CN114480721B

CN114480721B - Method for identifying whether melon variety to be detected is thin-skin melon or thick-skin melon and special SNP primer combination thereof

Info

Publication number: CN114480721B
Application number: CN202210269488.2A
Authority: CN
Inventors: 张建; 温常龙; 杨静静; 张晓飞; 夏昌选; 赵泓; 郭凌; 周蒙蒙
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2023-02-03
Anticipated expiration: 2042-03-18
Also published as: CN114480721A

Abstract

The invention discloses a method for identifying whether a melon variety to be detected is a thin-skin melon or a thick-skin melon and a special SNP primer combination thereof. The SNP primer set consists of 2 primer sets. Each primer set consists of 3 primer sequences for amplifying one SNP site. The nucleotide sequences of the primers in the 2 primer groups are sequentially shown as a sequence 1 to a sequence 6 in a sequence table. The SNP primer combination provided by the invention can be used for identifying whether the melon variety to be detected is a thin-skin melon or a thick-skin melon, and has important application value for determining the population property of the melon in the agricultural production and variety breeding processes. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

Method for identifying whether melon variety to be detected is thin-skin melon or thick-skin melon and special SNP primer combination thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for identifying whether a melon variety to be detected is a thin-skin melon or a thick-skin melon, and a special SNP primer combination thereof.

Background

The melon (Cucumis melo L.) is an annual herb plant of the genus Cucumis of the family Cucurbitaceae, has rich nutrition, and is a worldwide fruit with good color, fragrance and taste. China is one of the important origins of melons and is also the country with the largest production and export quantity in the world. Domestic breeders generally classify melon varieties into thick-skinned melons and thin-skinned melons: the muskmelon with thick peel has big pulp, is sweet and delicious, and is one of high-grade fruits; the thin-skin muskmelon is crisp, tender and juicy or has little juice when being cooked, and the skin and the pulp can be eaten, so the thin-skin muskmelon is popular with wide consumers.

Because the cultivation environment and the commercial application of the thin-peel muskmelon and the thick-peel muskmelon are greatly different, the population property of the muskmelon must be determined in the agricultural production and variety breeding processes. However, with the continued cross breeding and gene communication between the two types of pellicle melon and cantaloupe melon, it has not been possible to distinguish pellicle and cantaloupe melon simply by comparing seed sizes. According to statistics, the melon varieties applied and registered in China exceed 2000 parts, the traditional field phenotype identification is time-consuming and labor-consuming, and the identification requirement of the current quantity increase cannot be met. Therefore, a method for simply and quickly distinguishing the thin-skin muskmelon and the thick-skin muskmelon needs to be established as soon as possible, and technical support is provided for the seed selection and the population identification of the muskmelon.

The SNP is taken as a third-generation molecular marker, is widely distributed in a genome, has one SNP per 1000bp on average, is stable in heredity and is easy to detect automatically. KASP (Kompetitive Allele Specific PCR), a competitive Allele Specific PCR technology, is a common method for SNP typing, has the characteristics of high stability, accuracy and low cost, and is widely applied to high-throughput molecular assisted breeding and variety identification.

Disclosure of Invention

The invention aims to identify whether the melon variety is a thin-skin melon or a thick-skin melon.

The method firstly protects the SNP primer combination, and can comprise a primer group 1 for amplifying the SNP05 locus of the melon genome and/or a primer group 2 for amplifying the SNP28 locus of the melon genome;

SNP05 site is 33254142 th nucleotide on chromosome 1;

the SNP28 site is the 25077228 nucleotide on the 11 th chromosome;

the positions of SNP05 site and SNP28 site on the chromosome were determined based on alignment of the cucumis melo DHL92 reference genomic sequence, the version number of which is v3.5.1.

In the SNP primer combination, the primer group 1 can be composed of SEQ ID NO:1, and the forward primer 1F1 shown in SEQ ID NO:2 and the forward primer 1F2 shown in SEQ ID NO:3, and a reverse primer 1R shown in the specification. The primer set 2 may consist of SEQ ID NO:4, forward primer 2F1 shown in SEQ ID NO:5 and the forward primer 2F2 shown in SEQ ID NO:6, and a reverse primer 2R.

