SNP molecular marker primers for SD6, a gene associated with resistance to panicle sprouting in rice, and their application

Abstract

This invention provides SNP molecular marker primers for the rice germination resistance gene SD6. The nucleotide sequence of the rice germination resistance gene SD6 is shown in SEQ ID NO.1. The SNP molecular marker is a single nucleotide mutation T / C at position 1857 bp in the coding region of the SD6 gene. The SNP molecular marker primers for the SD6 gene are a primer set composed of specific and non-specific primers, and their nucleotide sequences are shown in SEQ ID NO.2-5. This invention also provides a method for genotyping the rice germination resistance gene SD6 using the aforementioned SNP molecular marker primers. This method can rapidly, accurately, cost-effectively, and with high throughput detect the dormant genotype of the rice SD6 gene. The SNP molecular marker primers for the rice germination resistance gene SD6 of this invention can be used to identify rice plants resistant to germination, and have practical guiding significance for breeding and screening germplasm resources of rice plants resistant to germination.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding technology, specifically relating to an SNP molecular marker primer for the rice anti-sprouting gene SD6 and its application. Background Technology

[0002] Seed dormancy and germination are crucial characteristics for plants to adapt to their environment and ensure their survival and reproduction. During crop domestication, the focus has often been on high yield, high quality, disease and pest resistance, and tolerance to adverse conditions, while ensuring consistent seed germination at sowing. This has often neglected the preservation of adequate seed dormancy, leading to pre-harvest sprouting in many gramineous crops. Pre-harvest sprouting, also known as pre-harvest sprouting, is a phenomenon where some cereal crops germinate on the panicle during the late stages of maturity when exposed to prolonged periods of high temperatures and rainy weather. In recent years, with global warming, rainy weather during the late stages of crop maturity has led to frequent occurrences of pre-harvest sprouting disasters. Reports indicate that in southern China, the rice harvest season often coincides with high temperatures and heavy rainfall, with approximately 6% of the sown area of ​​conventional rice and as much as 20% of the sown area of ​​hybrid rice experiencing pre-harvest sprouting. In Japan and South Korea, most major japonica rice varieties are not resistant to pre-harvest sprouting, with severe pre-harvest sprouting resulting in annual losses of between $8 billion and $10 billion. Sprouting of ears not only leads to reduced crop yield and quality, but also seriously affects the quality of crop seed production.

[0003] Public reports indicate the existence of a homologous allele, SD6 (LOC_Os06g06900), between Nipponbare and Kasalath. A single-base T-to-C mutation at position 1857 bp in the coding region of this allele leads to panicle budding resistance in rice. SD6 is a negative regulator of seed dormancy, and the sd6 mutation significantly increases the number of grains per panicle, making SD6 a potential candidate for both panicle budding resistance and yield increase. Therefore, developing molecular markers to detect rice genotypes using the SNP site of the SD6 allele is of significant reference value for molecular breeding of rice panicle budding resistance and screening of rice germplasm resources. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing an SNP molecular marker primer for the rice anti-sprouting gene SD6 and its application. The SNP molecular marker primer for the rice anti-sprouting gene SD6 is a primer set composed of specific primers and non-specific primers. This SNP molecular marker primer can be used to identify rice plants resistant to pre-sprouting, and has guiding significance for breeding and screening germplasm resources of rice resistant to pre-sprouting, which is conducive to realizing the efficient application of gene SD6 in molecular breeding for rice pre-sprouting resistance.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a SNP molecular marker primer for the rice anti-sprouting gene SD6, wherein the nucleotide sequence of the rice anti-sprouting gene SD6 is shown in SEQ ID NO.1; the SNP molecular marker of the SD6 gene is a single nucleotide mutation T / C at 1857bp in the coding region of the SD6 gene; the SNP molecular marker primer for the SD6 gene is a primer set consisting of two specific primers and two non-specific primers;

[0006] The specific primers include an upstream primer SD6-ID and a downstream primer SD6-IS, the nucleotide sequence of the upstream primer SD6-ID is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer SD6-IS is shown in SEQ ID NO.3;

[0007] The nonspecific primers include an upstream primer SD6-OS and a downstream primer SD6-OD. The nucleotide sequence of the upstream primer SD6-OS is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer SD6-OD is shown in SEQ ID NO.5.

[0008] This invention also provides an application of SNP molecular marker primers for the rice anti-sprouting gene SD6. The SNP molecular marker primers are used to detect the genotype of the rice anti-sprouting gene SD6. The detection method includes the following steps:

[0009] S1. Extract genomic DNA from the rice to be tested;

[0010] S2. Using the rice genomic DNA extracted in S1 as a template, PCR amplification was performed using the primer set to obtain the PCR product.

