A method for identification and purity of sweet corn seed No. 3 based on SSR markers

Abstract

The present invention relates to a primer composition for identifying the purity of "Hetian No. 3" corn seeds and a method for identifying the purity of "Hetian No. 3" corn seeds using the primer composition. The identification method of the present invention directly analyzes the genetic material DNA of the target object, and the result does not depend on the technician's understanding of the agronomic traits of the sample, nor is it affected by the environment. It solves the problem of data inaccuracy caused by human factors and environmental factors in the previous technology, saves identification costs, shortens identification time, and is more accurate and reliable for the identification of new varieties.

Description

Technical Field

[0001] The invention relates to the field of plant variety detection, and in particular to a method for identifying the purity of sweet No. 3 corn seeds. Background Art

[0002] In recent years, my country's sweet corn industry has developed rapidly. Domestic scientific research institutions have successively cultivated many excellent varieties, mainly concentrated in Guangdong, Yunnan, Hainan, the middle and lower reaches of the Yangtze River, central China and Northeast China. In 2018, the national sweet corn planting area reached 400,000 hm2. 2 , surpassing the United States to become the country with the largest sweet corn planting area in the world. According to the national division of corn planting areas, the southeast region is listed as a key area for the development of fresh corn. Among them, residents in the Yangtze River Delta region have always had a high pursuit of healthy and nutritious life. In particular, in recent years, the demand for sweet corn has been increasing, and the consumption of sweet corn has increased year by year. The active market demand has driven the development of sweet corn production in the Yangtze River Delta region. The Yangtze River Delta region, headed by Shanghai, has gradually become an important sweet corn production, processing and consumption area in my country. Focusing on the relevant planning for the development of urban efficient ecological agriculture, the sweet corn industry with characteristics and obvious competitive advantages has become a key development and support for agricultural advantage and characteristic industries.

[0003] "Hetian No. 3" is a sweet corn variety approved by the state in 2020. Its kernels are neatly arranged, beautiful in appearance, yellow in color, good cob capping, moderately tight bracts and emerald green in color, and strong in plant growth. It is suitable for spring planting in the southeastern fresh corn type area in Anhui and Jiangsu provinces south of the Huaihe River, Shanghai, Zhejiang, Jiangxi, Fujian, Guangdong, Guangxi Autonomous Region and Hainan Province. In recent years, it has been favored by growers in modern agricultural production in the suburbs and cities of Shanghai, with significant economic benefits.

[0004] The purity of hybrid corn seeds seriously affects their quality and yield, and is an important indicator for identifying the quality of hybrid corn seeds. In the past, the purity of hybrid corn varieties was usually identified by field plot planting identification. Although this method is relatively accurate, the identification process requires high professional quality of operators and requires rich field production experience. In addition, the identification cycle is long, the identification cost is high, and the efficiency is low. It is now difficult to meet the requirements of the market development of hybrid corn seeds.

[0005] SSR (Simple Sequence Repeats) is a type of tandem repeat sequence of dozens of nucleotides composed of several nucleotides as repeating units. It is a molecular marker technology based on specific primer PCR that has been developed in recent years. SSR marker technology uses upstream and downstream primers designed with relatively conservative single-copy sequences on both sides of the repeat sequence to present the variation of repeat sequences in different materials through PCR amplification and gel electrophoresis. SSR molecular identification technology does not rely on the understanding of the characteristics of the variety, nor is it affected by planting, so the identification results are more reliable. At present, this technology has been widely used.

[0006] In view of the urgency of effectively identifying the purity of corn seeds and the limitations of existing methods for identifying the purity of corn seeds, a "Hetian No. 3" corn seed purity identification method with short identification time and accurate and reliable results has become a technical problem that needs to be urgently solved in this field. Summary of the invention

[0007] To solve the above technical problems, the present invention includes the following aspects:

[0008] The first aspect of the present invention provides a primer composition for identifying the purity of "Hetian No. 3" corn seeds, which comprises 4 pairs of primers, and the names of the primers are bnlg439w1, bnlg1940k7, bnlg2291k4 and bnlg2305k4 respectively.

[0009] Preferably, the sequences of the primers are SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.15 and SEQ ID NO.16, and SEQ ID NO.19 and SEQ ID NO.20.

