Rice SNP molecular marker and application thereof

Abstract

The present application relates to the field of plant molecular breeding, and particularly relates to a rice SNP molecular marker and application thereof. The SNP site is located at 19358817bp of the 2nd chromosome of rice, and the polymorphism is T or A. The present application designs corresponding KASP primers aiming at the SNP site, and detects the polymorphism of the SNP site by using the primers to determine whether the rice grains have high resistant starch content. The SNP molecular marker provided by the present application can effectively identify the genotype of rice starch branching enzyme SBE3 . The SNP molecular marker provided by the present application has the advantages of simple operation, low cost, short period, etc. When applied to molecular marker assisted selection, the breeding of high resistant starch rice varieties can be accelerated, the breeding period of high resistant starch rice varieties can be significantly shortened, the breeding cost can be reduced, and the application value in the field of health food is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular breeding, and particularly relates to a rice SNP molecular marker and application thereof. BACKGROUND

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

[0003] Rice is a typical starch-rich food and is the main source of daily calories for most people in the world, especially in Asia. Most of the current rice varieties belong to high glycemic index (full name: glycemic index, GI), which can induce health problems of blood glucose disorders due to high heat intake. A large number of researchers have been exploring ways to reduce the glycemic index of rice. From the perspective of nutrition, starch can be divided into rapidly digestible starch, slowly digestible starch and resistant starch. Rapidly digestible starch can be rapidly digested and absorbed in the small intestine, and has a high blood glucose response; slowly digestible starch is slowly digested in the small intestine, and has a low blood glucose response; resistant starch cannot be hydrolyzed by amylase in the small intestine and does not cause a blood glucose response, but can ferment with volatile fatty acids in the colon of humans and has important physiological effects. Resistant starch can increase satiety without increasing calories, which is beneficial for diet control of diabetes patients, and therefore is theoretically beneficial for diabetes patients. However, the content of resistant starch in ordinary rice is very low, and the content of resistant starch in hot rice is generally less than 1%, and the content of resistant starch in cold rice is only 1% to 2.1%, so breeding rice varieties with high resistant starch is of great significance for the prevention and treatment of diabetes in China.

[0004] Traditional rice resistant starch breeding detects the content of resistant starch in the grain after the rice is ripe, but due to the complexity of the determination of the content of resistant starch, the cost is high, and the traditional breeding efficiency is low. Molecular marker-assisted selection breeding is to develop molecular markers or functional markers closely linked to genes related to the content of rice resistant starch, and to analyze the genotype in the offspring, so as to 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, and shortening the breeding cycle, which has important significance for improving the nutritional quality of rice. SUMMARY

[0005] The application provides a rice SNP molecular marker and application thereof, and the SNP molecular marker can be used for detecting high-resistance starch rice.

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

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

[0008] When the polymorphism of the SNP molecular marker corresponds to A, the rice is high-resistance starch rice.

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

[0010] The nucleotide sequence of the specific primer C2-19358817-wFAM is as follows:

[0011] 5'-GAAGGTGACCAAGTTCATGCTGCTGAAAGTCATGATCAAGCA

[0012] CT-3' (SEQ ID NO. 2).

[0013] The nucleotide sequence of the specific primer C2-19358817-mHEX is as follows:

[0014] 5'-GAAGGTCGGAGTCAACGGATTGCTGAAAGTCATGATCAAGCA

[0015] CA-3' (SEQ ID NO. 3).

[0016] The universal primer C2-19358817-COM nucleotide sequence is: 5'-GTAACCTTGTCC

[0017] ATCAACCAGAA-3' (SEQ ID NO. 4).

[0018] The specific primer C2-19358817-wFAM is connected with FAM fluorescent label, and the specific primer C2-19358817-mHEX is connected with HEX fluorescent label, and other fluorescent labels can also be selected according to needs.

[0019] In a third aspect, the present application provides a kit comprising any of the above SNP molecular markers or primer combinations.

