CN109628627B - Development and application of SNP (single nucleotide polymorphism) marker of broad-spectrum rice blast resistance gene Pigm of rice - Google Patents
Development and application of SNP (single nucleotide polymorphism) marker of broad-spectrum rice blast resistance gene Pigm of rice Download PDFInfo
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Abstract
The invention provides development and application of an SNP marker of broad-spectrum rice blast resistance gene Pigm of rice, which comprises an SNP molecular marker K _060508 tightly linked with the rice blast resistance gene Pigm, wherein the SNP marker detects that the base at the 10421726 th site of the No. 6 chromosome of the rice is G or A, and the Primer combination of the SNP marker developed based on the KASP technology is Primer X, Primer Y and Primer C. The invention uses KASP technology to rapidly classify the gene of the SNP marker which is closely linked with the rice blast resistance gene Pigm, and can be applied to commercial molecular breeding with high, medium and low flux; meanwhile, the selection efficiency for developing the SNP marker phenotype reaches 100 percent, and the broad-spectrum rice blast resistant gene Pigm can be rapidly and accurately detected in different germplasm resources such as indica rice, japonica rice and the like; the molecular marker-assisted selection with prospect can be carried out in the early breeding stage, the field planting scale of breeding groups is reduced, the breeding cost is reduced, and the breeding process is accelerated.
Description
Technical Field
The invention relates to the field of molecular markers and crop breeding, in particular to development and application of SNP markers of broad-spectrum rice blast resistance genes Pigm of rice.
Background
The rice blast caused by Magnapothie oryzae is a worldwide rice crop disease, the global rice yield loss caused by the rice blast can reach 11-30% every year, and long-term practice shows that the cultivation of disease-resistant varieties (lines) is the most economic and effective measure for preventing and treating the rice blast at present. In recent decades, with the development of molecular genetics, molecular marker assisted selection plays a very important role in rice disease resistance breeding, and efficient molecular markers are used for assisting selection of single plants containing target genes for hybridization, so that breeding of target characters can be accurately performed, the field planting scale of breeding groups can be reduced, the breeding period can be shortened, and the breeding cost can be saved.
Currently, by genetic analysis, about 100 rice blast resistance genes have been identified and named in rice, of which 26 have been successfully cloned (Liu et al, 2014). The Pigm gene is related to rice leaf blast resistance, is a broad-spectrum anti-blast gene, has a wider anti-spectrum than the accepted broad-spectrum resistance genes Pi1, Pi2 and Pi3, and the donor flos mume No. 4 of the Pigm gene has high resistance or immunity to 29 strong pathogenic strains collected from different regions and countries. Pigm is located in the 70Kb interval between chromosome 6 markers C5483 and C0428 of rice, cosegregated with markers S29742, C24901, and PC22705, and contains 13 gene clusters of NBS-LRR type disease-resistant genes (Deng et al, 2006), 2 of which are functional proteins PigmR and PigmS. PigmS can compete with PigmR to form a heterodimer to inhibit PigmR-mediated broad-spectrum disease resistance, reduce the evolution selection pressure of pathogenic bacteria and slow down the pathogenic evolution of the pathogenic bacteria on PigmR, so that Pigm-mediated disease resistance has lasting broad spectrum and is a more potential rice blast resistance gene (Deng et al, 2017).
In addition, Pigm is located at the Pi2/9 site and is a complex allele with Pi2, Pi9, Pi-zt, Piz and Pi50, but the respective gene sequences and resistance profiles have certain differences, so that the Pigm needs to be accurately distinguished from other complex alleles to realize the utilization of Pigm. However, most of the molecular markers used for detecting the Pigm at present are SSR, InDel or CAPS markers, the markers cannot effectively distinguish the Pigm from other multiple alleles and cannot completely distinguish donors and acceptors of the Pigm, and misjudgment is easily caused by detection results. In addition, the detection process of the markers such as SSR, InDel or CAPS requires the steps of enzyme digestion, gel electrophoresis and the like, the operation is complex, the experiment cost is high, the detection flux is small, and the environmental pollution and the harm to human bodies are easily caused. The development of the specific SNP molecular marker of the Pigm and the establishment of a high-throughput and environment-friendly detection system have important significance for promoting the application of the Pigm in commercial breeding.
