CN115491429A - Detection primer and detection kit for rice wx gene and application of detection kit - Google Patents

Abstract

The invention discloses a detection primer and a detection kit for rice wx gene and application thereof. Aiming at the rice wx gene functional region, sequence difference of waxy rice and non-waxy rice wx genes is found out through sanger dideoxy sequencing technology to determine a waxy allelic specific molecular marker, two pairs of wx gene detection primers with nucleotide sequences shown as SEQ ID No.1-SEQ ID No.4 are designed, the number and the size of fragments of PCR products are detected through electrophoresis, whether the detected rice sample contains wx genes or not is judged, the contained wx genes are heterozygous or homozygous allelic types is judged, and then auxiliary breeding of the wx genes, quick judgment of wx gene homozygotes and heterozygotes and identification of the purity of the waxy rice varieties are realized. The invention obviously reduces the detection cost and provides technical support for the cultivation of new glutinous rice varieties, the purification and rejuvenation of core germplasm and the purity identification of production seeds.

Description

Detection primers, detection kits and applications of rice wx gene

technical field

The present invention relates to detection primers and detection kits for the rice wx gene. The present invention further relates to the aided breeding of the rice wx gene detection primers for the rice wx gene, rapid judgment of wx gene homozygotes and heterozygotes, or identification of the purity of glutinous rice varieties The invention belongs to the field of molecular markers and detection primers and applications of rice wx gene.

Background technique

Rice is one of the important grain crops in China. The national rice planting area accounts for about 30% of the grain crop area, and the output is close to half of the total grain output. Because of its good edible quality, glutinous rice is widely used in health and nourishing food, wine making, cake making, cold drinks and medicine, etc., and is deeply loved by the people.

Since waxyness is a recessive trait, factors such as mixing and powdering during production can easily cause the loss or degradation of waxyness, which is often fatal to the maintenance of the species. In order to ensure the specificity, consistency and stability of conventional varieties, it is necessary to purify and rejuvenate the varieties so as to maintain the excellent seed properties of the varieties. The usual methods are the strain cycle method and the "three-year three-field" method. The core of these two methods is to select "typical single plant" or "typical single panicle", which is based on empirical phenotype selection, and there is a certain degree of blindness. . In the process of waxy rice breeding, most of the segregating generations are in a heterozygous state due to the waxy character, and the amylose content cannot accurately reflect whether the waxy character is homozygous or not. Accurate analysis can only be carried out after the strain is stable, which greatly increases the chance of breeding. workload.

Starch synthesis in rice is controlled by the waxy gene (Wx) encoding the starch synthase (granulaebound starch synthase I, GBSS I) bound to starch granules. The allelic variation of Wx gene causes the difference of amylose content in different rice varieties. Amylose content in glutinous rice varieties is generally lower than 2%, which belongs to the recessive mutation of Wx gene (wx). Previous studies have shown that the glutinous rice reported so far is due to a 23bp insertion in the second exon region of the Wx gene, resulting in a frameshift mutation in the expression of the Wx gene. Sun Huaqin, Tian Zhixi and other scholars have designed functional molecular markers to distinguish waxy/non-waxy varieties based on this locus difference. But at the same time, Sun Huaqin et al. pointed out that since the base sequence of the wx gene functional region has a high GC content, GC Buffer and high-fidelity enzyme (LA Taq) suitable for GC rich structure amplification should be selected in the PCR reaction, and a higher setting should be set. The annealing temperature is higher, and the detection cost is higher. The marker Wx M1 designed by Tian Zhixi et al. for the functional region of the wx gene selected an annealing temperature of 58°C during the PCR reaction, and many bands appeared during electrophoresis detection, making it difficult to determine the results.

Therefore, it is an urgent technical problem to be solved to design detection primers for the rice wx gene functional region with strong specificity, high sensitivity and low detection cost.

Contents of the invention

One of the purposes of the present invention is to provide rice wx gene detection primers;

The second object of the present invention is to provide a detection kit containing the rice wx gene detection primer.