[0011] S3. After separating the PCR product obtained in S2 on a 4% polyacrylamide gel, silver staining was performed, followed by color development in NaOH formaldehyde solution. The band pattern was photographed and stored on a gel electrophoresis apparatus. If the PCR product has only one band and its length is 237 bp, then the tested rice contains homozygous rice panicle germination SD6. Nip Gene; if the PCR product contains only one band and is 152 bp in length, then the tested rice contains homozygous rice anti-spillage SD6. Kasa Genes; if there are two PCR products with sizes of 237bp and 152bp respectively, then the rice being tested is heterozygous and contains both the SD6 gene for panicle sprouting and anti-sprouting.

[0012] Preferably, the PCR amplification reaction system described in S2 is as follows: 2 μL 10× Buffer, 0.2 μL 10 mmol / L dNTP, 0.5 μL 5 μmol / L SD6-ID, 0.5 μL 5 μmol / L SD6-OD, 0.5 μL 5 μmol / L SD6-IS, 0.5 μL 5 μmol / L SD6-OS, 1 μL rice genomic DNA to be tested, 0.15 μL 5 U / μL Taq DNA polymerase, and ddH2O to a final volume of 20 μL;

[0013] Preferably, the PCR amplification reaction conditions described in S2 are: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 72℃ for 30 s, 35 cycles, and a final extension at 72℃ for 1 min.

[0014] Preferably, the method for preparing the formaldehyde solution of NaOH in S3 is as follows: add 0.5 mL of formaldehyde to 100 mL of NaOH aqueous solution with a concentration of 0.5 mol / L.

[0015] This invention also provides another application of SNP molecular marker primers for the rice anti-sprouting gene SD6, which are used to identify rice plants resistant to sprouting.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] 1. This invention provides SNP molecular marker primers for the rice SD6 gene, which is associated with resistance to panicle sprouting. Using these primers, the dormant genotypes of the rice SD6 gene can be detected rapidly, accurately, cost-effectively, and with high throughput. Compared to conventional KASP genotyping techniques, this invention eliminates the need for special reagents and fluorescence PCR instruments. It requires only Taq enzyme (used for standard PCR amplification), conventional PCR instruments, and polyacrylamide gel electrophoresis to identify the alleles responsible for resistance to panicle sprouting in rice. This invention has a low application threshold and low cost.

[0018] 2. The SNP molecular marker primers of the rice anti-sprouting gene SD6 of the present invention can be used to identify rice plants resistant to pre-sprouting. By the difference in electrophoretic bands of different alleles, the genotype of rice resistant to pre-sprouting can be qualitatively identified. The differences in the amplified band patterns of different alleles are obvious, and the identification is simple. It has practical guiding significance for breeding rice resistant to pre-sprouting and screening germplasm resources of rice resistant to pre-sprouting, and is conducive to realizing the efficient application of gene SD6 in molecular breeding of rice pre-sprouting resistance.

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1This is a primer design diagram for the SNP molecular marker of the SD6 gene in Example 1 of the present invention.

[0021] Figure 2 This is an electrophoresis diagram of the Nipponbare and Kasalath rice SD6 genotypes in Example 3 of this invention.

[0022] Figure 3 This is an electrophoresis diagram of the SD6 genotype of different rice varieties in Example 4 of the present invention.

[0023] Figure 4 This is an electrophoresis diagram of the SD6 genotype of the Kasalath / 9311 hybrid F2 population in Example 5 of this invention. Detailed Implementation

[0024] Example 1

[0025] This embodiment describes the design of SNP molecular marker primers for SD6, a gene related to rice panicle and sprouting resistance.

[0026] The gene associated with resistance to panicle sprouting in rice is SD6 (LOC_Os06g06900), and its nucleotide sequence is shown in SEQ ID NO.1. According to publicly available reports, the T / C single nucleotide mutation at position 1857 bp in the SD6 coding region is the cause of the anti-panicle sprouting phenotype in rice. Therefore, primers were designed and developed for this SNP molecular marker.

[0027] The primers designed in this invention require high specificity. The selection criteria are: primer length 18–22 bp, and GC content generally 40–60%. The designed primers are first subjected to BLAST on RiceESTDataBase (http: / / redb.ncpgr.cn / modules / redbtools / blast.php) to eliminate primers with low specificity. Finally, the SNP molecular marker primer set of the rice anti-spillage and sprouting-related gene SD6 is obtained, including 2 specific primers and 2 non-specific primers.

[0028] Two specific primers, SD6-ID and SD6-IS, are complementary to the SNP site at their 3′ ends. Two non-specific primers, SD6-OS and SD6-OD, are located 218 bp and 133 bp apart on either side of the SNP site. Using this primer set, the target fragment was amplified, producing bands of 237 bp and 152 bp. The 237 bp fragment represents the rice panicle sprouting genotype, while the 152 bp fragment represents the rice panicle sprout resistance genotype.

[0029] The nucleotide sequence of the upstream primer SD6-ID in the specific primers is shown in SEQ ID NO.2. The introduction of a mismatch at the sixth base from the end of the 3′ end enhances the specificity of the primer. The nucleotide sequence of the downstream primer SD6-OD is shown in SEQ ID NO.3. The nucleotide sequence of the upstream primer SD6-OS in the non-specific primers is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer SD6-IS is shown in SEQ ID NO.5.

[0030] Figure 1 This is a diagram of the SNP molecular marker primer design strategy for the SD6 gene in this embodiment.

[0031] Example 2

[0032] This embodiment describes a method for genotyping the rice anti-spillage gene SD6. The method is as follows:

[0033] S1. Genomic DNA was extracted from rice leaves using the SDS method;

[0034] S2. Using the rice genomic DNA extracted in S1 as a template, PCR amplification was performed using the primer set to obtain the PCR product.

[0035] The PCR amplification reaction system was as follows: 2 μL 10× Buffer, 0.2 μL 10 mmol / L dNTP, 0.5 μL 5 μmol / L SD6-ID, 0.5 μL 5 μmol / L SD6-OD, 0.5 μL 5 μmol / L SD6-IS, 0.5 μL 5 μmol / L SD6-OS, 1 μL rice genomic DNA to be tested, 0.15 μL 5 U / μL Taq DNA polymerase, and ddH2O to a final volume of 20 μL.

[0036] The PCR amplification reaction conditions were: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 72℃ for 30 s, 35 cycles, and a final extension at 72℃ for 1 min.

[0037] S3. The PCR product obtained in S2 was separated on a 4% polyacrylamide gel and silver-stained. Then, it was developed in a formaldehyde solution containing NaOH. The band patterns were photographed and stored using a gel electrophoresis apparatus. There were significant differences in the band patterns of the rice with and without the anti-spillage germination gene. If the PCR product contained only one band with a length of 237 bp, the tested rice contained homozygous anti-spillage SD6. Nip Gene; if the PCR product shows only one band and is 152 bp in length, then the rice sample contains homozygous SD6 for resistance to pre-harvest sprouting. Kasa Gene; if there are two PCR products with sizes of 237bp and 152bp respectively, then the rice being tested has a heterozygous genotype and contains SD6. Kasa Genes and SD6Nip Gene.

[0038] The method for preparing the formaldehyde solution of NaOH is as follows: add 0.5 mL of formaldehyde to 100 mL of NaOH aqueous solution with a concentration of 0.5 mol / L.

[0039] Example 3

[0040] This embodiment describes the application of SNP molecular marker primers to detect the SD6 genotype of rice resistant to panicle sprouting. The rice materials used are: the weakly dormant variety Nipponbare and the strongly dormant variety Kasalath; the detection method is the method described in Example 2.

[0041] Figure 2 This is an electrophoresis image of the SD6 genotype detection of Nipponbare and Kasalath in this embodiment. Lane 1 represents Nipponbare rice, lane 2 represents Kasalath rice, and lane 3 represents the marker (Takaradl1000). The results show that Nipponbare amplified a 237bp band, indicating that Nipponbare carries the panicle germination SD6. Nip The gene; Kasalath amplified a 152bp band, indicating that Kasalath carries the anti-spillage SD6 gene. Kasa Genes. Therefore, the SNP molecular marker primers of this invention can be used for the efficient detection of different genotypes of the SD6 gene.

[0042] Example 4

[0043] This example demonstrates the use of SNP molecular marker primers for identifying the SD6 genotype in rice varieties. The rice materials used included: Kasalath, Zhehexiang 2, Jiahexiang 1, Nanjing 46, Ningxiangjing 9, Shanghai Normal University 19, Zhejing 100, Zhehujing 25, Zhehujing 26, Jia 67, Jiahe 247, Chunjiang 157, Xiushui 121, Xiushui 134, and Zhejiang University Yuanxiangjing. The identification method employed was the detection method described in Example 2 to determine whether different rice varieties carried the anti-spillage SD6 genotype. Kasa Genetic testing was performed.

[0044] Figure 3This is an electrophoresis diagram of the SD6 genotype of rice plants in this embodiment. Lane 1 is the marker (Takaradl1000), and lanes 2-16 are Kasalath, Zhehexiang 2, Jiahexiang 1, Nanjing 46, Ningxiangjing 9, Shanghai Normal University 19, Zhejing 100, Zhehujing 25, Zhehujing 26, Jia 67, Jiahe 247, Chunjiang 157, Xiushui 121, Xiushui 134, and Zheda Yuanxiangjing, respectively. The results show that only Kasalath amplified a 152bp band, indicating that Kasalath carries the SD6 genotype resistant to panicle sprouting. Kasa Genes; the remaining 14 conventional japonica rice varieties amplified 237bp bands, indicating that these 14 conventional japonica rice varieties SD6 Nip Genetically homozygous, does not carry SD6 Kasa Gene.