[0020] In a fourth aspect, the present application provides the above SNP molecular markers, primer combinations or kits for any of the following applications:

[0021] (1) application in identifying high resistant starch rice;

[0022] (2) application in rice molecular marker assisted breeding;

[0023] (3) application in identifying rice starch branching enzyme gene SBE3 genotype;

[0024] (4) application in cultivating high resistant starch rice or non-high resistant starch rice;

[0025] (5) application in improving rice germplasm resources and improving rice nutritional quality;

[0026] (6) application in preparing rice varieties for preventing or relieving diabetes.

[0027] In a fifth aspect, the present application provides a method for identifying high resistant starch rice, comprising the following steps:

[0028] Taking the DNA of the rice sample to be tested as a template, 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.

[0029] Preferably, the PCR reaction system 10 μl is composed of 5 μl of 2xFlu-ASPCR Mix for KASP (Changsha Chuandun Biological Technology Co., Ltd.), 0.15 μl of 10 μM nucleotide sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.3 μl of 10 μM nucleotide sequence as shown in SEQ ID NO: 4, 2 μl of DNA (50-100 ng / ul) of the rice sample to be tested as a template, and the rest is ultrapure water, totally 10 μl.

[0030] and / or the PCR amplification reaction procedure is: 94℃ pre-denaturation 3min, 94℃ denaturation 20S, 65℃-57℃ annealing and extension 40S cycle 10 times, the temperature of each cycle of annealing and extension decreases by 0.8℃, 94℃ denaturation 20S, 57℃ annealing and extension 40S cycle 28 times.

[0031] Further preferably, in the amplification result, the rice with base type A of the above SNP molecular marker is identified as high-starch resistant rice by the KASP primer combination, and the rice with base type T is not high-starch resistant rice.

[0032] In a sixth aspect, the present application provides a method for identifying the genotype of the starch branching enzyme gene SBE3 of rice, which uses the KASP primer combination or the kit described above to perform PCR amplification on the DNA of the rice sample to be tested; and detects the polymorphism of the SNP molecular marker, if the polymorphism is A, then the genotype of the starch branching enzyme gene SBE3 of the rice is AA, and if the polymorphism is T, then the genotype of the starch branching enzyme gene SBE3 of the rice is TT.

[0033] The present application has the following advantages:

[0034] The SNP molecular marker provided by the present application can effectively identify the genotype of SBE3 , which has the advantages of simple operation, low cost, short cycle, etc., and is applied to molecular marker assisted selection of rice, significantly shortens the breeding cycle of high-starch resistant rice breeding, and has high application value.

[0035] The detection method of the present application uses SNP molecular markers and combines KASP detection technology to quickly, accurately and high-throughput detect the genotype of SBE3 in rice materials, and accelerate the breeding of high-starch resistant rice varieties.

[0036] The application of the SNP molecular marker and the detection method provided by the present application can detect the genotype of SBE3 in rice seedlings by KASP primer combination, and then quickly screen whether the rice material is high-starch resistant rice, which saves a lot of manpower and material resources compared with the traditional method of detecting the content of resistant starch in grains at the mature stage of rice. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0038] Figure 1 In Example 1 gfls1 Mutant gene localization and candidate gene sequence analysis.

[0039] Figure 2 In Example 2, Radiation 9811, gfls1 Genotyping diagram of the mutant and its F1 hybrid SBE3. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Radiation 9811 is from the China National Rice Research Institute.

[0041] Example 1. gfls1 Mutant gene cloning

[0042] In the preliminary research, a mutant strain of rice variety Zhongzao 35 that was sensitive to low temperatures during grain filling was discovered in the EMS mutagenesis population. gfls1 The average temperature during the grain-filling period was 23.7℃, and its thousand-grain weight was 13.34±0.26g, while the thousand-grain weight of the parent variety, Zhongzao 35, was 23.62±0.44g. gfls1 The thousand-seed weight of the mutant seeds decreased by 44% compared to Zhongzao 35. To determine... gfls1 Genetic types were constructed respectively. gfls1 The F2 segregating population obtained by crossing the mutant with Zhongzao 35 and Fu 9811 showed that the seeds of all F1 hybrids had normal phenotypes. F2 seeds were obtained after self-pollination of F1. The segregation ratio of normal grains and incomplete grains in the F2 population was investigated at low temperature. The results, tested by the Kelvin method, showed that the segregation ratio of normal grains and incomplete grains was 3:1, indicating that the mutant trait is controlled by a pair of recessive nuclear genes.

