KASP Molecular Marker of Rice Brittle Culm Gene OsBC1.1A and Its Application

Abstract

The present application discloses a KASP molecular marker of rice brittle culm gene OsBC1.1A and its application. In the KASP molecular marker, a base T is inserted after the 82nd position of the coding region of OsBC1 in rice, and the displacement of the coding gene sequence causes the rice to produce a brittle culm phenotype. Genotyping is performed according to the KASP molecular marker of the rice brittle culm gene OsBC1.1A provided by the present application. After PCR amplification, enzyme digestion and electrophoresis are not required, and multiple samples can be detected with high throughput, greatly improving the detection efficiency. It provides a high-throughput and low-cost rapid detection method for breeding to screen rice breeding materials carrying the brittle culm gene, and accelerates the breeding process.

Description

Technical Field

[0001] This application relates to the technical field of rice breeding, and particularly relates to a KASP molecular marker of rice brittle culm gene OsBC1.1A and its application. Background Art

[0002] The brittleness of rice straw is caused by the reduction of cellulose and the increase of hemicellulose. The brittle straw is an excellent source of feed for cattle and sheep. The brittle straw has good palatability, is easy to chew and digest for ruminants, and its nutritional value is also relatively improved.

[0003] Screening the gene that causes brittle culm in rice has very important application value for cultivating brittle culm rice. At present, there is no report on the application of molecular marker-assisted selection breeding for this rice gene in China. In addition, the traditional molecular markers are complicated to operate, difficult to achieve batch and accurate detection, which will seriously hinder the large-scale application of this gene in the process of rice molecular marker-assisted (MAS) breeding.

[0004] Developing functional gene markers for rice brittle culm genes and establishing an efficient and environmentally friendly detection system are of great significance for promoting the application of this gene in commercial breeding.

[0005] The KASP technology does not require synthesizing specific fluorescent primers for each SNP locus. Based on its unique ARM PCR principle, all locus detections finally use universal fluorescent primers for amplification, which greatly reduces the reagent cost of LGC KASP. It has both high-standard accuracy and reduced usage cost, and has better locus adaptability than Taqman. The detection results are consistent with the phenotypes, and the detection process does not require electrophoresis, reducing the pollution of the experimental operation process to the environment and the harm to the human body. Therefore, LGC KASP has very good applications in medical and agricultural detections. Therefore, it is of great significance to develop a rice brittle culm gene marker suitable for detecting SNP loci by the KASP method. Summary of the Invention

[0006] In view of this, the purpose of this application is to provide a KASP molecular marker of rice brittle culm gene OsBC1.1A and its application to solve one of the above technical problems to a certain extent.

[0007] In the first aspect, the embodiment of this application discloses a KASP molecular marker of rice brittle culm gene OsBC1.1A, and a base T is inserted after the 82nd position of the coding region of OsBC1 of rice in the KASP molecular marker.

[0008] In the embodiment of this application, the KASP molecular marker causes the coding gene sequence of the OsBC1 coding region of rice to shift, resulting in the brittle culm phenotype of rice.

[0009] In a second aspect, embodiments of the present application provide a set of primers for amplifying the KASP molecular marker sequence described in the first aspect, and the sequences are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3.

[0010] In a third aspect, embodiments of the present application provide a method for screening brittle-stem rice using the KASP molecular marker described in the first aspect and the primers described in the second aspect, including the following steps:

[0011] Extract the DNA of the rice sample to be tested;

[0012] Perform PCR amplification using the primers described in the second aspect to obtain a PCR product;

[0013] Perform genotyping detection on the amplified sample to screen out the rice samples with the brittle-stem rice mutation gene OsBC1.1A;

[0014] Verify the screening results.

[0015] In a fourth aspect, embodiments of the present application provide a kit for detecting the KASP molecular marker, and the kit contains the primers described in the second aspect and other reagents for amplifying the KASP molecular marker.

