A SNP marker related to peach cold resistance, a primer set and its application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a SNP marker, a primer set, and applications thereof related to peach cold resistance. The SNP marker is named KH001, which is located at the 13,291,544th base pair of chromosome 4, where the base is C or G; the SNP marker is abbreviated as Chr0413,291,544,C / G. The present invention also relates to a primer set for genotyping the SNP marker. The SNP marker and primer set can be used as auxiliary selection tools for early screening of peach varieties with good cold resistance, which is of great value for improving peach cultivation efficiency and adapting to agricultural production under cold climate conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a SNP marker related to peach cold resistance, a primer set and applications thereof. Background Art

[0002] Peach (Prunus persica L.) is a deciduous fruit tree belonging to the genus Prunus, subgenus Amygdalus, Rosaceae. Temperature is a key factor influencing peach growth, development, and geographic distribution (Li et al., 2023). Winter temperatures in northern China are relatively low, making peach trees susceptible to freezing damage, resulting in severe losses in peach production. Breeding peach varieties for cold tolerance and elucidating the molecular mechanisms of peach cold tolerance have important theoretical and practical implications for ensuring safe wintering and expanding peach cultivation areas.

[0003] SLAF-Seq (Specific-locus amplified fragment sequencing) is a high-throughput, high-resolution SNP (single nucleotide polymorphism) marker identification and typing technology that has been widely used in the construction of high-density plant genetic maps, trait QTL mapping, genome-wide selection, and genome-wide association studies (Zhu et al., 2021). KASP (Kompetitive allele-specific PCR) is a widely used SNP typing technology. It uses two different upstream PCR primers designed based on the differences in known biallelic SNP loci. SNP marker typing is performed based on the color of the terminal fluorescence after the PCR reaction, making the detection convenient and efficient (Burow et al., 2019).

[0004] Currently, molecular-assisted selection systems have been established for many economic traits of peach, such as the genes for yellow and white flesh (Adami et al., 2013), the pollen sterility gene Ps (Dirlewanger et al., 2006), the red flesh gene bf (Werner et al., 1998; Gillen et al., 2005), and the fruit ripening gene Sr ( -Lillo et al., 2015). However, there are few reports on molecular-assisted selection for peach traits related to cold resistance, such as disease resistance, and progress has been slow. Identifying genes associated with cold resistance or closely linked molecular markers and using them to guide the breeding of cold-resistant peach varieties is a key issue that needs to be addressed in peach breeding. Summary of the Invention

[0005] (1) Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a SNP marker, a primer set and its application related to peach cold resistance. The SNP marker can be used as an auxiliary selection tool for early screening of peach varieties with good cold resistance, which is of great value for improving peach cultivation efficiency and adapting to agricultural production in cold climate conditions.

[0007] (2) Technical solution

[0008] In a first aspect, the present invention provides a SNP marker associated with peach cold resistance, which is named KH001 and is located at the 13,291,544th base pair of chromosome 4, where the base is C or G; abbreviated as Chr0413,291,544,C / G.

[0009] In a second aspect, the present invention provides a primer set comprising: an upstream primer KH001-AlleleX, an upstream primer KH001-AlleleY, and a common primer KH001-Common;

[0010] The upstream primer KH001-AlleleX can specifically bind to the DNA fragment carrying the G allele at the SNP marker site;

[0011] The upstream primer KH001-AlleleY can specifically bind to the DNA fragment carrying the C allele at the SNP marker site;

[0012] The common primer KH001-Common can bind to the conserved sequence downstream of the SNP marker site;

[0013] Among them, the sequence of KH001-AlleleX is:

[0014] 5-GAAGGTGACCAAGTTCATGCTCTCTACCTCTACGTGTATTCG ATCC-3;

[0015] The sequence of KH001-AlleleY is:

[0016] 5-GAAGGTCGGAGTCAACGGATTCTCTACCTCTACGTGTATTCG ATCG-3;

[0017] The sequence of KH001-Common is:

[0018] 5-TTTATGTAGCTTTCAGCCCAATTTGG-3.

[0019] In a third aspect, the present invention provides the use of the above-mentioned SNP markers and primer sets in identifying and screening peach varieties with cold resistance.

[0020] According to a preferred embodiment of the present invention, among the SNP markers, plants with the GG genotype or the CG genotype exhibit cold resistance, while plants with the CC genotype exhibit no cold resistance.

[0021] In a fourth aspect, the present invention provides a kit for identifying and screening peach varieties with cold resistance, the kit comprising the primer set.

[0022] According to a preferred embodiment of the present invention, the kit further includes two fluorescent tags; one fluorescent tag corresponds to the amplification product of the KH001-AlleleX primer, and the other fluorescent tag corresponds to the amplification product of the KH001-AlleleY primer. For example, the fluorescent tag FAM corresponds to the amplification product of the KH001-AlleleX primer, while the fluorescent tags HEX or VIC correspond to the amplification product of the KH001-AlleleY primer.

