Molecular marker for detecting specific mutation of rice high-resistance starch gene sbe3-rs and application of molecular marker

Abstract

The invention discloses a molecular marker for detecting specific mutation of a rice high-resistance starch gene sbe3-rs and application of the molecular marker. The molecular marker is developed and designed according to the fact that single SNP mutation (T-C) exists at the 16th exon of an sbe3-rs gene, the molecular marker is CAPS-SBE3 and is composed of a CAPS-SBE3-F primer pair and a CAPS-SBE3-R primer pair, the nucleotide sequence of the CAPS-SBE3-F primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of the CAPS-SBE3-R primer pair is shown as SEQ ID NO.2. The invention further discloses a detection method of the molecular marker. The molecular marker is high in specificity, the sizes of fragments of a PCR amplification product before and after enzyme digestion are controlled within 200-bp, electrophoresis detection can be carried out through high-resolution polyacrylamide gel, during electrophoresis, the migration rate is high, the band difference is obvious, and a large batch of rice materials (gt; 200) can be detected at the same time. The high-resistance starch gene sbe3-rs can be quickly, simply, conveniently and accurately identified and subjected to genotype analysis by utilizing the molecular marker, and the molecular marker can be well applied to breeding of new varieties of high-resistance starch rice and identification and screening of germplasm resources of the high-resistance starch rice.

Description

Technical Field

[0001] The present invention relates to the technical fields of rice genetic breeding and molecular biology, and more specifically, to a molecular marker for detecting specific mutations of a high resistant starch gene in rice sbe3-rs and its application. Background Art

[0002] Starch is the main source of human food and energy. After entering the human body, most of it is enzymatically hydrolyzed and digested and absorbed in the small intestine. A small part can escape enzymatic hydrolysis and enter the large intestine, where it is fermented and utilized by microorganisms and finally excreted from the body. The former is digestible starch (DS), and the latter is resistant starch (RS) (Wu Wei et al., 2006). Resistant starch has many important physiological functions such as lowering blood glucose, lowering blood lipids, promoting intestinal health, and promoting the digestion and absorption of minerals to increase nutrition (Wei et al., 2009; Shu et al., 2012; Maki et al., 2012; Zhou et al., 2014). Compared with those on a low resistant starch diet, individuals on a high resistant starch diet have less insulin response, which is of great significance for diabetic patients to control postprandial blood glucose levels. Especially for non-insulin-dependent patients, consuming high resistant starch foods can delay or inhibit the rise of postprandial blood glucose and effectively control and improve the condition of diabetic patients (Maki et al., 2012; Zhu Ping et al., 2015; Yang Ruifang et al., 2015). At present, the exploration of high resistant starch rice germplasm resources and the breeding and improvement of varieties have become research hotspots in hybrid rice breeding.

[0003] In recent years, rice genetic and breeding scientists at home and abroad have been committed to improving the resistant starch content of rice through means such as chemical mutagenesis, radiation mutagenesis, and traditional breeding, and a series of new rice varieties with high resistant starch content have been obtained (Fang Changyun et al., 2015). Among them, the mutant "Jiangtangdao 1" obtained by the Shanghai Academy of Agricultural Sciences using microspore culture technology is the first new functional japonica rice variety in China with a resistant starch content in grains more than 20 times higher than that of ordinary rice, and the major gene controlling the synthesis of resistant starch has been mapped sbe3-rs(Yang et al., 2012; 2016). Due to the low yield, low milling rate, and high production cost of "Jiangtangdao 1", its popularization and utilization are to a certain extent restricted. By using traditional pedigree breeding combined with a molecular marker-assisted selection breeding system, with "Jiangtangdao 1" as the donor parent, the Shanghai Academy of Agricultural Sciences further selected new high-resistant starch rice lines "Youtangdao 2" and "Youtangdao 3" that take both yield and quality into account. Their resistant starch contents are as high as 13.1% and 13.2% respectively, and they passed the Shanghai Crop Variety Approval in 2019 and 2020 respectively (Yang Ruifang et al., 2020; 2022).

