CN112501346A - SNP molecular marker related to rice bacterial leaf blight resistance, detection primer pair and application - Google Patents
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Abstract
The invention provides an SNP molecular marker related to rice bacterial leaf blight resistance, a detection primer pair and application, and relates to the technical field of crop genetic breeding. The SNP molecular marker is obtained by a whole genome association analysis method, the SNP molecular marker is positioned at 27792921bp nucleotide single base mutation on 11 th chromosome of a rice reference genome and at 26220673bp nucleotide single base mutation on 11 th chromosome, and the gene positioned nearby is a gene for coding an NBS-LRR type disease-resistant protein domain, so that the SNP molecular marker is proved to be related to rice disease resistance. By utilizing the SNP molecular marker provided by the invention, a resistance gene obviously associated with bacterial leaf blight resistance can be found, and a detection method expressed in a resistance material is established, so that a gene resource is provided for subsequent rice disease resistance breeding.
Description
Technical Field
The invention belongs to the technical field of crop genetic breeding, and particularly relates to a SNP molecular marker related to rice bacterial leaf blight resistance, a detection primer pair and application.
Background
Rice is one of the most important food crops in the world, and the yield loss of the rice caused by diseases is more than 20% every year. Bacterial blight of rice caused by Xanthomonas oryzae oryza sativa (Xoo) is the most important bacterial disease in rice production in the world, and is one of the three major diseases in current rice production. Xoo causes bacterial blight through colonization and spread in vascular tissues of rice leaves, thereby affecting photosynthesis and ultimately causing severe yield loss in rice. The breeding practice shows that digging resistance gene and breeding disease-resistant variety is the most economic and effective means for preventing and treating bacterial blight.
A total of 44 rice bacterial leaf blight Resistance genes (R Gene) were found, including 11 cloned rice bacterial leaf blight R genes, Xa1, Xa3/Xa26, Xa4, Xa5, Xa10, Xa13, Xa21, Xa23, Xa25, Xa27, and Xa41 (t). The R gene-mediated plant disease-resistant defense mechanism is mainly characterized in that after the corresponding avirulence protein coded by a pathogenic microorganism avirulence gene (Avr) is recognized by the antiviral protein coded by the gene self-antiviral gene, a disease-resistant signal path in a plant body is further activated, local cell and tissue allergic necrosis (HR) reaction is generated at an infected part, and finally infection and further diffusion of pathogenic bacteria are resisted. Plants infected with microbial pathogens induce a defense response by recognizing pathogen molecules by plant immune receptors that appear intracellularly to encode protein conserved structures (NBS-LRR), where NLR structures can recognize effectors within plant cells transmitted by pathogens, and thus the reported R genes mostly include bacterial leaf blight resistance proteins encoding NBS-LRR classes, such as the cloned Xa1 and Xa21 genes. However, the major resistance gene of bacterial blight is difficult to be directly applied to breeding for disease resistance due to narrow disease resistance spectrum or recessive gene. According to the hypothesis of gene-to-gene, the interaction of the R gene and the avr gene is expressed as the specificity resistance of the variety to the race, and the continuous emergence and evolution of the new physiological race of pathogenic bacteria directly threatens the vertical resistance of the existing resistance gene. Therefore, a new technical means is urgently needed to discover more major resistance genes of bacterial blight and apply the major resistance genes to rice disease resistance breeding.
Disclosure of Invention
In view of the above, the present invention aims to provide a SNP molecular marker related to rice bacterial leaf blight resistance, a detection primer pair and an application thereof, to determine a brand-new molecular marker related to rice bacterial leaf blight resistance, and to obtain a resistance gene significantly related to bacterial leaf blight resistance, so as to provide a gene resource for subsequent rice breeding for disease resistance.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SNP molecular marker related to rice bacterial blight resistance, which comprises SNP _11_27792921_ A _ G and/or SNP _11_26220673_ C _ T;
the molecular marker SNP _11_27792921_ A _ G is located at 27792921bp on the 11 th chromosome of rice, and a single-base mutation AG of nucleotides exists;
the molecular marker SNP _11_26220673_ C _ T is located at 26220673bp on the 11 th chromosome of rice, and has a single nucleotide mutation CT.