The third object of the present invention is to apply the rice wx gene detection primers to the auxiliary breeding of rice wx gene, the rapid judgment of wx gene homozygote and heterozygote, or the identification of the purity of glutinous rice varieties.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention firstly provides rice wx gene detection primers, any pair selected from primer pair (1) or primer pair (2), wherein, said primer pair (1) consists of a nucleotide sequence of SEQ ID No.1 Shown primer and nucleotide sequence are the primer composition shown in SEQ ID No.2; Described primer pair (2) by nucleotide sequence is the primer shown in SEQ ID No.3 and nucleotide sequence is SEQ ID The primer composition shown in No.4.

The present invention further provides a rice wx gene PCR detection kit, comprising: Taq enzyme, dNTP, Mg ²⁺ , deionized water and detection primers; wherein, the detection primers are designed with the rice wx gene functional region as the target The detection primer; As a preferred specific embodiment, the detection primer is selected from any pair in the primer pair (1) or the primer pair (2), and the primer pair (1) consists of a nucleotide sequence of The primer and nucleotide sequence shown in SEQ ID No.1 are primers shown in SEQ ID No.2 and form; Described primer pair (2) is the primer and nucleotide sequence shown in SEQ ID No.3 by nucleotide sequence The sequence is the primer composition shown in SEQ ID No.4.

The invention also includes the application of the rice wx gene detection primers in the auxiliary breeding of the rice wx gene, the quick judgment of the homozygote and the heterozygote of the wx gene, the identification of the purity of the glutinous rice variety, and the like.

The present invention provides a method for using the rice wx gene detection primer to determine whether there is a wx gene or a homozygote and a heterozygote of the wx gene in a rice sample, comprising:

(1) extracting the DNA of the rice sample to be detected;

(2) using the extracted DNA as a template, using the primer pair (1) or the primer pair (2) as PCR amplification primers to establish a PCR amplification system for PCR amplification;

(3) Utilize primer (1) to amplify, if the amplification result has only 140bp band, then the rice sample to be detected contains wx gene homozygote; If the amplification result only has 117bp band, then the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 140bp and 117bp at the same time, the rice sample to be detected is a heterozygous plant containing the wx gene;

Utilize the primer pair (2) to amplify, if the amplification result has only 199bp band, then the rice sample to be detected contains wx gene homozygote; If the amplification result only has 176bp band, then the rice sample to be detected does not contain wx gene ; If the amplification result contains two bands of 199bp and 176bp at the same time, the rice sample to be detected is a heterozygous plant containing the wx gene.

In order to transfer the wx gene into the rice material with excellent characters, the present invention also provides a method of using the rice wx gene detection primer to carry out rice wx gene assisted breeding, comprising:

Use excellent rice lines to cross and backcross glutinous rice materials containing wx gene, and use primer pair (1) (wx-1) or primer pair (2) (wx-2) to amplify the single plant wx of each hybrid (backcross) generation gene locus, and carry out polyacrylamide gel electrophoresis to the amplified PCR product; (2) use primer (1) to amplify, if the amplification result contains a 140bp band, then it can be judged that there is wx gene in the sample, use primer (2) Amplification. If the amplification result contains a 199bp band, it can be judged that there is a wx gene in the sample; thereby assisting in the selection of individual plants containing the waxy gene (wx) and having excellent agronomic traits.

The present invention also provides a method for quickly judging the homozygote of the wx gene in a single plant of a self-segregation population by using the rice wx gene detection primer, comprising:

(1) Extract the DNA of the single plant of the self-bred segregation population to be detected; (2) Use the extracted DNA as a template, and use the primer pair (1) or primer pair (2) as PCR amplification primers to establish a PCR amplification system for PCR Amplify; (3) use the primer pair (1) to amplify, if the amplification result only has a 140bp band, then the individual plant of the detection sample is a homozygous individual plant carrying the wx gene, if the amplification result contains two bands of 140bp and 117bp at the same time band, then the individual plant of the test sample is a heterozygous strain carrying the wx gene; use the primer pair (2) wx-2 to amplify, if the amplification result only has a 199bp band, then the individual plant of the test sample is a homozygous individual plant carrying the wx gene , if the amplification result contains two bands of 199bp and 176bp at the same time, it is detected that the individual plant of the sample is a heterozygous strain carrying the wx gene.