[0043] Using radiation 9811 / gfts1 Gene mapping was performed on the F2 segregating population obtained from hybridization. Whole-genome resequencing analysis was conducted to analyze the relationship between Radiance 9811 and... gfls1The SNPs were identified, and then KASP molecular markers evenly distributed across the 12 rice chromosomes were developed for polymorphism screening, yielding 96 pairs of polymorphic KASP markers evenly distributed across the 12 chromosomes between parents. Polymorphic KASP primers were used to analyze mutant plants. The results showed that KASP markers S5 and S6 on rice chromosome 2 were linked to the mutant trait gene, with 5 and 12 recombinant plants respectively, and a physical distance of 1627 kb between the two markers. Further narrowing of the molecular structure was then conducted. gfls1 To determine the gene mapping interval, a new KASP marker was developed to densify the region between S5 and S6. The F2 mapping population was expanded, and the mapping interval was shortened using chromosome walking. Fine mapping was performed using these molecular markers and 1216 F2 mutant single plants. It was found that recombination single plants marked with markers S8 and S10 both had a value of 1, while recombination single plants marked with marker S9 had a value of 0. Therefore, the gene was finely mapped to the region between S8 and S10, with a physical distance of 142 kb. Figure 1 ). Analysis of candidate genes using RGAP (http: / / rice.uga.edu / cgi-bin / gbrowse / rice / #search) revealed 16 genes in this region, 12 of which had functional annotations. Using Radiance 9811 and gfls1 Variation analysis of this region using whole-genome sequencing data revealed the following in candidate regions: gfls1 The mutant starch branching enzyme gene SBE3 The presence of a single-base T deletion in exon 16 of (LOC_Os02g32660) causes premature termination of translation of the frameshift mutant protein. Figure 1 Existing literature indicates that... SBE3 The loss of function reduces the synthesis of amylopectin, resulting in underdeveloped grains. This is related to... gfls1 The mutant's phenotype is consistent, therefore... gfls1 The defective grain phenotype of the mutant may be due to... SBE3 This is caused by a lack of functionality.

[0044] According to previous reports SBE3 Functional loss also leads to an increase in resistant starch content in rice, so we... gfls1 The resistant starch content of the mutant grains was tested using the method specified in industry standard NY / T 2638-2014, "Determination of Resistant Starch in Rice and Rice Products - Spectrophotometric Method". The results showed... gfls1 The mutant grains contained 15.6% resistant starch, significantly higher than the 3.99% resistant starch content of the wild-type Zhongzao 35, further validating the presence of the rice starch branching enzyme gene. SBE3 The lack of is the cause gfls1The reasons for the poor grain development and increased resistant starch content in mutant rice under low temperature conditions. Given the important role of rice with high resistant starch content in the prevention and treatment of diabetes, we utilized... gfls1 The mutant was used as a donor hybrid to improve the backbone parent of hybrid rice, and targeted... gfls1 In mutants SBE3 We will determine the genotype, develop SNP molecular markers, and conduct marker-assisted selection breeding to improve the breeding efficiency of high-resistant starch rice varieties.

