Rice SNP molecular marker and application thereof

Abstract

The invention relates to the field of plant molecular breeding, in particular to a rice SNP (Single Nucleotide Polymorphism) molecular marker and application thereof. The SNP site is located at the 19358817bp position of the second chromosome of the rice, and the polymorphism is T or A. The SNP site is a nucleotide sequence shown in the description. According to the invention, a corresponding KASP primer is designed aiming at the SNP site, and the primer is used for detecting the polymorphism of the SNP site to judge whether rice grains have high resistant starch content or not. The SNP molecular marker provided by the invention can effectively identify the genotype of the rice starch branching enzyme SBE3. The SNP molecular marker provided by the invention has the advantages of simplicity in operation, low cost, short period and the like, can accelerate the cultivation of the high-resistant starch rice variety when being applied to molecular marker-assisted selection, remarkably shortens the breeding period of the high-resistant starch rice variety, reduces the breeding cost, and has higher application value in the field of health food.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular breeding, and in particular to a rice SNP molecular marker and application thereof. Background Art

[0002] Diabetes is the second killer among modern diseases, and its harm to the human body is second only to cancer. In recent years, with the improvement of people's living standards, the prevalence of diabetes among adults in my country has also increased significantly, and is higher than the global average. As a chronic lifelong disease, diabetes has a long treatment process and high requirements for disease management. Diet therapy plays an important role in disease management. Diet control mainly controls sugar intake, while also controlling the amount and calories of the diet, and trying to choose foods with a low glycemic index (GI).

[0003] Rice is a typical starch-rich food and the main source of daily calories for most people in the world, especially in Asia. Most of the current rice varieties are high glycemic index (GI) rice, which can induce health problems caused by high calorie intake, such as blood sugar disorders. A large number of researchers have been exploring ways to reduce the glycemic index of rice. From a nutritional point of view, starch can be divided into rapidly digestible starch, slowly digestible starch and resistant starch. Rapidly digestible starch can be quickly digested and absorbed in the small intestine, with a high blood sugar response; slowly digestible starch is slowly digested in the small intestine, with a low blood sugar response; resistant starch cannot be hydrolyzed by amylase in the small intestine and does not cause a blood sugar response, but can react with volatile fatty acids in the human colon to ferment, and has an important physiological role. Resistant starch increases satiety without increasing calories, which is beneficial to the dietary control of diabetic patients, so in theory it should be beneficial for diabetic patients. However, the resistant starch content of ordinary rice is very low. The resistant starch content in hot rice is generally less than 1%, and only 1% to 2.1% in cold rice. Therefore, the breeding of rice varieties with high resistant starch is of great significance for the prevention and treatment of diabetes in my country.

[0004] Traditional rice resistant starch breeding is to test the resistant starch content of rice grains after they are full-grown. However, due to the complexity and high cost of resistant starch content determination, traditional breeding efficiency is low. Molecular marker-assisted selection breeding is to develop molecular markers or functional markers that are closely linked to genes related to rice resistant starch content, and conduct genotype analysis in the offspring. It can quickly and accurately identify whether the breeding material contains high resistant starch genes, greatly improving the efficiency and accuracy of rice resistant starch breeding, saving costs, shortening the breeding cycle, and is of great significance to improving the nutritional quality of rice. Summary of the invention

[0005] The invention provides a rice SNP molecular marker and application thereof. The SNP molecular marker can be used to detect high-resistant starch rice.

[0006] In a first aspect, the present invention provides a rice SNP molecular marker, wherein the polymorphic site of the SNP molecular marker is located at 19358817 bp of the starch branching enzyme gene on chromosome 2 of rice. SBE3 The reference genome version number is Os-Nipponbare-Reference-IRGSP-1.0, and the polymorphism of the SNP molecular marker is T or A.

[0007] The present invention further provides a rice SNP molecular marker, wherein the SNP molecular marker comprises a nucleotide sequence as shown in SEQ ID .1, wherein the polymorphism at the 1796th position from the 5' end of the nucleotide sequence is T or A.

[0008] The polymorphism of the above SNP molecular marker corresponds to A, which means it is high-resistant starch rice.

[0009] In a second aspect, the present invention provides a set of KASP primer combinations, comprising two specific primers C2-19358817-wFAM and C2-19358817-mHEX, and a universal primer C2-19358817-COM, the nucleotide sequence of the specific primer C2-19358817-wFAM is shown in SEQ ID NO.2, the nucleotide sequence of the specific primer C2-19358817-mHEX is shown in SEQ ID NO.3, and the nucleotide sequence of the universal primer C2-19358817-COM is shown in SEQ ID NO.4.