[0010] Preferably, the information of the primers is shown in the following table:

[0011]

[0012] Preferably, the primer composition further comprises the following primers, whose names are umc2007y4, phi053k2, umc1705w1, bnlg161k8, bnlg1702k1, umc1545y2, umc1125y3, phi065k9, umc1492y13, umc1506k12, phi96100y1, umc1536k9, bnlg1520K1, umc1999y3, umc2115k3, phi299852y2, umc2160k3, phi233376y1 and umc2084w2, respectively.

[0013] The second aspect of the present invention provides a kit for identifying the purity of "Hetian No. 3" corn seeds, which comprises the above-mentioned 4 pairs of primers.

[0014] The third aspect of the present invention provides a method for identifying the purity of "Hetian No. 3" corn seeds, which comprises the following steps:

[0015] (1) Grinding the corn seeds to be tested and extracting genomic DNA using a DNA magnetic bead method;

[0016] (2) using 4 pairs of primers to perform PCR amplification on the extracted DNA to obtain amplified products, wherein the names of the primers are bnlg439w1, bnlg1940k7, bnlg2291k4 and bnlg2305k4;

[0017] (3) detecting the amplified product by capillary electrophoresis;

[0018] (4) Compare the obtained electrophoresis results with the electrophoresis results obtained by amplifying "Hetian No. 3" corn seeds using the above primer pair to determine whether the sample contains a fingerprint consistent with "Hetian No. 3", thereby identifying whether the corn seeds are "Hetian No. 3".

[0019] Preferably, the PCR amplification system in step (2) is:

[0020] Element Single reaction volume (μl) HotStart Mix 2X 10 F-primer 0.1 R-primer 0.1 Template (concentration at 20ng / μl) 2 water 7.8 total 20

[0021] Preferably, the PCR reaction conditions in step (2) are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 35 s, extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min, and storage of the amplified product at 4°C.

[0022] Preferably, the electrophoresis buffer in step (3) is 1×TAE and the voltage is 100V.

[0023] The third aspect of the present invention provides the use of the above primer combination or the above kit in identifying the purity of "Hetian No. 3" corn seeds.

[0024] The fourth aspect of the present invention provides the use of the above primer combination in the preparation of a kit for identifying the purity of "Hetian No. 3" corn seeds.

[0025] The beneficial effects of the present invention are:

[0026] (1) The method of the present invention screened out 40 pairs of core primers with high polymorphism and good repeatability based on 20 core germplasms and 20 extended germplasms. These primers are suitable for SSR fingerprint data collection and variety identification of corn inbred lines and single crosses. The SSR molecular marker detection technology based on capillary electrophoresis has the advantages of high throughput, low cost, and high flexibility. Using the capillary electrophoresis detection technology platform, the corresponding analysis was performed based on 40 pairs of SSR marker primers. The DNA fingerprint (marker-genotype) of the new corn variety "Hetian No. 3" was established for the first time, and the differences between varieties were quickly and accurately identified at the DNA level, which safeguarded the variety rights and provided strong technical support for the promotion and planting of the new variety.

[0027] (2) The present invention uses 40 pairs of SSR standard primers to conduct genotypic analysis on the hybrid corn variety "Hetian No. 3" and its parents (SC13035×SC13019). The 40 SSR marker-genotype fingerprints contain 23 heterozygous sites. Through comparative analysis experiments with other varieties and comprehensive evaluation, 4 pairs of primers with stronger specificity than other varieties were screened, and specific markers that can distinguish "Hetian No. 3" from its parents were established. The hybrid corn "Hetian No. 3" seed purity molecular identification technology method was established, which can identify self-pollinated seedlings, backcross seedlings and heteromorphic plants.

[0028] (3) The identification method of the present invention directly analyzes the genetic material DNA of the target object. The result does not depend on the technician's understanding of the agronomic traits of the sample, nor is it affected by environmental interference. It solves the problem of inaccurate data caused by human factors and environmental factors brought about by previous technologies, saves identification costs, shortens identification time, and makes the identification of new varieties more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the capillary electrophoresis diagram of the SSR marker of the primer P01 (bnlg439w1) of the present invention;

[0030] Figure 2 This is the capillary electrophoresis diagram of the SSR marker of the primer P04 (bnlg1940k7) of the present invention;

[0031] Figure 3 This is the capillary electrophoresis diagram of the SSR marker of the primer P08 (bnlg2291k4) of the present invention;

[0032] Figure 4 This is the capillary electrophoresis diagram of the SSR marker of the primer P10 (bnlg2305k4) of the present invention;

[0033] Figure 5 This is a comparison of the capillary electrophoresis diagrams of SSR markers of the primer P03 (umc2007y4) of the present invention in different varieties of corn. DETAILED DESCRIPTION

[0034] The present invention is further described by way of test examples, but the present invention is not limited to the following test examples.