[0045] Example 5

[0046] This example demonstrates the use of SNP molecular marker primers to identify F2 hybrid rice populations resistant to panicle sprouting. The hybrid parents are Kasalath and 9311, with Kasalath being SD6, which is resistant to panicle sprouting. Kasa Genetically homozygous, 9311 is the SD6 variety that sprouts from the ear. Nip The hybrid F2 population consisted of 20 homozygous offspring. The identification method used was the detection method described in Example 2 to determine whether the hybrid F2 population carried SD6. Kasa Genetic testing was performed.

[0047] Figure 4 This is an electrophoresis image of the Kasalath / 9311 hybrid F2 population in this embodiment, where lane 1 is the marker (Takaradl 1000), and lanes 2-21 represent the Kasalath / 9311 hybrid F2 population. The results show that lanes 3, 6, 10, 11, 16, and 18 each contain only one PCR product band with a length of 152 bp, indicating that these six rice plants are resistant to panicle sprouting (SD6). Kasa The genes were homozygous, and the PCR product bands in lanes 2, 8, 12, 17, and 21 were only one band long, with a length of 237 bp, indicating that the five rice SD6 plants were homozygous. Nip The PCR products from lanes 4, 5, 7, 9, 13, 14, 15, 19, and 20 showed two bands with lengths of 152 bp and 237 bp, respectively, indicating that these nine rice plants simultaneously carried the SD6 heterozygous gene. Kasa Genes and SD6 Nip Gene.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A gene related to rice resistance to panicle sprouting SD6 The application of SNP molecular marker primers is characterized by, The SNP molecular marker primers were used to detect the rice anti-heading and sprouting gene. SD6 genotype; The rice anti-heading and sprouting related genes SD6 The nucleotide sequence is shown in SEQ ID NO.1; SD6 The SNP molecular marker primers for a gene consist of a primer set composed of two specific primers and two non-specific primers. The specific primers include an upstream primer SD6-ID and a downstream primer SD6-IS, the nucleotide sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of which is shown in SEQ ID NO.

3. The non-specific primers include an upstream primer SD6-OS and a downstream primer SD6-OD, the nucleotide sequence of which is shown in SEQ ID NO.4, and the nucleotide sequence of which is shown in SEQ ID NO.

5. The detection of rice anti-heading and sprouting genes SD6 The genotyping method includes the following steps: S1. Extract genomic DNA from the rice to be tested; S2. Using the rice genomic DNA extracted in S1 as a template, PCR amplification was performed using the primer set to obtain the PCR product. S3. After separating the PCR product obtained in S2 on a 4% polyacrylamide gel, silver staining was performed, followed by color development in NaOH formaldehyde solution. The band pattern was photographed and stored on a gel electrophoresis apparatus. If the PCR product has only one band and its length is 237 bp, then the tested rice contains homozygous rice panicle germination. SD6 Nip Gene; if the PCR product contains only one band and is 152 bp in length, then the tested rice contains homozygous rice resistance to panicle germination. SD6 Kasa Genes; if two PCR products are present, with sizes of 237bp and 152bp respectively, then the rice sample contains a heterozygous genotype and also contains... SD6 Genes that promote ear sprouting and genes that resist ear sprouting.

2. The application according to claim 1, characterized in that, The PCR amplification reaction system described in S2 is as follows: 2 μL 10×Buffer, 0.2 μL 10 mmol / L dNTP, 0.5 μL 5 μmol / L SD6-ID, 0.5 μL 5 μmol / L SD6-OD, 0.5 μL 5 μmol / L SD6-IS, 0.5 μL 5 μmol / L SD6-OS, 1 μL rice genomic DNA to be tested, 0.15 μL 5 U / µL Taq DNA polymerase, and ddH2O to a final volume of 20 μL. The PCR amplification reaction conditions are as follows: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 72℃ for 30 s, for 35 cycles, with a final extension at 72℃ for 1 min.

3. The application according to claim 1, characterized in that, The method for preparing the formaldehyde solution of NaOH described in S3 is as follows: add 0.5 mL of formaldehyde to 100 mL of NaOH aqueous solution with a concentration of 0.5 mol / L.

4. A rice anti-sprouting gene as described in claim 1 SD6 The application of SNP molecular marker primers is characterized by, The rice anti-heading and sprouting related genes SD6 SNP molecular marker primers were used to identify rice plants resistant to panicle sprouting.

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