[0010] The specific primer C2-19358817-wFAM nucleotide sequence is: 5'-GAAGGTGACCAAGTTCATGCTGCTGAAAGTCATGATCAAGCA CT-3' (SEQ ID NO. 2).

[0011] The specific primer C2-19358817-mHEX nucleotide sequence is: 5'-GAAGGTCGGAGTCAACGGATTGCTGAAAGTCATGATCAAGCA CA-3' (SEQ ID NO. 3).

[0012] The nucleotide sequence of the universal primer C2-19358817-COM is: 5'-GTAACCTTGTCC ATCAACCAGAA-3' (SEQ ID NO. 4).

[0013] The specific primer C2-19358817-wFAM is connected with a FAM fluorescent marker, and the specific primer C2-19358817-mHEX is connected with a HEX fluorescent marker. Other fluorescent markers can also be selected as needed.

[0014] In a third aspect, the present invention provides a kit comprising any one of the above-mentioned SNP molecular markers or primer combinations.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned SNP molecular marker, primer combination or kit in any of the following: (1) Application in identification of high resistant starch rice; (2) Application in rice molecular marker-assisted breeding; (3) Application in identifying the genotype of rice starch branching enzyme gene SBE3; (4) Application in breeding high-resistant starch rice or non-high-resistant starch rice; (5) Application in improving rice germplasm resources and improving the nutritional quality of rice; (6) Use in the preparation of rice varieties for preventing or alleviating diabetes.

[0016] In a fifth aspect, the present invention provides a method for identifying high resistant starch rice, comprising the following steps: The DNA of the rice sample to be tested is used as a template and the KASP primer combination or kit is used to perform PCR amplification on the DNA of the rice sample to be tested; and the resistant starch content phenotype of the rice sample to be tested is determined according to the amplification result.

[0017] Preferably, the PCR reaction system 10 μl is calculated as follows: 2×Flu-ASPCR Mix for KASP (Changsha Chuangchen Biotechnology Co., Ltd.) 5 μl, 10 μM nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3 0.15 μl each, 10 μM nucleotide sequence as shown in SEQ ID NO: 4 0.3 μl, DNA template (50-100 ng / ul) of the rice sample to be tested 2 μl, and the rest is ultrapure water, a total of 10 μl; And / or the PCR amplification reaction program is: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 20 seconds, annealing and extension at 65°C-57°C for 40 seconds for 10 cycles, the temperature of annealing and extension is reduced by 0.8°C in each cycle, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 40 seconds for 28 cycles.

[0018] Further preferably, in the amplification result, the KASP primer combination identifies the rice with the base type A of the SNP molecular marker as high-resistant starch rice, and identifies the rice with the base type T as not high-resistant starch rice.

[0019] In a sixth aspect, the present invention provides a method for identifying the genotype of the rice starch branching enzyme gene SBE3, using the above-mentioned KASP primer combination, or the above-mentioned kit to perform PCR amplification on the DNA of the rice sample to be tested; detecting the polymorphism of the SNP molecular marker, if the polymorphism is A, the genotype of the rice starch branching enzyme gene SBE3 is AA, if the polymorphism is T, the genotype of the rice starch branching enzyme gene SBE3 is TT.

[0020] The present invention has the following beneficial effects: The SNP molecular markers provided in this application can effectively identify SBE3 The genotype has the advantages of simple operation, low cost and short cycle. It is used in rice molecular marker-assisted selection, which can significantly shorten the breeding cycle of high-resistant starch rice and has high application value.

[0021] The detection method of this application uses SNP molecular markers and combines KASP detection technology to quickly, accurately and high-throughput detect rice materials. SBE3 genotypes to accelerate the breeding of high-resistant starch rice varieties.

[0022] The application of the SNP molecular markers provided in this application and the detection method thereof can be carried out by using the KASP primer combination in the rice seedling stage. SBE3 By conducting genotype testing on the rice, we can quickly identify whether the rice material is high-resistant starch rice. Compared with the traditional method of testing the resistant starch content of rice grains during the maturity period, this method saves a lot of manpower and material costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 In Example 1 gfls1 Mutant gene localization and candidate gene sequence analysis.

[0025] Figure 2 In Example 2, radiation 9811, gfls1 SBE3 genotyping diagram of mutants and their hybrid F1 generation. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Radiation 9811 comes from the Chinese National Rice Research Institute.

