Primer group for identifying KASP marker for albinism of branches caused by mutation of rice wpb1 gene locus, detection product and application of primer group for identifying KASP marker for albinism of branches caused by mutation of rice wpb1 gene locus

Abstract

The invention discloses a primer group for identifying a KASP marker for albinism caused by mutation of a rice wpb1 gene locus, a detection product and application, the KASP marker takes an SNP locus closely related to a rice branch albinism gene as a reference, the SNP locus comprises: a 11371610 bp locus located at a rice first chromosome, the corresponding mutation genotype being C / C homozygosis, and a 11371610 bp locus located at a rice second chromosome, the corresponding mutation genotype being C / C homozygosis, the mutation genotype being C / C homozygosis, the mutation genotype being C / C homozygosis, the mutation genotype being C / C homozygosis, and the mutation genotype being C / C homozygosis; through the primer group and the detection product comprising the primer group, the rice material with wpb1 mutation can be rapidly screened and identified, and the primer group and the detection product have important significance in research of auxiliary breeding, variety identification, seed production and impurity removal and the like of ornamental rice.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers in molecular biology, in particular to a primer set, a detection product and an application of a KASP marker for identifying branch and stem albino lesions caused by mutation of a rice wpb1 gene site. Background Art

[0002] Colored rice refers to rice with special colors in its tissues or organs. There are three main categories: one is colored leaves, which are purple, red, yellow, white, white and green stripes, etc.; the second is colored rice, which is rice with black, purple, red, yellow and green seed coats; the third is colored husks, which are purple-black, brown, golden yellow, brown, etc., but the color of the leaves and rice is the same as that of ordinary rice. Some rice varieties have colored genes. Rice breeders use traditional breeding methods to cross rice carrying colored genes with ordinary rice, and they can breed new colored rice varieties with stable target traits. Among them, rice stalks that are white are also a type of colored rice. In the process of breeding experts studying albino seedlings, they also studied the related factors that lead to the whitening of rice stalks.

[0003] In his paper "A study on the mechanism of rice wpb1 mutation causing branch albinism and offspring seedling albinism causing death", Dr. Cai Zhongquan revealed the development and genetic rules of branch albinism and seedling albinism caused by wpb1 mutation, located the QTL that interacts with wpb1 mutation to cause seedling albinism, and analyzed the formation mechanism of branch albinism and seedling albinism by multi-omics. The main results are as follows: The wpb1 mutation phenotype and its genetic rules Rice white branch albinism is a new type of branch albinism, the branch remains white and in the tillering stage There are white-striped young leaves; white peduncles and white-striped leaves are recessive traits. In the F2 population of the white peduncles mutant and Nipponbare, the green peduncles and white peduncles are separated in a 3:1 ratio; there is no green tissue in the white peduncles, and no normal chloroplasts with thylakoid structure; the color difference of the peduncles begins in the third stage of young spike differentiation, but the obvious white and green phenotypes differentiate in the seventh stage of young spike differentiation; the proplastids of the white peduncles proliferate less in the third to fifth stages of young spike differentiation, and cannot form thylakoid structures normally during the development process of transformation to chloroplasts in the sixth and seventh stages of young spike differentiation. Summary of the invention

[0004] The invention discloses a primer set, a detection product and an application of a KASP marker for identifying a rice wpb1 gene site mutation that causes branch and stalk albino disease. The primer set and the detection product comprising the primer set can be used to quickly screen and identify rice varieties with wpb1 mutations, which is of great significance to the research on auxiliary breeding, variety identification, seed production and weed removal of ornamental rice.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] A primer set for identifying a KASP marker caused by a mutation in a rice wpb1 gene locus leading to branch and stalk albino disease, wherein the KASP marker uses a SNP locus closely related to a rice branch and stalk albino disease gene as a reference, wherein the SNP locus includes: a 11371610 bp locus located on the first chromosome of rice, and the corresponding mutant genotype is C / C homozygous;

[0007] The SNP site is amplified and identified by primers as shown in SEQ ID No. 1 to SEQ ID No. 3 in the sequence listing.

[0008] The present invention also provides a detection reagent, a kit or a gene chip containing the above-mentioned KASP-labeled primer set.

[0009] The present invention also provides the use of a primer set or a detection product containing the above-mentioned KASP marker in auxiliary breeding of ornamental rice or in improvement of rice germplasm resources.

[0010] The present invention also provides the use of a primer set containing the above-mentioned KASP marker or the detection product of claim 2 in identifying rice varieties with mutations in the wpb1 gene locus.

[0011] The KASP-marked primer set and the detection product comprising the primer set of the present invention can quickly screen and identify rice varieties with wpb1 gene site mutations, with high specificity and sensitivity, and are of great significance for auxiliary breeding, variety identification, and seed production and weed removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram showing the detection results of identifying parents and individual plants in the BC2F2 population using the method of Example 2. DETAILED DESCRIPTION

[0013] The technical scheme of the present invention is described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical scheme of the present invention. The following examples and accompanying drawings are only used for exemplary description and cannot be construed as limiting the present invention. The reagents or materials used in the embodiments, if not otherwise specified, are all derived from commercial channels. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0014] Example 1 Primer design for KASP marker

[0015] The KASP marker uses SNP sites closely related to the rice albino gene as references. The SNP sites include: a 11371610bp site located on the first chromosome of rice, where a base substitution from T to C occurs, and the corresponding mutant genotype is C / C homozygous;

[0016] The specific primer pairs are:

[0017]

[0018]

[0019] In the above primer sequences, two forward primers and one reverse primer are designed for each SNP site, and each forward primer is provided with its own fluorescent linker sequence.

