN112176083B (en) | Functional molecular marker of wheat powdery mildew resistance related gene Pm41 and application thereof | |
| CN111793712A (en) | Application of single nucleotide polymorphism substance of wheat SNP IWA586 site | |
| KR20180055075A (en) | Single nucleotide polymorphism marker for selecting bacterial wilt-resistant or sensitive pepper cultivar and uses thereof | |
| CN111378781A (en) | Molecular marker primer for quickly and efficiently identifying salt-tolerant gene SKC1 of rice and application | |
| CN111607664B (en) | Application of SNP molecular marker on 1DS chromosome related to wheat stripe rust | |
| CN112501344B (en) | SNP molecular marker for detecting bacterial leaf blight resistance gene Xa23 and application thereof | |
| WO2020082314A1 (en) | Oryza sativa green gene chip and application | |
| CN111778346B (en) | Molecular marker for detecting stripe rust resistance QTL QYr.hbas-4BL.1 and using method | |
| CN111635957B (en) | Molecular marker for detecting wheat stripe rust resistance QTL and application of molecular marker in disease resistance breeding | |
| CN110229925B (en) | Potato late blight disease-resistant gene diagnosis primer and design method thereof | |
| CN114606335A (en) | Development and application of KASP molecular marker of sugarcane mosaic virus disease resistance gene of corn | |
| CN110592260B (en) | Competitive allele specific polymerase chain reaction marker of hard wheat adult plant leaf rust resistant locus and application thereof | |
| CN107653341B (en) | KASP marker for detecting powdery mildew resistance gene of aegilops tauschii and application thereof | |
| KR20170010204A (en) | Molecular marker for selecting powdery mildew resistance gene in watermelon | |
| CN113637790B (en) | KASP molecular marker of stripe rust resistance gene YrAS2388R, primer, kit and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |