A primer for identifying the purity of seeds of Brassica rapa hybrid "Xueyin 1573" and its application

Abstract

The invention discloses a specific primer for identifying the purity of seeds of Chinese cabbage moss hybrid "Xueyin 1573", the primer sequence of which is SEQ ID No.1 ～ 2. The present invention further discloses a method for identifying the purity of seeds of the Chinese cabbage moss hybrid "Xueyin 1573" using the above primers. The specific primers and methods provided by the present invention, the banding type obtained in the identification of seeds of the Chinese cabbage moss hybrid "Xueyin 1573" is the complementary banding type of the parents. After verification by the field seed production results, the primers have good stability and are consistent with the field test results, and can identify the purity of the Chinese cabbage moss hybrid "Xueyin 1573". Moreover, the identification method of the present invention can complete the seed purity identification work of the Chinese cabbage moss hybrid "Xueyin 1573" within 3-4 hours, has the advantages of high efficiency, low cost, easy operation, and no environmental impact, which is beneficial to the quality control of the seeds of the Chinese cabbage moss hybrid "Xueyin 1573" and the further promotion of the variety.

Description

Technical Field

[0001] The invention relates to a primer for identifying the purity of seeds of a Chinese cabbage moss hybrid "Xueyin 1573" and application thereof, belonging to the field of biotechnology. Background Art

[0002] Chinese cabbage, also known as Chinese cabbage heart, Chinese cabbage tip and cabbage heart, has many varieties, including white cabbage, green cabbage (i.e. rapeseed), wax cabbage, purple cabbage and mustard cabbage, etc. It is cool in nature and sweet in taste, with different properties and tastes, but they are all the flower mosses and tender leaves of biennial or biennial herbaceous vegetables of the cruciferous family.

[0003] Cabbage sprouts are the sprouts that grow from the core of Chinese cabbage. The tip of the sprout is pinched and eaten. It is one of the important vegetable varieties during autumn and winter. It is cultivated all over the country, especially in Hunan, Hubei, Anhui, Jiangsu, Zhejiang, Jiangxi and other places with large areas.

[0004] The Chinese cabbage moss hybrid "Xueyin 1573" is an early-maturing excellent Chinese cabbage moss hybrid bred by Wuhan Chuanhe Agricultural Technology Co., Ltd. It is suitable for autumn planting in the Yangtze River Basin. It has the characteristics of short growth period, early moss, strong disease resistance, etc. The extracted cabbage moss has the advantages of tender green color, sweet and crisp taste, and small moss leaves. In recent years, the cultivation area of ​​this variety has been continuously expanding, and the demand for seeds has also increased year by year.

[0005] The value of Chinese cabbage moss seeds is high, and the purity of seeds is the main indicator to directly measure the quality of seeds. Reduced seed purity will significantly reduce the yield and product quality of Chinese cabbage moss, and may even cause immeasurable economic losses. Therefore, each batch of production seeds should be identified before leaving the warehouse to strictly control quality. However, the traditional seed purity identification method is based on the field morphological observation of plants after sowing, which has a long cycle, a large workload, and is easily affected by environmental and seasonal factors, resulting in deviations in the results.

[0006] In recent years, with the rapid development of modern genetics and molecular biology technology, molecular markers based on DNA polymorphism are gradually becoming a powerful tool for analyzing biological genetic diversity. Because of their advantages of being unaffected by the environment, short testing cycle, large number of markers to choose from, and high-throughput testing and analysis, they have been widely used for seed purity identification and variety authenticity identification. Microsatellite (SSR) marker technology has the advantages of co-dominance, good polymorphism, good repeatability, and simple experimental operation, and is widely used. It is currently one of the most commonly used markers in crop variety identification and seed purity identification research. Summary of the invention

[0007] The purpose of the present invention is to provide a pair of SSR primers and applications for identifying the purity of seeds of Chinese cabbage moss hybrid "Xueyin 1573". To achieve this purpose, the present invention adopts the following technical solutions:

[0008] A pair of SSR primers for identifying the purity of seeds of Chinese cabbage moss hybrid "Xueyin 1573", wherein the nucleotide sequence of the primers is as shown in the sequence table SEQ ID No.1 ～ 2.

[0009] The present invention further discloses a method for identifying the purity of seeds of Chinese cabbage moss hybrid "Xueyin 1573" by using the primers, comprising the following steps:

[0010] (1) Extracting genomic DNA from the Chinese cabbage moss hybrid “Xueyin 1573” and its parents;

[0011] (2) Using the genomic DNA of the above sample as a template, the sequence list SEQ ID No. ～ 2 was used for PCR amplification, and the reaction system used was: 10 μL system, including: 2× Taq DNA polymerase Mix 5 μL, upstream and downstream primers 10 μmol / L each 0.5 μL, genomic DNA template of the tested Chinese cabbage moss hybrid (50 ng / μL) 1 μL, double distilled water to make up 10 μL;

[0012] The amplification program used was: 94°C pre-denaturation for 4 min, 35 amplification cycles (94°C for 45 sec, 55°C for 30 sec, 72°C for 45 sec); 72°C extension for 5 min, 4°C storage;

[0013] (3) Identification of PCR amplification products: The products obtained by the above amplification were separated by 10% polyacrylamide gel electrophoresis and stained by silver staining. The electrophoresis bands of the PCR amplification products of the sample to be tested contained both the bands of the amplification products of the male material and the bands of the amplification products of the female material, indicating that the sample was the seeds of the Chinese cabbage moss hybrid "Xueyin 1573";

[0014] (4) Calculation of seed purity: The results of the above silver staining were statistically analyzed as follows: seed purity (%) of the identified Chinese cabbage moss hybrid “Xueyin 1573” = (total number of identified seeds - number of non-hybrids - number of foreign hybrids) / total number of identified seeds × 100%.

[0015] The invention further discloses the application of the identification method in rapidly identifying the purity of seeds of the Chinese cabbage moss hybrid "Xueyin 1573".

[0016] The beneficial effects of the present invention are as follows: the pair of primers provided by the present invention has significant specificity, and the provided identification method is simple, rapid, stable, reliable, and low-cost, and can complete the purity identification of "Xueyin 1573" seeds within 3-4 hours, which can effectively ensure the seed quality of "Xueyin 1573", prevent business risks, and has high commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a partial electrophoresis diagram of the purity of 200 samples of the hybrid of Chinese cabbage moss "Xueyin 1573" using SSR primers; M represents DNA Marker, P1 represents the male material, P2 represents the female material, and the rest are the hybrid materials F1 to be tested. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. The experimental methods used in the following examples are conventional methods unless otherwise specified; the instruments, materials and reagents used, unless otherwise specified, can be obtained from commercial sources. The male and female materials used can be obtained from Wuhan Chuanhe Agricultural Technology Co., Ltd., and the Chinese cabbage moss hybrid "Xueyin 1573" is commercially available.

[0019] Example 1

[0020] Screening of SSR primers for purity identification

[0021] The published cabbage genome was downloaded from the Brassica database website (http: / / brassicadb.cn / # / ), the specific version was Brassica rapa cv.Chiifu V3.5, and the genomic SSR loci were searched and analyzed using MISA (https: / / webblast.ipk-gatersleben.de / misa / ) software under default parameters. The SSR loci and the sequences 150 bp upstream and downstream were selected, and 20 pairs of SSR primers were designed using Primer 5 software (Table 2).

[0022] Table 2 Screening of primers suitable for purity identification of "Xueyin 1573"

[0023]

[0024]

[0025] The polymorphism of primers was screened by DNA of the hybrid, maternal and paternal "Xueyin 1573". Through repeated experiments, multiple analyses and comparisons, it was finally found that the primer combination 22-201F / 22-201R had the best amplification effect. The primers were used as specific primers, and the genomic DNA of the sample to be tested was used as a template for PCR amplification. The amplified products were separated by electrophoresis and stained for detection. The results showed that a specific band of 170bp could be amplified in the paternal material, and a specific band of 182bp could be amplified in the maternal material. The specific bands of 170bp and 182bp could be amplified simultaneously in the hybrid material of the Chinese cabbage moss "Xueyin 1573".