[0020] Example 2 Identification method

[0021] The genomic DNA of the rice samples to be identified was extracted respectively, and the genomic DNA was used as a template and KASP-labeled primers were used for PCR amplification to obtain PCR amplification products. PCR amplification was performed in an ABI7500 real-time fluorescence quantitative PCR instrument, and after the PCR was completed, the instrument could perform genotyping based on the fluorescence signal.

[0022] The rice material to be identified comes from the backcross generation population of the white-branched peduncle mutant (wpb1) material and the wild-type green-branched peduncle material Nipponbare (NIP). Nipponbare (NIP) was used as a hybrid parent and a recurrent parent, and the F2 segregation positioning population was constructed: NIP was used as the female parent, the white-branched peduncle mutant was used as the male parent, artificial emasculation and hybridization were performed to obtain hybrid seeds, the hybrid seeds were planted to obtain F1 generation plants, and their seeds were harvested to obtain F2 generation seeds. The backcross population BC2F2 was constructed. The white-branched peduncle single plant isolated from the F2 segregation generation was selected as the female parent, NIP was used as the male parent, and emasculation and hybridization were performed; NIP was used as the male parent and backcrossed with the white-branched peduncle single plant of the BC1F2 generation to obtain BC2F1; BC2F1 was planted and self-pollinated to obtain BC2F2 population seeds. 28 single plants from the BC2F2 population were randomly selected and planted with one plant each of Nipponbare and Huazhan as controls as the rice materials to be identified.

[0023] The above method for obtaining DNA from the leaves of the sample to be tested is as follows: (1) Collect an appropriate amount of young leaves, grind them into powder with liquid nitrogen, and put 0.4 g into a 1.5 mL centrifuge tube (pre-cooled at -20°C); (2) Preheat 1.5×CTAB to 95°C, add 1 mL to the centrifuge tube containing the leaf powder, and mix well (to prevent freezing and thawing); (3) Immediately place in a 65°C water bath for 30 minutes, and turn upside down once every 5 minutes; (4) Centrifuge for 5 minutes; (5) Pipette about 600 μL of the supernatant, add an equal volume (600 μL) of chloroform / isoamyl alcohol (24:1), and turn upside down several times until the lower liquid phase turns dark green; (6) Centrifuge for 5 minutes; (7) Take 450 μL of the supernatant and put it in a new 1.5 mL centrifuge tube, add 1 mL of 95% ethanol and 45 μL of 10M NH4AC, mix well, and place at room temperature for 10 minutes. (8) Centrifuge at 12000g for 10 min, remove the supernatant, wash the precipitate with 75% EtOH, and dry it naturally for about 30 min. (9) Add 50 μL TE or sterile water (containing 20 μg / RNase), place at 4°C overnight, and after the DNA is dissolved, test the DNA concentration and quality.

[0024] A mixed primer was formed according to the ratio of upstream typing primer 1: upstream typing primer 2: downstream universal primer = 1:1:3, and the final concentration of the primer was 10 μM.

[0025] The PCR reaction system is a 10 μL system: containing 5.0 μL of 2×KASP Master mix, 1.8 μL of mixed primers, 1.0 μL of 30-50 ng / μL rice sample DNA template, and 10 μL of ddH2O.

[0026] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 5 s, annealing and extension at 55°C for 15 s, 40 cycles; and final extension at 72°C for 10 min.

[0027] A no-template control was set up on each 96-well plate. After the PCR reaction was completed, the fluorescence data was read using a qPCR instrument at a temperature below 40°C. The FAM fluorescent tag sequence was read at an excitation light of 485 nm and an emission light of 520 nm, and the HEX fluorescent tag sequence was read at an excitation light of 535 nm and an emission light of 556 nm. The genotype of the 11371610 bp site was determined based on the typing results.

[0028] The identification results are as follows Figure 1 , as shown in Table 1:

[0029] Table 1 Detection results and phenotypes of individual plants in parents and BC2F2 population

[0030]

[0031]

[0032] The results in Table 1 show that Nipponbare and Hanazan have T / T at the site of 11371610bp, and the phenotype is green stalks; 7 individuals in the BC2F2 population have no mutation detected at the site of 11371610bp, and the phenotype is green stalks, 14 individuals have C / T as the mutant genotype at the site of 11371610bp, and the phenotype is green stalks, and 7 individuals have C / C as the mutant genotype at the site of 11371610bp, and the phenotype is white stalks. This shows that the genotype results obtained by the detection in this embodiment are consistent with the rice phenotype, and have high specificity and sensitivity.

Claims

1. A method for identifying rice wpb1 A primer set for KASP markers in which a gene locus mutation leads to branch and stem albino disease, characterized in that: The KASP marker uses a SNP site closely related to the rice stalk albino gene as a reference, and the SNP site includes: a 11371610 bp site located on the first chromosome of rice, and the corresponding mutant genotype is C / C homozygous; The SNP site is amplified and identified by primers as shown in the sequence listing SEQ ID No.1 to SEQ ID No.

3.

2. A detection product, characterized in that: A detection reagent, a kit or a gene chip containing the primer set for the KASP marker according to claim 1.

3. Use of the KASP-marked primer set of claim 1 or the detection product of claim 2 in assisted breeding of ornamental rice or in improvement of rice germplasm resources.

4. The KASP-labeled primer set of claim 1 or the detection product of claim 2 is used to identify wpb1 Application in rice varieties with mutations in gene loci.

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