KASP molecular marker of rice mannan synthase gene CSLA12 and application thereof

Abstract

The application discloses a KASP molecular marker of a rice mannose synthase gene CSLA12 and application thereof, and relates to the technical field of biology.The KASP molecular marker is KASP_781, the KASP_781 site is a SNP site in a rice genome, is located at the 22901781th position of a 9th chromosome of the rice, and the polymorphism is A or C.The application also provides application of the KASP molecular marker in detection of rice endosperm mannose content.The application applies KASP technology to genotype identification of a rice mannose synthase CSLA12 gene, and has the advantages of simple operation, low cost, short detection period, stable marker, green environmental protection and the like, and effectively solves the problems of low efficiency and high cost of an existing detection method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a KASP molecular marker for the rice mannan synthase gene CSLA12 and its application. Background Technology

[0002] Rice dietary fiber is mainly found in the husk, bran, and endosperm cell walls, including cellulose, hemicellulose, pectin, resistant starch (RS), and lignin. During rice polishing, the dietary fiber content decreases significantly (approximately 0.1%-1.5% in polished rice and 3%-4% in brown rice), while whole grains (such as green rice and red rice) retain the bran and germ, resulting in higher dietary fiber content. Dietary fiber content directly affects the processing characteristics of rice. High-fiber varieties have higher flour content and loosely arranged starch granules, making them more prone to breakage during milling, leading to a lower head rice yield. However, moderate dietary fiber can improve the texture of cooked rice, such as increasing stickiness and elasticity, while also slowing down starch digestion and prolonging satiety. Therefore, researching the regulation of rice dietary fiber genes to improve the breeding efficiency of multi-purpose, high-quality rice has become one of the important directions of rice research in my country.

[0003] Mannose is an important component of plant cell wall polysaccharides, and it affects the processing and eating quality of rice by regulating the structure of cell wall polysaccharides. For example, mannose participates in the synthesis of β-glucan and hemicellulose, affecting the hardness and cooking properties of rice grains.

[0004] The rice mannose synthase gene mainly belongs to the cellulose synthase-like protein (CSL) family, which is a member of the glycosyltransferase 2 (GT2) superfamily, and possesses a conserved D, D, D, QXXRW motif. Based on sequence homology and function, 11 members have been identified so far (Wang, LQ, et al. (2010). Expression profiling and integrative analysis of the CESA / CSL superfamily in rice. BMC Plant Biology. doi:10.1186 / 1471-2229-10-282), including CSLA1, CSLA2, CSLA3, CSLA4, CSLA5, CSLA6, CSLA7, CSLA8, CSLA9, and CSLA11. Among them, the mannose synthase gene CSLA12, identified as OsMnS1 by GWAS, has been reported to significantly affect grain mannose content and chalkiness. The gene has a 10 bp insertion at position 22901956 on chromosome 9 of indica rice, which allows the gene to synthesize mannan in the endosperm. This invention found that a mutation at position 22901781 can cause the gene to lose its function, thereby leading to a decrease in mannan content in indica rice grains.

[0005] Molecular markers can select target plants at the DNA level at any stage of plant development, thus overcoming many drawbacks of traditional breeding and becoming an effective way to solve the problem of difficult variety selection. However, although many linkage markers of rice mannan-related genes can be used for marker-assisted selection, they are mostly RFLP markers, SSR markers, InDel markers, or enzyme digestion markers, which have low detection efficiency and are not suitable for high-throughput molecular detection platforms. Therefore, developing low-cost, high-throughput detectable molecular markers is an urgent need to promote the identification of the rice mannan synthase CSLA12 gene, increase the accuracy of rice mannan dietary fiber breeding, and improve breeding efficiency. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, this invention provides a KASP molecular marker for the rice mannan synthase gene CSLA12 and its application. This marker has advantages such as simple operation, low cost, short detection cycle, stable labeling, and environmental friendliness, effectively solving the problems of low efficiency and high cost in existing detection methods.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide the application of the KASP molecular marker of the rice mannan synthase gene CSLA12 in the detection of mannose content in rice endosperm.

[0008] Furthermore, the aforementioned KASP molecular marker is KASP_781, located at the SNP site at position 22901781 on rice chromosome 9, with polymorphisms of A or C.

[0009] Furthermore, the above application is at least one of the following (1)-(4): (1) Application in detecting rice genotypes; (2) Application in the preparation, identification or auxiliary preparation and identification of rice endosperm high-mannose or low-mannose products; (3) Application in rice assisted breeding.

[0010] (4) Application in the preparation of rice-assisted breeding products.

[0011] Furthermore, reagents or kits containing the aforementioned primer set.

[0012] Furthermore, the application of the aforementioned primer sets, reagents, or kits in the detection of mannose content in rice endosperm.