In the SNP primer combination, the primer group 1 can be composed of SEQ ID NO:1, forward primer 1F1 shown at positions 22 to 43 from the 5' end, SEQ ID NO:2 from the 22 nd to the 44 th positions from the 5' end, 1F2 and SEQ ID NO:3, and a reverse primer 1R shown in the figure. The primer set 2 may consist of SEQ ID NO:4 forward primer 2F1 shown at positions 22 to 41 from the 5' end, SEQ ID NO:5 forward primer 2F2 shown at positions 22 to 42 from the 5' end and SEQ ID NO:6, and a reverse primer 2R.

In any of the above primer sets, the molar ratio of the primer named as "F1" to the primer named as "F2" to the primer named as "R" may be specifically 2.

Any one of the SNP primer combinations described above may consist of the primer set 1 and/or the primer set 2.

In the above, the nucleotide sequence shown in 1 st to 21 st positions from the 5' end of the sequence 1 in the sequence table is a fluorescent tag sequence (i.e., FAM fluorescent tag sequence), and the fluorescent signal is blue in particular. The nucleotide sequence shown in 1 st to 21 th positions from the 5' end of the sequence 2 in the sequence table is also a fluorescent label sequence (namely a HEX fluorescent label sequence), and the fluorescent signal is red.

A kit containing any one of the SNP primer combinations also belongs to the protection scope of the invention.

The preparation method of the kit also belongs to the protection scope of the invention. The preparation method of the kit comprises the step of packaging each primer in any one of the primer groups separately.

The application of the kit also belongs to the protection scope of the invention. The application of the kit can be identifying whether the melon variety to be detected is thin-skin melon or thick-skin melon.

The application of any one of the SNP primer combinations in the preparation of the kit for identifying the melon variety to be detected as the thin-skin melon or the thick-skin melon also belongs to the protection scope of the invention.

The application of any one of the SNP primer combinations in identifying whether the melon variety to be detected is a thin-skin melon or a thick-skin melon also belongs to the protection scope of the invention.

The invention also provides a method for identifying whether the melon variety to be detected is a thin-skin melon or a thick-skin melon, which comprises the following steps: detecting the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site, and then judging as follows: if the genotype based on the SNP05 locus is TT homozygous and/or the genotype based on the SNP28 locus is CC homozygous, the melon variety to be detected is identified or suspected to be identified as the thin-skin melon; if the genotype based on the SNP05 locus is GG homozygous type and/or the genotype based on the SNP28 locus is AA homozygous type, the melon variety to be detected is identified or suspected to be identified as the muskmelon;

SNP05 site is 33254142 th nucleotide on chromosome 1;

the SNP28 site is the 25077228 nucleotide on the 11 th chromosome;

the location of SNP05 and SNP28 sites on the chromosome was determined based on alignment of the cucumis melo DHL92 reference genome sequence, the version number of the cucumis melo DHL92 reference genome sequence being v3.5.1.

In the above method, the step of detecting the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site may be as follows:

(1) Taking the genome DNA of the melon variety to be detected as a template, and carrying out PCR amplification by adopting any one of the primer group 1 and/or any one of the primer group 2 to obtain a PCR amplification product;

(2) And (3) after the step (1) is finished, detecting the fluorescent signal of the PCR amplification product by using an instrument, and obtaining the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site according to the color of the fluorescent signal.

In the method, the step of detecting the genotype of the melon variety to be detected based on the SNP05 locus and/or the SNP28 locus comprises the following steps:

(1) Taking the genome DNA of the melon variety to be detected as a template, and carrying out PCR amplification by adopting any one of the primer groups 1 and/or any one of the primer groups 2 to obtain a PCR amplification product;

(2) Taking the PCR amplification product obtained in the step (1) and sequencing;

(3) And (3) obtaining the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site according to the sequencing result obtained in the step (2).

In any of the above methods, the reaction procedure of "performing PCR amplification using any of the above primer sets 1 and/or any of the above primer sets 2" may specifically be: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s and 61-55 ℃ (touch down program is selected, the temperature is reduced by 0.6 ℃ per cycle), 1min, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. If the fluorescence signal is weak after PCR amplification is finished and data analysis is influenced, cycles (denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min and 5 cycles) can be added until the result is satisfactory.

