CN109517923B - Method for identifying authenticity of cucumber variety and special SNP primer combination thereof - Google Patents

Abstract

The invention discloses a method for identifying the authenticity of cucumber varieties and a special SNP primer combination thereof. The SNP primer combination provided by the invention consists of 32 primer groups. Each primer set consists of 3 primer sequences and is used for amplifying one SNP site. The nucleotide sequences of the primers in the 32 primer groups are sequentially shown as a sequence 1 to a sequence 96 in a sequence table. The SNP primer combination provided by the invention can be used for early identification of cucumber varieties in seed or seedling stage, so that the authenticity of the varieties is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for the protection of cucumber germplasm resources and new varieties. The method provided by the invention can be used for identifying unknown cucumber varieties and also can be used for identifying the authenticity of known varieties. 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 authenticity of cucumber variety and special SNP primer combination thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a method for identifying the authenticity of a cucumber variety and a special SNP primer combination thereof.

Background

The cucumber is an important vegetable crop in China, the cultivation area reaches 139.6 million hectares, and the cucumber accounts for 6.2 percent of the vegetable cultivation area in China. The variety approved by the nation and each province increases year by year. According to registration methods of non-main crop varieties, the agricultural department of 4 months in 2017 publishes a first registration catalogue of non-main crop varieties, wherein cucumber ranks. At present, more than 1000 varieties are identified, identified and registered nationwide, but the number of cucumber varieties on the market far exceeds the number. As the cucumber breeding enterprises are small and dispersed, the variety approval among provinces and cities is not coordinated, the variety management of the cucumber cannot be effectively followed up, and a plurality of fake and shoddy varieties exist, wherein the fake and shoddy varieties are mostly derived and derived similar varieties. In addition, the management of an improved seed production base is disordered, the phenomena of stealing and breeding, bought article species are rampant, the same-name heterogeneous phenomenon is serious, and the quality accident of seeds occurs sometimes. According to the requirements of 'non-main crop variety registration guide' the DNA detection result can be directly submitted if the related characters related in the variety characteristic description and the variety DUS test report have definite related genes. Therefore, establishing a set of standards for effectively identifying the authenticity and purity of cucumber variety resources based on DNA fingerprints is very important.

In recent years, SNPs have been extensively highlighted as third-generation molecular markers due to their advantages such as large number, wide distribution, and genetic stability. With the development of high-throughput sequencing technology and the continuous reduction of sequencing cost, large-scale cucumber re-sequencing becomes possible. Based on analysis of cucumber mutation group information, more stable and efficient SNP sites can be mined. And developing a specific primer by adopting an allele competitive specific PCR method, and finally obtaining the genotype of the sample at the SNP site.

At present, the DNA molecular detection for identifying the cucumber varieties in China is mainly based on an SSR molecular marking method, but the research and development of SSR primers do not refer to the information of cucumber genome variation groups, so that the unreal variation condition exists; the SSR primers are small in screening variety quantity and cannot represent the sales varieties in the current market in China; in addition, the method is limited by that unreal, false positive and false negative results are easily caused by an SSR detection mode; can not meet the requirements of automation, high flux and large scale. SNPs have several advantages over SSR markers: the variation is clear, stable and easy to detect, and the authenticity is high in accuracy; millions of SNPs are available for selection per crop; the method is suitable for high-throughput, low-cost and automatic rapid detection, and can reduce human errors; SNP typing does not require a control variety, and results are presented with accurate bases.

Disclosure of Invention

The invention aims to identify cucumber varieties.

The invention firstly protects the SNP locus combination, which can comprise 32 SNP loci of cucumber genome; the 32 SNP sites are as follows: the CuSNP01 site is the 1976800 th nucleotide on the No. 1 chromosome; the CuSNP02 site is the 8433330 th nucleotide on the No. 1 chromosome; the CuSNP03 site is the 13502481 th nucleotide on the No. 1 chromosome; the CuSNP04 site is the 17508118 th nucleotide on the No. 1 chromosome; the CuSNP05 site is the 25296763 th nucleotide on the No. 1 chromosome; the CuSNP06 site is the 1200640 th nucleotide on chromosome 2; the CuSNP07 site is the 2563528 th nucleotide on the No. 2 chromosome; the CuSNP08 site is the 5891488 th nucleotide on chromosome 2; the CuSNP09 site is the 6045153 th nucleotide on chromosome 2; the CuSNP10 site is the 11947823 th nucleotide on chromosome 2; the CuSNP11 site is 40726 th nucleotide on chromosome 3; the CuSNP12 site is the 1001975 th nucleotide on the 3 rd chromosome; the CuSNP13 site is the 10713052 th nucleotide on the 3 rd chromosome; the CuSNP14 site is the 14161884 th nucleotide on the 3 rd chromosome; the CuSNP15 site is the 18678235 th nucleotide on the 3 rd chromosome; the CuSNP16 site is the 27913661 th nucleotide on the 3 rd chromosome; the CuSNP17 site is the 16975775 th nucleotide on the 4 th chromosome; the CuSNP18 site is the 21068185 th nucleotide on the 4 th chromosome; the CuSNP19 site is the 2133319 th nucleotide on the chromosome 5; the CuSNP20 site is the 4054461 th nucleotide on the chromosome 5; the CuSNP21 site is the 8103814 th nucleotide on the chromosome 5; the CuSNP22 site is the 9582207 th nucleotide on the chromosome 5; the CuSNP23 site is the 17245080 th nucleotide on the chromosome 5; the CuSNP24 site is the 195377 th nucleotide on the 6 th chromosome; the CuSNP25 site is the 2397556 th nucleotide on chromosome 6; the CuSNP26 site is the 18079846 th nucleotide on the 6 th chromosome; the CuSNP27 site is the 20929474 th nucleotide on the 6 th chromosome; the CuSNP28 site is the 24439780 th nucleotide on the 6 th chromosome; the CuSNP29 site is the 2085399 th nucleotide on the 7 th chromosome; the CuSNP30 site is the 4866176 th nucleotide on the 7 th chromosome; the CuSNP31 site is the 6739101 th nucleotide on the 7 th chromosome; the CuSNP32 site is the 15415870 th nucleotide on chromosome 7.

The SNP site combination can specifically consist of the 32 SNP sites.