The present invention also provides a method for identifying the purity of glutinous rice varieties using the rice wx gene detection primer, comprising:

(1) extract the DNA of the glutinous rice variety to be detected;

(2) using the extracted DNA as a template, using the primer pair (1) or the primer pair (2) as PCR amplification primers to establish a PCR amplification system for PCR amplification;

(3) Use the primer pair (1) to amplify. If the result of electrophoresis shows that only the 140bp band is a single glutinous rice plant carrying a homozygous wx gene, the one containing two bands of 140bp and 117bp is a single glutinous rice plant carrying a heterozygous wx gene. strains, those containing only the 117bp band were mixed non-glutinous rice plants;

Utilize the primer pair (2) to amplify, if the electrophoresis result has only 199bp band, it is the single plant of glutinous rice carrying the homozygous wx gene, and the single plant containing two bands of 199bp and 176bp is the single plant of glutinous rice carrying the heterozygous wx gene, Those containing only the 176bp band are mixed non-glutinous rice plants;

(4) Calculate the rice sample purity according to the following formula:

In the formula:

P—sample purity;

N _T — is the number of seeds for inspection (number of seedlings, number of plants);

N _D — is the seed number (number of seedlings, number of plants) judged to be mixed.

As a preferred embodiment of the present invention, the PCR amplification reaction system includes: 0.2 μl of Taq enzyme, 2 μl of dNTP, 2 μl of Mg ²⁺ , 14 μl of DNA template, 1 μl of upstream primer, 1 μl of downstream primer, 5.8 μl of deionized water μl, 5×Buffer4 μl.

As a preferred specific embodiment of the present invention, the amplification program of the PCR amplification: after pre-denaturation at 95°C for 5 minutes, 1 minute at 94°C, 30s at 67°C, 40s at 72°C, 35 cycles, extension at 72°C for 10 minutes, 12 Store at ℃.

Aiming at the wx gene functional region, the present invention uses sanger dideoxy sequencing technology to find out the sequence difference between glutinous rice and non-glutinous rice wx gene, and develops wx gene-specific functional markers wx-1 and wx-2, and utilizes PCR technology for the markers Amplify the site, detect the number and size of PCR product fragments by electrophoresis, and judge whether the tested rice sample contains the wx gene and whether the wx gene contained is heterozygous or homozygous allele, and then realizes the assisted breeding of the wx gene , rapid judgment of homozygous and heterozygous wx genes and identification of the purity of glutinous rice varieties.

The invention develops waxy allele-specific molecular markers for sequence differences on the second exon of the wx gene of glutinous rice/non-glutinous rice varieties, and realizes genotyping of glutinous rice and non-glutinous rice by optimizing the reaction system and reaction program Accurate distinction significantly reduces testing costs, and provides technical support for the cultivation of new varieties of glutinous rice, the purification and rejuvenation of core germplasm, and the purity identification of production species.

The invention involves key term definitions and abbreviations

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell.Probes 8: 91-98 (1994)).

Gene molecular marker: An effective genetic marker closely related to a certain trait developed based on the sequence difference of a certain gene.

Description of drawings

Fig. 1 Base sequence of the second exon region of Wx gene.

Fig. 2 Flow chart of molecular-assisted selection of glutinous rice genes.

Figure 3 marks the electrophoresis pattern of wx-1; M: 20bp DNA standard molecular weight. 1-8 are glutinous rice varieties, respectively: Wankennuo 1, Wankennuo 2, Wankennuo 5, Wankennuo 1116, Zhennuo 19, Xuanjingnuo 1, Guangmingnuo 1, Wandao 68; 9-23 are non-waxy varieties, namely: Wankenjing 2, Wankenjing 3, Wankenjing 8, Wankenjing 516, Wankenjing 11036, Wankenjinqing, Zhendao 14, Zhendao 15, Zhendao 18, Dangyujing 2, Dangyujing 10, Changnongken 1, Wanjing 22, Tianjing 2 and Ningjing 3.