[0023] Among them, plants with GG and CG genotypes showed cold resistance, while plants with CC genotype were not cold-resistant.

[0024] It is understandable that the kit includes, in addition to the primer set and two fluorescent labels, some commonly used reagents and consumables, such as DNA extraction reagents, PCR reaction mixtures, control samples, reaction plates and sealing films, auxiliary reagents (sterile water or TE buffer), etc.

[0025] In a fifth aspect, the present invention provides a method for identifying and screening peach varieties with cold resistance, comprising:

[0026] (1) Extracting genomic DNA from the peach tree to be tested;

[0027] (2) using the genomic DNA of the peach tree to be tested as a template and performing a PCR amplification reaction using the primer set;

[0028] (3) Analyze PCR amplification products and identify and screen peach varieties with cold resistance.

[0029] According to a preferred embodiment of the present invention, in step (3), when the analysis result is GG genotype or CG genotype, the peach tree to be tested is judged to be a cold-resistant variety, and when the analysis result is CC genotype, the peach tree to be tested is judged to be a non-cold-resistant variety.

[0030] In step (3), the determination is made based on the fluorescence color development:

[0031] When only the fluorescent label corresponding to the amplification product of the KH001-AlleleX primer is colored, the peach tree to be tested is judged to be of GG genotype; GG genotype is a cold-resistant variety;

[0032] When the fluorescent labels of the amplification products corresponding to the KH001-AlleleX primer and the KH001-AlleleY primer are colored, it is judged to be the CG genotype; the CG genotype is a cold-resistant variety;

[0033] When only the fluorescent label corresponding to the amplification product of the KH001-AlleleY primer develops color, the peach tree to be tested is determined to be of the CC genotype; the CC genotype is a non-cold-resistant variety.

[0034] In this application, the SNP marker reference genome version number is V2.0.a1.

[0035] (3) Beneficial effects

[0036] A single-nucleotide polymorphism (SNP) marker tightly linked to peach cold resistance, provided by this invention, was tested using KASP (Kompetitive allele-specific PCR) technology in 117 individual plants and their parents from a hybrid population. The results showed that the KH001 marker was tightly linked to peach cold resistance, with plants carrying the homozygous GG or heterozygous CG genotypes more likely to exhibit cold resistance, while plants carrying the homozygous CC genotype were less likely to be cold-resistant. Therefore, this newly discovered SNP marker, KH001, could be used as an auxiliary selection tool for early screening of peach varieties with improved cold resistance, which is of great value for improving peach cultivation efficiency and adapting them to agricultural production in cold climates.

[0037] The SNP markers of the present invention not only deepen researchers' understanding of cold resistance mechanisms, but also provide a new means for plant breeding, which is of great practical value, especially for breeders who want to cultivate peach varieties that can grow well in colder environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is an example of assessing the frost damage level of peach hybrid trees.

[0039] Figure 2 This is an example of assessing frost damage levels for one-year-old branches of a peach hybrid population.

[0040] Figure 3 To type the molecular marker KH001 in peach hybrid populations and parents.

[0041] Figure 4 The average freezing damage grade of the three genotypes of plants marked with KH001 in the hybrid population. DETAILED DESCRIPTION

[0042] The present invention uses SLAF-Seq to locate peach cold resistance QTLs, and develops a KASP marker with strong specificity and high stability that is tightly linked to peach cold resistance in the main effect QTL interval, providing a powerful tool for molecular-assisted selection breeding of peach cold resistance.

[0043] The present invention relates to a SNP marker tightly linked to the cold resistance of peach and its application. Using 117 individual plants and their parents from an F1 hybrid population of 'Donghe No. 1' (a highly cold-resistant variety) x '21st Century' (a non-cold-resistant variety) as materials, a field freezing damage phenotypic survey and evaluation was conducted for three consecutive years. Specific-locus amplified fragment sequencing (SLAF-seq) technology was used to finely map the cold resistance QTLs (quantitative trait loci) of peach cold resistance. A major cold resistance QTL was located between Marker 349841 and Marker 350041. The physical locations of the two markers (Marker 349841 and Marker 350041) were located at positions 13,123,282 and 13,304,122 on chromosome 4 of the peach V2.0.a1 genome, respectively. Based on the parental variant loci obtained by resequencing the two varieties (Donghe No. 1 and 21st Century), a single nucleotide polymorphism (SNP) within the QTL interval (Chr0413,291,544, C / G; the SNP is located at base pair 13,291,544 on chromosome 4; at this position, some individuals may have cytosine (C) and others may have guanine (G)) was developed into the molecular marker KH001. In production practice, this molecular marker KH001 can be used for marker typing using the KASP technology. The results of this marker typing are closely linked to peach cold resistance and offer advantages such as high reproducibility, high stability, low cost, and simple operation. Therefore, this marker KH001 can be used for molecular-assisted selection breeding.