[0013] A method for detecting the mannose content in rice endosperm, characterized by comprising the following steps: S1. Extract the genomic DNA of the rice to be tested; S2. Using the rice genomic DNA to be tested as a template, KASP reaction detection was performed using the above primer set; S3. If the genotype corresponding to the SNP site is AA, the rice to be tested is low-mannose rice. If the genotype corresponding to the SNP site is CC, the rice to be tested is high-mannose rice. If the genotype corresponding to the SNP site is H, the rice to be tested is heterozygous rice with a high-mannose phenotype.

[0014] In summary, the present invention has the following beneficial effects: 1. This invention provides the development and application of a KASP marker tightly linked to the 781 locus of the rice mannan synthase CSLA12 gene. The molecular marker is KASP_781, a KASP marker tightly linked to the rice mannan synthase CSLA12 gene. This marker is developed based on KASP technology and can perform high-throughput detection of the 22901781st base on chromosome 9 of the maize genome. This invention applies KASP technology to identify the genotype of the rice mannan synthase CSLA12 gene, which has the advantages of simple operation, low cost, short detection cycle, stable marker, and environmental friendliness. It can accurately detect the 781 locus of the rice mannan synthase CSLA12 gene, which is of great significance for promoting functional breeding of rice with different mannose contents.

[0015] 2. This invention discloses the development method and application of the KASP molecular marker for the rice mannan synthase gene CSLA12, belonging to the field of biotechnology. It involves KASP achieving genotyping by specifically recognizing gene loci using fluorescent probes, and can be used to detect SNP sites. Compared with molecular markers such as SSR, RFLP, and InDel, KASP markers offer advantages such as rapid detection, low cost, and ease of large-scale application. KASP markers do not require genotyping based on DNA fragment size, eliminating the need for traditional gel electrophoresis, which is relatively cumbersome, low-throughput, and expensive. This makes it more suitable for the rapidly developing high-throughput molecular detection platforms. Therefore, developing a low-cost KASP molecular marker for the rice mannan synthase gene CSLA12, suitable for high-throughput molecular detection platforms, is of great significance for promoting the application of molecular marker technology and improving the breeding efficiency and level of rice in my country. Attached Figure Description

[0016] Figure 1 To detect the genotypes of 33 tested rice varieties using the molecular marker KASP_781. Detailed Implementation

[0017] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0018] Example 1 Primer Design and KASP Reaction 1. Primer design Flanking sequences of 50 bp each were extracted from the KASP_781 site. Ten sets of KASP primers were designed using Primer 5.0 software. After whole-genome BLAST analysis of rice using NCBI, one set of KASP primers with good polymorphism was selected for further validation. The specific KASP primers used for detecting KASP_781 are as follows: Primer A1F1: 5'-gaaggtgaccaagttcatgctGGGTGGAAGGAGCGGAC-3' (SEQ ID No. 1, the lowercase part is the specific fluorescent tag sequence FAM); Primer A2F2: 5'-gaaggtcggagtcaacggattGGGTGGAAGGAGCGGAA-3' (SEQ ID No. 2, the lowercase part is the specific fluorescent tag sequence HEX); Primer R: 5'-CTCAGGCTCGCCCGCA-3' (SEQ ID No. 3) 2. DNA extraction Genomic DNA was extracted from rice leaves using the conventional CTAB method.

[0019] 3. KASP reaction KASP labeling amplification and reaction system: Detection using Bio-Rad CFX7600 real-time PCR instrument: The 10 μL PCR quantitative PCR instrument reaction system included: 125 ng genomic DNA, 0.14 μL primer mixture (preferred primer mixture ratio: 36 μM each for forward primers A1F1 and A2F2, 90 μM for reverse primer R, and a 1:1:1 mixture of forward and reverse primers), and 5 μL of KASP PCRMixPlus from Dongsheng Biotechnology Co., Ltd.; following the Bio-Rad CFX7600 instrument operation manual, the sample table was edited, the running program was executed, and the fluorescence intensity data of FAM (excitation wavelength 485 nm, emission wavelength 520 nm) and HEX (excitation wavelength 535 nm, emission wavelength 556 nm) were read and saved.

[0020] The above reaction system is the preferred reaction system for Bio-Rad CFX7600. Other reasonable reaction systems can also achieve the same detection purpose.

[0021] The above are recommended detection methods. Other detection methods that can achieve the same detection purpose can also be applied to the molecular marker-assisted breeding process of the above markers.

[0022] The 2×KASP Mix consists of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B, as well as high-fidelity Taq enzyme, dNTPs, and Mg. 2+ The components are as follows: Fluorescent probe A has the nucleotide sequence 5'-GAAGGTGACCAAGTTCATGCT-3', with a FAM fluorescent group attached to its 5' end; Fluorescent probe B has the nucleotide sequence 5'-GAAGGTCGGAGTCAACGGATT-3', with a HEX fluorescent group attached to its 5' end; Quenching probe A has the nucleotide sequence 5'-AATCCGTTGACTCCGACCTTC-3', with a BHQ quenching group attached to its 3' end; Quenching probe B has the nucleotide sequence 5'-AGCATGAACTTGGTCACCTTC-3', with a BHQ quenching group attached to its 3' end.

