CN109468400B - Rice blast resistance gene Pi36 codominant molecular marker and application thereof - Google Patents
Rice blast resistance gene Pi36 codominant molecular marker and application thereof Download PDFInfo
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Abstract
The invention provides a rice blast resistance gene Pi36 codominant molecular marker, which comprises a marker SNP1 and/or a marker SNP2, wherein a polymorphic site contained in the marker SNP1 is positioned at a base corresponding to the rice Nipponbare No. 8 chromosome 2886707, and the base is G or A; the polymorphic site contained in the marker SNP2 is located at the position corresponding to 2888118 bases of the Nipponbare 8 chromosome of rice, and the bases are G or A. Specific amplification primers are respectively designed aiming at the molecular markers, and the Pi36 genotype is detected through PCR amplification. The molecular marker disclosed by the invention is co-dominant, can effectively distinguish 3 different genotypes, can be used for breeding rice blast resistant rice, shortens the breeding period of the rice blast resistant rice, accelerates the breeding speed, reduces the breeding cost, and has the advantages of simplicity in operation, low cost, short period and the like.
Description
Technical Field
The invention belongs to the fields of molecular biology and plant molecular breeding, and particularly relates to a rice blast resistance gene Pi36 codominant molecular marker and application thereof.
Background
Rice, as a main food and economic crop in China, is threatened by diseases and insect pests throughout the year, wherein the rice blast is one of the most serious diseases. In each rice production area in China, rice blast occurs every year, the yield can be reduced by 10% -20% when the disease and pest are prevalent, and can reach 40% -50% in more serious areas, even the rice is not harvested. The harm of rice blast can be relieved by using a large amount of rice blast resistant medicaments, but the cost of rice production is greatly increased, the environment is polluted, and the practice shows that the breeding of rice blast resistant varieties is the most economic and effective measure for controlling the rice blast. However, due to the large number of physiological races and fast variation of rice blast, newly-proposed disease-resistant varieties lose resistance soon after planting, thereby reducing the utilization value. Therefore, the discovery and the utilization of different rice blast resistance genes to culture rice varieties with different disease resistance genes play an important role in breeding broad-spectrum and durable rice blast resistance varieties.
In conventional disease-resistant breeding, resistance identification is complicated, more manpower and cost are required to be invested, and identification is easily influenced by external conditions to cause inaccurate results, so that the breeding efficiency is low. Molecular marker assisted breeding is a modern breeding technology which utilizes functional markers closely linked with functional genes or in genes to select target shapes in later generations by combining genotype and phenotypic identification. The method can greatly shorten the breeding period, improve the breeding efficiency and save a large amount of labor and material cost. Indica rice variety Kasalath shows stable and high-level resistance to a part of physiological races of rice blast isolated in China. The rice blast resistance gene Pi36(GenBank: DQ900896.1) was cloned from kasalath by map-based cloning method, and Pi36 was located in the 8 th chromosome short arm and is a single copy gene constitutively expressed and encodes a 1056 amino acid protein product. The clone of the disease-resistant gene Pi36 has important significance for developing Pi36 gene functional markers for molecular marker-assisted breeding and further breeding varieties with different rice blast resistance levels.
Disclosure of Invention
The invention aims to provide a rice blast resistance gene Pi36 codominant molecular marker and application thereof.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a rice blast resistance gene Pi36 co-dominant molecular marker (Pi36-RS) comprising a marker SNP1 and/or a marker SNP2, wherein the marker SNP1 contains a polymorphic site at a position corresponding to the position of chromosome 2886707 of Nipponbare 8 of rice, wherein the position is G or A; the polymorphic site contained in the marker SNP2 is located at the position corresponding to 2888118 bases of the Nipponbare 8 chromosome of rice, and the bases are G or A.
Wherein the Japanese sunny genome version number of the rice is IRGSP 1.0.
Preferably, the nucleotide sequence of the marker SNP1 is shown as SEQ ID NO. 1 (the 20 th base is the SNP site, n is g or a), and the nucleotide sequence of the marker SNP2 is shown as SEQ ID NO. 2 (the 46 th base is the SNP site, n is g or a).
In a second aspect, the invention provides ARMS-PCR primers for detecting the molecular marker, wherein the primers for detecting the marker SNP1 include Pi36-RR and Pi36-RF, and the nucleotide sequences are shown in SEQ ID NO. 3-4;
the primers used for detecting the marker SNP2 include Pi36-SF and Pi36-SR, and the nucleotide sequences thereof are shown in SEQ ID NO. 5-6.
In a third aspect, the present invention provides a detection reagent or kit comprising the primer Pi36-RR/Pi36-RF and the primer Pi36-SF/Pi 36-SR.