With the development of genomics technology, the melon genome has been published and the whole genome resequencing of more than 1000 melon resources is completed, and abundant big data of a variation group provides a marker resource for screening the specific genetic locus of the melon population. And screening population-specific genetic loci from the whole genome by using resequencing data of germplasm resources of the cucumis melo and the cucumis melo, and further developing an SNP primer combination for identifying the cucumis melo and the cucumis melo. The SNP primer combination provided by the invention can be used for identifying whether the melon variety to be detected is a thin-skin melon or a thick-skin melon, and has important application value for determining the population property of the melon in the agricultural production and variety breeding processes. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simple operation, manpower and material resource saving and the like, and has a very wide application prospect.

Drawings

FIG. 1 shows the PCA clustering results of 111 melon representative resource resequencing data.

FIG. 2 shows the result of partial SNP typing for identifying the melon variety to be tested by using the primer set 1.

FIG. 3 shows the result of partial SNP typing for identifying the melon variety to be tested using the primer set 2.

FIG. 4 shows the result of partial SNP typing for identifying the melon variety to be tested by using the primer set 1.

FIG. 5 shows the result of partial SNP typing for identifying the melon variety to be tested by using the primer set 2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 acquisition of SNP primer combination for identifying whether melon variety to be tested is thin-peel melon or thick-peel melon

1. Discovery of 31 SNP sites

According to the method, based on the re-sequencing data of 111 parts of melon representative resources, perfect SNP sites with 50bp of two wings of SNP without other variation and with specific genome are screened, and finally 31 SNP sites are obtained. These 111 parts of melon representative resources represent a wide genetic diversity of melons, including 57 parts of muskmelon and 54 parts of cantaloupe. The results of PCA clustering of 111 melon representative resource resequencing data are shown in FIG. 1.

Specifically, the screening criteria for SNP sites are as follows: firstly, selecting SNP sites with MAF of more than 0.3, heterozygosity of less than 0.1, deletion rate of less than 0.1 and conserved sequence of 50bp at two wings (without InDel, SSR and other SNPs) in a whole genome to obtain 11498 melon perfect SNPs; then, non-synonymous SNPs were screened for calculating the SNP variation frequency between cantaloupe and cantaloupe, and finally, a total of 31 SNP sites with a SNP frequency difference of 1 between the two populations were obtained.

The basic information of 31 SNP sites is detailed in Table 1 (the germplasm resource corresponding to the thin-peel melon is agrestis, and the germplasm resource corresponding to the thick-peel melon is melo). Wherein the position of the SNP locus on the chromosome is determined based on the comparison of the reference genome sequence of the melon DHL92, and the version number of the reference genome sequence of the melon DHL92 is V3.5.1 (the download address is http:// curbitangenetics. Org/ftp/genome/melon/v3.5.1 /).

TABLE 1.31 basic information of SNP sites

2. Obtaining of SNP primer combination for identifying whether melon variety to be detected is thin-skin melon or thick-skin melon

Based on the 31 SNP sites found in the step one, the inventor designs and synthesizes 31 SNP primer combinations and carries out SNP typing on 24 melon varieties of the known pellicle melon or the muskmelon. According to the SNP genotype result, 2 SNP loci, namely SNP05 and SNP28, which have higher homozygous genotype proportion and 100 percent linkage with the thin-skin melon or the thick-skin melon are obtained.

The SNP primer combination consists of a primer group 1 and/or a primer group 2. The primer set for amplifying SNP05 was primer set 1. The primer set for amplifying SNP28 was primer set 2. Each primer set consists of 3 primer sequences for amplifying one SNP site. The nucleotide sequences of the respective primers in the primer set 1 and the primer set 2 are shown in column 4 in Table 2.

TABLE 2 SNP primer combinations for identifying whether the melon variety to be tested is a thin-skin melon or a thick-skin melon

Note: single underlined is FAM fluorescent tag sequence and double underlined is HEX fluorescent tag sequence.

