SNP (Single Nucleotide Polymorphism) marker related to cold resistance of peaches, primer group and application of SNP marker

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an SNP (Single Nucleotide Polymorphism) marker related to cold resistance of peaches, a primer group and application of the SNP marker. The SNP marker is named as KH001 and is located at the 13th, 291 and 544th basic group pairs of a No.4 chromosome, and the basic groups at the positions are C or G; the SNP marker is abbreviated to be Chr0413, 291, 544, C / G. The invention further discloses an application of the SNP marker. The invention also relates to a primer group for carrying out genetic typing identification on the SNP marker. The SNP marker and the primer group can be used as auxiliary selection tools, are used for early screening peach tree varieties with relatively good cold resistance, and have important values for improving the peach tree cultivation efficiency and adapting to agricultural production under a cold climate condition.

Description

Technical Field

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

[0002] Peach (Prunus persica L.) is a deciduous fruit tree belonging to the genus Prunus, subgenus Amygdalus, Rosaceae. Temperature is an important factor affecting the growth, development and geographical distribution of peach trees (Li et al., 2023). The temperature in northern China is relatively low in winter, and peach trees are very susceptible to low temperature damage, causing serious losses to peach production. Breeding peaches for cold resistance and analyzing the molecular mechanism of peach cold resistance have important theoretical and practical guiding significance for the safe wintering of peach trees and the expansion of 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, which has been widely used in the construction of high-density genetic maps of plants, trait QTL positioning, whole-genome selection and whole-genome association studies (Zhu et al., 2021). KASP (Kompetitive allele specific PCR) is a widely used SNP typing technology. It designs two different upstream PCR primers based on the differences in SNP sites on known biallelic genes, and performs SNP marker typing based on the color of the terminal fluorescence after the PCR reaction. The detection is convenient and efficient (Burow et al., 2019).

[0004] At present, molecular-assisted selection systems have been established for many economic traits of peach, such as peach yellow flesh and white flesh genes (Adami et al., 2013), pollen sterility gene Ps (Dirlewanger et al., 2006), red flesh gene bf (Werner et al., 1998; Gillen et al., 2005), fruit ripening period-related gene Sr, etc. ( -Lillo et al., 2015). However, there are few reports on molecular-assisted selection breeding for peach cold resistance and other disease resistance-related traits, and progress is relatively slow. Finding peach cold resistance-related genes or molecular markers closely linked to them and using them to guide the breeding of cold-resistant peach varieties is one of the key issues that need to be solved 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 an application thereof 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 under cold climate conditions.

[0007] (II) 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 show cold resistance, while plants with the CC genotype show 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 comprises two fluorescent labels; one fluorescent label corresponds to the amplification product of KH001-AlleleX primers, and the other fluorescent label corresponds to the amplification product of KH001-AlleleY primers. For example, the fluorescent label FAM corresponds to the amplification product of KH001-AlleleX primers, and the fluorescent label HEX or VIC corresponds to the amplification product of KH001-AlleleY primers.

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

[0024] It can be understood that, in addition to the primer set and two fluorescent labels, the kit also includes some common 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, it is determined that the peach tree to be tested is of the GG genotype; the 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 is colored, it is determined that the peach tree to be tested is of the CC genotype; the CC genotype is a non-cold-resistant variety.

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

[0035] (III) Beneficial effects

[0036] The present invention provides a SNP marker closely linked to peach cold resistance. The KASP technology (Kompetitive allele specific PCR competitive allele specific PCR technology) is used to perform typing tests in 117 hybrid populations and parents. The results show that the KH001 marker is closely linked to the peach cold resistance trait. Plants with homozygous gene GG or heterozygous gene CG genotypes are more likely to show cold resistance, while homozygous gene CC genotypes tend to be non-cold resistant. Therefore, this newly discovered SNP marker KH001 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 under cold climate conditions.

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

[0038] Figure 1 This is an example of assessing the degree of frost damage to trees in a peach hybrid population.

[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 characterize the molecular marker KH001 in peach hybrid populations and parents.

[0041] Figure 4 The average freezing injury 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 closely linked to peach cold resistance in the main effect QTL interval, providing a powerful tool for peach cold resistance molecular assisted selection breeding.

[0043] The present invention relates to a SNP marker tightly linked to cold resistance of peach and application thereof. 117 individual plants and parents of the F1 hybrid population of 'Donghe No. 1' (high cold resistance variety) × '21st Century' (non-cold resistance variety) are used as materials to conduct field freezing injury phenotype investigation and evaluation for three consecutive years, and the cold resistance QTL (quantitative trait locus) of peach is finely located by using SLAF-seq (Specific-locus amplified fragment sequencing) technology. A major cold resistance QTL is located between Marker349841 and Marker350041, and the physical positions of the two markers (Marker349841 and Marker350041) are respectively located at 13,123,282 and 13,304,122 of chromosome 4 of peach V2.0.a1 genome. Based on the information of the parental variation sites obtained by resequencing the parents (i.e., 'Donghe No. 1' and '21st Century'), a SNP in the QTL interval (Chr0413,291,544, C / G; i.e., the SNP is located at the 13,291,544th base pair of chromosome 4; at this position, some individuals may have cytosine (C), while other individuals may have guanine (G)) was developed into a molecular marker KH001. In production practice, the molecular marker KH001 can be used for marker typing by KASP technology. The marker typing result is closely linked to the cold resistance of peach, and has the advantages of good repeatability, high stability, low cost, and simple operation. Therefore, the marker KH001 can be used for molecular assisted selection breeding.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of application of the present invention. Unless otherwise specified, the examples are carried out according to conventional experimental conditions, such as Sambrook et al.'s Molecular cloning laboratory manual (Sambrook J & Russell D.W., Molecular cloning: A laboratory manual (3rd ed.), 2001), or according to the recommended conditions in the manufacturer's instructions. All reagents involved in the examples can be purchased through commercial channels.