The invention also provides SNP primer combinations, which can comprise a primer set 1 for amplifying the CuSNP01, a primer set 2 for amplifying the CuSNP02, a primer set 3 for amplifying the CuSNP03, a primer set 4 for amplifying the CuSNP04, a primer set 5 for amplifying the CuSNP05, a primer set 6 for amplifying the CuSNP06, a primer set 7 for amplifying the CuSNP07, a primer set 8 for amplifying the CuSNP08, a primer set 9 for amplifying the CuSNP09, a primer set 10 for amplifying the CuSNP010, a primer set 11 for amplifying the CuSNP11, a primer set 12 for amplifying the CuSNP12, a primer set 13 for amplifying the CuSNP13, a primer set 14 for amplifying the CuSNP14, a primer set 15 for amplifying the CuSNP15, a primer set 16 for amplifying the CuSNP16, a primer set 5817 for amplifying the CuSNP17, a primer set 18 for amplifying the CuSNP2, and a primer set 5 for amplifying the CuSNP 3819, A primer set 20 for amplifying the CuSNP20, a primer set 21 for amplifying the CuSNP21, a primer set 22 for amplifying the CuSNP22, a primer set 23 for amplifying the CuSNP23, a primer set 24 for amplifying the CuSNP24, a primer set 25 for amplifying the CuSNP25, a primer set 26 for amplifying the CuSNP26, a primer set 27 for amplifying the CuSNP27, a primer set 28 for amplifying the CuSNP28, a primer set 29 for amplifying the CuSNP29, a primer set 30 for amplifying the CuSNP30, a primer set 31 for amplifying the CuSNP31, and a primer set 32 for amplifying the CuSNP 32.

In the SNP primer combination, the primer set 1 can be composed of a forward primer 01F1 shown in a sequence 1, a forward primer 01F2 shown in a sequence 2 and a reverse primer 01R shown in a sequence 3. The primer group 2 can be composed of a forward primer 02F1 shown in a sequence 4, a forward primer 02F2 shown in a sequence 5 and a reverse primer 02R shown in a sequence 6. The primer group 3 can be composed of a forward primer 03F1 shown in a sequence 7, a forward primer 03F2 shown in a sequence 8 and a reverse primer 03R shown in a sequence 9. The primer group 4 can be composed of a forward primer 04F1 shown in a sequence 10, a forward primer 04F2 shown in a sequence 11 and a reverse primer 04R shown in a sequence 12. The primer group 5 can be composed of a forward primer 05F1 shown in a sequence 13, a forward primer 05F2 shown in a sequence 14 and a reverse primer 05R shown in a sequence 15. The primer group 6 can be composed of a forward primer 06F1 shown in a sequence 16, a forward primer 06F2 shown in a sequence 17 and a reverse primer 06R shown in a sequence 18. The primer group 7 can be composed of a forward primer 07F1 shown in a sequence 19, a forward primer 07F2 shown in a sequence 20 and a reverse primer 07R shown in a sequence 21. The primer group 8 can be composed of a forward primer 08F1 shown in a sequence 22, a forward primer 08F2 shown in a sequence 23 and a reverse primer 08R shown in a sequence 24. The primer group 9 can be composed of a forward primer 09F1 shown in a sequence 25, a forward primer 09F2 shown in a sequence 26 and a reverse primer 09R shown in a sequence 27. The primer group 10 can be composed of a forward primer 10F1 shown in sequence 28, a forward primer 10F2 shown in sequence 29 and a reverse primer 10R shown in sequence 30. The primer set 11 can be composed of a forward primer 11F1 shown in a sequence 31, a forward primer 11F2 shown in a sequence 32 and a reverse primer 11R shown in a sequence 33. The primer set 12 can be composed of a forward primer 12F1 shown in sequence 34, a forward primer 12F2 shown in sequence 35, and a reverse primer 12R shown in sequence 36. The primer set 13 can be composed of a forward primer 13F1 shown in sequence 37, a forward primer 13F2 shown in sequence 38, and a reverse primer 13R shown in sequence 39. The primer set 14 can be composed of a forward primer 14F1 shown in sequence 40, a forward primer 14F2 shown in sequence 41, and a reverse primer 14R shown in sequence 42. The primer set 15 can be composed of a forward primer 15F1 shown in sequence 43, a forward primer 15F2 shown in sequence 44, and a reverse primer 15R shown in sequence 45. The primer set 16 may be composed of a forward primer 16F1 shown in sequence 46, a forward primer 16F2 shown in sequence 47, and a reverse primer 16R shown in sequence 48. The primer set 17 can be composed of a forward primer 17F1 shown in sequence 49, a forward primer 17F2 shown in sequence 50, and a reverse primer 17R shown in sequence 51. The primer set 18 can be composed of a forward primer 18F1 shown in sequence 52, a forward primer 18F2 shown in sequence 53, and a reverse primer 18R shown in sequence 54. The primer set 19 can be composed of a forward primer 19F1 shown in sequence 55, a forward primer 19F2 shown in sequence 56, and a reverse primer 19R shown in sequence 57. The primer set 20 can be composed of a forward primer 20F1 shown in sequence 58, a forward primer 20F2 shown in sequence 59, and a reverse primer 20R shown in sequence 60. The primer set 21 can be composed of a forward primer 21F1 shown in sequence 61, a forward primer 21F2 shown in sequence 62 and a reverse primer 21R shown in sequence 63. The primer set 22 may be composed of a forward primer 22F1 shown in sequence 64, a forward primer 22F2 shown in sequence 65, and a reverse primer 22R shown in sequence 66. The primer set 23 can be composed of a forward primer 23F1 shown in sequence 67, a forward primer 23F2 shown in sequence 68, and a reverse primer 23R shown in sequence 69. The primer set 24 can be composed of a forward primer 24F1 shown in a sequence 70, a forward primer 24F2 shown in a sequence 71 and a reverse primer 24R shown in a sequence 72. The primer set 25 can be composed of a forward primer 25F1 shown in sequence 73, a forward primer 25F2 shown in sequence 74, and a reverse primer 25R shown in sequence 75. The primer set 26 can be composed of a forward primer 26F1 shown in sequence 76, a forward primer 26F2 shown in sequence 77, and a reverse primer 26R shown in sequence 78. The primer set 27 may consist of a forward primer 27F1 shown in sequence 79, a forward primer 27F2 shown in sequence 80, and a reverse primer 27R shown in sequence 81. The primer set 28 can consist of a forward primer 28F1 shown in sequence 82, a forward primer 28F2 shown in sequence 83, and a reverse primer 28R shown in sequence 84. The primer group 29 can be composed of a forward primer 29F1 shown in a sequence 85, a forward primer 29F2 shown in a sequence 86 and a reverse primer 29R shown in a sequence 87. The primer set 30 may consist of a forward primer 30F1 shown in sequence 88, a forward primer 30F2 shown in sequence 89, and a reverse primer 30R shown in sequence 90. The primer group 31 can be composed of a forward primer 31F1 shown in a sequence 91, a forward primer 31F2 shown in a sequence 92 and a reverse primer 31R shown in a sequence 93. The primer set 32 may consist of a forward primer 32F1 shown in sequence 94, a forward primer 32F2 shown in sequence 95, and a reverse primer 32R shown in sequence 96.