[0004] Since the determination process of resistant starch content is complex, time-consuming, and costly, it has become an important limiting factor affecting the breeding and genetic research of high-resistant starch rice. Using molecular marker-assisted selection breeding is one of the most effective means (Wang Lin et al., 2009). Relevant research shows that resistant starch is controlled by genotype (Yang Shuming et al., 2012). Currently, the main research on the development and utilization of molecular markers closely linked to resistant starch content and the gene mapping related to controlling resistant starch content are as follows: In 2008, Mou Fanggui et al. reported that in the hybrid combination II-32B / RS111, RM72 located on chromosome 8 has a certain linkage relationship with the synthesis of resistant starch. Luo Xi et al. (2014) conducted gene mapping related to resistant starch content through the population constructed by Gongmi 3hao / Nipponbare, and screened out 3 pairs and 6 pairs of microsatellite markers that may be linked to the resistant starch content locus on rice chromosome 1 and chromosome 6 respectively. Yang et al. (2012) first mapped the major QTL locus controlling the resistant starch content in rice by forward genetic means using Jiangtangdao 1 as the research object. sbe3-rs And based on the single nucleotide polymorphism (SNP) mutation (T-C) site of this gene at the 16th exon, a sbe3-rs CAPS / SpeI functional marker within the gene locus was developed. By establishing a molecular marker-assisted breeding system for new rice varieties with high resistant starch content, it has become an important measure to reduce costs and accelerate popularization and utilization, which is of great significance for improving the breeding efficiency of high-resistant starch content rice and promoting its breeding process.

[0005] Although the CAPS / SpeI marker can be used for sbe3-rs the identification of major genes and the analysis of their genotypes, due to the relatively large molecular weights of its PCR amplification fragment (571-bp) and enzyme digestion products (375-bp and 196-bp), it is only suitable for detection using traditional agarose gel electrophoresis. The band migration rate is slow during the electrophoresis process, and it cannot detect a large number of rice materials at one time. Its identification process is time-consuming and laborious. Therefore, for sbe3-rsFor a specific gene mutation type, it is very necessary to design and develop a new type of molecular marker that has a fast detection rate, can be detected by high-resolution polyacrylamide gel electrophoresis, and can simultaneously perform sbe3-rs gene identification and genotype analysis on a large number of rice breeding materials at one time, which is of great significance for improving the detection efficiency and promoting the breeding process of high-resistant starch hybrid rice. SUMMARY OF THE INVENTION

[0006] In view of the above problems, the present invention provides a molecular marker for detecting specific mutations of high-resistant starch genes in rice sbe3-rs and its application.

[0007] The present invention adopts the following technical solutions: A molecular marker for detecting specific mutations of high-resistant starch genes in rice sbe3-rs The specific mutation is that sbe3-rs there is a single SNP mutation (T-C) at the 16th exon of the gene coding region, that is, the 1796th base of the CDS sequence is mutated from T to C. The molecular marker is CAPS-SBE3, which consists of the primer pair CAPS-SBE3-F and CAPS-SBE3-R. The nucleotide sequence of CAPS-SBE3-F is as shown in SEQ ID NO.1, and the nucleotide sequence of CAPS-SBE3-R is as shown in SEQ ID NO.2.

[0008] Furthermore, the present invention also provides the application of the above molecular marker in the breeding of new varieties of high-resistant starch rice and the identification and screening of high-resistant starch rice germplasm resources.

[0009] Even further, the present invention also provides a method for detecting specific mutations of high-resistant starch genes in rice sbe3-rs , which includes the following steps: Step S1: Extract the whole genome DNA of rice leaves; Step S2: Using the whole genome DNA of rice leaves in Step S1 as a template, perform PCR amplification on the rice genome with the molecular marker-specific special primer combination CAPS-SBE3-F and CAPS-SBE3-R; Step S3: Use restriction endonuclease Spe I to digest the PCR amplification product in Step S2; Step S4: Separate and detect the digested product in Step S3 by 8% non-denaturing polyacrylamide gel electrophoresis, with a voltage of 120 v and a time of 60 min; Step S5: Judge whether the detected rice material contains the major gene of high-resistant starch according to the electrophoresis band pattern sbe3-rs, if a 195-bp specific electrophoresis band appears, it is determined that the material contains one high resistant starch gene with a single SNP mutation (T-C) occurring in exon 16, and its rice has a high resistant starch content, belonging to the high resistant starch rice variety; if a 140-bp specific electrophoresis band appears, it is determined that the material does not contain sbe3-rs the high resistant starch gene, and its rice has a low resistant starch content, belonging to the low resistant starch rice variety; if both a 195-bp and a 140-bp specific electrophoresis band are present, it is determined that the material is heterozygous at the sbe3-rs gene locus, and its rice also has a low resistant starch content, belonging to the low resistant starch rice variety. sbe3-rs In a preferred embodiment, the reaction system for the above PCR amplification is a 10 μL system, including: 5.0 μL of 2×F8 PCR MasterMix, 1.0 μL of an equal volume mixture of CAPS-SBE3-F and CAPS-SBE3-R, 0.8 μL of DNA template, and 3.2 μL of ddH2O.