Disclosure of Invention
The invention discloses a molecular marker of broad-spectrum rice blast resistance gene Pigm of rice and application thereof, wherein the molecular marker is an SNP marker closely linked with the broad-spectrum rice blast resistance gene Pigm, and the marker is developed based on KASP technology and can detect 10421726 th base of 6 th chromosome of Nipponbare genome of rice variety in high flux. The invention applies the KASP technology to carry out genotyping on the SNP molecular marker, has the advantages of simple and convenient operation, low cost, short detection period, good marker stability and the like, can accurately distinguish Pigm from other closely-linked or allelic rice blast resistant genes, can accurately detect Pigm in a low-generation group, improves the breeding efficiency, shortens the breeding period and has important significance for improving the resistance of rice blast.
Specifically, the development process of the molecular marker of the rice blast resistance gene Pigm is shown in figure 1. According to the report of the literature, the Pigm gene is located in 10387509-10390465 interval of the rice chromosome 6, and SNP sites at two sides of the gene interval and the vicinity of the gene interval are discovered. And (3) extracting flanking sequences from the selected SNP sites, and performing primer design on the flanking sequences by using an online primer design website BatchPrimer 3. For these candidate SNP markers, KASP reaction verification was performed on the Pigm gene donor material, Valsa officinalis No. 4, and other 22 rice varieties without Pigm, and the SNP marker K _060508 which was coseparated from the Pigm donor material and had good amplification effect was selected. Natural population verification is carried out on the selected SNP markers linked with the resistance genes by using about 190 parts of materials, and the Pigm gene locus detected by the invention is proved to be a high-specificity resistance locus. The offspring of the colony generated by crossing the Pigm donor parent flos mume No. 4 and CO39 is used for phenotype verification, and the feasibility and the accuracy of the invention are verified again.
In order to solve the problems in the prior art, the invention aims to provide an SNP (single nucleotide polymorphism) molecular marker K _060508 tightly linked with a rice blast resistance gene Pigm and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
an SNP molecular marker closely linked with a rice blast resistance gene Pigm, wherein the molecular marker is an SNP marker K _060508 coseparated with the rice blast resistance gene Pigm, and the SNP marker detects that the base at the 10421726 th site of the No. 6 chromosome of rice is G or A;
a primer combination for detecting the SNP marker K _060508 includes: (1) two specific primers: primer X: 5'-CCAAGCAAATTACCACAACG-3', respectively; PrimerY: 5'-CCAAGCAAATTACCACAACAGC-3', respectively; (2) one universal primer: primer C: 5'-TTGTTATACGGTTTAATTAAGGTGA-3', see table 1.
TABLE 1 tagged sequence Listing
A kit for detecting SNP molecular markers for the rice blast resistance gene Pigm, which comprises a primer combination as described above.
The molecular marker is applied to detection of rice blast resistance genes Pigm, assisted breeding of the rice blast resistance genes Pigm and breeding of rice blast resistance rice resources.
Further, the applications include Pigm typing using KASP or anchoring Pigm in gene chips.
Further, the method comprises the following steps:
s1, extracting the genome DNA of the rice sample;
s2, using the rice genome DNA as a template, and carrying out KASP reaction detection by using the primer combination; wherein, the two specific primers are respectively connected with different fluorescent joints;
s3, if only detecting the fluorescent signal corresponding to the fluorescent joint connected with the Primer Y, the detection site is a basic group A, and the rice sample is judged to be the pure type carrying the rice blast resistance gene Pigm; if only detecting the fluorescent signal corresponding to the fluorescent joint connected with the Primer X, determining that the rice sample is a pure type without rice blast resistance gene Pigm if the detection site is a base G; if two kinds of fluorescence are detected simultaneously, the rice sample is judged to be a heterozygote of the rice blast resistance gene Pigm.
Further, the 5' segment of the specific primer is connected with FAM or HEX fluorescent linker sequence.
Still further, the application of the molecular marker in the disease resistance assisted breeding of rice comprises the following steps:
s1, extracting the genome DNA of the rice sample;
s2, using the rice genome DNA as a template, and carrying out KASP reaction detection by using the primer combination; wherein, the two specific primers are respectively connected with different fluorescent joints;
s3, selecting and breeding the rice sample which detects the fluorescence signal corresponding to the fluorescent joint connected with the primer PrimeRY. The invention has the beneficial effects that:
(1) the SNP marker developed by the invention is closely linked with the Pigm gene, and after the phenotype identification of separated group rice blast, the phenotype selection efficiency of the developed SNP marker is verified to reach 100 percent, and the broad-spectrum rice blast resistant gene Pigm can be rapidly and accurately detected in different germplasm resources such as indica rice, japonica rice and the like.