Example 2 and example 1 validation of the SNP primer combinations developed

The basic information for 144 melon varieties tested in this example is shown in columns 1 to 4 of Table 3. 144 tested melon varieties are common market varieties. According to the phenotype, 39 tested melon varieties were thin-skinned melons, and 105 tested melon varieties were thick-skinned melons.

TABLE 3.144 basic information of the melon varieties tested

1. Acquisition of genomic DNA of test melon variety

Genomic DNAs of the rootlets (rootlets of mixed 10 seeds) of 144 melon varieties to be tested were extracted by the CTAB method, respectively, to obtain genomic DNAs of the melon varieties to be tested.

The quality and the concentration of the genome DNA of the tested melon variety both need to meet the PCR requirement, and the standard of reaching standards is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; detecting that the ratio of A260 to A280 is about 1.8 and the ratio of A260 to A230 is more than 1.8 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of the genomic DNA of the melon variety to be tested is 10-30 ng/. Mu.L.

2. And respectively taking the genome DNA of 144 tested melon varieties as templates, and carrying out PCR amplification by adopting the primer group 1 or the primer group 2 to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F1" in the name, the primer containing "F2" in the name, and the primer containing "R" in the name was 2.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15min; modifying at 94 ℃ for 20s and 61-55 ℃ (touch down program is selected, and the temperature is reduced by 0.6 ℃ per cycle), and amplifying for 10 cycles for 1 min; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles.

3. After the step 2 is completed, when the temperature of each PCR amplification product is reduced to below 40 ℃, the fluorescence value is read through FAM and HEX light beam scanning of a microplate reader (the reading value of the FAM fluorescent label sequence is observed under the wavelength of 485nm of exciting light and 520nm of emitted light, the reading value of the HEX fluorescent label sequence is observed under the wavelength of 528nm of exciting light and 560nm of emitted light), and the genotype of 144 muskmelon varieties to be tested based on each SNP site is judged according to the color of the fluorescence signal. The specific judgment principle is as follows: if a certain test melon variety shows a blue fluorescent signal based on a certain SNP locus, the test melon variety is homozygotic based on the genotype of the SNP locus, wherein the complementary base is the 1 st base at the 3' end of the primer which amplifies the SNP locus and contains the F1 in the name; if a certain test melon variety shows a red fluorescent signal based on a certain SNP locus, the test melon variety is homozygotic based on the genotype of the SNP locus, wherein the complementary base is ' the 1 st base at the 3' tail end of a primer which amplifies the SNP locus and contains ' F2 ' in the name '; if a test melon variety shows a green fluorescent signal based on a certain SNP site, the test melon variety is heterozygous based on the genotype of the SNP site, one base is a complementary base of the 1 st base at the 3 'end of a primer which amplifies the SNP site and contains the F1 in the name, and the other base is a complementary base of the 1 st base at the 3' end of a primer which amplifies the SNP site and contains the F2 in the name.

If the fluorescence signal is weak after PCR amplification, which affects data analysis, cycles (denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min,5 cycles) may be added until the results are satisfactory.

The partial SNP typing results of the primer set 1 are shown in FIG. 2. The statistical results are shown in Table 3, column 5.

The partial SNP typing results of the primer set 2 are shown in FIG. 3. The statistical results are shown in column 6 of Table 3.

The result shows that the primer group 1 and the primer group 2 can obtain good typing effect in 144 melon varieties to be tested.

4. Efficiency evaluation for identifying 144 melon varieties to be tested as thin-skin melons or thick-skin melons by using primer group 1 or primer group 2

(1) And counting the genotypes of 144 tested melon varieties based on the SNP05 sites.

The result shows that 39 tested melon varieties with TT homozygous genotype based on SNP05 locus are judged to be thin-skin melons, and the 39 melons are all thin-skin melons with 100% of phenotype; 97 tested melons with genotype of GG homozygous type based on SNP05 locus are judged to be thick-skinned melons, and the 97 melons are all thick-skinned melons with phenotype of 100%.

(2) And counting the genotypes of 144 melon varieties to be tested based on the SNP28 sites.