In the SNP primer set, the primer set 1 may be composed of a forward primer 01F1 shown in the 22 nd to 46 th positions from the 5 'end of the sequence 1, a forward primer 01F2 shown in the 22 nd to 46 th positions from the 5' end of the sequence 2, and a reverse primer 01R shown in the sequence 3. The primer set 2 may be composed of a forward primer 02F1 shown in the 22 nd to 46 th positions from the 5 'end of the sequence 4, a forward primer 02F2 shown in the 22 nd to 43 th positions from the 5' end of the sequence 5, and a reverse primer 02R shown in the sequence 6. The primer set 3 may be composed of a forward primer 03F1 shown in 22 nd to 48 th positions from the 5 'end of the sequence 7, a forward primer 03F2 shown in 22 nd to 50 th positions from the 5' end of the sequence 8, and a reverse primer 03R shown in the sequence 9. The primer set 4 may be composed of a forward primer 04F1 shown in 22 nd to 52 th positions from the 5 'end of the sequence 10, a forward primer 04F2 shown in 22 nd to 51 th positions from the 5' end of the sequence 11, and a reverse primer 04R shown in the sequence 12. The primer set 5 may be composed of a forward primer 05F1 shown in positions 22 to 47 from the 5 'end of the sequence 13, a forward primer 05F2 shown in positions 22 to 47 from the 5' end of the sequence 14, and a reverse primer 05R shown in the sequence 15. The primer set 6 may be composed of a forward primer 06F1 shown in 22 th to 51 th positions from the 5 'end of the sequence 16, a forward primer 06F2 shown in 22 th to 51 th positions from the 5' end of the sequence 17, and a reverse primer 06R shown in 18 th positions. The primer set 7 may be composed of a forward primer 07F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 19, a forward primer 07F2 shown in 22 th to 48 th positions from the 5' end of the sequence 20, and a reverse primer 07R shown in the sequence 21. The primer set 8 may be composed of a forward primer 08F1 shown in 22 th to 48 th positions from the 5 'end of the sequence 22, a forward primer 08F2 shown in 22 th to 48 th positions from the 5' end of the sequence 23, and a reverse primer 08R shown in the sequence 24. The primer set 9 may be composed of a forward primer 09F1 shown in positions 22 to 48 from the 5 'end of the sequence 25, a forward primer 09F2 shown in positions 22 to 47 from the 5' end of the sequence 26, and a reverse primer 09R shown in the sequence 27. The primer set 10 may be composed of a forward primer 10F1 shown in 22 nd to 51 th from the 5 'end of the sequence 28, a forward primer 10F2 shown in 22 nd to 51 th from the 5' end of the sequence 29, and a reverse primer 10R shown in the sequence 30. The primer set 11 may be composed of a forward primer 11F1 shown in 22 nd to 46 th positions from the 5 'end of the sequence 31, a forward primer 11F2 shown in 22 nd to 45 th positions from the 5' end of the sequence 32, and a reverse primer 11R shown in 33 th positions. The primer set 12 may be composed of a forward primer 12F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 34, a forward primer 12F2 shown in 22 th to 47 th positions from the 5' end of the sequence 35, and a reverse primer 12R shown in the sequence 36. The primer set 13 may be composed of a forward primer 13F1 shown in 22 th to 43 th positions from the 5 'end of the sequence 37, a forward primer 13F2 shown in 22 th to 44 th positions from the 5' end of the sequence 38, and a reverse primer 13R shown in 39 th positions. The primer set 14 may be composed of a forward primer 14F1 shown in positions 22 to 49 from the 5 'end of the sequence 40, a forward primer 14F2 shown in positions 22 to 49 from the 5' end of the sequence 41, and a reverse primer 14R shown in the sequence 42. The primer set 15 may be composed of a forward primer 15F1 shown in the 22 nd to 48 th positions from the 5 'end of the sequence 43, a forward primer 15F2 shown in the 22 nd to 49 th positions from the 5' end of the sequence 44, and a reverse primer 15R shown in the sequence 45. The primer set 16 may be composed of a forward primer 16F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 46, a forward primer 16F2 shown in 22 th to 48 th positions from the 5' end of the sequence 47, and a reverse primer 16R shown in 48 th positions. The primer set 17 may be composed of a forward primer 17F1 shown in 22 nd to 51 th from the 5 'end of the sequence 49, a forward primer 17F2 shown in 22 nd to 52 th from the 5' end of the sequence 50, and a reverse primer 17R shown in 51. The primer set 18 may be composed of a forward primer 18F1 shown at positions 22 to 46 from the 5 'end of the sequence 52, a forward primer 18F2 shown at positions 22 to 44 from the 5' end of the sequence 53, and a reverse primer 18R shown at the sequence 54. The primer set 19 may be composed of a forward primer 19F1 shown at positions 22 to 48 from the 5 'end of the sequence 55, a forward primer 19F2 shown at positions 22 to 49 from the 5' end of the sequence 56, and a reverse primer 19R shown at the sequence 57. The primer set 20 may be composed of a forward primer 20F1 shown in 22 nd to 48 th positions from the 5 'end of the sequence 58, a forward primer 20F2 shown in 22 nd to 47 th positions from the 5' end of the sequence 59, and a reverse primer 20R shown in the sequence 60. The primer set 21 may be composed of a forward primer 21F1 shown in 22 nd to 43 th positions from the 5 'end of the sequence 61, a forward primer 21F2 shown in 22 nd to 42 th positions from the 5' end of the sequence 62, and a reverse primer 21R shown in 63. The primer set 22 may be composed of a forward primer 22F1 shown at positions 22 to 47 from the 5 'end of the sequence 64, a forward primer 22F2 shown at positions 22 to 46 from the 5' end of the sequence 65, and a reverse primer 22R shown at the sequence 66. The primer set 23 may be composed of a forward primer 23F1 shown at positions 22 to 46 from the 5 'end of the sequence 67, a forward primer 23F2 shown at positions 22 to 46 from the 5' end of the sequence 68, and a reverse primer 23R shown at the sequence 69. The primer set 24 may be composed of a forward primer 24F1 shown in 22 th to 43 th positions from the 5 'end of the sequence 70, a forward primer 24F2 shown in 22 th to 44 th positions from the 5' end of the sequence 71, and a reverse primer 24R shown in the sequence 72. The primer set 25 may be composed of a forward primer 25F1 shown in 22 th to 51 th from the 5 'end of the sequence 73, a forward primer 25F2 shown in 22 th to 51 th from the 5' end of the sequence 74, and a reverse primer 25R shown in the sequence 75. The primer set 26 may be composed of a forward primer 26F1 shown in the 22 nd to 43 th positions from the 5 'end of the sequence 76, a forward primer 26F2 shown in the 22 nd to 42 th positions from the 5' end of the sequence 77, and a reverse primer 26R shown in the sequence 78. The primer set 27 may be composed of a forward primer 27F1 shown in 22 th to 49 th positions from the 5 'end of sequence 79, a forward primer 27F2 shown in 22 th to 48 th positions from the 5' end of sequence 80, and a reverse primer 27R shown in sequence 81. The primer set 28 may be composed of a forward primer 28F1 shown at positions 22 to 54 from the 5 'end of the sequence 82, a forward primer 28F2 shown at positions 22 to 54 from the 5' end of the sequence 83, and a reverse primer 28R shown at the sequence 84. The primer set 29 may be composed of a forward primer 29F1 shown in 22 nd to 46 th positions from the 5 'end of the sequence 85, a forward primer 29F2 shown in 22 nd to 45 th positions from the 5' end of the sequence 86, and a reverse primer 29R shown in 87 th positions. The primer set 30 may be composed of a forward primer 30F1 shown in 22 nd to 52 th positions from the 5 'end of the sequence 88, a forward primer 30F2 shown in 22 nd to 52 th positions from the 5' end of the sequence 89, and a reverse primer 30R shown in the sequence 90. The primer set 31 may be composed of a forward primer 31F1 shown in 22 th to 54 th positions from the 5 'end of the sequence 91, a forward primer 31F2 shown in 22 th to 53 th positions from the 5' end of the sequence 92, and a reverse primer 31R shown in the sequence 93. The primer set 32 may be composed of a forward primer 32F1 shown in 22 nd to 56 th positions from the 5 'end of the sequence 94, a forward primer 32F2 shown in 22 nd to 56 th positions from the 5' end of the sequence 95, and a reverse primer 32R shown in 96.