[0010] In a preferred embodiment, the reaction program for the above PCR amplification is: pre-denaturation at 95.0°C for 3 min; denaturation at 95.0°C for 10 s, annealing at 57°C for 10 s, extension at 72°C for 10 s, for 35 cycles; extension at 72°C for 5 min, and preservation at 4°C.

[0011] In a preferred embodiment, the reaction system for the above enzyme digestion is a 20 μL system, including: 3.0 μL of the PCR product, 1.0 μL of

[0012] restriction endonuclease I, 2.0 μL of 10×M Buffer, and 14.0 μL of ddH2O, and enzyme digestion at 37°C for 2 h. Spe As can be seen from the above description of the present invention, compared with the prior art, the present invention has the following advantages:

[0013] First, the molecular marker CAPS-SBE3 (composed of the primer pair CAPS-SBE3-F and CAPS-SBE3-R) of the present invention has strong specificity and belongs to a CAPS marker using restriction endonuclease I. The fragment sizes of its PCR amplification product before and after enzyme digestion are both controlled within 200-bp, and it can be detected by electrophoresis using a high-resolution polyacrylamide gel. During electrophoresis, not only is the migration rate fast, the band differences are obvious, but it is also suitable for simultaneously detecting a large number of rice materials (>200) at one time, with higher efficiency. Spe

[0014] ​II. For the molecular marker of the present invention, during electrophoresis, since the fragment sizes before and after enzyme digestion of the PCR amplification products are relatively small, both are controlled within 200 bp, their band migration rates are fast, specificity is strong, and differences are more obvious. Using this marker, the major gene for high resistant starch can be identified and genotype analyzed quickly, simply and accurately. sbe3-rs for identification and genotype analysis.

[0015] III. For the detection of specific mutations of the rice high resistant starch gene using the molecular marker of the present invention, after PCR amplification, the amplification product is digested with the restriction endonuclease sbe3-rs I, and the digested product can be separated and detected by 8% non-denaturing polyacrylamide gel electrophoresis. The operation method is simple, the resolution is high, the experimental cost can be significantly reduced and the accuracy of identification can be improved. Spe

[0016] IV. Since the high resistant starch gene sbe3-rs is a recessive gene, only in the homozygous state can its rice grains show a relatively high resistant starch content. Using the molecular marker of the present invention, not only can it be identified in the early stage of breeding such as the seedling stage, but also the genotype individuals in the homozygous and heterozygous states can be distinguished. Therefore, it can well avoid the loss of genes in the heterozygous state sbe3-rs and thus accelerate the breeding efficiency of high resistant starch rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Identification of the high resistant starch gene of different materials by the molecular marker CAPS-SBE3 in Example 1. In the figure, M: 100 bp DNA Ladder Marker; 1: Youtang 2; 2: Youtang 3; 3: Minghui 2155; 4: Minghui 86; 5: Minghui 63; 6: Fuhui 673; 7: Minhui 3301; 8: R498; 9: Nipponbare; 10: 9311. sbe3-rs

[0018] Figure 2 Identification of the high resistant starch gene of different materials by the molecular marker CAPS / SpeI in Example 1. In the figure, M: DL2000 DNA Marker; 1: Youtang 2; 2: Youtang 3; 3: Minghui 2155; 4: Minghui 86; 5: Minghui 63; 6: Fuhui 673; 7: Minhui 3301; 8: R498; 9: Nipponbare; 10: 9311. sbe3-rs

[0019] Figure 3 For the identification of individual plants of the F2 generation from the cross of Youtang 3 and Zhongjiazao 17 by the molecular marker CAPS-SBE3 in Example 2 sbe3-rsGenotype identification. In the figure, P1: Youtang No. 3; P2: Zhongjiazao 17; F1: Zhongjiazao 17 / Youtang No. 3; 1 - 12: Heterozygous single plants with low resistant starch in the F2 generation; 13 - 17: Homozygous single plants with low resistant starch in the F2 generation; 18 - 20: Homozygous single plants with high resistant starch in the F2 generation.

