SNP molecular marker of rice blast resistance gene pi65, primer set and application thereof

Abstract

The application discloses a SNP molecular marker and primer group of rice blast resistance gene Pi65 and application, and the application utilizes the PARMS technology to carry out gene typing on the developed SNP marker, and the basic automation of DNA extraction, PCR system construction and fluorescence signal detection in the PARMS technology process can realize 96, 384, 1536 hole plate high-throughput detection, and is suitable for large-scale, high-throughput Pi65 gene identification and rice blast resistance resource screening. In the detection process, the application does not need complicated procedures such as enzyme cutting, electrophoresis and sequencing, reduces the pollution of PCR product aerosol and the use of toxic substances such as EB, and meanwhile, the application can carry out foreground selection and background selection in the early stage of molecular marker assisted breeding, improves the background recovery rate, reduces the scale of breeding population, speeds up the breeding process, and is favorable to the efficient and environment-friendly application of the Pi65 gene in commercial molecular breeding of rice.

Description

Technical Field

[0001] This invention relates to the field of rice breeding, and in particular to an SNP molecular marker, primer set, and application of the rice blast resistance gene Pi65. Background Technology

[0002] Rice is an important food crop, and rice blast can occur throughout its entire growth cycle, leading to complete crop failure and threatening food security. Utilizing the rice's own disease-resistant genes is the most economical, effective, and environmentally friendly way to control rice blast. The rice blast resistance gene Pi65 exhibits broad-spectrum resistance to rice blast fungus in different rice-growing regions of my country and has high utilization value in rice breeding.

[0003] Rice disease resistance breeding involves phenotypic selection of plants based on resistance identification, which is time-consuming, easily affected by environmental conditions, and prone to errors, resulting in low selection efficiency. Molecular marker-assisted selection (MAG) breeding, on the other hand, is simple and effective, reducing breeding costs, shortening the breeding cycle, and allowing for targeted multi-gene aggregation, thereby improving breeding efficiency and bringing significant socio-economic benefits. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an SNP molecular marker for the rice blast resistance gene Pi65, along with its primer set and applications.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0006] The first objective of this invention is to provide an SNP molecular marker for the rice blast resistance gene Pi65, wherein the SNP molecular marker is Pi65-1668, the polymorphic site of Pi65-1668 is located at the 28377994th base of Pi65 on rice chromosome 11, and the 28377994th base is C or G.

[0007] A second objective of this invention is to provide a primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi65. The primer set includes specific primers and universal primers. The specific primers include Pi65-1668Fc with nucleotide sequences as shown in SEQ ID NO.1 and Pi65-1668Fg with nucleotide sequences as shown in SEQ ID NO.2. The universal primer is Pi65-1668R with a nucleotide sequence as shown in SEQ ID NO.3.

[0008] Furthermore, the 5' end of the Pi65-1668Fc is connected to a FAM fluorescent sequence, and the 5' end of the Pi65-1668Fc is connected to a HEX fluorescent sequence.

[0009] The third objective of this invention is to provide an application of the SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1 in the identification of the rice Pi65 genotype.

[0010] The fourth objective of this invention is to provide a primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1 in the identification of the rice Pi65 genotype.

[0011] Specifically, the application of primer sets for detecting SNP molecular markers of the rice blast resistance gene Pi65 in identifying the rice Pi65 genotype includes the following steps:

[0012] S1. Extract genomic DNA from rice leaves;

[0013] S2. Using the genomic DNA extracted in step S1 as a template, the SNP molecular marker PI65-1668 is detected using the primer set described in claim 2 for detecting the SNP molecular marker Pi65, which is used to detect the rice blast resistance gene.

[0014] S3. If the polymorphic site of Pi65-1668 has a base of C, the rice sample tested is determined to be homozygous non-resistant Pi65 genotype; if the polymorphic site of Pi65-1668 has a base of G, the rice sample tested is determined to be homozygous resistant Pi65 genotype; if both bases C and G are detected at the polymorphic site of Pi65-1668, the rice sample tested is determined to be heterozygous resistant Pi65 genotype.

[0015] The fifth objective of this invention is to provide an application of the SNP molecular marker of the rice blast resistance gene Pi65 in rice breeding. Specifically, the application of a primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi65 in identifying the rice Pi65 genotype and selecting rice lines carrying the Pi65 gene for subsequent breeding.