[0044] The following examples illustrate the present invention but are not intended to limit its scope. Unless otherwise noted, all experiments were performed according to conventional experimental conditions, such as those described in Sambrook et al. (Sambrook J & Russell D.W., Molecular cloning: A laboratory manual (3rd ed.), 2001), or according to the manufacturer's instructions. All reagents used in the examples are commercially available.

[0045] Example 1

[0046] (1) Accurate assessment of peach frost damage level

[0047] Using 117 F1 hybrid plants of 'Donghe No. 1'×'21st Century' and their parents as materials, the frost damage grade was assessed based on the frost damage of the trees and branches in January every winter for three consecutive years. Grade 1 means no frost damage, Grade 2 means mild frost damage, and Grade 3 means severe frost damage. Figure 1 、 Figure 2 The average value of the frost damage grade of each plant in three consecutive years was calculated as the frost damage grade of the single plant.

[0048] (2) Extraction of genomic DNA

[0049] The CTAB method was used to extract genomic DNA from 117 individual plants in the F1 hybrid population and their parents.

[0050] The specific steps are as follows:

[0051] 1. Grind the plant material into powder under liquid nitrogen. Take about 1 g of powder and add it to a 2 mL centrifuge tube pre-cooled with liquid nitrogen.

[0052] 2. Quickly add 1 mL of CTAB solution preheated to 65°C into a 2 mL centrifuge tube containing the powder (add 2% mercaptoethanol before preheating the CTAB solution) and shake thoroughly with a vortex shaker until the solution and powder are thoroughly mixed.

[0053] 3. Place the shaken 2 mL centrifuge tube in a 65°C water bath for 30 min, invert it 5-8 times every 10 min to mix, and centrifuge it at 12,000 rpm at 4°C for 10 min.

[0054] 4. All the following steps were performed on ice. 800 μL of supernatant was added to a new 2 mL centrifuge tube. An equal volume of CI (chloroform: isoamyl alcohol = 24:1) was added and the tube was mixed by inversion for 5 minutes.

[0055] 5. Repeat step (4) once (this time only aspirate 600 μL of supernatant).

[0056] 6. Centrifuge at 12,000 rpm for 10 min at 4°C. Pipette 400 μL of the supernatant into a new 1.5 mL centrifuge tube. Add 2 / 3 volume of isopropanol and 1 / 10 volume of sodium acetate solution. Mix by inverting 5-8 times. Place in a -20°C refrigerator to settle for 1 h.

[0057] 7. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Pour off all the solution. The DNA will accumulate at the bottom of the tube. Add 1 mL of 75% ethanol pre-cooled to -20°C and wash 5-8 times by inverting the tube.

[0058] 8. Repeat step (7) once.

[0059] 9. Centrifuge at 12,000 pmr for 10 min at 4°C. Pour off all the solution, centrifuge briefly, remove the remaining liquid with a pipette, and place in a fume hood for 5-10 min until all the ethanol evaporates.

[0060] 10. Add 100 μL of sterile water and then 10 μL of 3‰ RNase to dissolve the DNA precipitate and remove residual RNA. Store at -20°C.

[0061] (3) Development of peach cold resistance SNP markers

[0062] Based on the major QTL interval and parental resequencing data, SNPs were screened within the QTL interval, requiring no other variants within 100 bp upstream or downstream of the site. Variant sites that met these criteria were developed into KASP markers. Each marker consisted of two upstream primers (denoted by AlleleX and AlleleY) and a common downstream primer (denoted by Common). The nucleotides at the 3' termini of the two upstream primers corresponded to the two variant types of the variant site, respectively, and the amplified product was 50-70 bp in length. Blast comparisons were then performed using the Peach Genome V2.0.a1 reference genome database to ensure the specificity of the amplified product. Through these steps, a SNP site (Chr0413, 291, 544, C / G) within the major peach cold resistance QTL was developed into a KASP marker, designated KH001. The genotype of this marker in both the parents, 'Donghe No. 1' and '21st Century', was CG. In the offspring of the hybrid population, individuals with the CG and GG genotypes of this marker tended to be cold-resistant, while individuals with the CC genotype tended to be non-cold-resistant. The primer sequences for KH001 are shown in Table 1.