[0020] Figure 4 For the F2 generation single plants of the cross between Youtang No. 3 and Zhongjiazao 17 by the molecular marker CAPS / SpeI in Example 2 sbe3-rs Genotype identification. In the figure, P1: Youtang No. 3; P2: Zhongjiazao 17; F1: Zhongjiazao 17 / Youtang No. 3; 1 - 12: Heterozygous single plants with low resistant starch in the F2 generation; 13 - 17: Homozygous single plants with low resistant starch in the F2 generation; 18 - 20: Homozygous single plants with high resistant starch in the F2 generation; M: DL2000 DNA Marker. Specific implementation manners

[0021] The following describes the specific implementation manners of the present invention with reference to examples. To fully understand the present invention, many details are described below. However, for those skilled in the art, the present invention can be implemented without these details. For well-known components, methods, and processes, no further detailed description will be given below.

[0022] Example 1 The 10 rice materials used in the following examples include 2 high resistant starch rice varieties, Youtang No. 2 and Youtang No. 3 (whose resistant starch contents are as high as 13.1% and 13.2% respectively, and both contain the major gene for high resistant starch sbe3-rs ), and 8 rice varieties with relatively low resistant starch contents, Minghui 2155, Minghui 86, Minghui 63, Fuhui 673, Minhui 3301, R498, Nipponbare, and 9311. Among them, the first 6 varieties are rice restorer line parents with excellent comprehensive agronomic traits and wide applications. Youtang No. 2 and Youtang No. 3 are provided by the Shanghai Academy of Agricultural Sciences, and the 6 excellent restorer lines are independently selected by the Sanming Academy of Agricultural Sciences, the Fujian Academy of Agricultural Sciences, Sichuan Agricultural University and other units respectively.

[0023] This example provides a molecular marker for detecting specific mutations of the high resistant starch gene in rice sbe3-rs where the specific mutation is sbe3-rs a single nucleotide site polymorphism mutation (T - C) at the 16th exon of the gene coding region. The molecular marker is designed and developed based on the sbe3-rs polymorphism mutation of the gene at this site. This molecular marker is CAPS - SBE3, which consists of the primer pair CAPS - SBE3 - F and CAPS - SBE3 - R. The nucleotide sequence of CAPS - SBE3 - F is as shown in SEQ ID NO.1, and the nucleotide sequence of CAPS - SBE3 - R is as shown in SEQ ID NO.2.

[0024] The following is the molecular markers of the present invention for the high-resistance starch gene in rice sbe3-rs The specific steps for detecting specific mutations are as follows: Step S1: extracting whole genome DNA from leaves of 10 individual rice plants; Step S2: Using the whole-genome DNA from rice leaves obtained in Step S1 as a template, PCR amplification of the rice genome was performed using the marker-specific primer combination, CAPS-SBE3-F and CAPS-SBE3-R. The 10 μL PCR amplification reaction system consisted of 5.0 μL of 2×F8 Fast PCR MasterMix (Vazyme), 1.0 μL of an equal volume mixture of CAPS-SBE3-F and CAPS-SBE3-R (10 μmol / L), 0.8 μL of DNA template, and 3.2 μL of ddH2O. The PCR amplification protocol was as follows: initial denaturation at 95.0°C for 3 min; 35 cycles of denaturation at 95.0°C for 10 s, annealing at 57°C for 10 s, and extension at 72°C for 10 s; extension at 72°C for 5 min, and storage at 4°C.

[0025] Step S3: Using restriction enzymes Spe I. Enzyme digestion of the PCR amplification product in step S2; the enzyme digestion reaction system is a 20 μL system, including: 3.0 μL (1 μg) PCR amplification product, 1.0 μL Spe I restriction endonuclease, 2.0 μL of 10×M Buffer, 14 μL of ddH2O, and digest at 37℃ for 2 h.

[0026] Step S4: Separate and detect the enzyme digestion products of step S3 by 8% non-denaturing polyacrylamide gel electrophoresis at a voltage of 120 V for 60 min.

[0027] The electrophoresis results of 10 rice varieties after amplification and detection of the specific functional marker CAPS-SBE3 designed and developed by the present invention are as follows: Figure 1 shown.