The invention also provides a group of primer pairs for detecting the SNP molecular markers, wherein the primer pairs for detecting the molecular markers SNP _11_27792921_ A _ G comprise an upstream primer 1F and a downstream primer 1R, the nucleotide sequence of the upstream primer 1F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer 1R is shown as SEQ ID NO. 2;
the primer pair for detecting the molecular marker SNP _11_26220673_ C _ T comprises an upstream primer 2F and a downstream primer 2R, wherein the nucleotide sequence of the upstream primer 2F is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer 2R is shown as SEQ ID NO. 4.
The invention also provides a kit for detecting the SNP molecular marker, and the kit comprises the primer pair.
The invention also provides the application of the SNP molecular marker or the primer pair or the kit in detecting the bacterial leaf blight resistance of rice.
The invention also provides a method for detecting rice bacterial leaf blight resistance, which comprises the steps of taking rice genome DNA as a template, carrying out PCR detection by using the primer pair or the kit, sequencing a PCR product, and if the position of the molecular marker SNP _11_27792921_ A _ G is an A base, determining the molecular marker as an AA genotype, wherein the resistance grade is 0 grade or 1 grade; if the molecular marker SNP _11_27792921_ A _ G is G base, the gene is GG genotype, and the resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is C base, the molecular marker is CC genotype, and the resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is T base, the gene type is TT, and the resistance grade is 0 grade or 1 grade.
Preferably, the PCR detection system is 25 μ l, 2 XKOD Buffer 12.5 μ l, KOD0.25 μ l, upstream and downstream primers 0.5 μ l, DNA template 2 μ l, double distilled water to 25 μ l. .
Preferably, the PCR program for detecting the molecular marker SNP _11_27792921_ a _ G includes: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, extension at 72 ℃ for 30s, and 35 cycles; extending at 72 deg.C for 10min, and keeping the temperature at 4 deg.C
The PCR program for detecting the molecular marker SNP _11_26220673_ C _ T comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 65.7 ℃ for 20s, extension at 72 ℃ for 30s, 35 cycles; extending for 10min at 72 ℃, and keeping the temperature at 4 ℃.
The invention also provides the application of the SNP molecular marker or the primer pair or the kit in rice breeding.
The invention also provides a molecular marker-assisted breeding method for rice disease resistance, which utilizes the primer pair or the kit to detect the SNP molecular marker so as to determine the bacterial leaf blight resistance of the rice of the variety/line to be detected.
The invention also provides a method for detecting the expression of the rice disease-resistant molecular marker in a resistant material, which is used for detecting the expression quantity of the SNP molecular marker in the rice genome of the variety/strain to be detected.
The invention provides an SNP molecular marker related to rice bacterial leaf blight resistance, which is obtained by a whole genome association analysis (GWAS) method, and the SNP molecular marker is positioned at 27792921bp nucleotide single base mutation (named: LOC _ Os11g45930) on the 11 th chromosome of a rice reference genome (Os-Nipponbare-reference-IRGSP-1.0) and at 26220673bp nucleotide single base mutation (named: LOC _ Os11g43420) on the 11 th chromosome, and the genes positioned nearby the SNP molecular marker are genes encoding NBS-LRR type disease-resistant protein domains, and are presumed to be related to rice disease resistance. By utilizing the SNP molecular marker provided by the invention, a resistance gene obviously associated with bacterial leaf blight resistance can be found, and a detection method expressed in a resistance material is established, so that a gene resource is provided for subsequent rice disease resistance breeding.
Drawings
FIG. 1 shows the bacterial leaf blight phenotype and lesion length of synB/Zhonghui 9308 inoculated with a physiological strain CR 4;
FIG. 2 is the frequency distribution of rice bacterial leaf blight spot length under CR4 physiological strain;
FIG. 3 is a genome-wide linkage disequilibrium attenuation map;
FIG. 4 is a graph of genome-wide association (GWAS) analysis on rice genome-wide for bacterial blight resistance; wherein: the abscissa represents the rice chromosome number; ordinate represents-log10A P value;
FIG. 5 is a relative quantitative expression of molecular markers; wherein: the abscissa represents two parents: synephrine early B (denoted XB) and zhonghui 9308 (denoted R9308); the ordinate represents the relative expression amount.
Detailed Description
The invention provides an SNP molecular marker related to rice bacterial blight resistance, which comprises SNP _11_27792921_ A _ G and/or SNP _11_26220673_ C _ T;
the molecular marker SNP _11_27792921_ A _ G is located at 27792921bp on the 11 th chromosome of rice, and a single-base mutation AG of nucleotides exists;
the molecular marker SNP _11_26220673_ C _ T is located at 26220673bp on the 11 th chromosome of rice, and has a single nucleotide mutation CT.