[0063] Table 1: Primer set sequences of molecular marker KH001 and numbers in the sequence listing

[0064]

[0065] Example 2

[0066] Genotyping of a hybrid population of 117 peach trees was performed using the SNP markers developed in Example 1. The specific steps are as follows:

[0067] S1, extract the genomic DNA of the peach tree to be tested; The extraction process can be seen in step (two) of Example 1;

[0068] S2, using the genomic DNA of the peach tree to be tested as a template, using the primer set to perform a PCR amplification reaction;

[0069] When performing PCR amplification, the PCR reaction system is shown in Table 2.

[0070] Table 2: PCR reaction system

[0071]

[0072]

[0073] PCR amplification reaction conditions are as follows:

[0074] (1) Pre-denaturation: 4°C, 15 minutes;

[0075] (2) First step PCR reaction (gradual cooling and annealing):

[0076] Initial denaturation: 94°C, 20 seconds

[0077] Annealing: Starting from 62°C, decrease the temperature by 1°C each cycle, maintaining each temperature for 60 seconds, for a total of 10 cycles. Cycle 1: 62°C, 60 seconds; Cycle 2: 61°C, 60 seconds; Cycle 3: 60°C, 60 seconds... Cycle 10: 53°C, 60 seconds.

[0078] (3) Second step PCR reaction (conventional cycle):

[0079] Initial denaturation: 94°C, 20 seconds

[0080] Annealing: 52°C, 60 seconds

[0081] Extension: 72°C, 30 seconds

[0082] The above steps were repeated for 35 cycles.

[0083] S3. Analyze PCR amplification products, identify and screen peach varieties with cold resistance.

[0084] During the analysis process, fluorescence signal scanning was used for analysis, and the fluorescence signal was read by the Matrix Scanner high-throughput microplate fluorescence scanning system to achieve labeling typing.

[0085] When the typing result is GG genotype or CG genotype, the peach tree to be tested is judged to be a cold-resistant variety. When the analysis result is CC genotype, the peach tree to be tested is judged to be a non-cold-resistant variety.

[0086] According to the above method, the experiment showed that the KH001 marker was well typed in the 117 hybrid populations, and was divided into 3 genotypes ( Figure 3 ), the ratio is GG:CG:CC=30:55:32, χ 2 The test showed that the ratio was consistent with Mendel's 1:2:1 segregation ratio (χ 2 =0.49<χ 2 0.05=5.99, that is, the actual chi-square value is less than the critical value of the chi-square distribution when the significance level α is set to 0.05). The average freezing damage levels of these three genotypes in the hybrid population are: GG = 1.30, CG = 1.33, CC = 2.09. The freezing damage levels of individuals with GG and CG genotypes are significantly lower than those of individuals with CC genotype ( Figure 4 The above results indicate that the KH001 marker is closely linked to the cold resistance trait of peach and can be used for molecular marker-assisted selection.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting SNP markers in identifying and screening peach varieties with cold resistance, characterized in that: The SNP marker, named KH001, is located at position 13,291,544 of chromosome 4, where the base is C or G, and the peach genome version is V2.0.a1. The peach variety is a hybrid of 'Donghe No. 1' and '21st Century'. Plants with the GG or CG genotype exhibit cold resistance, while plants with the CC genotype are not cold-resistant.

2. A primer set, characterized in that: The primer set includes: upstream primer KH001-AlleleX, upstream primer KH001-AlleleY and common primer KH001-Common; Among them, the sequence of KH001-AlleleX is: 5-GAAGGTGACCAAGTTCATGCTCTCTACCTCTACGTGTATTCGATCC-3; The sequence of KH001-AlleleY is: 5-GAAGGTCGGAGTCAACGGATTCTCTACCTCTACGTGTATTCGATCG-3; The sequence of KH001-Common is: 5-TTTATGTAGCTTTCAGCCCAATTTGG-3.

3. The use according to claim 1, characterized in that The reagent comprises the primer set according to claim 2.

4. A kit for identifying and screening peach varieties with cold resistance, characterized in that: The kit comprises the primer set according to claim 2.

5. The kit according to claim 4, characterized in that The kit also includes two fluorescent tags; one fluorescent tag corresponds to the amplification product of the KH001-AlleleX primer, and the other fluorescent tag corresponds to the amplification product of the KH001-AlleleY primer.

6. A method for identifying and screening peach varieties with cold resistance, characterized in that: include: (1) Extracting genomic DNA from the peach trees to be tested; (2) using the genomic DNA of the peach tree to be tested as a template, and performing a PCR amplification reaction using the primer set described in claim 2; (3) Analyze PCR amplification products to identify and screen peach varieties with cold resistance; the peach varieties are hybrid offspring of 'Donghe No. 1' and '21st Century'; the peach genome version is V2.

0. a1; The analysis results are judged as follows: when the genotype is GG or CG, the peach tree to be tested is judged to be a cold-resistant variety; when the analysis result is CC genotype, the peach tree to be tested is judged to be a non-cold-resistant variety.

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