CN107630103B - CAPS molecular marker method for identifying rice varieties and application - Google Patents

Abstract

The invention discloses a CAPS molecular marking method for identifying indica rice and japonica rice varieties and application thereof. The method comprises the following steps: s1, extracting a rice sample genome DNA; s2, carrying out PCR amplification on the genomic DNA of the step S1 by using the primer of claim 3; s3, subjecting the PCR product obtained in the step S2 toHhaI, carrying out electrophoresis after enzyme digestion to obtain a CAPS mark; s4, judging an electrophoresis result: if the electrophoresis band of 440bp is obtained, the rice sample is indica rice, and if the electrophoresis band of 220bp is obtained, the rice variety is japonica rice. The CAPS molecular marking method disclosed by the invention has high specificity, and can conveniently and accurately distinguish germplasm resources of indica rice and japonica rice of rice cultivars. Meanwhile, in practical application, the molecular marker provided by the invention only needs PCR combined enzyme digestion, and is low in cost, high in flux and high in specificity, thereby being very suitable for production practice.

Description

CAPS molecular marker method for identifying rice varieties and application

Technical Field

The invention belongs to the technical field of plant biology. More particularly, relates to a CAPS molecular marker method for identifying rice varieties and application thereof.

Background

The rice cultivars are mainly cultivated rice in Asia, and they are classified into indica rice (indica rice)OryzasalivasubspIndica) And japonica rice (1)OryzasalivasubspJapanica) According to planting conditions and production requirements of different regions, thousands of indica rice and japonica rice varieties are developed and cultivated in China. To ensure high and stable yield, the seed purity of rice varieties is one of the most critical factors. Traditionally, the purity of rice varieties is identified in different growth periods such as seedling period, heading period, and wax ripening period. The characteristics identified mainly have three aspects, namely plant characteristics, ear characteristics and grain characteristics, but are greatly influenced by the experience of an identifier in actual production application. Furthermore, with the development of modern breeding, the phenotypic characters of some varieties are greatly different from those of classical indica rice and japonica rice, for example, the grains of some japonica rice are not wide and short, thick, oval or oval but rather biased to be long and narrow, and the like. Therefore, in the production practice, it is necessary to find a method for rapidly and accurately identifying the difference between indica rice and japonica rice. The molecular marker identification is a high-efficiency and accurate method aiming at the genetic difference of rice genetic information DNA sequences between indica rice and japonica rice. SNP (single nucleotide polymorphism) markers and CAPS (clean amplified polymorphic sequence) technology developed according to the SNP markers can obtain more abundant polymorphisms which cannot be shown by other molecular marker technologies (RafalsiA. applications of single nucleotide polymorphisms in microorganisms. CurrorOpin Plant Biol, 2002, 5(2) 94-100).

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the traditional rice indica type rice and japonica rice variety identification technology and providing a CAPS molecular marking method capable of quickly and accurately identifying whether rice is indica type rice or japonica rice variety. In practical application, the molecular marker provided by the invention only needs PCR combined enzyme digestion, has low cost, high flux and high specificity, and is very suitable for production practice.

The invention aims to provide a specific molecular marker V14SNP1 of a rice V14 gene.

Another objective of the invention is to provide a primer for detecting the molecular marker.

The invention further aims to provide a CAPS molecular marker method for identifying indica rice and japonica rice varieties.

The above object of the present invention is achieved by the following technical solutions:

a specific molecular marker V14SNP1 of a paddy rice V14 gene is shown in SEQ ID NO: 1, the sequence in indica is shown as SEQ ID NO: 2, respectively.

The specific molecular marker V14SNP1 is highly conserved in japonica rice varieties and common wild rice, indica rice and Nivara wild rice, and the V14SNP1 has a SNP site difference of G ← → C in 222bp of sequences in japonica rice and indica rice.

Thus, SEQ ID NO: 1 and SEQ ID NO: 2 in the invention, the application of the specific molecular marker V14SNP1 in the identification of indica rice and japonica rice varieties is also within the protection scope of the invention.