(2) The SNP marker developed by the invention is combined with KASP genotyping technology, can be applied to commercial molecular breeding with high, medium and low throughput, does not need complicated procedures such as enzyme digestion, electrophoresis, sequencing and the like in the detection process, reduces the pollution of aerosol and the use of toxic substances such as EB and the like, directly detects the basic group, has the accuracy not influenced by the length of an amplified fragment, is simple, convenient, rapid and accurate, has high automation degree, greatly improves the gene breeding efficiency and reduces the cost.
(3) The SNP marker can perform foreground selection and background selection in the seedling stage, reduce the field planting scale of breeding groups, shorten the breeding period and accelerate the breeding process.
Drawings
FIG. 1 is a flow chart of the development of the molecular marker Pigm of the blast-resistant gene of the invention.
FIG. 2 is a diagram of the detection of natural population typing using the molecular marker K _ 060508.
Detailed Description
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings. The experimental methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
Example 1 preparation of molecular marker of anti-Rice blast Gene Pigm
1. Primer design
According to related documents, the Pigm gene position is anchored in 10387509-10390465 interval of the rice chromosome 6, 50kb is expanded towards both sides by taking the gene interval as the center, SNP locus extraction is carried out according to 3000 parts of rice re-sequencing data of International Rice institute, a flanking sequence is extracted from the selected SNP locus, and primer design is carried out on the SNP locus by utilizing an online primer design website BatchPrimer3(http:// probes. pw. usda. gov/BatchPrimer3 /). Each group is marked with three primers, and the 5' ends of two specific primers are respectively connected with FAM and HEX fluorescent joints. The primers were synthesized by Invitrogen corporation.
If the sample PCR product only detects a fluorescent signal corresponding to the primer PrimerX, the detection site is a base G, and the rice sample to be tested does not contain the Pigm gene; if only the primer PrimerY corresponding to the fluorescent signal is detected, the detection site is a base A, and the rice sample to be tested is judged to contain homozygous Pigm gene; if two fluorescence signals are detected simultaneously, the detection site is G: and A, judging that the rice to be detected contains heterozygous Pigm genes.
2. DNA extraction: extracting genome DNA from rice leaf and adopting simplified CTAB method.
3. KASP reaction test
The KASP reaction assay was performed on the LGC SNPline genotyping platform. 20ng of DNA sample was added to the microplate, dried and added to the KASP reaction mixture, and the reaction system is shown in Table 2. PCR amplification is completed in a water bath thermal cycler, and the Touchdown 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 20 seconds, annealing at 65-57 ℃ and extending for 60 seconds 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 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles. After the reaction is finished, a scanner Pherastar is used for reading fluorescence data of the KASP reaction product, and the result of fluorescence scanning can be automatically converted into a graph.
The LGC SNpline genotyping platform used in the invention and the consumable materials of the reagents matched with the platform are purchased from LGC company in the United kingdom.
TABLE 2 reaction System for KASP detection
4. Tagging typed data
Marker K _060508 KASP primary screening assays were performed on the Pigm donor material, oryzanol No. 4, and on 21 other rice varieties without Pigm, with the results shown in table 3. The detection result of the Pigm donor material flos Pruni mume No. 4 at the K _060508 test site is a base A, and 20 rice varieties without Pigm, no matter other rice blast resistant gene donors or susceptible materials, detect a base G at the test site. The SNP marker K _060508 co-separated from the Pigm donor material and having good amplification effect was selected.
TABLE 3 Primary sifting data labeled K _060508
| Material | Description of the materials | Detection of |
| 75-1- | Pi9 donor | G |
| C101 | Pi2 donor | G |
| Two eight | Pi50 donor | G |
| Fukun | Pi-z donor | G |
| Gumei 4 | Pigm donor | A |
| Fujin | Pi-z + Pi-sh donors | G |
| BL6 | Pi1+ Pi2 Donor | G |
| Asom | Pathogenic material | G |
| Cpslo | Pathogenic material | G |
| Xiangzao (Xiangzao) | Pathogenic material | G |
| Yuanfeng tea | Pathogenic material | G |
| Lijiang river | Pathogenic material | G |
| CO39 | Pathogenic material | G |
| Xiang short | Pathogenic material | G |
| Kasal | Pathogenic material | G |
| Yang (Yang-Yang) | Pathogenic material | G |
| Minghui (Minghui) | Pathogenic material | G |
| Ⅱ | Pathogenic material | G |
| Middle 9B | Pathogenic material | G |
| Huang Hua | Pathogenic material | G |
| Gold (Au) | Pathogenic material | G |
| Japanese | Pathogenic material | G |
Example 2 population of molecular markers for the blast-resistant Gene Pigm and marker phenotype validation
1. Natural population verification
To test the specificity and utility of the marker in the present invention, 187 materials were tested and verified using the marker K _ 060508. 187 materials include known homozygous Pigm gene-containing varieties, donors containing other rice blast resistance genes, common materials, common hybrid rice and core rice breeding materials. The results of typing marked in natural populations are shown in FIG. 2, 4 varieties known to contain Pigm gene were detected as homozygous Pigm genotype with rice blast resistance, 2 hybrid rice were detected as heterozygous Pigm genotype, 6 materials had no amplification signal, and the rest donors, common materials, common hybrid rice and core rice breeding materials containing other rice blast resistance genes were detected as homozygous Pigm genotype without rice blast resistance.
