Primer group for detecting SNP (Single Nucleotide Polymorphism) molecular marker of rice blast resistance gene Pi-ty269 (t) and application of primer group

Abstract

The invention belongs to the field of rice breeding, and particularly discloses a primer group for detecting SNP (Single Nucleotide Polymorphism) molecular markers of a rice blast resistance gene Pi-ty269 (t) and application of the primer group, and the primer group comprises specific primers and universal primers of the SNP molecular markers Pi-ty269 (t)-313, Pi-ty269 (t)-315 and Pi-ty269 (t)-325. Genetic typing is carried out on the developed SNP marker by utilizing the PARMS technology, DNA extraction, PCR system construction, fluorescence signal detection and the like in the PARMS technology flow are basically automatic, 96, 384 and 153 6 pore plate high-throughput detection can be realized, and the primer and the method are suitable for large-scale and high-throughput Pi-ty269 (t) gene identification and rice blast resistance resource screening.

Description

Technical Field

[0001] The invention relates to the field of rice breeding, in particular to a primer set and application thereof for detecting a SNP molecular marker of a rice blast resistance gene Pi-ty269(t). Background Art

[0002] Rice is an important food crop. Rice blast can occur throughout the growth process of rice. In severe cases, it can lead to a total crop failure, threatening food security. Using rice's own disease-resistant genes is the most economical and effective way to prevent and control rice blast, and it is also environmentally friendly. Tongyu 269 is a conventional rice variety with high resistance to rice blast selected by Tonghua Academy of Agricultural Sciences, and it has shown high resistance to rice blast in the field.

[0003] Rice disease resistance breeding is to select plants by phenotypic selection through resistance identification, which is time-consuming and easily restricted by environmental conditions. The identification results are prone to errors and the selection efficiency is low. Molecular marker-assisted selection breeding is simple and effective, which can reduce breeding costs and shorten the breeding cycle. It can also carry out targeted multi-gene aggregation, improve breeding efficiency, and bring huge social and economic benefits.

[0004] Chinese patent application number CN201910998274.7 discloses a rice blast resistance gene RMg39 and its application. The total DNA of the rice variety Huanghuazhan (HHZ) is used as a template, and the nucleotide fragment obtained by PCR amplification of the primers TA3 ID NO.1 and TA3ID NO.2 with the following base sequences: TA3 ID NO.1 (5'-3'): TGCTCAAAGAATTGGGAGG TA3ID NO.2 (5'-3'): CATGGGAAATATACGCAAGCAG. The obtained PCR product is digested with restriction endonuclease Hae III and presents a specific band-type molecular marker with Pita3.

[0005] The detection process of the rice blast resistance gene requires cumbersome procedures such as enzyme cutting, electrophoresis and sequencing, which will increase the contamination of PCR product aerosols and the use of toxic substances such as EB. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a primer set and application thereof for detecting SNP molecular markers of rice blast resistance gene Pi-ty269(t).

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] One of the technical solutions of the present invention is a primer set for detecting a SNP molecular marker of a rice blast resistance gene Pi-ty269(t), the primer set comprising a specific primer and a universal primer, the SNP molecular marker consisting of Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325, the polymorphic sites of the SNP molecular markers Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 are located at bases 31386403, 31548744 and 32520322 of chromosome 4 of rice Nipponbare (MSU7.0), the base 31386403 is T or G, the base 31548744 is G or A, and the base 32520322 is G or A;

[0009] The specific primers of the SNP molecular marker Pi-ty269(t)-313 include Pi-ty269(t)-313Ra with a nucleotide sequence as shown in SEQ ID NO.1 and Pi-ty269(t)-313Rc with a nucleotide sequence as shown in SEQ ID NO.2, and the universal primer is Pi-ty269(t)-313F with a nucleotide sequence as shown in SEQ ID NO.3;

[0010] The specific primers of the SNP molecular marker Pi-ty269(t)-315 include Pi-ty269(t)-315Rc with a nucleotide sequence as shown in SEQ ID NO.4 and Pi-ty269(t)-315Rt with a nucleotide sequence as shown in SEQ ID NO.5, and the universal primer is Pi-ty269(t)-315F with a nucleotide sequence as shown in SEQ ID NO.6;

