A set of SNP molecular markers for identifying sand pear varieties and its application

Abstract

This invention relates to the field of molecular biology and discloses a set of SNP molecular markers for identifying sand pear varieties. The SNP molecular marker set is a specific SNP molecular marker set, comprising 30 SNP markers. It also provides a KASP primer set for amplifying the SNP molecular marker set. The KASP primer set consists of detection primers for the aforementioned 30 SNP loci, with each SNP locus having two forward primers and one reverse primer. This invention has the following advantages and effects: it employs the latest international SNP marker technology and KASP genotyping technology for variety identification, and compared to variety identification techniques based on field trials, it is not affected by human subjectivity, climate, or season; compared to SSR molecular markers, the results are more accurate, the detection process is more convenient, and it does not require the use of toxic chemical reagents such as acrylamide. This invention fills the gap in sand pear variety identification based on SNP marker technology and can be effectively used for constructing a DNA fingerprint database of sand pear varieties and for rapid variety identification.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a set of SNP molecular markers for identifying sand pear varieties and their applications. Background Technology

[0002] Pear is the second most cultivated deciduous fruit tree in my country, holding 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 harvest area and total output were 864,920 hectares and 16 million tons, respectively, accounting for 66.9% and 69.2% of the global total, ranking first in the world. However, with increasingly frequent exchanges between different units regarding sand pear varieties, phenomena such as synonyms for the same species or different species with the same name are inevitable. Furthermore, the current management of the sand pear seedling market is not perfect, and the arbitrary alteration of variety names and speculation on varieties frequently occur, greatly damaging the interests of breeders. Therefore, finding an economical, efficient, and accurate method for variety identification is of great significance for 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 new methods for identifying sand pear varieties due to their advantages such as short cycle time, insensitivity to environmental influences, and high-throughput detection. Among them, SSR (simple sequence repeat) and SNP (single nucleotide polymorphism) molecular markers have been designated as two preferred marker methods by the UPOV BMT molecular testing guidelines and the domestic "General Rules for DNA Fingerprinting Methods for Plant Variety Identification" (NY / T2594-2016). Currently, SSR and other molecular marker methods are commonly used in sand pear variety identification. However, SSR detection is prone to inaccurate, false positive, and false negative results; it cannot meet the requirements of automation, high throughput, and large-scale testing. Compared with SSR markers, SNPs have several advantages: clear, stable, and easy-to-detect variations; high accuracy and reliability; millions of SNPs available for each crop; suitable for high-throughput, low-cost, automated, and rapid detection; SNP genotyping does not require control varieties, presenting results with accurate base pairs, reducing human error; and SNP detection does not require the use of toxic chemical reagents such as acrylamide. 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) is a technique for precise genotyping of SNP loci based on specific matching of primer terminal bases. It boasts advantages such as higher efficiency, flexibility, accuracy, and lower cost, and is widely used in SNP genotyping of crops such as wheat, corn, rice, cotton, cabbage, grapes, and peaches. Therefore, leveraging the advantages of SNP molecular marker technology, developing a set of SNP molecular markers suitable for identifying sand pear varieties can scientifically address current market challenges, 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 this invention is to provide a set of SNP molecular markers for identifying sand pear varieties and their applications.

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

[0007]

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

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

[0010] The sand pear variety-specific SNP molecular marker combination described in this invention can be used for: constructing a sand pear variety DNA fingerprint database and identifying sand pear varieties.

[0011] This invention also provides a method for constructing a DNA fingerprint library of sand pear varieties, comprising the following steps:

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

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

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

[0015] (1) Extract DNA from the varieties to be tested and suspected varieties;

[0016] (2) Using the DNA in step (1) as a template, add a specific primer combination and PCR premix for identifying the SNP site combination of sand pear varieties, perform PCR amplification, detect the fluorescence signal of the PCR amplification product, and obtain the genotype of the sand pear variety to be tested at the SNP site based on the fluorescence signal color.

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

[0018] a) Varieties with ≥2 different loci are considered different varieties;

[0019] b) Varieties with ≤1 different loci are suspected to be the same variety.

[0020] The beneficial effects of this invention are as follows: The technical solution of this invention adopts the latest international SNP marker technology and KASP genotyping technology for variety identification. Compared with variety identification techniques based on field trials, it is not affected by human subjectivity, climate, or season. Compared with SSR molecular markers, the results are more accurate, the detection process is more convenient, and it does not require the use of toxic chemical reagents such as acrylamide. This 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 databases and rapid variety identification. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The distinguishing efficiency of different combinations of markers on 586 sand pear samples;

[0023] Figure 2 A schematic diagram showing the distribution of 30 SNP markers on the chromosome;

[0024] Figure 3 To use the primers corresponding to the SNP locus 7, we conducted KASP genotyping analysis on 20 nationally registered sand pear varieties. Detailed Implementation

[0025] The technical solution of the present invention will now be clearly and completely described with reference to specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Development of SNP molecular markers in sand pear

[0027] Using the '20th Century' assembled genome of *Pyrus pyrifolia* (PPY_r1.0) as a reference genome, genome resequencing was performed on 586 *Pyrus pyrifolia* germplasm accessions, yielding a total of 11,459.90 G reads. The sequencing Q30 was 95.60%, and the GC content was 37.66%. Variation detection analysis identified 32,453,152 SNPs and 4,079,097 Indels. Genotyping and data quality control were performed on variant sites to obtain accurate and high-quality variant sites. Quality control included read coverage, sequencing depth, deletion rate, phylogenetic analysis, minimum allele frequency (MAF), Hardy-Weinberg equilibrium, and outlier samples.