A primer group for detecting the molecular marker V14SNP1, wherein the sequence of the primer is shown as SEQ ID NO: 3. SEQ ID NO: 4 is shown in the specification;

an upstream primer: 5'-TGAGAAACTTACCATTGGC-3' (SEQ ID NO: 3);

a downstream primer: 5'-AAGCATCCTCTTCATATCCTTG-3' (SEQ ID NO: 4).

SEQ ID NO: 3 and SEQ ID NO: 4 can be used as CAPS marker primers for identifying rice varieties, so that the application of the primer group in identifying indica rice and japonica rice varieties is also in the protection scope of the invention.

A CAPS molecular marker method for identifying indica rice and japonica rice varieties of rice comprises the following steps:

s1, extracting a rice sample genome DNA;

s2, the primer pair SEQ ID NO: 3 and SEQ ID NO: 4, performing PCR amplification on the genomic DNA obtained in the step S1;

s3, subjecting the PCR product obtained in the step S2 toHhaI, carrying out electrophoresis after enzyme digestion to obtain a CAPS mark;

s4, judging an electrophoresis result: if the electrophoresis band of 440bp is obtained, the rice sample is indica rice, and if the electrophoresis band of 220bp is obtained, the rice variety is japonica rice.

Preferably, the PCR reaction system is 10 XPCR Buffer 10 uL, 10mmol/L dNTPs 0.2 uL, 0.1 umol/L PCR primers are 1 uL respectively, the high fidelity PCR polymerase is 5U, the DNA template is 0.2 ug, and the deionized water is supplemented to 100 uL.

Preferably, the PCR reaction procedure is pre-denaturation at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15sec, 52 ℃ for 20sec, and 72 ℃ for 30 sec; extension at 72 ℃ for 5 min.

Compared with the indica rice type and japonica rice type sequences, the molecular marker V14SNP1 has a SNP locus difference of G ← → C; the japonica rice has restriction endonuclease in adjacent sequenceHhaI, restriction enzyme recognition site: GCGC. The SNP mutation GCGG of indica rice at the enzyme cutting recognition site causes that restriction endonuclease cannot be usedHhaI, enzyme digestion recognition. Thus is inHhaI, after enzyme digestion and recognition of the PCR fragments, the japonica rice fragments are digested into DNA fragments with the size of 220bp, and the indica rice still keeps DNA fragments with the size of 440 bp.

The molecular marker V14SNP1 can also be used for detecting and identifying the purity of rice parents, so that the application of the molecular marker V14SNP1 in the purity detection of indica rice and japonica rice parents is also within the protection scope of the invention.

Meanwhile, the application of the CAPS molecular marker method in the purity detection of indica rice and japonica rice parents is also within the protection scope of the invention.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a CAPS molecular marker method for identifying indica rice and japonica rice varieties of rice. The CAPS molecular marking method has high specificity, and can conveniently and accurately distinguish germplasm resources of indica rice and japonica rice of rice cultivars. Meanwhile, in practical application, the molecular marker provided by the invention only needs PCR combined enzyme digestion, and is low in cost, high in flux and high in specificity, thereby being very suitable for production practice.

Drawings

FIG. 1 shows the PCR amplification of CAPS-tagged primersHhaI, enzyme cutting result chart. Wherein, indica rice variety 1E93-11 parts of 7 parts of nante, Guang Lu dwarf No. 4 parts of 7 parts of 93-11 parts of 7 parts of 93-11 parts of 7; the japonica rice varieties 1-7 are Nipponbare, Taizhong 65, Zhonghua 11, agricultural reclamation 58, Yunyuan No. 23, Huevening No. 3 and Jindao No. 1 respectively. Carrying out PCR amplification on the products of the above varietiesHhaI, enzyme digestion results show that fragments of indica rice and Nivara wild rice cannot be digested, and fragments of japonica rice varieties and common wild rice are digested into DNA fragments of 220 bp.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 molecular marker and detection primer for identifying Rice