In a fourth aspect, the invention provides any one of the following applications of the molecular marker SNP1, SNP2, primer Pi36-RR/Pi36-RF, primer Pi36-SF/Pi36-SR or a detection reagent or kit containing the primer:
1) the application in the genotype identification of rice blast resistance gene Pi 36;
2) the application in identifying and screening rice blast resistant rice germplasm resources;
3) the application in molecular marker assisted breeding of rice blast resistant rice varieties.
In a fifth aspect, the present invention provides a method for genotyping rice blast resistance gene Pi36, comprising the steps of:
(1) extracting the genome DNA of the rice to be detected;
(2) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and using a primer Pi36-RR/Pi36-RF and a primer Pi36-SF/Pi 36-SR;
(3) the amplification products were analyzed.
The PCR reaction system in step (2) of the method is as follows: 1ul 10 XPCR reaction buffer, 0.8ul 10mM dNTP, 10uM each primer 0.15ul, 2.5U/ul Taq DNA polymerase 0.1ul, 2ul DNA template, ddH2And O is supplemented to 10 ul.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 56-58 ℃ (preferably 56 ℃) for 45 seconds, extension at 72 ℃ for 45 seconds, for 30-35 cycles (preferably 32 cycles); extension at 72 ℃ for 5 minutes.
Preferably, step (3) analyzes the amplification product by agarose gel electrophoresis.
The step (3) is specifically as follows: if the amplification product has a characteristic band of 345bp, determining that the rice to be detected is homozygous for the Pi36 disease-resistant allele; if the amplification product has a 433bp characteristic band, determining that the rice to be detected is homozygous for the Pi36 susceptible allele; if the amplified product has two banding patterns of 345bp and 433bp, the rice to be detected is judged to be the heterozygous genotype.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention designs the molecular marker based on the sequence difference in the Pi36 gene, and has the advantages of no genetic exchange and high accuracy.
And secondly, the amplification products of the molecular markers have obvious difference, can be directly used for agarose gel electrophoresis detection, and are convenient, quick and low in cost.
And thirdly, the molecular marker of the invention is co-dominant, and can effectively distinguish 3 different genotypes, and the method can be used for breeding rice blast resistant rice, shortening the breeding period of the rice blast resistant rice, accelerating the breeding speed, reducing the breeding cost, and has the advantages of simple operation, low cost, short period and the like.
Drawings
FIG. 1 is an alignment chart of the Pi36 gene sequence of a known rice variety containing the allele of rice blast resistance or rice blast infection Pi 36. Among them, Kasalath in A and B is a rice variety known to contain a Pi36 disease-resistant allele, and 9311, Nipponbare, IR64, Shuhui 498 and 02428 are rice varieties known to contain a Pi36 disease-sensitive allele.
FIG. 2 is a detection electrophoretogram of a rice breeding material in example 4 of the present invention. Wherein, M: DL1000DNA marker, wherein lanes 1-26 correspond to kasalath, Huazhan, 638S, Fengyou, Yandao 1531, Huahui 284, Chenghui 19, Huarun No. 2, Hui 3728, Mf63, Hujing 5, 02428, Hujing 6, Yanjing 5507, HONGJING 35, YUPINGXIANG, Yujing 0618, Huizjing 602, Zhendao 819, Liaolian Sal 287, Jiangsu Jing 2, farmlands 31, Fukuniski, Zhejiang 75, Hanliangyou No. 1, and Wanliangyou 385 in sequence.
FIG. 3 is an electrophoretogram of F2 population detection in example 5 of the present invention. Wherein, M: DL1000DNA marker, lanes 1 and 2 are disease resistant and disease susceptible parents, kasalath and 9311, respectively, lanes 3-38 are F2 population single plants constructed by using kasalath/9311, the genotype of each material is marked above the corresponding lane, S represents the type of disease susceptible allele, R represents the type of disease resistant allele, and H represents the type of heterozygosity.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 Pi36 Gene molecular marker development
The rice blast resistance gene Pi36 is located on the short arm of the No. 8 chromosome of rice, and two specific SNP loci are found by sequencing the known rice blast resistance or rice blast susceptibility rice variety Pi36 genome regions of Kasalath, 9311, Nipponbare and the like and online comparison analysis of NCBI databases, wherein the SNP1 is located at the base of the No. 8 chromosome 2886707 of the Nipponbare sequence, the Pi36 disease resistance allele is G, and the susceptible allele is A (as shown in figure 1A); SNP2 is located on chromosome 2888118 of Nipponbare sequence No. 8, and disease-resistant allele of Pi36 is G, and disease-susceptible allele is A (shown in FIG. 1B). Two pairs of dominant markers are developed aiming at the two SNP loci and are respectively used for identifying disease-resistant alleles and disease-susceptible alleles, and the identification of different genotypes of Pi36 can be realized by using the two pairs of dominant markers in a combined manner.