Figure 4 marks the wx-2 electrophoresis pattern; M: 20bp DNA standard molecular weight. 1-8 are glutinous rice varieties, respectively: Wankennuo 1, Wankennuo 2, Wankennuo 5, Wankennuo 1116, Zhennuo 19, Xuanjingnuo 1, Guangmingnuo 1, Wandao 68; 9-23 are non-waxy varieties, namely: Wankenjing 2, Wankenjing 3, Wankenjing 8, Wankenjing 516, Wankenjing 11036, Wankenjinqing, Zhendao 14, Zhendao 15, Zhendao 18, Dangyujing 2, Dangyujing 10, Changnongken 1, Wanjing 22, Tianjing 2 and Ningjing 3.

Fig. 5 Marker wx-1, wx-2 for assisted breeding of wx gene self-bred progeny; M: 20bp DNA standard molecular weight; 1-21: detection of single plant; 1, wx gene donor (Xuanjingnuo No. 7); 2. Wuyujing 3; 9, 18, 21: homozygous wx gene; 5, 6, 12, 13, 15, 20: heterozygous wx gene; the rest are single plants without wx gene.

Fig. 6 utilizes mark wx-1, wx-2 to carry out purity detection to two glutinous rice varieties respectively;

▲: WX gene heterozygous strain ★: No WX gene hybrid strain.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Sequence comparison of rice wx gene functional regions and design and verification of wx gene detection primers

1. Extraction of rice genomic DNA by CTAB method

The materials include 8 glutinous rice varieties including Wankennuo 1 and Zhennuo 19 and 15 non-glutinous rice varieties including Wankenjing 2 and Wanjing 22. Cut about 0.1g of the young leaves of the test material from each variety, transfer them into a 2.0mL centrifuge tube, add 2 grinding beads into the centrifuge tube, freeze them in liquid nitrogen, grind them fully to powder with a tissue grinder, and then add 800 μL 65 ℃ preheated CTAB extraction buffer (81.7gNaCl and 20g CTAB fully dissolved in appropriate amount of water, then add 1mol/L Tris-HCl 100mL, 0.5mol/LEDTA 40mL, dilute to 1000mL, store at 4℃.), incubate at 65℃ 1h, mix by inverting every 15min; add an equal volume of chloroform/isoamyl alcohol (24:1), mix gently, let stand at room temperature for 10min; centrifuge at 12,000rpm for 10min, absorb the supernatant and transfer to another 1.5mL centrifuge Add an equal volume of isopropanol to the tube, mix well, place at -20°C for 30min, centrifuge at 4°C, 12,000rpm for 10min, discard the supernatant, add 500μL of 75% ethanol to wash the precipitate; centrifuge at 12,000rpm for 10min, discard the ethanol, and After drying, add 50 μL sterilized water to fully dissolve. The extracted genomic DNA was measured by an implen Nanophotometer ultra-micro ultraviolet spectrophotometer to measure the concentration of each sample, and the OD ₂₆₀ /OD ₂₈₀ ratio was detected. After passing the test, the concentration was uniformly diluted to 50ng/μL with sterile water and stored at -20°C for later use.

2. Sequence determination of wx gene functional region

2.1 DNA sequencing primer sequence of wx gene functional region

Table 1 Wx gene sequencing primer information

2.2 PCR amplification

The rice genomic DNA was amplified using sequencing primers, and the composition of each component in the amplification system is shown in Table 2.

Table 2 PCR reaction system

Note: The reaction volume can be selected between 10-50μL, and the original concentration of template DNA can be between 20-200ng/μL.

The PCR reaction program was: 95°C pre-denaturation for 5 min, 94°C for 1 min, 55/58°C for 30 s, 72°C for 40 s, 35 cycles, 72°C extension for 10 min, and 12°C storage. PCR amplification products were detected by 1.5% agarose electrophoresis.