[0035] Experimental Example 1: Determination of SSR marker-genotype fingerprint of corn variety "Hetian No. 3"

[0036] 1. Extraction of Maize Genomic DNA

[0037] Twenty "Hetian No. 3" corn seeds were taken from each sample, placed in a 2ml round-bottom centrifuge tube, and two steel balls were added. The seeds were ground with a QIAGEN grinder at the maximum frequency for 20 minutes to prepare the extracted DNA. The genomic DNA was extracted using the seed DNA magnetic bead method. The extraction kit was produced by Changchun Zhiang Biotechnology Co., Ltd., model GO-GPLF-FX96. The instruments used included Thermo FLEX nucleic acid extractor, mixing elf, centrifuge and metal bath / water bath / air bath. After DNA extraction, it was detected by 1.0% agarose gel electrophoresis, the electrophoresis buffer was 1×TAE, and the voltage was 100V; DNA purity and concentration were measured using NanoDrop2000c UV-visible spectrometer (Thermo Company), diluted to 50ng / μL working solution for use, and stored in a refrigerator at 4℃.

[0038] 2. Primers

[0039] The sequence information of the corn SSR core primers P01-P40 used in this test example is shown in Table 1 below.

[0040] Table 1 Names and sequences of core SSR primers in maize

[0041]

[0042]

[0043]

[0044]

[0045] 3. PCR reaction system and procedure

[0046] PCR system standard configuration:

[0047]

[0048]

[0049] PCR reaction conditions were set as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 35 s, and extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min, and the amplified product was stored at 4°C.

[0050] 4. Capillary electrophoresis analysis

[0051] Electrophoresis was performed according to the instructions. All data analysis was based on QIAxcel Screen Gel version 1.4 software. The software system automatically analyzed the raw data of the fingerprint to determine the alleles of each locus. The capillary electrophoresis diagram of some SSR markers is shown in the figure below. Figure 1-4 shown.

[0052] 5. DNA fingerprint of corn varieties

[0053] Through the capillary electrophoresis SSR molecular marker analysis technology platform, 40 SSR markers were used to analyze "Hetian No. 3" and three other varieties (glutinous corn N1, glutinous corn N2, sweet corn N3). The specific data are shown in Table 2-1 and Table 2-2. The DNA fingerprint of the variety can be established through the analysis of differential sites, laying a solid foundation for the protection of variety rights.

[0054] Table 2-1 Grain patterns of SSR markers of sweet corn No. 3 and glutinous corn N1

[0055]

[0056]

[0057]

[0058] Table 2-2 Patterns of SSR markers of waxy corn N2 and sweet corn N3

[0059]

[0060]

[0061]

[0062] Note: “--” in Tables 2-1 and 2-2 above indicates missing data.

[0063] 6. DNA fingerprint of the corn variety “Hetian No. 3”

[0064] Through the fingerprint analysis of the above 40 pairs of SSR markers, the DNA fingerprint (marker-genotype) of the corn variety "Hetian No. 3" was determined, as shown in Table 3 below.

[0065] Table 3 Marker fingerprint of corn variety "Hetian No. 3" (marker-genotype)

[0066] serial number mark genotype serial number mark genotype P01 bnlg439w1 CD P21 umc1147y4 AA P02 umc1335y5 AA P22 bnlg1671y17 AA P03 umc2007y4 AC P23 phi96100y1 AB P04 bnlg1940k7 BC P24 umc1536k9 CD P05 umc2105k3 CC P25 bnlg1520K1 BC P06 phi053k2 AB P26 umc1489y3 AA P07 phi072k4 CC P27 bnlg490y4 BB P08 bnlg2291k4 CD P28 umc1999y3 AC P09 umc1705w1 AB P29 umc2115k3 BC P10 bnlg2305k4 CD P30 umc1429y7 BB P11 bnlg161k8 BC P31 bnlg249k2 BB P12 bnlg1702k1 BC P32 phi299852y2 BC P13 umc1545y2 AC P33 umc2160k3 AC P14 umc1125y3 BC P34 umc1936k4 BB P15 bnlg240k1 AA P35 bnlg2235y5 AA P16 phi080k15 BB P36 phi233376y1 AB P17 phi065k9 BC P37 umc2084w2 AB P18 umc1492y13 AB P38 umc1231k4 AA P19 umc1432y6 AA P39 phi041y6 AA P20 umc1506k12 CD P40 umc2163w3 BB