[0045] Example 1

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

[0047] The 117 F1 hybrid plants of 'Donghe No. 1'×'21st Century' and their parents were used as materials. The frost damage grade was evaluated in January every winter for three consecutive years according to the frost damage of the tree and branches. Grade 1 was no frost damage, grade 2 was mild frost damage, and grade 3 was 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] (II) Extraction of genomic DNA

[0049] The CTAB method was used to extract genomic DNA from 117 individual plants of 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, and add about 1 g of powder into 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 use a vortex shaker to fully shake the solution and powder until they 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 well, and centrifuge it at 12,000 rpm at 4°C for 10 min.

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

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

[0056] 6. Centrifuge at 12000 rpm for 10 min at 4°C, pipette 400 μL of 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, and place in a -20°C refrigerator to settle for 1 hour.

[0057] 7. Centrifuge at 12000 rpm for 10 min at 4°C, pour out all the solution, at this time the DNA will gather at the bottom of the centrifuge tube. Add 1 mL of 75% ethanol pre-cooled at -20°C, and wash upside down 5-8 times.

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

[0059] 9. Centrifuge at 12,000 pmr for 10 min at 4°C, pour out all the solution, centrifuge briefly, aspirate 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 sterile water and then 10 μL 3‰ RNase to dissolve the DNA precipitate and remove residual RNA, and store at -20°C.

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

[0062] According to the main effect QTL interval and the resequencing data of the parents, SNP sites were screened in the QTL interval, and it was required that there were no other variant sites within the 100bp interval upstream and downstream of the site. The variant sites that met the requirements were developed into KASP markers. A single marker contained two upstream primers (denoted by AlleleX and AlleleY) and one universal downstream primer (denoted by Common). The nucleotides at the 3' ends of the two upstream primers corresponded to the two variant types of the variant site, and the amplification length of the product was 50-70bp. Then, the peach genome V2.0.a1 reference genome database was used for blast comparison to ensure the specificity of the amplified product. Through the above steps, a SNP site (Chr0413,291,544,C / G) in the main effect QTL of peach cold resistance was developed into a KASP marker, named KH001. The genotype of this marker in the parents'Donghe No. 1' and'21st Century' was CG type. In the offspring of the hybrid population, the CG and GG genotype individuals of this marker tend to be cold-resistant, while the CC genotype individuals tend to be non-cold-resistant. The primer sequence of KH001 is 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] The SNP markers developed in Example 1 were used to perform genotyping on a hybrid population of 117 peach trees. The specific steps are as follows:

[0067] S1, extracting the genomic DNA of the peach tree to be tested; the extraction process can refer to step (ii) of Example 1;

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

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

[0070] Table 2: PCR reaction system

[0071]

[0072]

[0073] The PCR amplification reaction conditions are as follows:

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

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

[0076] Pre-denaturation: 94°C, 20 seconds

[0077] Annealing: starting from 62°C, decreasing by 1°C in each cycle, maintaining each temperature for 60 seconds, for a total of 10 cycles. 1st cycle: 62°C, 60 seconds; 2nd cycle: 61°C, 60 seconds; 3rd cycle: 60°C, 60 seconds... 10th cycle: 53°C, 60 seconds.

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

[0079] Pre-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 proved 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 separation 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 the three genotypes in the hybrid population are: GG = 1.30, CG = 1.33, CC = 2.09, and the freezing damage levels of GG and CG genotype individuals are significantly lower than those of CC genotype individuals ( Figure 4 The above results show 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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. A SNP marker associated with peach cold resistance, characterized in that: The SNP marker is named KH001, and is located at the 13,291,544th base pair of chromosome 4, where the base is C or G.

2. A primer set, characterized in that: The primer set includes: upstream primer KH001-AlleleX, upstream primer KH001-AlleleY and common primer KH001-Common; The upstream primer KH001-AlleleX can specifically bind to the DNA fragment carrying the G allele at the SNP marker site of claim 1; The upstream primer KH001-AlleleY can specifically bind to the DNA fragment carrying the C allele at the SNP marker site of claim 1; The common primer KH001-Common can bind to the conserved sequence downstream of the SNP marker site of claim 1; Among them, the sequence of KH001-AlleleX is: 5-GAAGGTGACCAAGTTCATGCTCTCTACCTCTACGTGTATTCG ATCC-3; The sequence of KH001-AlleleY is: 5-GAAGGTCGGAGTCAACGGATTCTCTACCTCTACGTGTATTCG ATCG-3; The sequence of KH001-Common is: 5-TTTATGTAGCTTTCAGCCCAATTTGG-3.

3. Use of the SNP marker according to claim 1 or the primer set according to claim 2 in identifying and screening peach varieties with cold resistance.

4. The use according to claim 3, characterized in that: Plants with GG or CG genotypes showed cold resistance, while plants with CC genotypes were not cold resistant.

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

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

7. A method for identifying and screening peach varieties with cold resistance, characterized in that: include: (1) extracting genomic DNA from the peach tree 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 and identify and screen peach varieties with cold resistance.

8. The method according to claim 7, characterized in that 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.

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