3. Sequence comparison of wx gene functional regions

SeqMan in the DNAstar software was used to complete the assembly of multiple fragments in the sequencing results, and the Clustal Omega software was used to perform multiple sequence alignments to find out the difference sites of wx gene specificity.

4. Design and verification of primers for wx gene detection

4.1 Primer design for wx gene detection

According to the unique differences in the functional regions of the wx gene, PCR detection primers wx-1 and wx-2 were designed using Primer Premier 5 software, and the sequences of the selected primers were compared on NCBI-blast to test their specificity.

Table 3 Functional marker information of waxy gene wx

4.2wx genetic testing verification

Use wx-1 and wx-2 primers to amplify glutinous rice and non-glutinous rice varieties respectively, and refer to Table 2 for the PCR reaction system.

The reaction program was as follows: 95°C pre-denaturation for 5 minutes, 94°C for 1 min, 67°C for 30 s, 72°C for 40 s, 35 cycles, 72°C extension for 10 min, 12°C storage. The product is separated by 10% polyacrylamide gel electrophoresis, not only the existence of the wx gene can be accurately judged, but also the homozygote and heterozygote of the wx gene can be judged.

Using the primer wx-1 to amplify, the result is that only the 140bp band contains the wx gene homozygote, only the 117bp band does not contain the wx gene, and the single plant containing two bands of 140bp and 117bp is the heterozygous strain containing the wx gene .

Using the primer wx-2 to amplify, the results showed that only the 199bp band contained the wx gene homozygote, only the 176bp band did not contain the wx gene, and the individual plants containing both 199bp and 176bp bands were heterozygous for the wx gene.

4.wx gene assisted breeding

In order to transfer the wx gene into the rice material with excellent traits, the excellent rice line is used to cross and backcross the glutinous rice material containing the wx gene, and the primer wx-1 or wx-2 is used to amplify the single plant wx of each hybrid (backcross) generation Gene loci, and polyacrylamide gel electrophoresis was performed on the amplified PCR product to determine the presence or absence of the wx gene, so as to assist in the breeding of individual plants containing the waxy gene (wx) and excellent agronomic traits.

5. Rapid judgment of wx gene homozygosity

According to the difference between markers wx-1 and wx-2 on the amplification product digestion bands of individual plants in self-segregation populations, it was judged whether the tested individual plants contained wx gene and whether the wx gene was homozygous.

Using the primer wx-1 to amplify, the result is that only the 140bp band is the homozygous individual plant carrying the wx gene, and the individual plant containing two bands of 140bp and 117bp is the heterozygous strain carrying the wx gene. Using the primer wx-2 to amplify, the result is that only the 199bp band is homozygous for the wx gene, and the single with 199bp and 176bp bands is the heterozygous for the wx gene.

Therefore, when identifying self-segregation populations: if the marker wx-1 is used to amplify, the electrophoresis result shows only a single plant with a 140bp band; if the marker wx-2 is used to amplify, the electrophoresis result shows only a single plant with a 199bp band , are all single plants carrying homozygous wx gene.

Test example 1 uses wx gene detection primers to carry out wx gene assisted breeding application test

1. Genomic DNA was extracted from 8 glutinous rice materials and 15 non-glutinous rice materials by CTAB method:

1) About 100 seeds were taken from each of the 23 varieties, and the seedlings were germinated in a light incubator at 28°C-32°C for about 1 week;

2) Cut a single seedling and place it in a 2.0mL centrifuge tube, add 2 grinding beads, freeze it in liquid nitrogen, grind it to powder with a tissue grinder, and process 5 individual plants for each material;

3) Add 800 μL of 65°C preheated CTAB extraction buffer (81.7g NaCl and 20g CTAB fully dissolved in an appropriate amount of water) into the centrifuge tube, then add 1mol/L Tris-HCl 100mL, 0.5mol/L EDTA 40mL, and dilute to 1000mL, store at 4°C.);

4) Water bath at 65°C for 1 hour, invert and mix several times every 15 minutes;

5) Add an equal volume of chloroform/isoamyl alcohol (24:1), mix gently, and let stand at room temperature for 10 minutes;

6) Centrifuge at 12,000rpm for 10min, absorb the supernatant and transfer to another 1.5mL centrifuge tube,

7) Add an equal volume of isopropanol, mix well and freeze at -20°C for 30 minutes.