[0016] Compared with existing technologies, this invention utilizes SNP molecular markers to identify the rice blast resistance gene Pi65 in rice materials and employs PARMS technology for Pi65 gene detection. This allows for rapid and accurate detection of the Pi65 gene in different germplasm resources, including indica and japonica rice. This invention uses PARMS technology for genotyping of the developed SNP markers. The PARMS process is largely automated, including DNA extraction, PCR system construction, and fluorescence signal detection, enabling high-throughput detection in 96,384, and 1536-well plates. It is suitable for large-scale, high-throughput Pi65 gene identification and screening of rice blast resistance resources. This invention eliminates the need for cumbersome procedures such as enzyme digestion, electrophoresis, and sequencing during detection, reducing PCR product aerosol contamination and the use of toxic substances like EB. Furthermore, it allows for foreground and background selection in the early stages of marker-assisted breeding, improving background recovery rates, reducing breeding population size, and accelerating the breeding process. This facilitates the efficient and environmentally friendly application of the Pi65 gene in commercial rice molecular breeding. Attached Figure Description

[0017] Figure 1 This is a map showing the SNP molecular marker positions of Pi65.

[0018] Figure 2 This is a flowchart illustrating the molecular marker development process according to an embodiment of the present invention.

[0019] Figure 3 This is a typing diagram of the molecular marker PARMS according to an embodiment of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The GeneMatrix genotyping platform and its supporting reagents and consumables were developed by Chengdu Hanchen Guangyi Technology Co., Ltd., and are my country's first professional fully automated high-throughput SNP detection hardware system.

[0022] Example 1: SNP molecular markers of rice blast resistance gene Pi65 and their applications

[0023] This embodiment discloses the SNP molecular marker of the rice blast resistance gene Pi65, namely Pi65-1668. The polymorphic site of Pi65-1668 is located at the 28377994th base of Pi65 on rice chromosome 11, and the 28377994th base is either C or G. The location of the SNP molecular marker Pi65-1668 on the rice chromosome is as follows. Figure 1 As shown.

[0024] like Figure 2 The diagram illustrates the molecular marker design process of this invention. The physical location of the cloned target gene Pi65 is determined, SNP sites and flanking sequences are extracted, primer sequences for the marker are designed and synthesized, and then the marker is screened and tested. The details are as follows:

[0025] 1. Primer design

[0026] Based on the location marker information in the literature, the physical location of Pi65 was determined to be the region 28376327-28379731 on chromosome 11 of Nipponbare (MSU7.0). SNP sites and flanking sequences of this region were extracted, and primers were designed using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). The primer designs for Pi65-1668 are shown in Table 1. Each marker group has three primers, with two specific primers having FAM and HEX fluorescent sequences attached to their 5' ends, respectively. The primers were synthesized by Invitrogen.

[0027] Table 1: Primer design for Pi65-1668

[0028]

[0029] 2. Sample testing

[0030] DNA extraction: Take a leaf about 1 cm long and wide and place it in a deep-well plate (1.2 ml for 96 wells). Add 100 μL of 0.3 M sodium hydroxide and grind the sample at 50 Hz for 1 min (Shanghai Jingxin tissue homogenizer). After grinding, centrifuge at 3000 rpm for 1 min, boil in water for 1 min, add 400 μL of 0.2 M Tris-HCl and mix well. Boil in water again for 1 min. After the water bath, centrifuge at 3000 rpm for 1 min. Take the supernatant and dilute it 10-30 times to use as a template for subsequent PCR amplification.

[0031] PARMS reaction assay: The PARMS reaction assay was performed on the GeneMatrix high-throughput genotyping platform. 20 ng of DNA sample was added to each microplate, followed by the PARMS reaction mixture (see Table 2). PCR amplification was performed in a water bath thermal cycler. The Touchdown PCR conditions were: 94℃ pre-denaturation for 15 minutes; Step 1 amplification reaction: 94℃ denaturation for 20 seconds, annealing and extension at 65℃–57℃ for 60 seconds, 10 cycles, with the annealing and extension temperatures decreasing by 0.8℃ per cycle; Step 2 amplification reaction: 94℃ denaturation for 20 seconds, annealing and extension at 57℃ for 60 seconds, 26 cycles.