[0027] Example 1. gfls1 Mutant gene cloning In the early stage of the research, it was found that there was a mutant with low temperature sensitivity in grain filling in the EMS-induced population of rice Zhongzao 35. gfls1 The average temperature during the grain filling period was 23.7℃, and the thousand-grain weight was 13.34±0.26g, while the thousand-grain weight of the parent Zhongzao 35 was 23.62±0.44g. gfls1 The thousand-grain weight of the mutant seeds was 44% lower than that of Zhongzao 35. gfls1 Genetic types were constructed gfls1 The F2 segregation population of the mutant was hybridized with Zhongzao 35 and Fu 9811. The seed phenotype of all F1 hybrid combinations was normal. F2 seeds were obtained after self-pollination of F1, and the segregation ratio of normal grains and underfilled grains under low temperature was investigated in the F2 population. The results of the Chi-square test showed that the segregation ratio of normal grains and underfilled grains was 3:1, indicating that the mutant trait was controlled by a pair of recessive nuclear genes.

[0028] Using radiation 9811 / gfts1 The F2 segregating population obtained by hybridization was used for gene mapping. Whole genome resequencing was used to analyze the relationship between 9811 and gfls1 Then, we developed KASP molecular markers that are evenly distributed on the 12 chromosomes of rice for polymorphism screening, and obtained 96 pairs of KASP markers that are evenly distributed on the 12 chromosomes and polymorphic between the parents. We used polymorphic KASP primers to analyze the mutant plants. The results showed that KASP markers S5 and S6 on chromosome 2 of rice were linked to the mutant trait gene, with 5 and 12 recombinant plants, respectively, and the physical distance between the two markers was 1627kb. In order to further narrow the gap, gfls1 The gene localization interval was developed by encrypting new KASP markers between S5 and S6, expanding the F2 localization population, and shortening the localization interval by chromosome walking. Using these molecular markers and 1216 F2 mutant plants for fine localization, it was found that the recombinant plants of markers S8 and S10 were both 1, and the recombinant plant of marker S9 was 0. Therefore, the gene was finely localized between S8 and S10, and the physical distance of the interval was 142 kb ( Figure 1). RGAP (http: / / rice.uga.edu / cgi-bin / gbrowse / rice / #search) was used to analyze candidate genes and 16 genes were found in this interval, of which 12 genes had functional annotations. gfls1 The whole genome sequencing data was used to perform variation analysis on this interval and found gfls1 Starch branching enzyme gene SBE3 There is a single T deletion in exon 16 of (LOC_Os02g32660), which causes premature termination of translation of the frameshift mutant protein ( Figure 1 ). Existing literature shows that SBE3 The functional loss of gfls1 The mutant phenotype is consistent with gfls1 The underfilled phenotype of the mutant may be due to SBE3 caused by the lack of functionality.

[0029] According to previous reports SBE3 Loss of function also leads to an increase in the resistant starch content of rice, so we gfls1 The mutant grains were tested for resistant starch according to the industry standard NY / T 2638-2014 "Spectrophotometric method for determination of resistant starch in rice and its products". gfls1 The resistant starch content of the mutant grains was 15.6%, which was significantly higher than that of the wild type Zhongzao 35 (3.99%), further confirming the role of the rice starch branching enzyme gene in starch branching. SBE3 The lack of gfls1 The reason why the mutant grains are not full and the resistant starch content is increased. Given the importance of rice with high resistant starch content in the prevention and treatment of diabetes, we used gfls1 Mutants are used as donors for hybridization to improve the backbone parents of hybrid rice, and gfls1 In mutant SBE3 genotype, develop SNP molecular markers, conduct molecular marker-assisted selection breeding, and improve the efficiency of breeding high-resistant starch rice varieties.