[0011] The specific primers of the SNP molecular marker Pi-ty269(t)-325 include Pi-ty269(t)-325Fg with a nucleotide sequence as shown in SEQ ID NO.7 and Pi-ty269(t)-325Fa with a nucleotide sequence as shown in SEQ ID NO.8, and the universal primer is Pi-ty269(t)-325R with a nucleotide sequence as shown in SEQ ID NO.9;

[0012] The 5' ends of Pi-ty269(t)-313Ra, Pi-ty269(t)-315Rc and Pi-ty269(t)-325Fg are connected to FAM groups respectively, and the 5' ends of Pi-ty269(t)-313Rc, Pi-ty269(t)-315Rt and Pi-ty269(t)-325Fa are connected to HEX groups respectively.

[0013] The second technical solution of the present invention is an application of a primer set for detecting SNP molecular markers of rice blast resistance gene Pi-ty269(t) in joint identification of rice Pi-ty269(t) genotype, comprising the following steps:

[0014] S1. Extract genomic DNA from the rice leaves to be tested;

[0015] S2. Using the genomic DNA extracted in step S1 as a template, using a primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi-ty269(t), and using PARMS technology to simultaneously detect the Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 molecular markers; if the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is A, and the base of the SNP site of Pi-ty269(t)-325 is A, it is determined that the rice to be tested is homozygous for the disease-resistant Pi-ty269(t). Genotype; if the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is G, and the base of the SNP site of Pi-ty269(t)-325 is G, the rice to be tested is judged to be a homozygous disease-susceptible Pi-ty269(t) genotype; if T and G are detected at the SNP site of Pi-ty269(t)-313 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-315 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-325 at the same time, the rice to be tested is judged to be a heterozygous disease-resistant Pi-ty269(t) genotype.

[0016] Compared with the prior art, the rice blast resistance gene Pi-ty269(t) of the present invention is subjected to SNP molecular markers, and the Pi-ty269(t) gene detection of rice materials is performed using the PARMS technical reaction, so that the rice blast resistance gene Pi-ty269(t) can be quickly and accurately detected in different germplasm resources such as indica rice and japonica rice. The present invention uses the PARMS technology to perform genotyping on the developed SNP markers, and the DNA extraction, PCR system construction and fluorescence signal detection in the PARMS technical process are basically automated, and 96, 384, 1536-well plate high-throughput detection can be achieved, which is suitable for large-scale, high-throughput Pi-ty269(t) gene identification and rice blast resistance resource screening. The present invention does not require cumbersome procedures such as enzyme cutting, electrophoresis and sequencing during the detection process, reduces the pollution of PCR product aerosol and the use of toxic substances such as EB, and can perform foreground selection and background selection in the early stage of molecular marker-assisted breeding, improve the background recovery rate, reduce the scale of the breeding population, and accelerate the breeding process, which is conducive to the efficient and environmentally friendly application of the Pi-ty269(t) gene in commercial rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the initial gene localization results of an embodiment of the present invention.

[0018] Figure 2 This is a diagram of the gene fine positioning results of an embodiment of the present invention.

[0019] Figure 3 The figure is a flow chart of molecular marker development according to an embodiment of the present invention.

[0020] Figure 4 This is a phenotyping diagram of the molecular marker PARMS according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0023] This embodiment discloses SNP molecular markers of the rice blast resistance gene Pi-ty269(t), wherein the SNP molecular markers are Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325, and the polymorphic sites of the SNP molecular markers Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 are located at bases 31386403, 31548744 and 32520322 of chromosome 4 of rice Nipponbare (MSU7.0), respectively, the base 31386403 is T or G, the base 31548744 is G or A, and the base 32520322 is G or A. The locations of Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 on rice chromosomes are as follows Figure 2 The specific design process is as follows. Figure 3 As shown, the physical interval of the Pi-ty269(t) gene was located, the SNP site and flanking sequence were extracted, the primer sequence of the marker was designed and synthesized, and the marker was screened and tested, as follows:

[0024] 1) Initial location of the gene

[0025] In the early stage, we constructed an F3 population with Tongyu 269 and Mongolian rice as parents, and planted it in the disease resistance identification nursery of Tonghua Academy of Agricultural Sciences in 2020 to investigate the incidence of leaf blight and panicle blast. The rice blast resistance gene carried by Tongyu 269 was located using the mixed pool sequencing BSA method, and a significant positioning interval was obtained according to the 99% confidence interval screening. The leaf blast resistance gene was located between bases 31,140,001-34,660,000 on chromosome 4, and the panicle blast gene was located between bases 27720001-35420000 on chromosome 4 ( Figure 1 ). Therefore, Tongyu 269 has a major rice blast resistance gene at the end of the long arm of chromosome 4, which has good resistance to both leaf blast and panicle blast.

[0026] 2) Fine positioning of genes

[0027] Tongyu 269 and Mongolian rice were resequenced, and 7 SNPs were screened in the initial positioning region (between bases 31140001 and 34660000 on chromosome 4). By using the map-based cloning method, it was determined that the rice blast resistance gene carried by Tongyu 269 was located between 31.3 and 32.3 Mb on chromosome 4 and was named Pi-ty269(t)( Figure 2 ).

[0028] 3) Development of linked markers

[0029] According to the positioning information, the SNP site and flanking sequence of the interval were obtained, and primers were designed using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). The primer designs of Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 are shown in Table 1. Each group of markers has three primers, and two of the specific primers are connected to FAM or HEX groups respectively. The primers were commissioned to be synthesized by Invitrogen.

[0030] Table 1

[0031]

[0032] 4) Sample testing

[0033] DNA extraction: Take a leaf with a length and width of about 1 cm and put it into a deep well plate (96 wells 1.2 ml), add 100 μL of 0.3 M sodium hydroxide, grind the sample at 50HZ for 1 min (Shanghai Jingxin tissue grinder), centrifuge at 3000 rpm for 1 min after grinding, boil in water bath for 1 min, add 400 μL of 0.2 M Tris-HCl to mix, boil in water bath for 1 min again, centrifuge at 3000 rpm for 1 min after the water bath is completed, take the supernatant and dilute 10-30 times as a template for subsequent PCR amplification.

[0034] PARMS reaction test: PARMS reaction test was performed on the GeneMatrix high-throughput genotyping platform. 20 ng DNA sample was added to the microwell reaction plate, and the PARMS reaction mixture was added. The reaction system is shown in Table 2. PCR amplification was completed in a water bath thermal cycler. The Touchdown PCR reaction conditions were: 94°C pre-denaturation for 15 minutes; the first step of amplification reaction, 94°C denaturation for 20 seconds, 65°C-57°C annealing and extension for 60 seconds, 10 cycles, and the temperature of annealing and extension was reduced by 0.8°C in each cycle; the second step of amplification reaction, 94°C denaturation for 20 seconds, 57°C annealing and extension for 60 seconds, 26 cycles.

[0035] Table 2

[0036]

[0037] After the PCR is completed, the PARMS reaction test is carried out on the GeneMatrix high-throughput genotyping platform. Specifically, the Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 molecular markers are detected simultaneously using the PARMS technology; if the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is A and the base of the SNP site of Pi-ty269(t)-325 is A, the rice to be tested is judged to be a homozygous disease-resistant Pi-ty269(t) genotype; if Pi-t If the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is G, and the base of the SNP site of Pi-ty269(t)-325 is G, the rice to be tested is judged to be a homozygous disease-susceptible Pi-ty269(t) genotype; if T and G are detected at the SNP site of Pi-ty269(t)-313 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-315 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-325 at the same time, the rice to be tested is judged to be a heterozygous disease-resistant Pi-ty269(t) genotype. After the reaction was completed, the fluorescence signal was read using a TECAN infinite M1000 microplate reader, and then the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) was used to analyze and convert the fluorescence signal to obtain a clear and intuitive typing diagram, and the genotype results were output according to different colors.

