SNP (Single Nucleotide Polymorphism) molecular marker set for identifying Chinese pear varieties and application of SNP molecular marker set

Abstract

The invention relates to the field of molecular biology, and discloses an SNP molecular marker set for identifying the variety of Chinese pears, the SNP molecular marker set is a specific SNP molecular marker set, the specific molecular marker set comprises 30 SNP markers, the invention also provides a KASP primer combination for amplifying the SNP molecular marker set, the KASP primer combination is composed of detection primers of the 30 SNP sites, and the KASP primer combination is used for amplifying the variety of Chinese pears. The specific primer for detecting each SNP site is composed of two forward primers and one reverse primer. The method has the following advantages and effects that compared with a variety identification technology based on field trials, the latest SNP marking technology and KASP genetic typing technology for international variety identification are not influenced by human subjectivity, climate environment and seasons; compared with an SSR molecular marker, the result is more accurate, the detection process is more convenient, and toxic chemical reagents such as acrylamide are not needed. The method makes up the blank of identifying the Chinese pear variety based on the SNP marker technology, and can be effectively used for Chinese pear variety DNA fingerprint database construction and rapid variety identification.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a SNP molecular marker set for identifying sand pear varieties and an application thereof. Background Art

[0002] Pear is the second largest deciduous fruit tree planted in my country and occupies an important position in the national economy. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), in 2020, China's pear harvesting area and total output were 864,920 hectares and 16 million tons, respectively, accounting for 66.9% and 69.2% of the world's total pear harvesting area and output, respectively, both ranking first in the world. However, with the increasing frequency of exchanges between sand pear varieties between different units, it is inevitable that the same thing has different names or the same name has different things. In addition, the current sand pear seedling market management is not perfect, and adverse effects such as arbitrary changes in variety names and speculation on varieties occur from time to time, which greatly damages the interests of breeders. Therefore, finding an economical, efficient and accurate variety identification method is of great significance to improving the management efficiency and variety protection capabilities of my country's sand pear germplasm resources.

[0003] In recent years, DNA molecular markers have provided a new means for the identification of sand pear varieties with the advantages of short cycle, no environmental influence, and high-throughput detection. Among them, SSR (simple sequence repeat) and SNP (single nucleotide polymorphism) molecular markers have been designated as the two preferred marker methods by the International Union for the Protection of New Varieties of Plants (UPOV) BMT Molecular Testing Guidelines and the domestic "General Principles of DNA Fingerprint Methods for Plant Variety Identification" (NY / T2594-2016). At present, molecular marker methods such as SSR are commonly used in the identification of sand pear varieties, but the SSR detection method is very likely to lead to untrue, false positive, and false negative results; it cannot adapt to the requirements of automation, high throughput, and large-scale. Compared with SSR markers, SNP has many advantages: the variation is clear, stable, and easy to detect, with high authenticity and accuracy; there are millions of SNPs to choose from for each crop; it is suitable for high-throughput, low-cost, and automated rapid detection; SNP typing does not require a reference variety, and the results are presented with accurate bases, which can reduce human errors; and no toxic chemical reagents such as acrylamide are required during the SNP detection process. However, to date, a SNP marker technology system that can be used to identify sand pear varieties has not been established.

[0004] KASP (kompetitive allele specific PCR) refers to a technology that accurately genotypes SNP sites based on the specific matching of primer terminal bases. It has the advantages of being more efficient, flexible, accurate, and low-cost, and is widely used in SNP typing of crops such as wheat, corn, rice, cotton, cabbage, grapes, and peaches. Therefore, based on the advantages of SNP molecular marker technology, developing a set of SNP molecular markers suitable for sand pear variety identification can scientifically respond to current market problems, provide technical support for sand pear variety management, and ensure the healthy and sustainable development of the sand pear industry. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker set for identifying sand pear varieties and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solution: a specific SNP molecular marker set for identifying sand pear varieties, the specific molecular marker set includes 30 SNP markers, and the site information of the SNP markers is shown in the following table:

[0007]

[0008] The physical positions of the SNP markers in the above table are determined based on the Pyrus serrata '20th Century' reference genome, with the genome version number being PPY_r1.0.

[0009] The second object of the present invention is to provide a KASP primer combination for amplifying the above-mentioned specific SNP molecular markers, wherein the KASP primer combination consists of detection primers for the above-mentioned 30 SNP sites, and the specific primers for detecting each SNP site consist of two forward primers and one reverse primer, and the sequence information thereof is shown in Table 1.

[0010] The sand pear variety-specific SNP molecular marker combination of the present invention can be used for: construction of a sand pear variety DNA fingerprint library and sand pear variety identification.