In any of the above primer sets, the molar ratio of the primer named as "F1", the primer named as "F2" and the primer named as "R" may be specifically 2:2: 5.

The SNP primer set may specifically include the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, the primer set 8, the primer set 9, the primer set 10, the primer set 11, the primer set 12, the primer set 13, the primer set 14, the primer set 15, the primer set 16, the primer set 17, the primer set 18, the primer set 19, the primer set 20, the primer set 21, the primer set 22, the primer set 23, the primer set 24, the primer set 25, the primer set 26, the primer set 27, the primer set 28, the primer set 29, the primer set 30, the primer set 31, and the primer set 32.

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 specifically blue. 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 separately packaging each primer in any one primer group.

The application of the kit also belongs to the protection scope of the invention. The application of the kit can be x3) or x 4): x3) identifying cucumber varieties; x4) identifying the authenticity of the cucumber variety.

The application of any one of the SNP site combinations or any one of the SNP primer combinations can be any one of x1) to x 4): x1) preparing a kit for identifying cucumber varieties; x2) preparing a reagent kit for identifying the authenticity of the cucumber variety; x3) identifying cucumber varieties; x4) identifying the authenticity of the cucumber variety.

The invention also discloses a method for identifying the variety of 241 cucumbers to be detected, which comprises the following steps: and respectively detecting the genotypes of the cucumber to be detected and 241 cucumber varieties based on the 32 SNP loci, and then judging as follows: if the genotypes of the cucumber to be detected based on the 32 SNP sites are completely consistent with the genotypes of a certain variety of the 241 cucumber varieties based on the 32 SNP sites, the cucumber to be detected and the cucumber variety belong to the same variety; and if the genotypes of the cucumber to be detected based on the 32 SNP sites and the genotypes of the 241 cucumber varieties based on the 32 SNP sites are different, the varieties of the cucumber to be detected and the 241 cucumber varieties are different.

In the above method, the step of detecting the genotypes of the cucumber to be detected and the 241 cucumber varieties based on the 32 SNP sites may be as follows:

(1) respectively taking the genome DNA of cucumber to be detected and the genome DNA of 241 cucumber varieties as templates, and respectively adopting the primer groups in any one of the SNP primer combinations to carry out PCR amplification to obtain PCR amplification products;

(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 genotypes of the cucumber to be detected and 241 cucumber varieties based on the 32 SNP sites according to the color of the fluorescent signal.

In the above method, the step of detecting the genotypes of the cucumber to be detected and 241 cucumber varieties based on the 32 SNP sites may be as follows:

(1) respectively taking the genome DNA of cucumber to be detected and the genome DNA of 241 cucumber varieties as templates, and respectively adopting the primer groups in any one of the SNP primer combinations to carry out PCR amplification to obtain PCR amplification products;

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

(3) and (3) obtaining the genotypes of the cucumber to be detected and 241 cucumber varieties based on the 32 SNP loci according to the sequencing result obtained in the step (2).

The invention also discloses a method for identifying the variety of 241 cucumbers to be detected, which comprises the following steps:

(1) taking the genome DNA of cucumber to be detected as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the SNP primer combinations to obtain PCR amplification products; taking the genome DNA of each cucumber variety in a standard cucumber variety population as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the SNP primer combinations to obtain PCR amplification products; the standard cucumber population consists of 241 cucumber varieties;

(2) and performing cluster analysis on each PCR amplification product obtained from the cucumber to be detected and the PCR amplification product corresponding to each standard cucumber variety, wherein the cucumber to be detected and which standard cucumber variety are in the same class in the cluster analysis, and the cucumber to be detected and the standard cucumber variety belong to the same variety.