The invention preferably finds the SNP molecular marker which is obviously related to the resistance of the bacterial blight by applying a genome wide association analysis (GWAS) method based on the combination of 139 high-generation recombinant inbred line groups generated by the hybridization of rice variety Xieqingzao B and Zhonghui 9308 and a high-density SNP molecular marker map, and the specific method preferably comprises the following steps: 1) 139 recombinant inbred line groups of the rice variety Xieqingzao B and Zhonghui 9308 are constructed and planted in experimental bases of the Chinese rice institute in 2019 and 2020; inoculating different physiological bacterial strains of the bacterial leaf blight at the booting stage of the rice, and investigating the length of lesion spots after a plurality of days;
2) extracting genome DNA of the recombinant inbred line population;
3) using Next-generation Sequencing technology (Next-generation Sequencing) to complete the resequencing work of the parents and the recombinant inbred line population;
4) carrying out SNP detection by using BWA software; quality control and filtration of sequencing raw data by the PLINK pair comprise filtering the Minimal Allele Frequency (MAF) <0.05, the genotype deletion rate (Geno) >0.2 and filtering the loci where the genotypes are not different in the parental population by PLINK software;
5) analyzing the group structure and group heredity of the recombinant inbred line by using software PLINK and TASSEL;
6) performing correlation analysis of phenotype, genotype and covariate by combining the length data of rice leaf spots and adopting EMMAX software to determine SNP (Single nucleotide polymorphism) associated with the rice bacterial leaf blight resistance; the gene was annotated with reference to the rice genome function annotation website (http:// rice. plant. msu. edu/cgi-bin/gbrowse/rice).
The SNP molecular marker associated with rice bacterial leaf blight resistance is obtained by using the GWAS method, the nucleotide single base mutation (named as LOC _ Os11g45930) at 27792921bp on the 11 th chromosome of a rice reference genome (Os-Nipponbare-reference-IRGSP-1.0) and the nucleotide single base mutation (named as LOC _ Os11g43420) at 26220673bp on the 11 th chromosome are positioned nearby genes which are genes for coding NBS-LRR type disease-resistant protein domains, and the SNP molecular marker is presumed to be related to rice disease resistance.
The invention also provides a group of primer pairs for detecting the SNP molecular markers, wherein the primer pairs for detecting the molecular markers SNP _11_27792921_ A _ G comprise an upstream primer 1F and a downstream primer 1R, the nucleotide sequence of the upstream primer 1F is shown as SEQ ID No.1 (5'-CGATGTGGCGTCTGGTCAA-3'), and the nucleotide sequence of the downstream primer 1R is shown as SEQ ID No.2 (3 '-GGATGGTGCTCCAGTGCTTG-5');
the primer pair for detecting the molecular marker SNP _11_26220673_ C _ T comprises an upstream primer 2F and a downstream primer 2R, wherein the nucleotide sequence of the upstream primer 2F is shown as SEQ ID NO.3 (5'-GAGTGATATTGTCTATCGCCGCCAT-3'), and the nucleotide sequence of the downstream primer 2R is shown as SEQ ID NO.4 (3 '-ATGGCGGCGATAGACAATATCACTC-5').
The Primer pair is preferably designed based on nucleotide sequences of positions before and after the molecular marker on NCBI by using software Primer 5.0.
The invention also provides a kit for detecting the SNP molecular marker, and the kit comprises the primer pair.
The invention determines the genotype of rice based on the SNP molecular marker, the primer pair or the kit, and carries out genotyping on the rice, thereby determining the resistance of the rice to the bacterial blight of the rice.
The invention also provides the application of the SNP molecular marker or the primer pair or the kit in detecting the bacterial leaf blight resistance of rice.
The invention also provides a method for detecting rice bacterial leaf blight resistance, which comprises the steps of taking rice genome DNA as a template, carrying out PCR detection by using the primer pair or the kit, sequencing a PCR product, and if the molecular marker SNP _11_27792921_ A _ G is an A base, determining the molecular marker as an AA genotype, wherein the corresponding resistance grade is 0 grade or 1 grade; if the molecular marker SNP _11_27792921_ A _ G is G base, the G base is GG genotype, and the corresponding resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is a C base, the molecular marker is a CC genotype, and the corresponding resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is T base, the gene type is TT, and the corresponding resistance grade is 0 grade or 1 grade.