8) Centrifuge at 12,000 rpm for 10 min at 4°C, and discard the supernatant.

9) Add 500 μL of 75% ethanol to wash the precipitate; discard the ethanol after centrifuging at 12,000 rpm for 10 min;

10) Repeat step 9

11) After drying at room temperature, add 50 μL of sterilized water, fully dissolve and set aside.

2. Amplification and detection of wx gene functional region differential sequence

1) Amplification primers for the functional region of the wx gene

The amplification primers for the functional region of the wx gene are as follows:

wx-1F:5'CGTCGTCGCTGCTCCGC3'

wx-1R:5'ACCGCTGCTGCTTGGGC 3'

wx-2F: 5'CGTCCCAGCTCGCCACCT 3'

wx-2R: 5'CACCGACCGCTGCTGCTT 3'

2) PCR amplification and electrophoresis detection

Add in sequence in 0.2mL thin-walled tubes, the reaction system is as follows:

Table 4 PCR reaction system

Amplification program: 95°C pre-denaturation for 5min, 94°C for 1min, 67°C for 30s, 72°C for 40s, 35 cycles, 72°C extension for 10min, 12°C storage.

3. Using the markers wx-1 and wx-2 to assist the selection of wx gene

1) Breeding process of material containing wx gene (Fig. 2).

2) Extraction of rice genomic DNA

Rice materials used: BC2F4 single plant selected by crossing Xuanjingnuo 7 and Wuyujing 3. Cut out about 0.1g of young rice leaves at the adult stage, and extract genomic DNA by CTAB method;

3) PCR amplification and electrophoresis detection of rice genomic DNA

Using primers wx-1 and wx-2 to amplify the genomic DNA of individual plants of Xuanjingnuo 7, Wuyujing 3 and BC2F4 populations, the PCR products were detected by 10% polyacrylamide gel electrophoresis.

4) Selection of offspring containing wx gene

The wx gene homozygous single plant, the marker wx-1 amplification only contained a 140bp band, the marker wx-2 amplification only contained a 199bp band; the wx gene heterozygous single plant, the wx-1 amplification contained both 140bp and 117bp Band, wx-2 amplification contains two bands of 199bp and 176bp. In the backcross progeny, the wx gene heterozygous individual plants were selected, and in the selfed progeny, the wx gene homozygous individual plants were selected (Fig. 5).

Test example 2 using wx gene detection primers to identify the purity of glutinous rice and non-glutinous rice hybrids

According to the difference in the electrophoresis bands of the amplified products of markers wx-1 and wx-2 between glutinous rice and non-glutinous rice varieties, the purity of glutinous rice varieties can be identified.

Using the primer wx-1 to amplify, the results of electrophoresis show that only the 140bp band is the single plant of glutinous rice carrying the homozygous wx gene, and the one containing two bands of 140bp and 117bp is the single plant of glutinous rice carrying the heterozygous wx gene, which only contains 117bp The band is a mixture of non-glutinous rice plants. Using the primer wx-2 to amplify, the results of electrophoresis show that only the 199bp band is a single plant of glutinous rice carrying a homozygous wx gene, and the single plant containing two bands of 199bp and 176bp is a single plant of glutinous rice carrying a heterozygous wx gene, which only contains The 176bp band is a mixture of non-glutinous rice plants (Figure 6).

The results of the rice sample purity test were calculated according to the following formula:

In the formula:

P—sample purity;

N _T — is the number of seeds for inspection (number of seedlings, number of plants);

N _D — is the seed number (number of seedlings, number of plants) judged to be mixed.

Claims (10)

1. The detection primer of the rice wx gene is characterized by being selected from any one of a primer pair (1) or a primer pair (2), wherein the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.