The result shows that 39 tested melon varieties with CC homozygous genotype based on SNP28 locus are judged to be thin-skin melons, and the 39 melons are all thin-skin melons with 100% of phenotype; 97 tested melons with AA homozygous genotypes based on the SNP28 loci are judged to be thick-skinned melons, and the 97 melons are all thick-skinned melons with 100% of phenotypes.

Therefore, the SNP primer combination developed in the embodiment 1 can identify whether the melon variety to be detected is thin-skin melon or thick-skin melon.

Example 3. Accuracy of identifying whether the melon variety to be tested is a thin-skin melon or a thick-skin melon by using the SNP primer combination developed in example 1

The muskmelon varieties to be detected are respectively 1 to 48 to be detected, and the total number is 48.

1. And (3) identifying whether each melon variety to be detected is a thin-skin melon or a thick-skin melon by adopting the SNP primer combination developed in the example 1. The method comprises the following specific steps:

(1) Planting seeds of a melon variety to be detected to obtain melon seedlings to be detected; taking leaves or roots of the melon seedlings to be detected, and respectively extracting genome DNA by adopting a CTAB method to obtain the genome DNA of the melon varieties to be detected.

(2) And (3) performing PCR amplification by using the genome DNA of the melon variety to be detected as a template and adopting the primer group 1 or the primer group 2 to obtain a PCR amplification product. In each PCR reaction system, the concentration ratio of the primer containing "F1" in the name, the primer containing "F2" in the name, and the primer containing "R" in the name was 2.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s and 61-55 ℃ (touch down program is selected, the temperature is reduced by 0.6 ℃ per cycle), 1min, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles.

(3) After the step (2) is finished, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm exciting light and 520nm emitting light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm exciting light and 560nm emitting light) to obtain the color of a fluorescence signal, and judging as follows: if the melon variety to be detected shows a blue fluorescent signal based on a certain SNP locus, the melon variety to be detected is homozygote based on the genotype of the SNP locus, wherein the complementary base is ' the complementary base which is used for amplifying the SNP locus and contains the 1 st base at the 3' end of the primer of ' F1 ' in the name '; if the melon variety to be detected shows a red fluorescent signal based on a certain SNP locus, the melon variety to be detected is homozygote based on the genotype of the SNP locus, wherein the complementary base is the 1 st base at the 3' end of the primer which amplifies the SNP locus and contains F2 in the name; if the melon variety to be tested shows green fluorescent signals based on a certain SNP locus, the melon variety to be tested is a heterozygous type based on the genotype of the SNP locus, one base is a complementary base of the 1 st base at the 3 'end of the primer which amplifies the SNP locus and contains F1 in the name, and the other base is a complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP locus and contains F2 in the name.

The partial SNP typing results of the primer set 1 are shown in FIG. 4 and column 2 of Table 4.

The partial SNP typing results of the primer set 2 are shown in FIG. 5 and column 3 of Table 4.

(4) After the step (3) is completed, the following judgment is carried out: identifying the melon as the cantaloupe if the genotype based on the SNP05 locus is TT homozygous and/or the genotype based on the SNP28 locus is CC homozygous; and identifying the cantaloupe if the genotype based on the SNP05 site is GG homozygous and/or the genotype based on the SNP28 site is AA homozygous.

TABLE 4

2. And respectively planting 48 melon varieties to be detected. And judging whether the 48 melon varieties to be detected are thin-peel melons or thick-peel melons according to the phenotypes.

Phenotypic statistics are shown in column 4 of table 4.

The results showed that the identification results of the SNP primer combination developed in example 1 were completely consistent with the phenotypic identification results.