[0028] At the same time, this example also uses the specific molecular marker CAPS / SpeI to identify the 10 rice varieties. sbe3-rs The identification of the gene was used as a control test, and its detection results were completely consistent with the molecular marker CAPS-SBE3 designed and developed by the present invention ( Figure 2 ).

[0029] like Figure 1 and Figure 2 As shown, containing sbe3-rsPCR amplification products of high resistant starch rice varieties Youtang 2 and Youtang 3 cannot be cut by restriction endonuclease Spe I, and a specific 195-bp band can be detected by the CAPS-SBE3 marker ( Figure 1 ), and a specific 571-bp band can be detected by the CAPS / SpeI marker ( Figure 2 ); PCR amplification products of 8 low resistant starch content rice varieties (Minghui 2155, Minghui 86, Minghui 63, Fuhui 673, Minhui 3301, R498, Nipponbare and 9311) can be cut by restriction endonuclease Spe I, and a specific 155-bp band with a smaller molecular weight can be detected by the CAPS-SBE3 marker ( Figure 1 ), while the CAPS / SpeI marker can detect two specific bands of 375-bp and 196-bp ( Figure 2 ). It shows that the molecular marker CAPS-SBE3 developed and designed for the sbe3-rs specific mutation of the present invention can be well used for the identification of the major gene of high resistant starch sbe3-rs .

[0030] Example 2 This example provides an application of a molecular marker for detecting a specific mutation of a high resistant starch gene in rice breeding of high resistant starch rice varieties. Specifically as follows: sbe3-rs (1) Rice materials (1) Rice materials The high resistant starch rice variety Youtang 3 containing the sbe3-rs gene, the low resistant starch rice variety Zhongjiazao 17 without the sbe3-rs gene, and the F1 generation obtained by crossing Youtang 3 and Zhongjiazao 17 as parents. After self-crossing the F1 generation, an F2 generation segregation population of 164 individual plants was obtained.

[0031] (2) Extraction of genomic DNA from rice leaves The genomic DNA of the leaves of the two parents, F1 and 164 F2 individual plants at the seedling stage was extracted by the improved CTAB method.

[0032] (3) PCR amplification and electrophoresis detection The reaction system for PCR amplification was a 10 μL system, including: 5.0 μL 2×F8 Fast PCR MasterMix (Vazyme), 1.0 μL of the equal volume mixture of CAPS-SBE3-F and CAPS-SBE3-R, 0.8 μL of DNA template, and 3.2 μL of ddH2O.

[0033] The PCR amplification reaction program was as follows: 95.0℃ pre-denaturation for 3 min; 95.0℃ denaturation for 10 s, 57℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃. Spe The enzyme digestion products were detected by 8% non-denaturing polyacrylamide gel electrophoresis at 120 V constant voltage for 60 min, stained with nucleic acid dye (Ultra GelRed), and photographed using a gel imaging system.

[0034] (4) Genotype analysis The existing molecular marker CAPS / SpeI and the specific functional marker CAPS-SBE3 designed and developed by the present invention were used to perform genotyping and identification of the two parents, Youtang 3 and Zhongjiazao 17, as well as the hybrid F1 and F2 generation plants, and the test results were compared and analyzed.

[0035] (5) Results and analysis The test results show that the present invention sbe3-rs The identification results of the specific functional marker CAPS-SBE3 for the genotype of the material in this example, which was independently developed and designed by the specific gene mutation, were completely consistent with the identification results of the existing molecular marker CAPS / SpeI. The PCR amplification product of Youtang No. 3 could not be identified by restriction endonucleases. Spe I cut, a 195-bp (CAPS-SBE3) or a 571-bp (CAPS / SpeI) electrophoresis band can be detected, showing a high-resistant starch band type; the PCR amplification product of Zhongjiazao 17 can be detected by restriction endonucleases Spe I cut, CAPS-SBE3 marker can detect a small molecular weight of 140-bp specific band, CAPS / SpeI marker can detect two specific bands of 375-bp and 196-bp, showing low-resistant starch band type; F1 showed a heterozygous band type ( Figure 3 and Figure 4 ). Among the 164 F2 plants, 35 plants showed high resistant starch homozygous band type, 86 plants showed heterozygous band type, and 43 plants showed low resistant starch homozygous band type. Figure 3 and Figure 4 As shown, the present invention is based on sbe3-rs The specific functional marker CAPS-SBE3 independently developed and designed for gene-specific mutations and the existing CAPS / SpeI marker were used to cross 20 F2 generation plants obtained by hybridizing Zhongjiazao 17 and Youtang 3. sbe3- rs Genotype electrophoresis test results: in the figure, 1-12 are heterozygous bands, 13-17 are homozygous bands of low-resistant starch, and 18-20 are homozygous bands of high-resistant starch.