[0026] Test materials

[0027] The materials used for primer screening included 2 individual plants of the hybrid, female parent and male parent of "Xueyin 1573". The materials used for purity identification of the hybrid of "Xueyin 1573" were 200 randomly selected seeds of "Xueyin 1573" for production of Chinese cabbage moss.

[0028] Sampling and DNA extraction

[0029] Seeds of the Chinese cabbage moss hybrid "Xueyin 1573" and its parents were sown in a plug tray. After germination, samples were taken into a 2 ml centrifuge tube, and a 5 mm diameter steel ball was added. 200 μL CTAB lysis buffer (20 g / L CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na2EDTA) was added. The plant tissue was ground (Shanghai Jingxin grinder), and lysed in a 65 ° C constant temperature water bath (Neslab) for 30 min. The subsequent DNA extraction and purification was carried out according to the resin-type genomic DNA purification kit (Sebason). After DNA extraction, the concentration was uniformly diluted to 50 ng / μL. The DNA extraction method can also be carried out according to a commercially available kit.

[0030] PCR amplification

[0031] Using the sequence list SEQ ID No.1 ～ 2 was used for PCR amplification, wherein the total volume of the PCR amplification reaction was 10 μL, and the amplification reaction system was: 5 μL of 2×Taq DNA polymerase Mix, 0.5 μL of each of the upstream and downstream primers at 10 μmol / L, 1 μL of the genomic DNA template (50 ng / μL) of the tested Chinese cabbage moss hybrid, and 10 μL of double distilled water; the PCR reaction program was 94°C pre-denaturation for 4 min, 35 amplification cycles (94°C 45 sec, 55°C 30 sec, 72°C 45 sec); 72°C extension for 5 min, and storage at 4°C;

[0032] Polyacrylamide gel electrophoresis

[0033] The PCR amplification products were separated by polyacrylamide gel electrophoresis. The concentration of non-denaturing polyacrylamide gel was 10%. The gel components were: 21 mL of deionized water, 3 mL of 10×TBE, 6 mL of 40% PAG, 30 μL of TMED, and 300 μL of 10% perthioamide. The voltage was set to 250 V, and the electrophoresis was performed for 1 hour. The power was turned off and the staining was performed. The staining process was: silver staining for 8 to 10 minutes, rapid water washing, NaOH color development for several minutes, until the bands were clear, and then water washing again.

[0034] Results Analysis

[0035] The development results were photographed and statistically analyzed to compare the characteristic bands of the Chinese cabbage moss hybrid "Xueyin 1573" and its parents. The ones that showed co-dominant complementary bands were the true "Xueyin 1573" hybrids, and the other bands were hybrids (see Figure 1 );

[0036] Purity Identification of “Xueyin 1573” Seeds

[0037] According to the statistical results of polyacrylamide gel electrophoresis: the electrophoresis band of the PCR amplification product of the sample to be tested contains both the band of the 170bp amplification product of the male material and the band of the 182bp amplification product of the female material, which is the seed of the Chinese cabbage moss hybrid "Xueyin 1573".

[0038] "Snow Silver 1573" seed purity (%) = (200-2-1) / 200×100% = 98.5%.

[0039] Conclusion: The present invention is used to identify the seed purity of the Chinese cabbage moss hybrid "Xueyin 1573", which has the advantages of being rapid and not affected by the environment. It can replace the traditional field identification method and be used to identify the seed purity of the commercial variety of the Chinese cabbage moss hybrid "Xueyin 1573".

[0040] Those skilled in the art should understand that the above-mentioned specific embodiments are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Application of a pair of SSR primers in identifying the seed purity of Chinese cabbage moss hybrid "Xueyin 1573", wherein the nucleotide sequences of the primers are shown in SEQ ID No.1-2, and the primers simultaneously amplify specific bands of 170 bp and 182 bp in the hybrid material of Chinese cabbage moss hybrid "Xueyin 1573".

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