The inventor finds that a molecular marker V14-SNP1 exists in rice, and the sequence of the molecular marker in japonica rice is shown as SEQ ID NO: 1, the sequence in indica is shown as SEQ ID NO: 2, the molecular markers are 440bp, and at 222bp, there is a SNP locus difference of G ← → C, and the molecular markers can be used for identifying indica rice and japonica rice varieties. The analysis shows that japonica rice has restriction enzyme in the adjacent sequence of the SNP siteHhaI, restriction enzyme recognition site: GCGC, and the SNP mutation GCGG of indica rice at the enzyme cutting recognition site causes that restriction endonuclease cannot be usedHhaI, enzyme digestion recognition, so that the indica rice and japonica rice varieties can be detected and identified by a CAPS molecular marking method, and the method can be used when being usedHhaAfter the molecular marker V14-SNP1 is identified by enzyme, the japonica rice variety is cut into a DNA fragment with the size of 220bp by enzyme, while the indica rice still keeps the DNA fragment with the size of 440bp, so that the indica rice and japonica rice varieties of rice are identified.

Meanwhile, by aligning the sequences of the molecular marker V14-SNP1 in japonica rice and indica rice, the following PCR amplification primers are designed as CAPS marker primers:

an upstream primer: 5'-TGAGAAACTTACCATTGGC-3' (SEQ ID NO: 3);

a downstream primer: 5'-AAGCATCCTCTTCATATCCTTG-3' (SEQ ID NO: 4).

The PCR reaction system is 10 muL of 10 XPCR Buffer, 0.2 muL of 10mmol/L dNTPs, 1 muL of 0.1 mumol/L PCR primers respectively, 5U of high-fidelity PCR polymerase, 0.2 mug of DNA template and 100 muL of deionized water.

The PCR reaction program is pre-denaturation at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15sec, 52 ℃ for 20sec, and 72 ℃ for 30 sec; extension at 72 ℃ for 5 min.

Example 2 CAPS molecular marker method for identifying indica rice and japonica rice varieties

The experimental materials of the CAPS molecular marker method for identifying the varieties of the indica rice and the japonica rice comprise common wild rice and Nivara wild rice; indica rice varieties are 93-11, dwarf south China, Guang-land short No. 4, Zhenshan 97, Minghui 63, Huangsi occupation and Huanghua occupation respectively; the japonica rice varieties are Nipponbare, Taizhong 65, Zhonghua 11, agricultural cultivation 58, Yujing 23, Huevening 3 and Jindao 1. The method comprises the following specific steps:

1. extraction of genomic DNA from rice samples

(1) Taking about 0.5g of rice leaves, cutting the leaves into pieces, putting the pieces into a precooled mortar, adding liquid nitrogen, grinding the leaves into powder, putting the powder into a 2 mL centrifuge tube, adding 800 mu L of 2 xCTAB extraction buffer solution, uniformly mixing, and carrying out water bath at 65 ℃ for 30 min;

(2) adding equal volume of chloroform, isoamyl alcohol and anhydrous alcohol (76: 4: 20), shaking for 10 min, and mixing well;

(3) centrifuging for 12min under the condition of 12000 r/min, transferring the supernatant into another 1.5mL centrifuge tube, adding isopropanol with the volume 0.6 times of that of the supernatant or absolute ethanol with the volume twice of that of the supernatant, mixing uniformly, centrifuging for 2min at 12000 r/min, removing the supernatant, rinsing twice with 0.5mL 70% ethanol, air-drying, and dissolving in 100-;

(4) mu.L of the mixture was used for the subsequent PCR amplification reaction.

2. PCR amplification reaction

Amplifying the rice sample genome DNA by using the CAPS labeled primer in the embodiment 1;

the PCR amplification reaction system is as follows: 10 μ L of 10 XPCR Buffer, 0.2 μ L of 10mmol/L dNTPs, 1 μ L of 0.1 μmol/L PCR primers respectively, 5U of high-fidelity PCR polymerase, 0.2 μ g of DNA template and 100 μ L of deionized water.