Thus, it can be seen that whether the melon is a thin-skin melon or a thick-skin melon can be completely identified by using the SNP primer set developed in example 1.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

<110> agriculture and forestry academy of sciences of Beijing City

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Claims

An SNP primer combination A, which consists of a primer set 1 for amplifying an SNP05 site of the melon genome and/or a primer set 2 for amplifying an SNP28 site of the melon genome;

SNP05 site is 33254142 th nucleotide on chromosome 1;

SNP28 position is 25077228 th nucleotide on chromosome 11;

the positions of the SNP05 site and the SNP28 site on the chromosome are determined based on the alignment of a muskmelon DHL92 reference genome sequence, and the version number of the muskmelon DHL92 reference genome sequence is V3.5.1;

the primer group 1 consists of SEQ ID NO:1, and the forward primer 1F1 shown in SEQ ID NO:2 and the forward primer 1F2 shown in SEQ ID NO:3, and a reverse primer 1R;

the primer group 2 consists of SEQ ID NO:4, forward primer 2F1 shown in SEQ ID NO:5 and the forward primer 2F2 shown in SEQ ID NO:6, and a reverse primer 2R.
An SNP primer combination B, which consists of a primer set 3 for amplifying an SNP05 site of the melon genome and/or a primer set 4 for amplifying an SNP28 site of the melon genome;

SNP05 site is 33254142 th nucleotide on chromosome 1;

SNP28 position is 25077228 th nucleotide on chromosome 11;

the positions of the SNP05 site and the SNP28 site on the chromosome are determined based on the alignment of a muskmelon DHL92 reference genome sequence, and the version number of the muskmelon DHL92 reference genome sequence is V3.5.1;

the primer group 3 consists of SEQ ID NO:1, forward primer 1F1 shown at positions 22 to 43 from the 5' end, SEQ ID NO:2 from position 22 to 44 from the 5' end and the forward primer 1F2 shown in SEQ ID NO:3, and a reverse primer 1R;

the primer group 4 consists of SEQ ID NO:4 forward primer 2F1 shown at positions 22 to 41 from the 5' end, SEQ ID NO:5 forward primer 2F2 shown at positions 22 to 42 from the 5' end and SEQ ID NO:6, and a reverse primer 2R.
3. Use of the SNP primer combination A of claim 1 or the SNP primer combination B of claim 2 for preparing a kit for identifying whether a melon variety to be tested is a thin-skin melon or a thick-skin melon.
4. The application of the SNP primer combination A of claim 1 or the SNP primer combination B of claim 2 in identifying whether a melon variety to be detected is a thin-skin melon or a thick-skin melon.
5. A method for identifying whether a melon variety to be detected is a thin-skin melon or a thick-skin melon comprises the following steps: detecting the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site, and then judging as follows: if the genotype based on the SNP05 locus is TT homozygous type and/or the genotype based on the SNP28 locus is CC homozygous type, identifying the melon variety to be detected as thin-peel melon or suspected identifying the melon variety to be detected as thin-peel melon; if the genotype based on the SNP05 locus is GG homozygous type and/or the genotype based on the SNP28 locus is AA homozygous type, identifying the melon variety to be detected as the muskmelon with thick skin or suspected identifying the melon variety to be detected as the muskmelon with thick skin;

SNP05 site is 33254142 th nucleotide on chromosome 1;

the SNP28 site is the 25077228 nucleotide on the 11 th chromosome;

the positions of SNP05 site and SNP28 site on the chromosome were determined based on alignment of the cucumis melo DHL92 reference genomic sequence, the version number of which is v3.5.1.
6. The method of claim 5, wherein: the steps for detecting the genotype of the melon variety to be detected based on the SNP05 locus and/or the SNP28 locus are as follows:

(1) Carrying out PCR amplification by using the genome DNA of a melon variety to be detected as a template and adopting the primer group 1 and/or the primer group 2 in the claim 1 to obtain a PCR amplification product;

(2) And (2) after the step (1) is finished, detecting the fluorescent signal of the PCR amplification product by using an instrument, and obtaining the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site according to the color of the fluorescent signal.
7. The method of claim 5, wherein: the steps for detecting the genotype of the melon variety to be detected based on the SNP05 locus and/or the SNP28 locus are as follows:

(1) Carrying out PCR amplification by using the genome DNA of a melon variety to be detected as a template and adopting the primer group 3 and/or the primer group 4 in the claim 2 to obtain a PCR amplification product;

(2) Taking the PCR amplification product obtained in the step (1) and sequencing;

(3) And (3) obtaining the genotype of the melon variety to be detected based on the SNP05 site and/or the SNP28 site according to the sequencing result obtained in the step (2).