[0016] Compared with the prior art, the present application has at least one of the following beneficial effects:

[0017] The present application relates to a KASP molecular marker of the rice brittle-stem gene OsBC1.1A and its application. The KASP molecular marker OsBC1.1A-KASP is designed according to the SNP locus where a base T is inserted after the 82nd position in the coding region of the OsBC1 gene. According to the OsBC1.1A-KASP marker provided by the present application for genotyping, after PCR amplification, there is no need for enzyme digestion and electrophoresis, and multiple samples can be detected by high throughput, greatly improving the detection efficiency, providing a high-throughput, low-cost and rapid detection method for breeding to screen rice breeding materials carrying the brittle-stem gene, and accelerating the breeding process. Description of the Drawings

[0018] Figure 1 It is a result diagram of genotyping a sample population using the OsBC1.1A-KASP molecular marker provided by the embodiments of the present application.

[0019] Figure 2 It is a sequencing peak diagram of the wild (WT) allele genotype provided by the embodiments of the present application.

[0020] Figure 3 It is a sequencing peak diagram of the heterozygous genotype provided by the embodiments of the present application.

[0021] Figure 4 This is the sequencing peak map of the homozygous genotype of OsBC1.1A provided by the embodiments of the present application. Detailed implementation manners

[0022] In order to make the objectives, technical solutions and advantages of the present application more clear and understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. Reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.

[0023] Example 1

[0024] 1. Rice materials

[0025] The materials used in this example include wild-type rice varieties and rice varieties with brittle culm gene phenotypes. The test materials are from the laboratory of Hefei Jiangu Biology Technology Co., Ltd.

[0026] 2. Extraction of rice leaf DNA

[0027] Rice leaves were sampled at the three-leaf stage. 100 mg of leaves were taken and added with liquid nitrogen for sufficient grinding. The ground powder was used to extract DNA with a plant genomic DNA extraction kit produced by Beijing Juhemei Biology Co., Ltd. A Nanodrop 2000 ultra-micro spectrophotometer produced by Thermo Fisher Scientific was used to measure the DNA concentration, and the DNA concentration was adjusted to 50 ng / ul.

[0028] 3. Design of KASP molecular marker primers

[0029] According to the comparison between the rice brittle culm mutant gene and the wild type, it was found that there is an SNP site at the 83rd position in the coding region. According to the sequences upstream and downstream of the SNP site, a set of primers was designed, namely the specific primer OsBC1.1A-KASP-FP for the brittle culm gene, the wild-type OsBC1 gene primer WT-FP, and a reverse universal primer WT-RP. Their primer sequences are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively. Among them, the 5' end of the OsBC1.1A-KASP-FP is a HEX fluorescent signal tag, and its tag sequence is shown in SEQ ID No.4; the 5' end of the WT-FP is a FAM fluorescent signal tag, and its tag sequence is shown in SEQ ID No.5.

[0030] 4. PCR amplification reaction

[0031] The extracted DNA was added to a 96-well plate, and a reaction system was prepared for PCR amplification reaction. The PCR reaction system is shown in Table 1, wherein KASP PCR MIX is a premix produced by LGC, with a product number of KBS-1016-001.

[0032] Table 1

[0033]

[0034] The PCR amplification program is shown in Table 2.

[0035] Table 2

[0036]

[0037] 5. KASP genotyping

[0038] After the reaction, the data was read using an ABI7500 fluorescence quantitative PCR instrument at 25°C for 5s+PlateRead. After the band reading was completed, the data was aggregated and analyzed using the software provided by the ABI7500.

[0039] 6. Sequencing verification

[0040] To verify the accuracy of the OsBC1.1A-KASP marker, 33 F1 samples were amplified by PCR and then sequenced. The sequencing results were compared. The OsBC1.1A allele had a T at the 83rd position in the gene coding region, while the wild (WT) allele had a C at the 83rd position in the gene coding region.