Example 2 design of primers for detection of the codominant molecular marker of the Pi36 Gene
Detection primers were designed for the two Pi36 gene-specific molecular markers developed in example 1, and the principle of primer design is as follows.
Firstly, designing a specific primer for amplifying a disease-resistant allele, designing a primer Pi36-RR by taking a complementary base C of a disease-resistant allele G base at an SNP1 locus as the 3 'end of a reverse primer, changing the C base to an A base at the 3 rd base of the 3' end to introduce mismatch, and designing a forward primer Pi36-RF matched with the primer upstream of the primer, wherein the primers Pi36-RR and Pi36-RF (SEQ ID NO:3-4) can only specifically amplify Pi36 disease-resistant allele to generate a 345bp strip, and the Pi36 disease-resistant allele has NO strip during amplification;
then, a specific primer for amplifying the disease-sensitive allele is designed, a forward primer Pi36-SF is designed by taking the disease-sensitive allele A at the site of SNP2 as the 3 'end, the base of the 3' 3 rd position is changed into a T base to introduce mismatch, a reverse primer Pi36-SR matched with the forward primer is designed at the downstream of the forward primer, the primer Pi36-SF and the Pi36-SR (SEQ ID NO:5-6) can only specifically amplify the disease-sensitive allele Pi36 to generate a 433bp band, and the disease-resistant allele Pi36 has NO band during amplification.
The primer sequences are shown in Table 1, the four primers are mixed to form a Pi36 codominant molecular marker detection primer group, and when an amplification product is only a 345bp strip, rice to be detected carries a Pi36 disease-resistant allele; when the amplification product only has a 433bp band, the rice to be detected carries Pi36 susceptible allele; when the amplified product has two banding patterns of 345bp and 433bp, the rice to be detected is Pi36 heterozygous genotype rice.
TABLE 1 Pi36 specific molecular marker primer sequences and related parameters
Example 3 establishment of method for detecting Rice blast resistance Gene Pi36 specific molecular marker
The reaction program and the reaction system of the PCR are designed according to the detection primers of two molecular markers Pi36 designed in example 2, and the following reaction program and system are determined through continuous optimization:
PCR reaction (10. mu.L): 1ul 10 XPCR reaction buffer, 0.8ul 10mM dNTP, 4 primers (10 uM) all 0.15uL, 0.1ul Taq DNA polymerase (2.5U/ul), 2ul DNA template, ddH2And O is supplemented to 10 ul.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds for 32 cycles; extension at 72 ℃ for 8 minutes.
Example 4 application of specific molecular marker of rice blast resistance gene Pi36 in detection of disease-resistant and disease-susceptible varieties of rice
1. Rice material
Pi36 donor parent Kasalath and 25 breeding parent materials or commercial rice varieties specifically include: huazhan, Yuzhenxiang, 638S, Fengyuazhan, Huahui 284, Chenghui 19, Huarun No. 2, hui 3728, Mf63, Hujing 5, 02428, Hujing 6, Yanjing 5507, hot jing 35, Yujing 0618, Huizjing 602, Zhendao 819, Liaoning 287, Jiangsu jing 2, Yandao 1531, agricultural cultivation 31, Fukuniski, Zhejiang 75, Hanliangyou No. 1 and Wanliangyou 385.
2. Extraction of rice genome DNA and primer synthesis
Genomic DNA of the above rice material was extracted by CTAB method and the primer sequences shown in Table 1 were synthesized.
3. PCR detection
The reaction system and reaction procedure for PCR are as described in example 3. The amplification products were electrophoresed in 2% agarose gel and the results were recorded by scanning with a gel imager.
4. Analysis of results
The electrophoresis results of the PCR amplification products are shown in FIG. 2, wherein the donor material Kasalath in lane 1 Pi36 specifically amplified a 345bp band, and the parent materials corresponding to lanes 2-26 all amplified a 433bp band. In order to verify the accuracy of the PCR detection result, the molecular marker target regions corresponding to Kasalath and 25 parent materials are subjected to sequencing comparison. The result shows that the molecular marker detection result is consistent with the sequencing result, and the primer provided by the invention can accurately identify the genotype of the rice blast resistant gene Pi36 and can realize accurate and efficient screening of rice variety resources.
Example 5 detection of Single Gene isolation of Rice blast resistance Gene Pi36 in F2 population
1. Rice material
Disease resistant and susceptible parents, kasalath and 9311, and 96 randomly selected F2 individuals in a F2 population constructed by two parental crosses.
2. Extraction and PCR detection of rice genome DNA
The primers, reaction procedures and system for the extraction of genomic DNA from rice and the detection by PCR were as described in example 4.