In any of the above methods, 241 cucumber varieties may be emerald green, Jiza 16, Zhongnong 20, Lu cucumber 9, Zhongnong 29, Dianna, Zhongnong 8F1, Beisha, Zhongnong 23F1, Jinbode 99-3F1, Zhongnong 17F1, Jinbode 99-2F1, Jinbode 99F1, Jinbode 99-1F1, Jinbode 98F1, Jinbode 98-1F1, Zhongnong 19F1, silver embryo 88F1, 128-generation hybrid, Zhongnong 6, Zhongnong 10, Beinong Jiaoxiu, Jing 106, transco cucumber F1, Ningxing Xingxing spring and autumn F1, Yameite, AMATA765, Jinshan old cucumber, Jingshansha 9, Jingyan 3, Jingyan 5, Mingganyi mini 4, Jing 4, Jingyan 4, Minjing Minqin jingganli, Jingmei Shi Miao, Jingmei Shi, Jingmei Bai Baihui Hai, Yuan, Beijing Kong Yuan Yufa, Beijing Kong fruit jelly, Beijing Kong Yuan Yu, Tangshan Cuiyao F1, refined Baiyesan, Tangchun 100, cucurbit, Aosguan, Manguan, Bao Chun and autumn F1, Jing Yan Linglong No. 2, Jing Yanhanbao No. 5, Youliang Wangzhongwang, Jing Yanxiamei, Jing Yangyun, Jing Feng 298, Jinyou No. 35, Jing Yanqimei, Jing Yanchun Mei, Luo Boelen No. 2, Luboqi No. 403, Beijing 403, Luxiu, Beijing 204, Xinjiang Quzhen No. four, Jing Yan Si No. four, Mici Wang CUCUMBER, pluck Baihui Diwang, Longquan-Longquan Green crown, XSBN4, XSBN6, XSBN8, XSBN11, XSBN16, XSBN1, XSBN2, XSBN3, XS 7, XSBN9, XSBN 29, XSBN12, XSBN 4642, XSBN # 19, Qinshinun No. 2-4619, Qijin No. 11-4623, Qishen No. 23, Qichun Xichun No. 11, XSBN 3-4619, Qichun No. 3-XSBN, Qichun No. 11, XSBN 3-XSBN, XS19, XSBN 3-XS19, Zusan No. 4, XSBN 3-, 13AC230, jade, LVCING, JINGZHUANMINI No. 1, LIANGLIANG, DONGONG 804, NING JIA No. 3, YUJIALOQUANJUA 09, HEHAN, YUNV, JIN you No. 12, JINCHUN No. 4, HUIZAO No. 6, BAIHAO No. 2, JIN you No. 2, DONGLI, JIN you No. 36, MC2065, ZHONG nong No. 15, North Japan star, Zhongnong No. 26, JIN you No. 38, JIN you No. 31, BOMEI No. 11, BOMEI No. 74, JINCHUN No. 3, JIN Green No. 3, JIZAO No. nine, TIANJIAO No. eight, SHUFENG, SIFENG, CUOLONG 9801, LVYINGYINGYINGYINGYANG, YINGQING No. 1, LUCUISHUNSU No. 10, JIN YOU No. 6913, QIANDE 117, QINGCHUN HUN No. 3, JIN YOU U, JIN YOU 308, P02, P KOU, P9930, BINANJIAO MEI JUN YE 008, BIDE MEI, BING ME, Jing AK42, Meifeng melon king, Jing AK18, Del LD-1, Del RE Te F16, Del RE Te Y8, Ding Hao Liang Zan Wang, Hei you Liang 999, jin Zheng A207, Lvbao Zhu No. 8, Del RE Te 727, Del RE Te 7876, Del RE LD-3, YOU LV Wang Zi, Jing Ming 118, Huiyang 15, Cyanin Dazhua, Mini No. 3, Jinwang 203, Cucumis sativus, Ldelubao, Shuojiu, Tianyi, Qijian No. 1, Lvyou 188, Chunxi F1, Qiangshenjie Jie, Xinjiang No. four, Xinxing Ming No. 2, Hei Jian No. 2, Xinjin Chun Yu Meili Xinjing Mao Xingqi Xin Li Xin Lu Yu Mei Xin, Gu Yu Li Xin Hu Lu Si Xin Si Lu No. 2, Mi Mei Xin Si Lu, Jing, White spirit No. 2, Junyou 66 cucumber, Delaut cucumber 136-7, Jingbaiyu super white leaf three, specially selected Jizai No. four, Jingyan green exquisite, Cucumis luteus, Tianjiao No. 7 Huanan type, Ruiguan No. two, Burseo green you cucumber, selected leaf three, Guyu Jiali, Guyu drought cucumber, Tong 52, Delaut cucumber L14-2, Delaut cucumber GZ1601, Delaut cucumber L14-5, Delaut cucumber, Korean cucumber 2-06-97-164, Shengfeng 908, Delaut 15-10 Mici cucumber, Jindong Ruizu 99 Mici cucumber, Zhongnong No. 106, Zhongnong No. 27, Jinyou No. 4, Jinyou No. 11, Jinza No. 2, Zhongnong No. 16, Jinyou 38, PI197088, H9, WI2757, Wue Mone, 13642014.11 and Z2982013.11.

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

The SNP primer combination provided by the invention can be used for early identification of cucumber varieties in seed or seedling stage, so that the authenticity of the varieties is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for the protection of cucumber germplasm resources and new varieties. The method provided by the invention can be used for identifying unknown cucumber varieties and also can be used for identifying the authenticity of known varieties. 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.

Drawings

FIG. 1 is a cluster plot of 241 cucumber varieties tested, built on 32 primer sets.

FIG. 2 is a graph showing the relationship between the number of SNP markers (i.e., the number of SNP sites) and the discrimination of 241 cucumber varieties to be tested.

FIG. 3 shows the SNP typing effect of 32 primer sets in a part of cucumber varieties tested.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 obtaining of SNP primer combination for identifying the Authenticity of cucumber varieties

Discovery of one, 32 SNP sites

The invention obtains 32 SNP loci based on the resequencing data of 49 cucumber representative resources. The 49 cucumbers are rich in resource types, include North China (7), Indian (18), Japanese (3), south China (2), European fruit (5), American processed (4), Xishuangbanna (5) and intermediate (5), basically comprise the main ecological types and agronomic traits of the cucumbers, reflect germplasm representativeness as much as possible, and have higher genetic diversity.

Specifically, the screening criteria for SNP sites are as follows: SNP loci with uniform positions, good polymorphism, small heterozygosity, MAF >0.3, good PCA clustering effect, high discrimination and two-wing 50bp sequence conservation (no InDel, no SSR and no other SNP) are selected in the whole genome range.

The basic information of the 32 SNP sites is detailed in columns 1 to 4 of Table 1. The position of the SNP locus on the chromosome is determined based on the comparison of cucumber 9930 reference genome sequences, and the version number of the cucumber 9930 reference genome sequence is V2 (download address: http:// cucurbitangencis. org/organissm/2).

TABLE 1.32 basic information of SNP sites

Second, obtaining of SNP primer combination for identifying cucumber variety authenticity

Based on the 32 SNP sites found in step one, the inventors of the present invention developed SNP primer combinations for identifying the authenticity of cucumber varieties with higher polymorphism information amount (i.e., PIC value, see column 5 in Table 1).

The SNP primer set consists of 32 primer sets, and the name of each primer set is shown in the 2 nd column in Table 2. Each primer set consists of 3 primer sequences and is used for amplifying one SNP site. The nucleotide sequences of the individual primers in the 32 primer sets are shown in column 4 of Table 2.

TABLE 2

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

Example 2 validation of SNP primer combinations developed in the examples

The basic information of the 241 cucumber varieties tested in this example is shown in table 3. The 241 tested cucumber varieties are common fine varieties or partially foreign introduced varieties.

Table 3.241 basic information of cucumber varieties tested

1. Acquisition of genomic DNA of cucumber varieties to be tested

The genome DNA of the leaf of 241 tested cucumber varieties (mixing true leaves of 30 seeds) is extracted by a CTAB method respectively to obtain the genome DNA of the tested cucumber varieties.

The quality and concentration of the genome DNA of the cucumber variety to be tested both need to meet the PCR requirement, and the standard of reaching the standard 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 cucumber variety to be tested was 10-30 ng/. mu.L.