When the PCR detection is carried out, the PCR detection system is 25 ul, preferably comprises 12.5 ul of 2 XKOD Buffer, 0.25 ul of KOD, 0.5 ul of upstream and downstream primers respectively, 2 ul of DNA template and 25 ul of double distilled water. In the present invention, the PCR program for detecting the molecular marker SNP _11_27792921_ a _ G preferably includes: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃, and preserving heat at 4 ℃; the PCR program for detecting the molecular marker SNP _11_26220673_ C _ T preferably comprises: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 65.7 ℃ for 20s, extension at 72 ℃ for 30s, 35 cycles; extending for 10min at 72 ℃, and keeping the temperature at 4 ℃.
The invention preferably grades the resistance of the rice to the bacterial leaf blight based on a plant disease research method of formula Zhongda, and the grading standard of the disease degree of the rice leaves is as follows: wherein, the 0 grade is immunity and has no disease spots; grade 1 is high resistance, and the length of the lesion spots is not more than 3 cm; grade 2 is moderate, and the length of the scab is less than 25% of the total leaf length; grade 3 is the neutral feeling, and the length of the lesion is less than 50 percent of the total leaf length; grade 4 is high, and the length of the lesion is less than 75% of the total leaf length; grade 5 is susceptible and full leaf. In the embodiment of the invention, the AA genotype corresponds to the length phenotype of the bacterial blight scab of 5.45 +/-2.5 cm, and the corresponding resistance grade is 0 grade or 1 grade; the length of a lesion spot corresponding to the GG genotype reaches 9.48 +/-4.34 cm, the corresponding resistance grade is grade 2 or grade 3, and the extremely significant level is reached; the CC genotype corresponds to a lesion length of 9.87 + -4.21 cm and a corresponding resistance rating of 2 or 3, whereas the TT genotype is a lesion length of 5.78 + -3.23 cm and a corresponding resistance rating of 0 or 1. The two SNP molecular markers have the effects of synergistically and positively regulating the resistance to the bacterial blight, and evolutionary analysis finds that the two NBS-LRR genes and the cloned 11 Xa genes are in different subgroups, so that the two new molecular markers have great utilization value in breeding of the bacterial blight resistance.
The invention also provides the application of the SNP molecular marker or the primer pair or the kit in rice breeding.
The invention also provides a molecular marker-assisted breeding method for rice disease resistance, which utilizes the primer pair or the kit to detect the SNP molecular marker so as to determine the bacterial leaf blight resistance of the rice of the variety/line to be detected. The method for determining the bacterial leaf blight resistance of the rice variety/line to be tested is preferably the same as the above method, and is not described herein again.
The invention also provides a method for detecting the expression of the rice disease-resistant molecular marker in a resistant material, which is used for detecting the expression quantity of the SNP molecular marker in the rice genome of the variety/strain to be detected.
The expression quantity is preferably detected by utilizing a qRT-PCR method, a quantitative primer utilized in the method is preferably designed according to a coding region sequence provided by a rice genome function annotation website, a primer pair of a gene LOC _ Os11g43420 comprises an upstream primer 3F and a downstream primer 3R, the nucleotide sequence of the upstream primer 3F is shown as SEQ ID No.5 (5'-CTTTATGCGAATCCTAACAAGACC-3'), and the nucleotide sequence of the downstream primer 1R is shown as SEQ ID No.6 (3 '-CGAGAAGAGCATCAAAACATCG-5'). The primer pair of the gene LOC _ Os11g45930 comprises an upstream primer 3F and a downstream primer 3R, wherein the nucleotide sequence of the upstream primer 3F is shown as SEQ ID No.7 (5'-CTTGTGCCATAGGGGAAAAGA-3'), and the nucleotide sequence of the downstream primer 1R is shown as SEQ ID No.8 (3 '-AAACGAATGCTGGGGTGC-5'). The upstream primer nucleic acid sequence of the reference gene Actin is preferably shown as SEQ ID NO.9 (5'-AGCTGCGGGTATCCATGAGA-3'), and the downstream primer nucleic acid sequence is preferably shown as SEQ ID NO.10 (5'-GCAATGCCAGGGAACATAGTG-3').