[0032] After PCR, the PARMS reaction was performed on the GeneMatrix high-throughput genotyping platform. After the reaction, the fluorescence signal was read using a TECAN Infinite M1000 microplate reader, and then the converted fluorescence signal was analyzed using the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) to obtain a clear and intuitive genotyping map, outputting the genotype results based on different colors.

[0033] Table 2: PARMS PCR reaction system

[0034]

[0035] Based on the above detection method, the initial screening reaction of rice varieties containing the Pi65 resistance-susceptibility genotype was verified using the SNP molecular marker Pi65-1668.

[0036] The donor variety of Pi65 gene showed a base G at the Pi65-1668 test site, indicating it is resistant to Pi65, while the susceptible controls all showed a base C, which is not resistant to Pi65. This demonstrates the accuracy of the Pi65 gene detection method in this invention.

[0037] 3. Application Examples

[0038] Natural group validation

[0039] Natural population validation of the SNP marker Pi65-1668 was performed using 247 accessions. As shown in Table 1, the 247 accessions included varieties known to contain homozygous Pi65 genes, F1 materials containing other rice blast gene donors, universally susceptible materials, common hybrid rice, core rice breeding, and core breeding materials.

[0040] Table 1

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] The markers in the natural population typing results are as follows Figure 3 As shown, 67 samples tested positive for the homozygous Pi65 genotype of rice blast resistance, 32 samples contained the susceptible Pi65 genotype, and the remaining samples showed unclear genotypes or no amplification results. This indicates that the SNP markers detected highly specific loci, and the Pi65 gene is not widely used in common rice breeding practices. For subsequent targeted improvement of rice blast resistance by introducing the Pi65 gene, Pi65-1668 can be used for efficient detection of the rice Pi65 gene.

[0048] Subsequently, rice lines carrying the Pi65 gene were selected for further breeding.

[0049] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker for the rice blast resistance gene Pi65, characterized in that: The SNP molecular marker is Pi65-1668, and the polymorphic site of Pi65-1668 is located at the 28377994th base of Pi65 on rice chromosome 11. The 28377994th base is either C or G.

2. A primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1, characterized in that: The primer set includes specific primers and universal primers, wherein the specific primers include Pi65-1668Fc with nucleotide sequences as shown in SEQ ID NO.1 and Pi65-1668Fg with nucleotide sequences as shown in SEQ ID NO.2; and the universal primer is Pi65-1668R with nucleotide sequences as shown in SEQ ID NO.

3.

3. The primer set according to claim 2 for detecting the SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1, characterized in that: The 5' end of the Pi65-1668Fc is connected to a FAM fluorescent sequence, and the 5' end of the Pi65-1668Fc is connected to a HEX fluorescent sequence.

4. The application of an SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1 in the identification of the rice Pi65 genotype.

5. The application of a primer set as described in claim 2 or 3 for detecting the SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1 in the identification of the rice Pi65 genotype.

6. The application of the primer set for detecting the SNP molecular marker of the rice blast resistance gene Pi65 according to claim 4 in identifying the rice Pi65 genotype, characterized in that, Includes the following steps: S1. Extract genomic DNA from rice leaves; S2. Using the genomic DNA extracted in step S1 as a template, the SNP molecular marker Pi65-1668 is detected using the primer set described in claim 2 for detecting the SNP molecular marker Pi65 of the rice blast resistance gene. S3. If the polymorphic site of Pi65-1668 has a base of C, the rice sample tested is determined to be homozygous non-resistant Pi65 genotype; if the polymorphic site of Pi65-1668 has a base of G, the rice sample tested is determined to be homozygous resistant Pi65 genotype; if both bases C and G are detected at the polymorphic site of Pi65-1668, the rice sample tested is determined to be heterozygous resistant Pi65 genotype.

7. The application of an SNP molecular marker of the rice blast resistance gene Pi65 as described in claim 1 in rice breeding.

8. The application of the SNP molecular marker of the rice blast resistance gene Pi65 according to claim 7 in rice breeding, characterized in that: The primer set described in claim 4 for detecting the SNP molecular marker Pi65, a rice blast resistance gene, is used to identify the Pi65 genotype in rice and select rice lines carrying the Pi65 gene for subsequent breeding.

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