Therefore, the SNP marker K _060508 detects that the Pigm gene locus is a high-specificity resistant locus, and can be conveniently and efficiently used for identifying whether the rice variety contains the Pigm gene.
2. Marker phenotype validation
F2 constructed using marker K _060508 on Pigm donor parent oryzalin No. 4 and acceptor parent CO 39: 3 families were tested. Meanwhile, in the rice seedling stage, 20F 2: the rice blast strain CHL506 is inoculated on 3 families and 2 parents, the disease condition is investigated, the phenotype data is completely matched with the genotype, and the feasibility and the accuracy of the invention are verified again (see table 4).
TABLE 4 genetic segregation population phenotypic and genotypic characterization
Although the present invention has been described in detail with reference to the foregoing general description and specific embodiments, the foregoing description is only illustrative of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
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Claims (3)
1. The application of an SNP molecular marker closely linked with a rice blast resistance gene Pigm in detecting the rice blast resistance gene Pigm is characterized in that the molecular marker is an SNP marker K _060508 which is co-separated from the rice blast resistance gene Pigm, the polymorphic base of the K _060508 is G or A, and the K _060508 is located at 10421726 th base of No. 6 chromosome of rice; the primer combination for detecting the K _060508 comprises: two specific primers: primer X: 5'-CCAAGCAAATTACCACAACG-3', respectively; primer Y: 5'-CCAAGCAAATTACCACAACA-3', respectively; one universal primer: primer C: 5'-TTGTTATACGGTTTAATTAAGGTGA-3' are provided.
2. The use according to claim 1, characterized in that the method for detecting SNP molecular markers closely linked to the rice blast resistance gene Pigm comprises the following steps:
s1, extracting the genome DNA of the rice sample;
s2, using the rice genome DNA as the template, using the primer combination of claim 1 to carry out KASP reaction detection; wherein, the two specific primers are respectively connected with different fluorescent joints;
s3, if only detecting the fluorescent signal corresponding to the fluorescent joint connected with the Primer Y, the detection site is a basic group A, and the rice sample is judged to be the pure type carrying the rice blast resistance gene Pigm; if only detecting the fluorescent signal corresponding to the fluorescent joint connected with the Primer X, determining that the rice sample is a pure type without rice blast resistance gene Pigm if the detection site is a base G; if two kinds of fluorescence are detected simultaneously, the rice sample is judged to be a heterozygote of the rice blast resistance gene Pigm.
3. Use according to claim 2, wherein the 5' stretch of Primer Y, Primer X is attached to a FAM or HEX fluorescent linker.
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| CN110257553B (en) * | 2019-08-05 | 2022-07-08 | 江苏省农业科学院 | KASP molecular marker method for identifying rice blast resistance gene Pigm |
| CN110408719B (en) * | 2019-08-05 | 2022-07-08 | 江苏省农业科学院 | Four-primer molecular marking method for identifying rice blast resistance gene Pigm |
| CN112522432A (en) * | 2020-12-17 | 2021-03-19 | 华智生物技术有限公司 | Molecular marker for assisted breeding of rice blast resistance gene Bsr-d1 and application thereof |
| CN113046349B (en) * | 2020-12-25 | 2021-11-16 | 华智生物技术有限公司 | SNP molecular marker combination for detecting rice Wx gene and application thereof |
| CN114395641A (en) * | 2022-01-19 | 2022-04-26 | 山东省农业科学院 | Primer pair and kit for detecting blast disease-resistant gene PigmR, application of primer pair and kit and detection method |
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