In the embodiment of the invention, preferably, the synephrine-senia B and the Heihui 9308 are sampled in the field at different time points before and after inoculation, total RNA is extracted by using Trizol reagent, cDNA after reverse transcription is used as a template, and quantitative PCR is carried out in CFX96(Bio-Rad) by using Fast Essentia DNA Green reagent of Roche. The invention detects whether the molecular marker isWhen expressed in resistant material, the qRT-PCR system used is 25. mu.l, preferably 2 xFast Essential DNA Green Master 12.5. mu.l, s quantitative PCR upstream and downstream primers 0.5. mu.l, cDNA template 0.5. mu.l, ddH2Supplementing O to 25 μ l; the reaction procedure preferably comprises 10min at 95 ℃; 40 cycles of 95 ℃ 20s, 60 ℃ 20s, 72 ℃ extension 20 s.
The following examples are provided to describe the SNP molecular markers, detection primer pairs and applications related to bacterial blight resistance of rice in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of a Rice population
The experimental population used in the invention is a super rice 'Jiouyou 9308' source recombinant inbred line, original parents of hybrid combination are Jiuqingzao B (XB in the figure) and Zhonghui 9308(R9308 in the figure), and the population is obtained by a continuous 13-generation single-grain transmission method. Wherein the XieQINGZANG B is female parent of bacterial leaf blight infection, and Zhonghui 9308 is male parent of disease resistance.
The Heyou 9308 recombinant inbred line population is planted in Fuyang test base (N30 degrees 32 'and E120 degrees 12') of the Chinese Rice research institute in 2019 and 2020, and comprises 139 recombinant inbred line lines and two original parents. All the recombinant inbred line material seeds are cultivated in seedling trays after single plant harvest, the plant is transplanted to a field after 30 days, 5 lines and 8 plants are planted in each row, and simultaneously, a rice bacterial leaf blight infection control variety of Jingang 30 is planted.
Identification and investigation of bacterial leaf blight resistance of rice
Bacterial leaf blight resistance identification adopts a leaf-cutting method, and a physiological bacterial strain of bacterial leaf blight (CR4, isolated from Zhejiang province, China) is inoculated at the rice booting stage. Culturing Bacillus subtilis in NA culture medium (yeast extract 1g, beef extract 3g, peptone 5g, sucrose 10g and agar powder 15g, adjusting pH to 6.0-6.5) at 28 deg.C for 2 days, diluting with sterile water to 10 deg.C9Bacteria/ml (OD 0.8). Each physiological strain was inoculated with 1 row, and each number was inoculated with at least 20 leaves fully expanded. After inoculation, the medicine is not taken, the rest is managed according to the conventional field, the length and the total length of the scab are measured after 15 days, as shown in figure 1, XieqingThe length of the early B lesion is 21.60 cm; zhonghui 9308 lesion length is 5.28 cm. The EXCEL calculates the normal distribution of lesion length of 139 recombinant inbred lines and parents, and as shown in FIG. 2, the lesion length of the recombinant inbred lines and the parents conforms to the normal distribution.
The resistance is graded according to the research method of plant diseases of the formula, and the grading standard of the disease degree of the sword leaves of the rice is as follows: wherein, the 0 grade is immunity and has no disease spots; grade 1 is high resistance, and the length of the lesion spots is not more than 3 cm; grade 2 is moderate, and the length of the scab is less than 25% of the total leaf length; grade 3 is the neutral feeling, and the length of the lesion is less than 50 percent of the total leaf length; grade 4 is high, and the length of the lesion is less than 75% of the total leaf length; grade 5 is susceptible and full leaf.
Three-gene typing
Extracting genome DNA from rice leaf tissues by adopting a CTAB method, and finishing the re-Sequencing work by using a Next-generation Sequencing technology (Next-generation Sequencing) in Tianjin Nuo Poa genesis company. Double affinity 139 recombinant inbred lines were used at 100 x and 10 x coverage, respectively. The Japanese acrylonitrile genome (os-Nipponbare-reference-IRGSP-1.0) was used as a reference genome Sequence, and the Sequence Reads (Sequnence Reads) and the Sequence Alignment (Sequence Alignment) of the reference genome were performed using BWA software. The sequencing data were screened by PLINK.