[0030] Example 2. gfls1 In mutant SBE3 Development and application of SNP molecular markers 1. SBE3 Development of SNP molecular markers according to gfls1 At 19358817 bp on chromosome 2 in the mutant, SBE3The base type at position 1796 in the coding region is A, while the base type at position 35 in the wild type is T. Based on the polymorphism of this site, KASP primers were designed using Primer5 software. The wild type type ( SBE3 The 5' end of the primer with the base type at position 1796 of the coding region being T) was labeled with FAM fluorescent marker, and the primer was labeled with C2-19358817-wFAM (SEQ ID NO.2). SBE3 The 5' end of the primer with base type A at position 1796 in the coding region was labeled with HEX fluorescent marker, and the primer was labeled C2-19358817-mHEX (SEQ ID NO.3), and the other one was labeled C2-19358817-COM (SEQ ID NO.4). The KASP primer sequences are as follows: C2-19358817-wFAM: 5'-GAAGGTGACCAAGTTCATGCTGCTGAAAGTCATGATCAAGCACT-3' C2-19358817-mHEX: 5'-GAAGGTCGGAGTCAACGGATTGCTGAAAGTCATGATCAAGCACA-3' C2-19358817-COM: 5'-GTAACCTTGTCCATCAACCAGAA-3' 2. SBE3 Application of SNP molecular markers (1) DNA extraction of samples to be tested a) Take 2-3 cm of rice leaves from the rice sample to be tested, cut them into pieces, put them into a 2 mL EP tube (which can be placed in a 65°C oven for drying), add 400 μL of TPS extract (TPS extract formula: 100 mL 1M Tris-HCL, 20 mL 0.5M EDTA, 74.55 g KCl, fixed to 1 L), add a 5 mm stainless steel bead, and put it into a tissue disruptor for disruption.

[0031] b) Incubate at 65°C for half an hour.

[0032] c) Place the centrifuge tube in a centrifuge, centrifuge at 12000 rpm for 3 minutes, and pipette 130uL of the supernatant into a 1.5mL centrifuge tube.

[0033] d) Add 130 μL of isopropanol (can be pre-chilled) to the supernatant and mix gently (can be frozen in a -20°C refrigerator for 30-60 min).

[0034] e) Centrifuge at 12,000 rpm for 5 minutes, discard the isopropanol, add 200 μL of the prepared 75% ethanol solution, mix thoroughly, let stand, centrifuge again to discard the alcohol solution, and place in a fume hood to air dry.

[0035] f) After air drying, add 100 μL of TE buffer to the centrifuge tube containing the dried DNA precipitate. After the DNA is dissolved, use an MBA 2000 UV / VS spectrometer (Perkin Elemer Co., Ltd.) to measure its concentration and quality. Then dilute the DNA stock solution with sterile water to a working solution of 50 ng / μL and store it in a refrigerator at 4°C for use in the next experiment.

[0036] (2) PCR amplification of SBE3 SNP molecular markers.

[0037] a) Configure the PCR amplification reaction system The PCR amplification reaction system (10 μl) consists of: 5 μl of 2×Flu-ASPCR Mix for KASP (Changsha Chuangchen Biotechnology Co., Ltd.), 0.15 μl of each of the 10 μM nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.3 μl of the 10 μM nucleotide sequence shown in SEQ ID NO: 4, 2 μl of the DNA template (50-100 ng / ul) of the rice sample to be tested, and the rest is ultrapure water, a total of 10 μl.

[0038] b) PCR amplification reaction PCR amplification program: pre-denaturation at 94°C for 3 min, 10 cycles (denaturation at 94°C for 20 s, annealing and extension at 65°C-57°C for 40 s), the temperature of annealing and extension decreased by 0.8°C in each cycle, 28 cycles (denaturation at 94°C for 20 s, annealing and extension at 57°C for 40 s) to obtain PCR amplification products.

[0039] (3) SBE3 genotype analysis of the samples to be tested The obtained PCR products were subjected to fluorescence scanning and genotyping using the CFX96 Touch real-time fluorescence quantitative PCR instrument of Bio-Rad Company, and the specific procedure was: 37°C, 1 min, Plate Read, End. If the HEX fluorescence signal was detected in the PCR product, the genotype of the SBE at position 1796 in the coding region was A / A, and the rice to be tested was high-resistant starch rice; if the FAM fluorescence signal was detected, the genotype of the SBE at position 1796 in the coding region was T / T, and the rice to be tested was not high-resistant starch rice; if both FAM and HEX fluorescence were detected, the genotype of the SBE3 at position 1796 in the coding region was heterozygous T / A, and the rice to be tested was not high-resistant starch rice.

[0040] Will gfls1 Seventeen strains of each of the mutant, Fu9811 and their hybrid F1 were selected and PCR amplified using the above-mentioned KASP primers. The amplified products were subjected to fluorescence scanning and genotyping. The results are as follows: Figure 2 As shown, the PCR product using Fu9811 as a template detected a FAM fluorescence signal, so the genotype of SBE3 in Fu9811 at position 1796 of the coding region was T / T, which was not a high-resistant starch rice. gfls1 The HEX fluorescence signal was detected in the PCR product with the mutant as the template. gfls1 The mutant SBE3 had a genotype of A / A at position 1796 in the coding region, which was a high-resistant starch rice; gfls1 The fluorescence signal of hybrid F1 of mutant and radiation 9811 is located between FAM and HEX, and there is a clear boundary to distinguish between FAM and HEX, so its genotype is heterozygous. The above test results are consistent with reality, indicating that the KASP primer combination C2-19358817-wFAM (SEQ ID NO.2), C2-19358817-mHEX (SEQ ID NO.3) and C2-19358817-COM (SEQ ID NO.4) designed for the polymorphism of the base at position 1796 of the SBE3 coding region, that is, T or A, can accurately distinguish different alleles of T or A.