[0036] The above results show that the specific functional molecular marker CAPS-SBE3 developed and designed by the present invention can efficiently, accurately and clearly identify the major gene for high resistant starch sbe3-rs and perform genotype analysis, and can be well applied to the breeding of new varieties of high resistant starch rice and the identification and screening of high resistant starch rice germplasm resources, thereby accelerating the breeding process of high resistant starch special rice.

[0037] The above is only the specific implementation manner of the present invention, but the design concept of the present invention is not limited thereto. Any non-substantive modification of the present invention using this concept shall fall within the scope of infringement of the protection scope of the present invention.

Claims

1. Detection of rice high resistant starch gene sbe3-rs Molecular marker for specific mutation, and the specific mutation is sbe3-rs There is a single SNP mutation at the 16th exon of the gene coding region, that is, the 1796th base of the CDS sequence is mutated from T to C, and its characteristics are as follows: The molecular marker is CAPS-SBE3, which consists of the primer pair CAPS-SBE3-F and CAPS-SBE3-R. The nucleotide sequence of CAPS-SBE3-F is shown as SEQ ID NO.1, and the nucleotide sequence of CAPS-SBE3-R is shown as SEQ ID NO.

2.

2. The molecular marker for detecting specific mutations of the rice high resistant starch gene according to claim 1 sbe3-rs , characterized in that: The application of the molecular marker in the breeding of new varieties of high-resistant starch rice and the identification and screening of high-resistant starch rice germplasm resources.

3. Detection method for high resistant starch gene of rice sbe3-rs Characterized by: Using the molecular marker as described in claim 1, comprising the following steps: Step S1: Extract the genomic DNA of rice leaves; Step S2: Using the genomic DNA of rice leaves in Step S1 as a template, perform PCR amplification on the rice genome using the molecular marker-specific primer combination CAPS-SBE3-F and CAPS-SBE3-R; Step S3: Use a restriction endonuclease Spe I to digest the PCR amplification product in Step S2; Step S4: Separate and detect the enzyme digestion products in Step S3 by 8% non-denaturing polyacrylamide gel electrophoresis, with a voltage of 120 v and a time of 60 min; Step S5: Determine whether the tested rice material contains the major gene for high resistant starch according to the electrophoretic band pattern sbe3-rs , if a specific electrophoretic band of 195-bp appears, it is determined that the material contains one high resistant starch gene with a single SNP mutation in the 16th exon sbe3-rs , and its rice has a high resistant starch content and belongs to the high resistant starch rice variety; if a specific electrophoretic band of 140-bp appears, it is determined that the material does not contain sbe3-rs the high resistant starch gene, and its rice has a low resistant starch content and belongs to the low resistant starch rice variety; if both 195-bp and 140-bp specific electrophoretic bands are present, it is determined that the material is heterozygous at the sbe3-rs gene locus, and its rice also has a low resistant starch content and belongs to the low resistant starch rice variety.

4. Detection method for the rice high resistant starch gene as claimed in claim 3 sbe3-rs characterized in that: The reaction system for the PCR amplification is a 10 μL system, including: 5.0 μL of 2×F8 PCR MasterMix, 1.0 μL of an equal-volume mixture of CAPS-SBE3-F and CAPS-SBE3-R, 0.8 μL of DNA template, and 3.2 μL of ddH2O.

5. Detection method for the rice high resistant starch gene as described in claim 4 sbe3-rs Characterized in that: The reaction program for the PCR amplification is: pre-denaturation at 95.0℃ for 3 min; denaturation at 95.0℃ for 10 s, annealing at 57℃ for 10 s, extension at 72℃ for 10 s, 35 cycles; extension at 72℃ for 5 min, and storage at 4℃.

6. Detection method for the rice high resistant starch gene as claimed in claim 5 sbe3-rs characterized in that: The reaction system for the enzyme digestion is a 20 μL system, including: 3.0 μL of the PCR product, 1.0 μL of Spe Restriction endonuclease I, 2.0 μL of 10× M Buffer, and 14.0 μL of ddH2O, and digest at 37 °C for 2 h.

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