[0067] From the information in Table 3 above, we can know that among the 40 SSR marker-genotype fingerprint maps, there are 23 heterozygous sites (see the genotypes of the bold marks), and then the markers with stronger specificity are selected from the heterozygous sites. Taking the molecular marker numbered P01 of "Hetian No. 3" as an example, its genotype is CD, which is different from the genotypes of its parents and the other three varieties, and is a molecular marker with stronger specificity; while the genotype of the molecular marker numbered P21 is AA, and the genotypes of its parents and the molecular marker "Sticky Corn N1" are also AA, so this marker is not specific. Based on the test results of Tables 2-1 and 2-2, through comparative analysis experiments and comprehensive evaluation with other varieties, 4 pairs of primers with stronger specificity compared with other varieties were screened out (see Table 4 below). Compared with the primers in Table 4, the specificity of other primers in different varieties is not significant (for example Figure 5 The specific fingerprints listed in Table 4 can therefore be used for variety identification and analysis of "Hetian No. 3", to quickly and accurately identify the differences between varieties at the DNA level and safeguard variety rights.

[0068] Table 4 Molecular marker information used to identify the corn variety “Hetian No. 3”

[0069] serial number Tag Name Sweet No.3 Remark P01 bnlg439w1 CD C:351bp; D:331bp P04 bnlg1940k7 BC B:364bp; C:377bp P08 bnlg2291k4 CD C:383bp; D:374bp P10 bnlg2305k4 CD C: 255bp; D: 282bp

[0070] Test Example 2: Screening primers for single seed detection results

[0071] 196 single seeds were randomly selected, and the selected single seeds of "Hetian No. 3" were used to verify the screened SSR primers. The SSR primer screening test was the same as the field experiment, and the results of the screening primers on the single seeds of the population were observed (see Table 5). The seed purity (%) was calculated as follows: number of hybrid seed samples / number of experimental samples × 100.

[0072] Table 5 Screening primers for single seed detection results

[0073]

[0074] As can be seen from Table 5 above, the average values ​​of the four pairs of primer combinations for field purity identification and molecular identification are basically consistent, indicating that the results of using the screened primers to identify the purity of "Hetian No. 3" seeds are credible.

[0075] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. Use of a primer combination in preparing a kit for identifying the purity of "Hetian No. 3" corn seeds, characterized in that: The primer composition consists of 4 pairs of primers, the names of the primers are bnlg439w1, bnlg1940k7, bnlg2291k4 and bnlg2305k4, and the sequences of the primers are SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 15 and SEQ ID NO. 16, and SEQ ID NO. 19 and SEQ ID NO.

20.

2. Application of a primer combination or a kit in identifying the purity of "Hetian No. 3" corn seeds, characterized in that: The primer composition consists of 4 pairs of primers, the names of the primers are bnlg439w1, bnlg1940k7, bnlg2291k4 and bnlg2305k4, and the sequences of the primers are SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 15 and SEQ ID NO. 16, and SEQ ID NO. 19 and SEQ ID NO. 20, respectively. The kit contains the primer composition.

3. A method for identifying the purity of "Hetian No. 3" corn seeds, characterized in that: The following steps are involved: (1) Grind the corn seeds to be tested and extract genomic DNA using the DNA magnetic bead method; (2) using four pairs of primers to perform PCR amplification on the extracted DNA to obtain amplified products, wherein the names of the primers are bnlg439w1, bnlg1940k7, bnlg2291k4 and bnlg2305k4, and the sequences of the primers are SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 15 and SEQ ID NO. 16, and SEQ ID NO. 19 and SEQ ID NO. 20, respectively; (3) Detecting the amplified product by capillary electrophoresis; (4) Compare the obtained electrophoresis results with the electrophoresis results obtained by amplifying the "Hetian No. 3" corn seeds using the above primer pair to determine whether the corn seeds to be tested contain fingerprints consistent with "Hetian No. 3", thereby identifying whether the corn seeds are "Hetian No. 3".

4. The method according to claim 3, characterized in that The PCR amplification system in step (2) is: 。 5. The method according to claim 3, characterized in that: The PCR reaction conditions in step (2) are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 35 s, and extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min, and storage of the amplified product at 4°C.

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