[0041] (4) Analysis of resistant starch content of the sample to be tested In order to further verify the detection effect of SNP molecular markers on SBE3 genotype, the above gfls1 The resistant starch content of the mutant, Fu9811 and their hybrid F1 rice was tested. The results of the resistant starch content test were consistent (Table 1).

[0042] This indicates that the SBE3 SNP molecular marker provided by the present invention is closely linked to the trait locus related to high resistant starch content, can accurately screen out individual plants with high resistant starch content, and can be used for molecular assisted breeding of high resistant starch rice to improve breeding efficiency.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rice SNP molecular marker, characterized in that: The polymorphic site of the SNP molecular marker is located at 19358817 bp of rice chromosome 2, and the polymorphism of the SNP molecular marker is T or A.

2. A rice SNP molecular marker, characterized in that: The SNP molecular marker comprises a nucleotide sequence as shown in SEQ ID NO.1, and the polymorphism at position 1796 from the 5' end of the nucleotide sequence is T or A.

3. The SNP molecular marker according to any one of claims 1 or 2, characterized in that The polymorphism of the SNP molecular marker corresponds to A, which indicates high-resistant starch rice.

4. A KASP primer combination, characterized in that: The KASP primer combination includes two specific primers C2-19358817-wFAM and C2-19358817-mHEX, and a universal primer C2-19358817-COM. The nucleotide sequence of the specific primer C2-19358817-wFAM is shown in SEQ ID NO.2, the nucleotide sequence of the specific primer C2-19358817-mHEX is shown in SEQ ID NO.3, and the nucleotide sequence of the universal primer C2-19358817-COM is shown in SEQ ID NO.

4.

5. A kit, characterized in that: It comprises the SNP molecular marker according to any one of claims 1 to 3 or the primer combination according to claim 4.

6. Any of the following uses of the SNP molecular marker according to any one of claims 1 to 3, the primer combination according to claim 4, or the kit according to claim 5: (1) Application in identification of high resistant starch rice; (2) Application in rice molecular marker-assisted breeding; (3) Application in identifying the genotype of rice starch branching enzyme gene SBE3; (4) Application in breeding high-resistant starch rice or non-high-resistant starch rice; (5) Application in improving rice germplasm resources and improving the nutritional quality of rice; (6) Use in the preparation of rice varieties for preventing or alleviating diabetes.

7. A method for identifying high resistant starch rice, characterized in that: The following steps are involved: Using the DNA of the rice sample to be tested as a template, using the KASP primer combination described in claim 4 or the kit described in claim 5 to perform PCR amplification on the DNA of the rice sample to be tested; The resistant starch content phenotype of the rice sample to be tested is determined according to the amplification result.

8. The method according to claim 7, characterized in that The PCR reaction system 10 μl is calculated as follows: 2×Flu-ASPCR Mix for KASP 5 μl, 10 μM nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3 0.15 μl each, 10 μM nucleotide sequence as shown in SEQ ID NO: 4 0.3 μl, 2 μl DNA template of the rice sample to be tested, and the rest is ultrapure water, a total of 10 μl; And / or the PCR amplification reaction program is: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 20 seconds, annealing and extension at 65°C-57°C for 40 seconds for 10 cycles, the temperature of annealing and extension is reduced by 0.8°C in each cycle, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 40 seconds for 28 cycles.

9. The method according to any one of claims 7 or 8, characterized in that: In the amplification results, the KASP primer combination identified the rice with the base type A of the SNP molecular marker as high-resistant starch rice, and identified the rice with the base type T as not high-resistant starch rice.

10. A method for identifying the genotype of rice starch branching enzyme gene SBE3, characterized in that: The DNA of the rice sample to be tested is amplified by PCR using the KASP primer combination described in claim 4 or the kit described in claim 5; the polymorphism of the SNP molecular marker described in any one of claims 1 to 3 is detected, and if the polymorphism is A, the genotype of the rice starch branching enzyme gene SBE3 is AA, and if the polymorphism is T, the genotype of the rice starch branching enzyme gene SBE3 is TT.

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