2. And respectively taking the genome DNA of 241 cucumber varieties to be tested as templates, and respectively adopting 32 primer groups to carry out PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer named "F1", the primer named "F2" and the primer named "R" was 2:2: 5.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, 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 completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading value is observed when the FAM fluorescent label sequence is at 485nm of exciting light and 520nm of emitting light, reading value is observed when the HEX fluorescent label sequence is at 528nm of exciting light and 560nm of emitting light), and judging the genotype of 241 cucumber varieties to be tested based on each SNP locus according to the color of a fluorescence signal. The specific judgment principle is as follows: if a certain cucumber variety to be tested shows a blue fluorescent signal based on a certain SNP locus, the cucumber variety to be tested is homozygous for the complementary base of the 1 st base at the 3' end of the primer which is used for amplifying the SNP locus and contains F1 in the name based on the genotype of the SNP locus; if a certain cucumber variety to be tested shows a red fluorescent signal based on a certain SNP locus, the cucumber variety to be tested 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 of the SNP locus; if a cucumber variety to be tested shows a green fluorescent signal based on a certain SNP site, the cucumber variety to be tested is of a hybrid type based on the genotype of the SNP site, one base is a complementary base of the 1 st base at the 3 'end of the 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 the primer which amplifies the SNP site and contains the F2 in the name.

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

Partial results are shown in FIG. 3. The results show that each primer group can obtain good typing effect in cucumber varieties to be tested.

4. Cluster analysis

And (3) carrying out cluster analysis on 241 cucumber varieties to be tested by utilizing MiniMarker and MEGA7 software according to the genotypes of the 241 cucumber varieties to be tested based on 32 SNP loci.

The cluster map of 241 cucumber species tested, established on 32 primer sets, is shown in fig. 1. The results show that the 241 cucumber species tested in table 3 can be completely distinguished by the 32 primer groups. Therefore, the SNP primer combination developed in the example 1 can be applied to the construction of a cucumber variety DNA fingerprint database and the identification of variety authenticity.

5. Evaluation of efficiency

The variety authenticity identification can reduce the workload by adopting a sequential analysis mode. The inventors of the present invention compared the relationship between the number of SNP markers (i.e., the number of primer sets) and the discrimination rate for discriminating 241 cucumber varieties to be tested.

The experimental results show (fig. 2) that the discrimination rate of the 32 primer sets (namely the number of the 32 SNP markers) in the 241 cucumber varieties to be tested reaches 100%.

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

<120> method for identifying cucumber variety authenticity and special SNP primer combination thereof

<160> 96

<170> PatentIn version 3.5

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<213> Artificial sequence

<400> 74

gaaggtcgga gtcaacggat tcaacaaaca aatggagctt taagtaaagt aa 52

<210> 75

<211> 32

<212> DNA

<213> Artificial sequence

<400> 75

gccttttgac tcgtatgttt tactttactt ta 32

<210> 76

<211> 43

<212> DNA

<213> Artificial sequence

<400> 76

gaaggtgacc aagttcatgc ttgaacagct gagggccatt gca 43

<210> 77

<211> 42

<212> DNA

<213> Artificial sequence

<400> 77

gaaggtcgga gtcaacggat tgaacagctg agggccattg cg 42

<210> 78

<211> 27

<212> DNA

<213> Artificial sequence

<400> 78

cacaacgtct tcctcttggc tagttat 27

<210> 79

<211> 49

<212> DNA

<213> Artificial sequence

<400> 79

gaaggtgacc aagttcatgc tcatatacac acaatacata gctgttgga 49

<210> 80

<211> 48

<212> DNA

<213> Artificial sequence

<400> 80

gaaggtcgga gtcaacggat tatatacaca caatacatag ctgttggg 48

<210> 81

<211> 31

<212> DNA

<213> Artificial sequence

<400> 81

ctctgccaga aacaaacaat aacttttgta a 31

<210> 82

<211> 54

<212> DNA

<213> Artificial sequence

<400> 82

gaaggtgacc aagttcatgc tatgaggaag aaatgagcat atatttattt gttt 54

<210> 83

<211> 54

<212> DNA

<213> Artificial sequence

<400> 83

gaaggtcgga gtcaacggat tatgaggaag aaatgagcat atatttattt gtta 54

<210> 84

<211> 30

<212> DNA

<213> Artificial sequence

<400> 84

gattaggagg ggtagaagtg attaatgaat 30

<210> 85

<211> 46

<212> DNA

<213> Artificial sequence

<400> 85

gaaggtgacc aagttcatgc tgatgtgtgc agatgacgat gataga 46

<210> 86

<211> 45

<212> DNA

<213> Artificial sequence

<400> 86

gaaggtcgga gtcaacggat tatgtgtgca gatgacgatg atagg 45

<210> 87

<211> 34

<212> DNA

<213> Artificial sequence

<400> 87

acttagtttg gaattattga taatttcctg caat 34

<210> 88

<211> 52

<212> DNA

<213> Artificial sequence

<400> 88

gaaggtgacc aagttcatgc tgtttattgg gtttatttaa actaagacat cg 52

<210> 89

<211> 52

<212> DNA

<213> Artificial sequence

<400> 89

gaaggtcgga gtcaacggat tgtttattgg gtttatttaa actaagacat cc 52

<210> 90

<211> 31

<212> DNA

<213> Artificial sequence

<400> 90

agagaagaag ccaaatatga atggaattct t 31

<210> 91

<211> 54

<212> DNA

<213> Artificial sequence

<400> 91

gaaggtgacc aagttcatgc ttctttttct cttttctttt ttctgacttc ttta 54

<210> 92

<211> 53

<212> DNA

<213> Artificial sequence

<400> 92

gaaggtcgga gtcaacggat tctttttctc ttttcttttt tctgacttct ttg 53

<210> 93

<211> 30

<212> DNA

<213> Artificial sequence

<400> 93

ctgaaacatg cgtacaacta aactaagcta 30

<210> 94

<211> 56

<212> DNA

<213> Artificial sequence

<400> 94

gaaggtgacc aagttcatgc ttttgtttcc aatgattaga agtatattta tatatc 56

<210> 95

<211> 56

<212> DNA

<213> Artificial sequence

<400> 95

gaaggtcgga gtcaacggat ttttgtttcc aatgattaga agtatattta tatatg 56

<210> 96

<211> 36

<212> DNA

<213> Artificial sequence

<400> 96

ctttgttcca attttctaaa ttttgttttt ccatta 36

Claims (6)