Four genome-wide association analysis and genome annotation
The PopLDdecay software calculated linkage disequilibrium attenuation and plotted the LD attenuation of this population across the entire genome (fig. 3). The Tassel software evaluates the influence of genetic relationship on the population of the recombinant inbred line to obtain a genetic relationship matrix (Kinship matrix). Genome-wide association analysis a Mixed Linear Model (MLM) with the addition of genetic relationship matrices as covariates was selected using the EMMAX software. Manhattan plots and Q-Q plots are drawn in the R language (FIG. 4). Bonferroni correction gave a significance level of p<10e-7。
And (3) searching genes located near the significant SNP markers according to the Japanese sunny sequence annotation map, wherein the searching range is in a 1000bp (+/-500 bp) genomic region of the site. And to the Rice genome functional annotation Website (http:// rice. plant biology. msu. edu/cgi-bin/gbrows)e/rice) gene annotation for this gene. The p value of the SNP marker SNP _11_27792921_ a _ G is p 2.70257E-05The p-value of the nearby gene LOC _ Os11g45930 and marker SNP _11_26220673_ C _ T is p-5.00378E-05And the nearby gene LOC _ Os11g 43420. The annotation information shows that the two genes contain NBS-LRR type anti-disease protein structural domains, and the evolution analysis shows that the genes different from the cloned genes indicate that the genes are novel rice bacterial blight resistance genes (see table 1).
TABLE 1 molecular marker Annotation information
Example 2
Design of target gene primer
Primers shown in SEQ ID NO. 1-SEQ ID NO.4 are respectively designed through an NCBI website according to nucleotide sequences at the front and rear positions of the markers SNP _11_27792921_ A _ G and SNP _11_26220673_ C _ T.
Two-molecule marked LOC _ Os11g45930 and LOC _ Os11g43420 locus genotypes are used for analyzing the resistance difference of 139 different genotypes of the recombined inbred lines with double affinity at the LOC _ Os11g45930 locus of the molecular marker in table 2, and the resistance difference of 139 different genotypes of the recombined inbred lines with double affinity at the LOC _ Os11g43420 locus of the molecular marker in table 3.
TABLE 2 molecular markers LOC _ Os11g45930 locus differential between different genotype groups
TABLE 3 differences between different genotype groups at LOC _ Os11g43420 site of molecular markers
The length phenotype of the bacterial leaf blight disease spots corresponding to the AA genotype is 5.45 +/-2.5 cm, and the corresponding resistance grade is 0 grade or 1 grade; the length of a lesion spot corresponding to the GG genotype reaches 9.48 +/-4.34 cm, the corresponding resistance grade is 2 grade or 3 grade, and the extremely significant level is reached. The length of the disease spot corresponding to the CC genotype is 9.87 +/-4.21 cm, and the corresponding resistance grade is 2 grade or 3 grade; the length of TT genotype lesion is 5.78 plus or minus 3.23cm, the corresponding resistance grade is 0 grade or 1 grade, and the extremely significant level is reached. The two molecular markers are shown to act as synergistic positive control of resistance to bacterial blight, consistent with the data in figure 1 of example 1.
Example 3
After the 139 recombinant inbred lines and the two parents are inoculated with physiological strains in 2019, the 78 families are homozygous for GG based on the 11_27792921_ A _ G SNP locus genotype, the rice bacterial leaf blight resistance grade of the 78 recombinant inbred line families is determined to be grade 2 or grade 3, and the accuracy rate reaches 76%.
After the 139 recombinant inbred lines and the two parents are inoculated with physiological strains in 2019, the 78 families are CC homozygotic based on the genotype of the 11_26220673_ C _ T SNP locus, the resistance level of the 78 recombinant inbred lines to the rice bacterial leaf blight is determined to be level 2 or level 3, and the accuracy rate reaches 76%.
A method for detecting the expression of rice disease-resistant molecular markers in resistant materials includes designing quantitative primers according to coding region sequences provided by a rice genome function annotation website (Table 4). The synqing zao B and zhonghui 9308 were sampled in the field at different time points before and after inoculation, total RNA was extracted using Trizol reagent, and cDNA after reverse transcription was used as template, and quantitative PCR was performed using Fast Essentia DNA Green reagent of Roche in CFX96(Bio-Rad) (fig. 5).