[0011] The present invention also provides a method for constructing a DNA fingerprint library of Pyrus communis varieties, the steps comprising:

[0012] (1) extracting DNA from the sand pear sample to be tested;

[0013] (2) Using the DNA in step (1) as a template, adding a specific primer combination and a PCR premix for identifying the SNP site combination of the sand pear variety, performing PCR amplification, detecting the fluorescent signal of the PCR amplification product, and obtaining the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescent signal, thereby constructing a sand pear variety DNA fingerprint library.

[0014] The present invention also provides a method for identifying sand pear varieties, the steps comprising:

[0015] (1) Extracting DNA from the tested species and suspected species;

[0016] (2) using the DNA in step (1) as a template, adding a specific primer combination and a PCR premix for identifying the SNP site combination of the sand pear variety, performing PCR amplification, detecting the fluorescent signal of the PCR amplification product, and obtaining the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescent signal;

[0017] (3) Compare the typing of the tested species and the suspected species, and make a judgment based on the test results:

[0018] a) If the number of difference loci between varieties is ≥2, they are different varieties;

[0019] b) If the number of difference sites between varieties is ≤ 1, the varieties are suspected to be the same

[0020] The beneficial effects of the present invention are as follows: the technical solution of the present invention adopts the latest SNP marker technology and KASP genotyping technology for variety identification in the world, which is not affected by human subjectivity, climate environment and season compared with variety identification technology based on field trials; the results are more accurate compared with SSR molecular markers, the detection process is more convenient and no toxic chemical reagents such as acrylamide are required. The present invention fills the gap in the identification of sand pear varieties based on SNP marker technology, and can be effectively used for the construction of sand pear variety DNA fingerprint library and rapid variety identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The discrimination efficiency of different marker combinations for 586 sand pear samples;

[0023] Figure 2 Schematic diagram of the distribution of 30 SNP markers on chromosomes;

[0024] Figure 3 In order to use the primers corresponding to the SNP site 7, 20 sand pear varieties that have passed the national variety registration were tested by KASP genotyping technology. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Development of Pyrus sandii SNP molecular markers

[0027] Using the Pyrus 'Twentieth Century' assembled genome (PPY_r1.0) as the reference genome, 586 Pyrus accessions were resequenced, and a total of 11,459.90G reads were obtained, with a sequencing Q30 of 95.60% and a GC content of 37.66%. A total of 32,453,152 SNPs and 4,079,097 Indels were obtained through variant detection analysis. The variant sites were typed and data quality controlled to obtain accurate and high-quality variant sites. The quality control content included: reads coverage sequencing depth, deletion rate, kinship detection, minimal allele frequency (MAF), Hardy-Weinberg equilibrium, outlier samples, etc.

[0028] Screening of core SNP combinations: Randomly extract different numbers of marker sets from the filtered site data set, extract several times, and generate several site combinations. Then calculate the discrimination of each marker combination, that is, the ratio of the number of samples with unique genotype combinations to the total number of samples. 5 to 15 marker combinations were extracted respectively, and each group was randomly repeated 100 times. The combination with the highest discrimination in each marker combination was selected to represent the discrimination of the marker combination of that number. 30 SNP sites that 100% distinguish all 586 samples were screened ( Figure 1 ), distributed on 17 chromosomes of the Pyrus sandii genome ( Figure 2 ), which was determined as the core SNP combination set, and primers were designed based on its preceding and following sequences to develop KASP markers (see Table 1).

[0029] Table 1 KASP primer combinations corresponding to the amplification of 30 SNP sites

[0030]

[0031]

[0032] Example 2: Construction of DNA fingerprint of Pyrus sutchuenensis

[0033] (1) Taking 20 registered varieties of Pyrus ovata as an example, genomic DNA of the registered varieties of Pyrus ovata was extracted;

[0034] (2) Design and synthesize a KASP primer combination based on the core SNP sites screened in Example 1, and use the designed KASP primer combination for PCR amplification. The PCR reaction system (10 μL) is 2×KASP Master Mix 5 μL, primer mix 2.5 μL and template DNA (10-20 ng·μL-1) 2.5 μL. The PCR reaction conditions are: 95°C for 10 min; 95°C for 20 s, 61-55°C for 60 s, 10 touchdown PCR cycles (0.6°C lowered for each cycle); the second round of 95°C for 20 s, 55°C for 60 s, 35 cycles; and finally, read the fluorescence signal data at 25°C.

[0035] (3) Detection of PCR amplification products: The judgment principle based on the fluorescence signal data is as follows: if a certain sand pear germplasm tested shows a blue fluorescence signal based on a certain SNP site, the genotype of the tested germplasm based on the SNP site is the homozygous type of "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and contains "F1" in its name"; if a certain sand pear germplasm tested shows a red fluorescence signal based on a certain SNP site, the genotype of the tested germplasm based on the SNP site is the homozygous type of "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site"; If a test germplasm shows a green fluorescence signal based on a SNP site, the genotype of the test germplasm based on the SNP site is heterozygous, one base is "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and contains "F1" in its name", and the other base is "the complementary base of the first base at the 3' end of the primer that amplifies the SNP site and contains "F2" in its name".