[0045] Example 2. gfls1 In mutants SBE3 Development and application of SNP molecular markers

[0046] 1. SBE3 Development of SNP molecular markers

[0047] according to gfls1 The mutant is located at 19358817 bp on chromosome 2, i.e. SBE3 The base type at position 1796 of the coding region is A, while the base type at position 35 in the wild type is T. Based on the polymorphism at this site, KASP primers were designed using Primer5 software. These primers will amplify the wild-type type (…). SBE3 A primer with a base type of T at position 1796 of the coding region is coupled with a FAM fluorescent label at its 5' end. This primer is labeled C2-19358817-wFAM (SEQ ID NO.2). This will amplify the mutant type ( SBE3 A HEX fluorescent label is added to the 5' end of the primer with base type A at position 1796 of the coding region. This primer is labeled C2-19358817-mHEX (SEQ ID NO.3). Another common primer is labeled C2-19358817-COM (SEQ ID NO.4). The KASP primer sequences are as follows:

[0048] C2-19358817-wFAM:

[0049] 5'-GAAGGTGACCAAGTTCATGCTGCTGAAAGTCATGATCAAGCACT-3'

[0050] C2-19358817-mHEX:

[0051] 5'-GAAGGTCGGAGTCAACGGATTGCTGAAAGTCATGATCAAGCACA-3'

[0052] C2-19358817-COM: 5'-GTAACCTTGTCCATCAACCAGAA-3'

[0053] 2. SBE3 Application of SNP molecular marker of

[0054] (1) DNA extraction of the sample to be tested

[0055] a) Take 2-3 cm of rice leaves from the rice sample to be tested, cut them into pieces, and put them into a 2 mL EP tube (which can be dried in a 65°C oven), add 400 μL of TPS extraction solution (TPS extraction solution formula: 100 mL of 1M Tris-HCL, 20 mL of 0.5M EDTA, 74.55g of KCl, and dilute to 1L), add a 5mm stainless steel bead, and put it into a tissue crusher for crushing.

[0056] b) Warm at 65°C for half an hour.

[0057] c) Put the centrifuge tube into the centrifuge, centrifuge at 12000 rpm for 3 minutes, and aspirate 130uL of supernatant into a 1.5mL centrifuge tube.

[0058] d) Add 130 μL of isopropanol (which can be pre-cooled) to the supernatant, mix gently (which can be frozen in a -20°C refrigerator for 30-60 min).

[0059] e) Centrifuge in the centrifuge, 12000 rpm, 5 minutes, discard the isopropanol, add 200 μL of prepared 75% ethanol solution, mix up and down, stand still, and then centrifuge to discard the alcohol solution and dry in the fume hood.

[0060] f) After drying, add 100 μL of TE buffer to the centrifuge tube containing the dried DNA precipitate, and after the DNA is dissolved, measure its concentration and quality with a MBA 2000 UV / VS spectrometer (Perkin ELemer Co., Ltd.). Then dilute the DNA mother liquor to 50ng / µL working solution with sterile water, and store it in a 4°C refrigerator for use in the next step of the experiment.

[0061] (2) PCR amplification of the SNP molecular marker of SBE3.

[0062] a) Configure the PCR amplification reaction system

[0063] The PCR amplification reaction system is 10 μl: 5 μl of 2x Flu-AS PCR Mix for KASP (Changsha Chuandun Biological Technology Co., Ltd.), 0.15 μl of 10 μM nucleotide sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.3 μl of 10 μM nucleotide sequence as shown in SEQ ID NO: 4, 2 μl of DNA template (50-100 ng / ul) of the rice sample to be tested, and the rest is ultrapure water, a total of 10 μl.

[0064] b) PCR amplification reaction

[0065] PCR amplification program: 94℃ pre-denaturation for 3 min, 10 cycles (94℃ denaturation for 20 s, 65℃~57℃ annealing and extension for 40 s), with the annealing and extension temperature decreasing by 0.8℃ in each cycle, 28 cycles (94℃ denaturation for 20 s, 57℃ annealing and extension for 40 s) to obtain PCR amplification products.

[0066] (3) SBE3 genotyping analysis of the test samples

[0067] The obtained PCR products were scanned and genotyped using a Bio-Rad CFX96 Touch real-time quantitative PCR instrument. The specific procedure was: 37℃, 1 min, Plate Read, End. If 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 tested rice was a highly resistant starchy rice. If FAM fluorescence signal was detected, the genotype of the SBE at position 1796 in the coding region was T / T, and the tested rice was not a highly resistant starchy 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 tested rice was not a highly resistant starchy rice.