[0038] 6) Natural group verification

[0039] According to the above detection method, the markers Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 were used to verify the PARMS initial screening reaction of 59 rice materials, including 31 rice varieties and 28 F3 populations of susceptible or resistant materials. The test results are shown in Table 3 and the typing diagram is shown in Figure 4 As shown. Among the 31 rice varieties, 10 materials were homozygous Pi-ty269(t) genotype materials for resistance to rice blast, and 21 were pure materials susceptible to rice blast. Among the 28 F3 materials, 8 were homozygous Pi-ty269(t) genotype materials for resistance to rice blast, 8 were heterozygous genotype materials, and 12 were pure materials susceptible to rice blast.

[0040] Table 3

[0041]

[0042]

[0043] Depend on Figure 4 As shown in Table 3, the SNP marker is a linked marker, and the Pi-ty269(t) gene has not been widely used in the common rice breeding tested. The Pi-ty269(t) gene was subsequently introduced to improve rice blast resistance. Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 can be used for the efficient detection of the rice Pi-ty269(t) gene.

[0044] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi-ty269(t), characterized in that: The primer set comprises a specific primer and a universal primer, the SNP molecular marker consists of Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325, the polymorphic sites of the SNP molecular markers Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 are respectively located at the 31386403rd, 31548744th and 32520322nd bases of chromosome 4 of rice Nipponbare (MSU7.0), the 31386403rd base is T or G, the 31548744th base is G or A, and the 32520322nd base is G or A; The specific primers of the SNP molecular marker Pi-ty269(t)-313 include Pi-ty269(t)-313Ra with a nucleotide sequence as shown in SEQ ID NO.1 and Pi-ty269(t)-313Rc with a nucleotide sequence as shown in SEQ ID NO.2, and the universal primer is Pi-ty269(t)-313F with a nucleotide sequence as shown in SEQ ID NO.3; The specific primers of the SNP molecular marker Pi-ty269(t)-315 include Pi-ty269(t)-315Rc with a nucleotide sequence as shown in SEQ ID NO.4 and Pi-ty269(t)-315Rt with a nucleotide sequence as shown in SEQ ID NO.5, and the universal primer is Pi-ty269(t)-315F with a nucleotide sequence as shown in SEQ ID NO.6; The specific primers of the SNP molecular marker Pi-ty269(t)-325 include Pi-ty269(t)-325Fg with a nucleotide sequence as shown in SEQ ID NO.7 and Pi-ty269(t)-325Fa with a nucleotide sequence as shown in SEQ ID NO.8, and the universal primer is Pi-ty269(t)-325R with a nucleotide sequence as shown in SEQ ID NO.9; The 5' ends of Pi-ty269(t)-313Ra, Pi-ty269(t)-315Rc and Pi-ty269(t)-325Fg are connected to FAM groups respectively, and the 5' ends of Pi-ty269(t)-313Rc, Pi-ty269(t)-315Rt and Pi-ty269(t)-325Fa are connected to HEX groups respectively.

2. Use of the primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi-ty269(t) as claimed in claim 1 in the joint identification of rice Pi-ty269(t) genotype.

3. The use of the primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi-ty269(t) according to claim 2 in the joint identification of the rice Pi-ty269(t) genotype, characterized in that: The steps include: S1. Extract genomic DNA from the rice leaves to be tested; S2. Using the genomic DNA extracted in step S1 as a template, using a primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi-ty269(t), and using PARMS technology to simultaneously detect the Pi-ty269(t)-313, Pi-ty269(t)-315 and Pi-ty269(t)-325 molecular markers; if the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is A, and the base of the SNP site of Pi-ty269(t)-325 is A, it is determined that the rice to be tested is homozygous for the disease-resistant Pi-ty269(t). Genotype; if the base of the SNP site of Pi-ty269(t)-313 is T, the base of the SNP site of Pi-ty269(t)-315 is G, and the base of the SNP site of Pi-ty269(t)-325 is G, the rice to be tested is judged to be a homozygous disease-susceptible Pi-ty269(t) genotype; if T and G are detected at the SNP site of Pi-ty269(t)-313 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-315 at the same time, and G and A are detected at the SNP site of Pi-ty269(t)-325 at the same time, the rice to be tested is judged to be a heterozygous disease-resistant Pi-ty269(t) genotype.

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