2. A rice wx gene PCR detection kit comprises: taq enzyme, dNTP, mg ²⁺ Deionized water and a detection primer; the method is characterized in that the detection primer is selected from any one of a primer pair (1) or a primer pair (2), and the primer pair (1) consists of a primer with a nucleotide sequence shown as SEQ ID No.1 and a primer with a nucleotide sequence shown as SEQ ID No. 2; the primer pair (2) consists of a primer with a nucleotide sequence shown as SEQ ID No.3 and a primer with a nucleotide sequence shown as SEQ ID No. 4.

3. Use of the detection primer of claim 1 for determining the presence of wx gene or homozygote and heterozygote of wx gene in a rice sample.

4. The use according to claim 3, comprising:

(1) Extracting DNA of a rice sample to be detected;

(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer according to claim 1;

(3) Amplifying by using the primer (1), and if the amplification result is only a 140bp strip, determining that the rice sample to be detected is a wx gene homozygote; if the amplification result is only 117bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 140bp and 117bp at the same time, the rice sample to be detected is a hybrid strain containing wx genes;

amplifying by using the primer pair (2), and if the amplification result is only 199bp bands, determining that the rice sample to be detected is a wx gene homozygote; if the amplification result is only 176bp band, the rice sample to be detected does not contain wx gene; if the amplification result contains two bands of 199bp and 176bp, the rice sample to be detected is a hybrid strain containing wx genes.

5. The use of the detection primer of claim 1 in wx gene-assisted rice breeding.

6. The use according to claim 5, comprising:

(1) Hybridizing and backcrossing a fine rice line and a glutinous rice material containing wx genes, amplifying wx gene sites of each hybridization or backcross generation individual plant by using the primer pair (1) or the primer pair (2) as claimed in claim 1, and performing polyacrylamide gel electrophoresis on the amplified PCR product;

(2) And (2) amplifying by using the primer (1), if the amplification result contains a 140bp strip, judging that wx genes exist in the sample, and if the amplification result contains a 199bp strip, judging that wx genes exist in the sample, so as to assist in breeding single plants which contain waxy genes and have excellent agronomic characters.

7. The use of the detection primer of claim 1 in determining whether a single plant of a rice selfed segregating population is a wx gene homozygote.

8. Use according to claim 7, characterized in that it comprises:

(1) Extracting DNA of a single plant of a self-copulated segregation population to be detected;

(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer;

(3) Amplifying by using a primer pair (1), if the amplification result only has 140bp bands, determining that the single detection sample plant is a homozygous single plant carrying wx genes, and if the amplification result simultaneously contains two bands of 140bp and 117bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes; amplifying by using a primer pair (2) wx-2, if the amplification result only has 199bp bands, determining that the single detection sample plant is a homozygous plant carrying wx genes, and if the amplification result simultaneously contains two bands of 199bp and 176bp, determining that the single detection sample plant is a heterozygous plant carrying wx genes.

9. The use of the detection primer as claimed in claim 1 in the identification of the purity of a waxy rice variety.

10. The use according to claim 9, comprising:

(1) Extracting DNA of a glutinous rice variety to be detected;

(2) Establishing a PCR amplification system for PCR amplification by taking the extracted DNA as a template and taking the primer pair (1) or the primer pair (2) as a PCR amplification primer according to claim 1;

(3) Amplifying by using a primer pair (1), if the electrophoresis result has only 140bp bands which are glutinous rice single plants carrying homozygous wx genes, and simultaneously contains two bands of 140bp and 117bp which are glutinous rice single plants carrying heterozygous wx genes, and only contains 117bp bands which are non-glutinous rice single plants mixed;

amplifying by using a primer pair (2), if the electrophoresis result has only 199bp bands which are glutinous rice single plants carrying homozygous wx genes, single plants containing two bands of 199bp and 176bp are glutinous rice single plants carrying heterozygous wx genes, and single plants containing only 176bp bands which are non-glutinous rice single plants are mixed;

(4) The purity of the rice sample is calculated according to the following formula:

in the formula:

p-sample purity;

N _T the number of seeds to be tested (number of seedlings, number of plants);

N _D the number of seeds (number of seedlings, number of plants) determined as a mixed plant.

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