[0068] Will gfls1 Seventeen mutants, 17 strains of radiated 9811, and 17 F1 hybrids were taken and PCR amplified using the above-mentioned KASP primers. The amplification products were then subjected to fluorescence scanning and genotyping. The results are as follows: Figure 2 As shown, the PCR product using Fu9811 as a template detected FAM fluorescence signal, therefore the SBE3 genotype of Fu9811 at position 1796 in the coding region is T / T, and it is not a highly resistant starchy rice; gfls1 The PCR product using the mutant as a template detected a HEX fluorescence signal, therefore gfls1 The mutant SBE3 has an A / A genotype at position 1796 in the coding region, indicating it is a highly resistant starchy rice. gfls1The fluorescence signal of the hybrid F1 of the mutant and Guang9811 is located in the middle of FAM and HEX, and has obvious boundary distinguishing from FAM and HEX, so the genotype is hybrid. The above detection results are all consistent with the actual situation, which shows that the SNP molecular marker designed according to the polymorphism of the base at the 1796th position of the SBE3 coding region, i.e. T or A, can accurately distinguish the different allelic genotypes of T or A by using 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).

[0069] (4) Analysis of the resistant starch content of the sample to be tested

[0070] In order to further verify the detection effect of the SNP molecular marker on the SBE3 genotype, the above-mentioned gfls1 The resistant starch content of the rice of the mutant, Guang9811 and their hybrid F1 is detected. The resistant starch content determination results are consistent (Table 1).

[0071]

[0072] Therefore, it is shown that the SNP molecular marker of SBE3 provided by the present application is closely linked to the high resistant starch content trait locus, can accurately screen single plants with high resistant starch content, and can be used for molecular assisted breeding of high resistant starch rice to improve the breeding efficiency.

[0073] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a rice SNP molecular marker in identifying a rice plant with high resistant starch content, characterized in that, The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, and the polymorphic base at the 1796th position from the 5' end is T or A; When the polymorphic base of the SNP molecular marker corresponds to A, the rice is high in resistant starch content.

2. A set of KASP primer combinations characterised in that, The KASP primer combination comprises two specific primers C2-19358817-wFAM and C2-19358817-mHEX and one universal primer C2-19358817-COM, the nucleotide sequence of the specific primer C2-19358817-wFAM is shown as SEQ ID NO. 2, the nucleotide sequence of the specific primer C2-19358817-mHEX is shown as SEQ ID NO. 3, and the nucleotide sequence of the universal primer C2-19358817-COM is shown as SEQ ID NO.

4.

3. A kit characterized in that, The primer combination of claim 2.

4. The use of any one of the primer combination of claim 2 or the kit of claim 3: (1) in identifying rice high in resistant starch content; (2) in breeding rice high in resistant starch content.

5. A method for identifying a rice plant having a high content of resistant starch, characterized in that, The method comprises the following steps: Using the DNA of the rice sample to be tested as a template, the KASP primer combination of claim 2 or the kit of claim 3 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.

6. The method of claim 5, wherein The PCR reaction system is 10 μl, which comprises 5 μl of 2xFlu-ASPCR Mix for KASP, 0.15 μl of 10 μM C2-19358817-wFAM and C2-19358817-mHEX primers respectively, 0.3 μl of 10 μM C2-19358817-COM primer, 2 μl of DNA template of the rice sample to be tested, and the rest is ultrapure water, and the total volume is 10 μl; The PCR amplification reaction program is: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 20 s, 65℃-57℃ annealing and extension for 40 s for 10 cycles, the temperature of annealing and extension is reduced by 0.8℃ for each cycle, 94℃ denaturation for 20 s, 57℃ annealing and extension for 40 s for 28 cycles.

7. The method according to any of claims 5 or 6, characterized in that, In the amplification result, the rice identified by the KASP primer combination as having the base type A of the SNP molecular marker is high in resistant starch content, and the rice identified as having the base type T is not high in resistant starch content.

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