[0041] result:

[0042] (1) Figure 1 As shown, according to the color classification of the two detected fluorescences, the genotype of the samples showing blue near the Allele1 axis is the wild (WT) allele type connected to the FAM fluorescent tag sequence, the genotype of the samples showing red near the Allele2 axis is the OsBC1.1A allele type connected to the HEX fluorescent tag sequence, and the genotype of the sample showing green in the middle is the heterozygous type. According to the genotyping results of the OsBC1.1A-KASP molecular marker, among the 33 rice samples, the genotype of 11 individual plants is the OsBC1.1A allele type; the genotype of 11 individual plants is the wild (WT) allele type, and the genotype of 11 individual plants is the heterozygous type.

[0043] (2) Figure 2 , 3 The peak diagrams of sequencing results of wild (WT) allele type and heterozygous single strain are shown respectively. Figure 4This is the sequencing peak map of the homozygous genotype of OsBC1.1A. The results of comparison with the genotyping results of the OsBC1.1A-KASP molecular marker are summarized in Table 3. Among the DNA samples of 33 rice samples, 11 samples showed the genotype of the OsBC1.1A allele, 11 samples showed the genotype of the wild (WT) allele, and 11 samples showed the heterozygous genotype. The results of the sequencing analysis were completely consistent with the results of genotyping using the OsBC1.1A-KASP molecular marker. This result indicates that the OsBC1.1A-KASP marker can accurately screen out individuals carrying the OsBC1.1A allele of the brittle culm genotype in the population.

[0044] Table 3

[0045]

[0046] Note: WT is the wild type; OsBC1.1A is the brittle culm genotype; H is the heterozygous type.

[0047] In summary, genotyping according to the OsBC1.1A-KASP marker provided by the present application provides a high-throughput and low-cost detection method for breeding and screening rice breeding materials carrying the brittle culm gene. The detection method is simple, fast, and has high accuracy.

[0048] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present application should be covered by the protection scope of the present application. Sequence Listing <110> Hefei Jiangu Biotechnology Co., Ltd. <120> KASP Molecular Marker of Rice Brittle Culm Gene OsBC1.1A and Its Application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 42 <212> DNA <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 1 gaaggtcgga gtcaacggat tggcgtatga tccgctggac ct 42 <210> 2 <211> 41 <212> DNA <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 2 gaaggtgacc aagttcatgc tgcgtatgat ccgctggacc c 41 <210> 3 <211> 25 <212> DNA <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 3 cccactttat cgtgatgttc ccctt 25 <210> 4 <211> 21 <212> DNA <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 4 gaaggtcgga gtcaacggat t 21 <210> 5 <211> 21 <212> DNA <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 5 gaaggtgacc aagttcatgc t 21

Claims

1. A set of KASP molecular marker primers for detecting the rice brittle culm gene OsBC1.1A, characterized in that, The primers are the specific primers OsBC1.1A-KASP-FP for the brittle culm gene, the primer WT-FP for the wild-type OsBC1 gene, and a reverse universal primer WT-RP, and their primer sequences are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively.

2. A method for screening brittle culm type rice using the primers described in claim 1, comprising the following steps: Extract the DNA of the rice sample to be tested; Perform PCR amplification using the primers described in claim 1 to obtain a PCR product. The 5' end of the specific primer OsBC1.1A-KASP-FP for the brittle culm gene is labeled with a HEX fluorescent signal, and the 5' end of the primer WT-FP for the wild-type OsBC1 gene is labeled with a FAM fluorescent signal; Perform genotyping detection on the amplified sample. According to the color classification of the two detected fluorescences, the genotype of the sample that aggregates and shows blue near the Allele1 axis is the wild (WT) allele genotype linked to the FAM fluorescent label sequence, the genotype of the sample that aggregates and shows red near the Allele2 axis is the OsBC1.1A allele genotype linked to the HEX fluorescent label sequence, and the genotype of the sample that shows green in the middle is the heterozygous type, and the rice samples with the brittle culm mutant gene OsBC1.1A are screened out; Verify the screening results.

3. A kit for detecting the KASP molecular marker of the rice brittle culm gene OsBC1.1A, characterized in that, The kit contains the primers described in claim 1 and other reagents for amplifying the KASP molecular marker.

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