3. Analysis of results
The electrophoresis results of the PCR amplification products of the rice blast resistant parent Kasalath, the rice blast susceptible parent 9311 and part of the F2 strains are shown in FIG. 3, lanes 1 and 2 are the disease resistant parent Kasalath and the disease susceptible parent 9311 respectively, lanes 3 to 38 are randomly selected part of F2 single strains constructed by utilizing two parents, the genotypes of all materials are marked below the corresponding lanes, S represents the type of the disease susceptible allele, R represents the type of the disease resistant allele, and H represents the heterozygous type. The results showed that 96F 2 individuals were tested, the segregation ratio of 3 different genotypes was 25SS:50H:21RR, and the Mendelian single gene segregation ratio (χ) was 1:2:1 by Chi-square test20.50 less than χ2 0.055.99), the marker is a co-dominant marker, two different homozygote and heterozygote genotypes can be distinguished, and the detection sites show single gene segregation at the same time.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (9)
1. The ARMS-PCR primer group for detecting the codominant molecular marker of the rice blast resistance gene Pi36 is characterized by comprising primers Pi36-RR and Pi36-RF for detecting a marker 1 and primers Pi36-SF and Pi36-SR for detecting a marker 2, wherein the nucleotide sequences of the primers Pi36-RR and Pi36-RF are shown as SEQ ID NO. 3-4, and the nucleotide sequences of the primers Pi36-SF and Pi36-SR are shown as SEQ ID NO. 5-6.
2. A detection reagent or kit comprising the primer set according to claim 1.
3. Any one of the following uses of the primer set of claim 1 or the detection reagent or kit of claim 2:
1) the application in the genotype identification of rice blast resistance gene Pi 36;
2) the application in identifying and screening rice blast resistant rice germplasm resources;
3) the application in molecular marker assisted breeding of rice blast resistant rice varieties.
4. The application of the rice blast resistance gene Pi36 codominant molecular marker comprises the following steps:
1) the application in the genotype identification of rice blast resistance gene Pi 36;
2) the application in identifying and screening rice blast resistant rice germplasm resources;
3) the application in molecular marker assisted breeding of rice blast resistant rice varieties;
wherein the rice blast resistance gene Pi36 codominant molecular marker consists of a marker 1 and a marker 2, the nucleotide sequence of the marker 1 is shown as SEQ ID NO. 1, wherein, the 20 th base n is an SNP locus, and n is g or a; the nucleotide sequence of the marker 2 is shown as SEQ ID NO. 2, wherein, the 46 th base n is an SNP site, and n is g or a.
5. A genotyping method of rice blast resistance gene Pi36 is characterized by comprising the following steps:
(1) extracting the genome DNA of the rice to be detected;
(2) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and the primer set according to claim 1;
(3) the amplification products were analyzed.
6. The method of claim 5, wherein the PCR reaction system used in step (2) is: 1 μ L of 10 XPCR reaction buffer, 0.8 μ L of 10mM dNTP, 0.15 μ L of 10 μ M primers, 0.1 μ L of 2.5U/. mu.L Taq DNA polymerase, 2 μ L DNA template, ddH2And O is supplemented to 10 mu L.
7. The method of claim 5, wherein the PCR reaction conditions used in step (2) are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 56-58 ℃ for 45 seconds, and extension at 72 ℃ for 45 seconds for 30-35 cycles; extension at 72 ℃ for 5 minutes.
8. The method of claim 5, wherein the step (3) comprises analyzing the amplification product by agarose gel electrophoresis.
9. The method according to any one of claims 5 to 8, wherein step (3) is in particular: if the amplification product has a characteristic band of 345bp, determining that the rice to be detected is homozygous for the Pi36 disease-resistant allele; if the amplification product has a 433bp characteristic band, determining that the rice to be detected is homozygous for the Pi36 susceptible allele; if the amplified product has two banding patterns of 345bp and 433bp, the rice to be detected is judged to be the heterozygous genotype.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821406A (en) * | 2006-03-06 | 2006-08-23 | 华南农业大学 | Resistance gene Pi 36 of rice blast and its use |
| CN106755475A (en) * | 2017-01-17 | 2017-05-31 | 中国水稻研究所 | A kind of method of early 39 rice blast resistance genes in identification super early rice |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821406A (en) * | 2006-03-06 | 2006-08-23 | 华南农业大学 | Resistance gene Pi 36 of rice blast and its use |
| CN106755475A (en) * | 2017-01-17 | 2017-05-31 | 中国水稻研究所 | A kind of method of early 39 rice blast resistance genes in identification super early rice |
Non-Patent Citations (2)
| Title |
|---|
| the in silico map-based cloning of pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich repeat gene that confers race-specific resistance to the blast fungus;Xinqiong Liu等;《Genetics》;20070831;第2541-2549页 * |
| 辽宁地区水稻资源抗稻瘟病基因的检测分析;王丽丽等;《植物遗传资源学报》;20171231;第325-339页 * |
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