1. The primer combination A comprises a primer group 1-a primer group 32;

the primer group 1 consists of a forward primer 01F1 shown in a sequence 1, a forward primer 01F2 shown in a sequence 2 and a reverse primer 01R shown in a sequence 3; the primer group 2 consists of a forward primer 02F1 shown in a sequence 4, a forward primer 02F2 shown in a sequence 5 and a reverse primer 02R shown in a sequence 6; the primer group 3 consists of a forward primer 03F1 shown in a sequence 7, a forward primer 03F2 shown in a sequence 8 and a reverse primer 03R shown in a sequence 9; the primer group 4 consists of a forward primer 04F1 shown in a sequence 10, a forward primer 04F2 shown in a sequence 11 and a reverse primer 04R shown in a sequence 12; the primer group 5 consists of a forward primer 05F1 shown in a sequence 13, a forward primer 05F2 shown in a sequence 14 and a reverse primer 05R shown in a sequence 15; the primer group 6 consists of a forward primer 06F1 shown in a sequence 16, a forward primer 06F2 shown in a sequence 17 and a reverse primer 06R shown in a sequence 18; the primer group 7 consists of a forward primer 07F1 shown in a sequence 19, a forward primer 07F2 shown in a sequence 20 and a reverse primer 07R shown in a sequence 21; the primer group 8 consists of a forward primer 08F1 shown in a sequence 22, a forward primer 08F2 shown in a sequence 23 and a reverse primer 08R shown in a sequence 24; the primer group 9 consists of a forward primer 09F1 shown in a sequence 25, a forward primer 09F2 shown in a sequence 26 and a reverse primer 09R shown in a sequence 27; the primer group 10 consists of a forward primer 10F1 shown in a sequence 28, a forward primer 10F2 shown in a sequence 29 and a reverse primer 10R shown in a sequence 30; the primer group 11 consists of a forward primer 11F1 shown in a sequence 31, a forward primer 11F2 shown in a sequence 32 and a reverse primer 11R shown in a sequence 33; the primer group 12 consists of a forward primer 12F1 shown in a sequence 34, a forward primer 12F2 shown in a sequence 35 and a reverse primer 12R shown in a sequence 36; the primer group 13 consists of a forward primer 13F1 shown in a sequence 37, a forward primer 13F2 shown in a sequence 38 and a reverse primer 13R shown in a sequence 39; the primer group 14 consists of a forward primer 14F1 shown in a sequence 40, a forward primer 14F2 shown in a sequence 41 and a reverse primer 14R shown in a sequence 42; the primer group 15 consists of a forward primer 15F1 shown in a sequence 43, a forward primer 15F2 shown in a sequence 44 and a reverse primer 15R shown in a sequence 45; the primer group 16 consists of a forward primer 16F1 shown in a sequence 46, a forward primer 16F2 shown in a sequence 47 and a reverse primer 16R shown in a sequence 48; the primer group 17 consists of a forward primer 17F1 shown in a sequence 49, a forward primer 17F2 shown in a sequence 50 and a reverse primer 17R shown in a sequence 51; the primer group 18 consists of a forward primer 18F1 shown in a sequence 52, a forward primer 18F2 shown in a sequence 53 and a reverse primer 18R shown in a sequence 54; the primer group 19 consists of a forward primer 19F1 shown in a sequence 55, a forward primer 19F2 shown in a sequence 56 and a reverse primer 19R shown in a sequence 57; the primer group 20 consists of a forward primer 20F1 shown in a sequence 58, a forward primer 20F2 shown in a sequence 59 and a reverse primer 20R shown in a sequence 60; the primer group 21 consists of a forward primer 21F1 shown in a sequence 61, a forward primer 21F2 shown in a sequence 62 and a reverse primer 21R shown in a sequence 63; the primer group 22 consists of a forward primer 22F1 shown in a sequence 64, a forward primer 22F2 shown in a sequence 65 and a reverse primer 22R shown in a sequence 66; the primer group 23 consists of a forward primer 23F1 shown in a sequence 67, a forward primer 23F2 shown in a sequence 68 and a reverse primer 23R shown in a sequence 69; the primer group 24 consists of a forward primer 24F1 shown in a sequence 70, a forward primer 24F2 shown in a sequence 71 and a reverse primer 24R shown in a sequence 72; the primer group 25 consists of a forward primer 25F1 shown in a sequence 73, a forward primer 25F2 shown in a sequence 74 and a reverse primer 25R shown in a sequence 75; the primer group 26 consists of a forward primer 26F1 shown in a sequence 76, a forward primer 26F2 shown in a sequence 77 and a reverse primer 26R shown in a sequence 78; the primer group 27 consists of a forward primer 27F1 shown in a sequence 79, a forward primer 27F2 shown in a sequence 80 and a reverse primer 27R shown in a sequence 81; the primer group 28 consists of a forward primer 28F1 shown in a sequence 82, a forward primer 28F2 shown in a sequence 83 and a reverse primer 28R shown in a sequence 84; the primer group 29 consists of a forward primer 29F1 shown in a sequence 85, a forward primer 29F2 shown in a sequence 86 and a reverse primer 29R shown in a sequence 87; the primer group 30 consists of a forward primer 30F1 shown in a sequence 88, a forward primer 30F2 shown in a sequence 89 and a reverse primer 30R shown in a sequence 90; the primer group 31 consists of a forward primer 31F1 shown in a sequence 91, a forward primer 31F2 shown in a sequence 92 and a reverse primer 31R shown in a sequence 93; the primer group 32 is composed of a forward primer 32F1 shown in a sequence 94, a forward primer 32F2 shown in a sequence 95 and a reverse primer 32R shown in a sequence 96.