The PCR amplification reaction is carried out on a DNA (deoxyribonucleic acid) amplification instrument, and the PCR reaction program is pre-denaturation at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15sec, 52 ℃ for 20sec, and 72 ℃ for 30 sec; extending at 72 deg.C for 5min, and storing at 4 deg.C;

3. enzyme digestion

For the PCR amplification product of the above rice sampleHhaI, enzyme digestion, electrophoresis of PCR amplification products after enzyme digestion on 1.2% agarose gel for 1.5h (5V/cm), staining by ethidium bromide (EtBr), observation by a gel imaging system and photographing and recording.

4. Results and analysis

The PCR amplification of 16 different rice material genome DNAs can amplify a very clear single band, 8 bands have the same size, and the result is stable and consistent after 3 times of repetition (as shown in FIG. 1A). Through positive and negative sequence determination, the lengths of 16 PCR products are 440 bp; furthermore, all primers used for PCR amplification were correctly detected at both ends. Wherein, the sequences of the japonica rice variety and the common wild rice are shown as SEQ ID NO: 1, the sequences of indica rice and Nivara wild rice are shown as SEQ ID NO: 2, respectively.

In thatHhaAfter the PCR fragment of the rice variety is identified by enzyme digestion, the japonica rice fragment is digested into a DNA fragment with the size of 220bp, while the indica rice still keeps the DNA fragment with the size of 440bp (as shown in figure 1B), so the CAPS molecular marker method can be used for identifying the indica rice and japonica rice varieties.

Sequence listing

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<120> CAPS molecular marker method for identifying rice variety and application thereof

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Claims (8)

1. The application of a specific molecular marker V14SNP1 of a rice V14 gene in identifying varieties of indica rice and japonica rice is characterized in that a nucleotide sequence of the V14SNP1 in indica rice is shown as SEQ ID NO: 1, the sequence in japonica rice is shown as SEQ ID NO: 2, respectively.

2. A primer set for detecting the molecular marker V14SNP1 of claim 1, wherein the sequence of the primer is as shown in SEQ ID NO: 3, SEQ ID NO: 4, respectively.

3. The use of the primer set according to claim 2 for identifying varieties of indica rice and japonica rice.

4. A CAPS molecular marker method for identifying indica rice and japonica rice varieties is characterized by comprising the following steps:

s1, extracting a rice sample genome DNA;

s2, carrying out PCR amplification on the genomic DNA of the step S1 by using the primer of claim 2;

s3, carrying out enzyme digestion on the PCR product obtained in the step S2 by Hha I and then carrying out electrophoresis to obtain a CAPS mark;

s4, judging an electrophoresis result: if the electrophoresis band of 440bp is obtained, the rice sample is indica rice, and if the electrophoresis band of 220bp is obtained, the rice variety is japonica rice.

5. The method of claim 4, wherein the PCR reaction system is 10 μ L of 10 XPCR Buffer, 0.2 μ L of 10mmol/L dNTPs, 1 μ L of 0.1 μmol/L PCR primers, 5U of high fidelity PCR polymerase, 0.2 μ g of DNA template, and 100 μ L of deionized water.

6. The method of claim 4, wherein the PCR reaction procedure is pre-denaturation at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15sec, 52 ℃ for 20sec, and 72 ℃ for 30 sec; extension at 72 ℃ for 5 min.

7. The application of a specific molecular marker V14SNP1 of a rice V14 gene in the purity detection of indica rice and japonica rice parents is characterized in that the nucleotide sequence of the V14SNP1 in indica rice is shown as SEQ ID NO: 1, the sequence in japonica rice is shown as SEQ ID NO: 2, respectively.

8. The CAPS molecular marker method of any one of claims 4-6, applied to purity detection of indica rice and japonica rice parents.

Images