TABLE 4 quantitative PCR primer sequences for different molecular marker genes
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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<400> 5
ctttatgcga atcctaacaa gacc 24
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
cgagaagagc atcaaaacat cg 22
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
cttgtgccat aggggaaaag a 21
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
gcaatgccag ggaacatagt g 21
Claims (10)
1. An SNP molecular marker related to rice bacterial blight resistance, which is characterized by comprising SNP _11_27792921_ A _ G and/or SNP _11_26220673_ C _ T;
the molecular marker SNP _11_27792921_ A _ G is located at 27792921bp on the 11 th chromosome of rice, and a single-base mutation AG of nucleotides exists;
the molecular marker SNP _11_26220673_ C _ T is located at 26220673bp on the 11 th chromosome of rice, and has a single nucleotide mutation CT.
2. The primer pair for detecting the SNP molecular marker of claim 1, wherein the primer pair for detecting the SNP molecular marker SNP _11_27792921_ A _ G comprises an upstream primer 1F and a downstream primer 1R, the nucleotide sequence of the upstream primer 1F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer 1R is shown as SEQ ID NO. 2;
the primer pair for detecting the molecular marker SNP _11_26220673_ C _ T comprises an upstream primer 2F and a downstream primer 2R, wherein the nucleotide sequence of the upstream primer 2F is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer 2R is shown as SEQ ID NO. 4.
3. A kit for detecting the SNP molecular marker according to claim 1, wherein the kit comprises the primer pair according to claim 2.
4. Use of the SNP molecular marker of claim 1, the primer pair of claim 2 or the kit of claim 3 for detecting bacterial blight resistance of rice.
5. A method for detecting bacterial blight resistance of rice, which is characterized in that rice genome DNA is used as a template, PCR detection is carried out by using the primer pair of claim 2 or the kit of claim 3, a PCR product is sequenced, if the molecular marker SNP _11_27792921_ A _ G is an A base, the molecular marker is an AA genotype, and the resistance grade is 0 grade or 1 grade; if the molecular marker SNP _11_27792921_ A _ G is G base, the gene is GG genotype, and the resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is C base, the molecular marker is CC genotype, and the resistance grade is grade 2 or grade 3; if the molecular marker SNP _11_26220673_ C _ T is T base, the gene type is TT, and the resistance grade is 0 grade or 1 grade.
6. The method of claim 5, wherein the PCR assay is performed in a system of 25. mu.l, 2 XKOD Buffer 12.5. mu.l, KOD 0.25. mu.l, upstream and downstream primers 0.5. mu.l, DNA template 2. mu.l, double distilled water to 25. mu.l.
7. The method according to claim 5 or 6, wherein the PCR program for detecting the molecular marker SNP _11_27792921_ A _ G comprises: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃, and preserving heat at 4 ℃;
the PCR program for detecting the molecular marker SNP _11_26220673_ C _ T comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 65.7 ℃ for 20s, extension at 72 ℃ for 30s, 35 cycles; extending for 10min at 72 ℃, and keeping the temperature at 4 ℃.
8. Use of the SNP molecular marker according to claim 1, the primer pair according to claim 2 or the kit according to claim 3 for rice breeding.
9. A molecular marker-assisted breeding method for rice disease resistance, which is characterized in that the primer pair of claim 2 or the kit of claim 3 is used for detecting the SNP molecular marker of claim 1 to determine the bacterial leaf blight resistance of rice of a variety/line to be detected.
10. A method for detecting the expression of a molecular marker for resisting rice diseases in a resistant material, which is characterized in that the expression quantity of the SNP molecular marker in the rice genome of a variety/strain to be detected is detected.
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| CN114410828A (en) * | 2022-03-07 | 2022-04-29 | 中国农业科学院作物科学研究所 | Primer composition for identifying or assisting in identifying rice bacterial leaf blight resistance and method thereof |
| CN117551667A (en) * | 2024-01-03 | 2024-02-13 | 中国农业科学院农业基因组研究所 | Application of RBB1 gene in preparing rice disease spot material and regulating and controlling rice bacterial infection resistance |
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| CN117551667A (en) * | 2024-01-03 | 2024-02-13 | 中国农业科学院农业基因组研究所 | Application of RBB1 gene in preparing rice disease spot material and regulating and controlling rice bacterial infection resistance |
| CN117551667B (en) * | 2024-01-03 | 2024-03-12 | 中国农业科学院农业基因组研究所 | Application of RBB1 gene in preparing rice disease spot material and regulating and controlling rice bacterial infection resistance |
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