[0036] (4) Construction of SNP fingerprint: The genotype set based on 30 SNP loci is the DNA fingerprint of the variety. The fingerprints of 20 registered sand pear varieties are shown in Table 2.

[0037] Table 2 SNP fingerprints of 20 registered varieties of sand pear

[0038]

[0039]

[0040]

[0041] Example 3: Identification of the 'Jinguo' pear variety

[0042] The 'Golden Fruit' pear is a pear variety that has been promoted by fruit tree nursery companies in Henan, Hubei and other places in recent years, but its origin is unknown. Some experts speculate that it is a Japanese pear variety 'Atago', so the 'Golden Fruit' pear variety was identified. The specific steps are as follows:

[0043] (1) Extract DNA from samples of 'Kinguo' and 'Atago';

[0044] (2) using the DNA in step (1) as a template, adding a specific primer combination and a PCR premix for identifying the SNP site combination of the sand pear variety, performing PCR amplification, detecting the fluorescent signal of the PCR amplification product, and obtaining the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescent signal;

[0045] (3) The genotypes of the SNP sites of 'Jinguo' and 'Atago' were compared. It was found that the genotypes of 'Jinguo' and 'Atago' in the fingerprint library were consistent at all SNP sites. It was preliminarily judged that 'Jinguo' and 'Atago' were suspected to be the same variety (Table 3).

[0046] Table 3 Identification of the cultivar of 'Jinguo' pear

[0047] SNP Golden Fruit Atago SNP Golden Fruit Atago SNP1 CA CA SNP16 CC CC SNP2 AA AA SNP17 GA GA SNP3 GT GT SNP18 TC TC SNP4 CC CC SNP19 TC TC SNP5 GC GC SNP20 AA AA SNP6 AA AA SNP21 CC CC SNP7 CC CC SNP22 CC CC SNP8 CC CC SNP23 GA GA SNP9 CG CG SNP24 CC CC SNP10 GG GG SNP25 GG GG SNP11 CC CC SNP26 TC TC SNP12 TA TA SNP27 AA AA SNP13 GG GG SNP28 TC TC SNP14 TA TA SNP29 AA AA SNP15 GC GC SNP30 GG GG

Claims

1. A SNP molecular marker set for identifying sand pear varieties, characterized in that: The SNP molecular marker set is a specific SNP molecular marker set, which includes 30 SNP markers, and the site information of the SNP markers is shown in the following table:

2. The SNP molecular marker set for identifying Pyrus communis varieties according to claim 1, characterized in that: The physical positions of the SNP markers in the table are determined based on the Pyrus serrata ‘Twentieth Century’ reference genome, with the genome version number being PPY_r1.

0.

3. A KASP primer combination for amplifying the SNP molecular marker set according to claim 1, characterized in that: The KASP primer combination consists of the detection primers of the above 30 SNP sites, and the specific primers for detecting each SNP site consist of two forward primers and one reverse primer, and their sequence information is shown in Table 1.

4. Use of the SNP molecular marker set for identifying sand pear varieties as claimed in claim 1 in the construction of a sand pear variety DNA fingerprint library.

5. Use of the SNP molecular marker set for identifying sand pear varieties as claimed in claim 1 in identifying sand pear varieties.

6. A method for constructing a DNA fingerprint library of Pyrus pyrifolia varieties using the SNP molecular marker set for identifying Pyrus pyrifolia varieties according to claim 1, characterized in that: The following steps are involved: (1) extracting DNA from a sand pear sample to be tested; (2) Using the DNA in step (1) as a template, adding the specific primer combination and PCR premix of the SNP site combination for identifying sand pear varieties in claim 3, performing PCR amplification, detecting the fluorescent signal of the PCR amplification product, and obtaining the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescent signal, thereby constructing a sand pear variety DNA fingerprint library.

7. A method for identifying Pyrus communis varieties using the SNP molecular marker set for identifying Pyrus communis varieties according to claim 1, characterized in that: The following steps are involved: (1) Extracting DNA from the tested species and suspected species; (2) using the DNA in step (1) as a template, adding the specific primer combination and PCR premix solution for identifying the SNP site combination of sand pear varieties according to claim 3, performing PCR amplification, detecting the fluorescent signal of the PCR amplification product, and obtaining the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescent signal; (3) Compare the typing of the tested species and the suspected species, and make a judgment based on the test results: a) If the number of difference loci between varieties is ≥2, they are different varieties; b) If the number of difference loci between varieties is ≤1, they are suspected to be the same variety.

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