[0023] Amplification program: 95℃ pre-denaturation for 1 min, 1 cycle; 95℃ denaturation for 15 s, 58-52℃ annealing for 15 s, 72℃ extension for 20 s, 10 cycles, with the annealing temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 20 s, 30 cycles; finally, read the plate for 60 s at a solution temperature of 30℃.

[0024] The experiment also included a blank control (NTC) in the reaction system without template DNA, with one or more blank controls in each plate.

[0025] Example 2: Analysis of Scanning Data The scan data were analyzed, and then the genotype of the KASP_781 locus in the rice genome was determined as follows (i.e., whether the 22901781st base on chromosome 9 of the maize genome is A or C). If the fluorescence signal data of the amplification product of the rice was analyzed by CFX Maestro Allelic... If the fluorescence signal of the amplification product of the rice sample is close to the X-axis (FAM signal) according to the genotyping analysis, then the genotype of the KASP_781 locus in the rice genome is CC homozygous (i.e., the 22901781st base of chromosome 9 of the rice genome is C homozygous). If the fluorescence signal data of the amplification product of the rice sample is close to the Y-axis (HEX signal) according to the genotyping software analysis, then the genotype of the KASP_781 locus in the rice genome is AA homozygous (i.e., the 22901781st base of chromosome 9 of the rice genome is A homozygous). If the fluorescence signal data of the amplification product of the rice sample is located between the X-axis and Y-axis (HEX and FAM signals) according to the genotyping software analysis, then the genotype of the KASP_781 locus in the rice genome is H heterozygous (i.e., the 22901781st base of chromosome 9 of the maize genome is H heterozygous). The sample shown in black in the lower left corner is the blank control.

[0026] Example 3: Labeling and Genotyping Data Analysis To verify the reliability of the KASP_781 marker, a mannose phenotype survey was conducted on 33 rice varietals, and the results are shown in Table 1. Genotyping of the materials was performed using a genotyping platform. The detection method, reaction system, and amplification procedure followed the optimized scheme described above, and the results are as follows: Figure 1 As shown. Figure 1 In the diagram, circle CC represents genotype CC (high mannose rice), triangle AA represents genotype AA (low mannose rice), and square represents genotype H.

[0027] Table 1. Mannose content and genotype information of 33 tested rice varieties.

[0028] Depend on Figure 1 As shown in Table 1, the amplification results indicate that the KASP_781 marker can produce stable PCR products in 33 materials, and both A and C alleles can be detected. Fifteen rice samples with high mannose content were identified as having the CC genotype, 16 as having the AA genotype, and two as having the H genotype. Therefore, it can be seen that the KASP_781 marker can be used for breeding rice with varying mannose levels.

[0029] Therefore, the KASP_781 marker described in this invention can be used for marker-assisted breeding of the rice mannose content gene CSLA12.

[0030] It should be noted that this invention constructs F4 recombinant inbred lines with high and low mannose parents and performs BSA mining to find an SNP site that is related to the mannose content of rice grains. This site is anchored at position 22901781 on rice chromosome 9.

[0031] (https: / / rapdb.dna.affrc.go.jp / , genome version IRGSP-1.0), the SNP site was named KASP_781, and its flanking sequence is shown in SEQ ID No.4. KASP_781 is located at position 51 of SEQ ID No.4, and the nucleotide sequence at this position is C or A.

[0032] The A genotype indicates that the nucleotide sequence of KASP_781 is homozygous for AA; the CC genotype indicates that the nucleotide sequence of KASP_781 is homozygous for C; and the H genotype indicates that the nucleotide types of KASP_781 are heterozygous for both A and C.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of primer set for detecting KASP molecular marker of rice mannan synthase gene CSLA12 in detecting mannose content in rice endosperm, wherein the nucleotide sequence of the primer set is shown in SEQ ID No.1-SEQ ID No.

3.

2. The application as described in claim 1, wherein the application is at least one of the following (1)-(3): (1) Application in the preparation and identification or auxiliary identification of rice endosperm high-mannose or low-mannose products; (2) Application in rice-assisted breeding; (3) Application in the preparation of rice-assisted breeding products.

3. A primer set for detecting the KASP molecular marker as described in claim 1, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID No. 1-SEQ ID No.

3.

4. A reagent or kit containing the primer set as described in claim 3.

5. The application of the primer set of claim 3, the reagent or kit of claim 4 in the detection of mannose content in rice endosperm.

6. A method for detecting mannose content in rice endosperm, characterized in that, Includes the following steps: S1. Extract the genomic DNA of the rice to be tested; S2. Using the rice genomic DNA to be tested as a template, KASP reaction detection was performed using the primer set described in claim 3; S3. If the genotype corresponding to the SNP site is AA, the rice to be tested is low-mannose rice. If the genotype corresponding to the SNP site is CC, the rice to be tested is high-mannose rice. If the genotype corresponding to the SNP site is H, the rice to be tested is heterozygous rice with a high-mannose phenotype.

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