[0028] Screening for core SNP combinations: Different numbers of marker sets were randomly selected from the filtered locus dataset, repeated several times to generate several locus combinations. The discriminative power of each marker combination was then calculated, which is the ratio of the number of samples with unique genotype combinations to the total number of samples. Five to 15 marker combinations were selected, and each group was randomly repeated 100 times. The combination with the highest discriminative power was selected to represent the discriminative power of that number of marker combinations. This process resulted in the selection of 30 SNP loci that were 100% distinguishable from all 586 samples. Figure 1 ), distributed across 17 chromosomes of the sand pear genome ( Figure 2 We identified it as the core SNP combinatorial set and designed primers based on its preceding and following sequences to develop KASP markers (see Table 1).

[0029] Table 1. KASP primer combinations for amplifying 30 SNP sites.

[0030]

[0031]

[0032] Example 2: Construction of DNA fingerprinting of sand pear

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

[0034] (2) KASP primer combinations were designed and synthesized based on the core SNP sites screened in Example 1, and PCR amplification was performed using the designed KASP primer combinations. The PCR reaction system (10 μL) consisted of 5 μL of 2×KASP Master Mix, 2.5 μL of primer mix, and 2.5 μL of template DNA (10–20 ng·μL⁻¹). The PCR reaction conditions were: 95℃ for 10 min; 95℃ for 20 s, 61–55℃ for 60 s, 10 falling PCR cycles (each cycle decreasing by 0.6℃); the second round was 95℃ for 20 s, 55℃ for 60 s, 35 cycles; finally, fluorescence signal data were read at 25℃.

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

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

[0037] Table 220 SNP fingerprint maps of registered sand pear varieties

[0038]

[0039]

[0040]

[0041] Example 3: Variety identification of 'Golden Fruit' pear

[0042] The 'Jinguo' pear is a pear variety that has been heavily promoted by fruit tree seedling enterprises in Henan and Hubei provinces in recent years, but its origin is unknown. Some experts speculate that it may be a Japanese pear variety called 'Atago'. Therefore, 'Jinguo' pear is being identified through variety identification. The specific steps are as follows:

[0043] (1) Extract DNA from 'Golden Fruit' and 'Atago' samples;

[0044] (2) Using the DNA in step (1) as a template, add a specific primer combination and PCR premix for identifying the SNP site combination of sand pear varieties, perform PCR amplification, detect the fluorescence signal of the PCR amplification product, and obtain the genotype of the sand pear variety to be tested at the SNP site based on the fluorescence signal color.

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

[0046] Table 3 Variety identification of 'Jin Guo' 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 KASP primer combination for identifying sand pear varieties, characterized in that, The KASP primer set consists of primers for detecting 30 SNP sites. The specific primers for detecting each SNP site consist of two forward primers and one reverse primer, and their sequence information is as follows: The site information for the 30 SNPs is shown in the table below: The physical locations of the SNP markers in the table are determined based on the 20th century reference genome of *Pyrus pyrifolia*, with genome version number PPY_r1.

0.

2. A method for constructing a DNA fingerprint database of sand pear varieties using the KASP primer combination for identifying sand pear varieties as described in claim 1, characterized in that, Includes the following steps: (1) Extract DNA from the sand pear sample to be tested; (2) Using the DNA in step (1) as a template, add the specific primer combination and PCR premix of the SNP site combination used to identify sand pear varieties in claim 1, perform PCR amplification, detect the fluorescence signal of the PCR amplification product, and obtain the genotype of the sand pear variety to be tested at the SNP site according to the color of the fluorescence signal, thereby constructing a sand pear variety DNA fingerprint library.

3. A method for identifying sand pear varieties using the KASP primer combination for identifying sand pear varieties as described in claim 1, characterized in that, Includes the following steps: (1) Extract DNA from the varieties to be tested and suspected varieties; (2) Using the DNA in step (1) as a template, add the specific primer combination and PCR premix of the SNP site combination used to identify sand pear varieties in claim 1, perform PCR amplification, detect the fluorescence signal of the PCR amplification product, and obtain the genotype of the sand pear variety to be tested at the SNP site according to the fluorescence signal color. (3) Compare the typing of the tested variety and the suspected variety, and make a judgment based on the test results: a) Varieties with ≥2 different loci are considered different varieties; b) Varieties with ≤1 different loci are suspected to be the same variety.

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