2. The primer combination B comprises a primer group 1-a primer group 32;

the primer group 1 consists of a forward primer 01F1 shown in 22 th to 46 th positions from the 5 'end of a sequence 1, a forward primer 01F2 shown in 22 th to 46 th positions from the 5' end of a sequence 2 and a reverse primer 01R shown in a sequence 3; the primer group 2 consists of a forward primer 02F1 shown in the 22 nd to 46 th positions from the 5 'end of the sequence 4, a forward primer 02F2 shown in the 22 nd to 43 th positions from the 5' end of the sequence 5 and a reverse primer 02R shown in the sequence 6; the primer group 3 consists of a forward primer 03F1 shown in 22 th to 48 th positions from the 5 'end of the sequence 7, a forward primer 03F2 shown in 22 th to 50 th positions from the 5' end of the sequence 8 and a reverse primer 03R shown in the sequence 9; the primer group 4 consists of a forward primer 04F1 shown in 22 th to 52 th positions from the 5 'end of the sequence 10, a forward primer 04F2 shown in 22 th to 51 th positions from the 5' end of the sequence 11 and a reverse primer 04R shown in the sequence 12; the primer group 5 consists of a forward primer 05F1 shown in 22 th to 47 th positions from the 5 'end of the sequence 13, a forward primer 05F2 shown in 22 th to 47 th positions from the 5' end of the sequence 14, and a reverse primer 05R shown in the sequence 15; the primer group 6 consists of a forward primer 06F1 shown in 22 th to 51 th positions from the 5 'end of the sequence 16, a forward primer 06F2 shown in 22 th to 51 th positions from the 5' end of the sequence 17, and a reverse primer 06R shown in the sequence 18; the primer group 7 consists of a forward primer 07F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 19, a forward primer 07F2 shown in 22 th to 48 th positions from the 5' end of the sequence 20, and a reverse primer 07R shown in the sequence 21; the primer group 8 consists of a forward primer 08F1 shown in 22 th to 48 th positions from the 5 'end of the sequence 22, a forward primer 08F2 shown in 22 th to 48 th positions from the 5' end of the sequence 23, and a reverse primer 08R shown in the sequence 24; the primer group 9 consists of a forward primer 09F1 shown in 22 th to 48 th positions from the 5 'end of the sequence 25, a forward primer 09F2 shown in 22 th to 47 th positions from the 5' end of the sequence 26, and a reverse primer 09R shown in the sequence 27; the primer group 10 consists of a forward primer 10F1 shown in 22 th to 51 th positions from the 5 'end of the sequence 28, a forward primer 10F2 shown in 22 th to 51 th positions from the 5' end of the sequence 29, and a reverse primer 10R shown in the sequence 30; the primer set 11 consists of a forward primer 11F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 31, a forward primer 11F2 shown in 22 th to 45 th positions from the 5' end of the sequence 32, and a reverse primer 11R shown in the sequence 33; the primer set 12 consists of a forward primer 12F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 34, a forward primer 12F2 shown in 22 th to 47 th positions from the 5' end of the sequence 35, and a reverse primer 12R shown in the sequence 36; the primer set 13 consists of a forward primer 13F1 shown in 22 th to 43 th positions from the 5 'end of the sequence 37, a forward primer 13F2 shown in 22 th to 44 th positions from the 5' end of the sequence 38, and a reverse primer 13R shown in the sequence 39; the primer set 14 is composed of a forward primer 14F1 shown in 22 nd to 49 th from the 5 'end of the sequence 40, a forward primer 14F2 shown in 22 nd to 49 th from the 5' end of the sequence 41, and a reverse primer 14R shown in the sequence 42; the primer group 15 consists of a forward primer 15F1 shown in 22 nd to 48 th positions from the 5 'end of the sequence 43, a forward primer 15F2 shown in 22 nd to 49 th positions from the 5' end of the sequence 44, and a reverse primer 15R shown in the sequence 45; the primer set 16 consists of a forward primer 16F1 shown in 22 th to 46 th positions from the 5 'end of the sequence 46, a forward primer 16F2 shown in 22 th to 48 th positions from the 5' end of the sequence 47, and a reverse primer 16R shown in 48 th position; the primer group 17 consists of a forward primer 17F1 shown in 22 nd to 51 th positions from the 5 'end of the sequence 49, a forward primer 17F2 shown in 22 nd to 52 th positions from the 5' end of the sequence 50, and a reverse primer 17R shown in 51; the primer set 18 consists of a forward primer 18F1 shown in the 22 nd to 46 th positions from the 5 'end of the sequence 52, a forward primer 18F2 shown in the 22 nd to 44 th positions from the 5' end of the sequence 53, and a reverse primer 18R shown in the sequence 54; the primer set 19 consists of a forward primer 19F1 shown in 22 nd to 48 th positions from the 5 'end of the sequence 55, a forward primer 19F2 shown in 22 nd to 49 th positions from the 5' end of the sequence 56, and a reverse primer 19R shown in the sequence 57; the primer set 20 consists of a forward primer 20F1 shown in 22 nd to 48 th positions from the 5 'end of the sequence 58, a forward primer 20F2 shown in 22 nd to 47 th positions from the 5' end of the sequence 59, and a reverse primer 20R shown in the sequence 60; the primer group 21 consists of a forward primer 21F1 shown in 22 nd to 43 th positions from the 5 'end of the sequence 61, a forward primer 21F2 shown in 22 nd to 42 th positions from the 5' end of the sequence 62, and a reverse primer 21R shown in the sequence 63; the primer set 22 consists of a forward primer 22F1 shown in the 22 nd to 47 th positions from the 5 'end of the sequence 64, a forward primer 22F2 shown in the 22 nd to 46 th positions from the 5' end of the sequence 65, and a reverse primer 22R shown in the sequence 66; the primer group 23 consists of a forward primer 23F1 shown in the 22 nd to 46 th positions from the 5 'end of the sequence 67, a forward primer 23F2 shown in the 22 nd to 46 th positions from the 5' end of the sequence 68, and a reverse primer 23R shown in the sequence 69; the primer set 24 consists of a forward primer 24F1 shown in 22 th to 43 th positions from the 5 'end of the sequence 70, a forward primer 24F2 shown in 22 th to 44 th positions from the 5' end of the sequence 71 and a reverse primer 24R shown in the sequence 72; the primer set 25 consists of a forward primer 25F1 shown in 22 th to 51 th positions from the 5 'end of the sequence 73, a forward primer 25F2 shown in 22 th to 51 th positions from the 5' end of the sequence 74, and a reverse primer 25R shown in the sequence 75; the primer set 26 consists of a forward primer 26F1 shown in 22 nd to 43 th positions from the 5 'end of the sequence 76, a forward primer 26F2 shown in 22 nd to 42 th positions from the 5' end of the sequence 77, and a reverse primer 26R shown in the sequence 78; the primer group 27 consists of a forward primer 27F1 shown in the 22 nd to 49 th positions from the 5 'end of the sequence 79, a forward primer 27F2 shown in the 22 nd to 48 th positions from the 5' end of the sequence 80, and a reverse primer 27R shown in the sequence 81; the primer set 28 consists of a forward primer 28F1 shown in the 22 nd to 54 th positions from the 5 'end of the sequence 82, a forward primer 28F2 shown in the 22 nd to 54 th positions from the 5' end of the sequence 83, and a reverse primer 28R shown in the sequence 84; the primer group 29 consists of a forward primer 29F1 shown in 22 nd to 46 th positions from the 5 'end of the sequence 85, a forward primer 29F2 shown in 22 nd to 45 th positions from the 5' end of the sequence 86, and a reverse primer 29R shown in 87 th positions; the primer group 30 consists of a forward primer 30F1 shown in 22 nd to 52 th positions from the 5 'end of the sequence 88, a forward primer 30F2 shown in 22 nd to 52 th positions from the 5' end of the sequence 89, and a reverse primer 30R shown in the sequence 90; the primer set 31 consists of a forward primer 31F1 shown in 22 th to 54 th positions from the 5 'end of the sequence 91, a forward primer 31F2 shown in 22 nd to 53 th positions from the 5' end of the sequence 92, and a reverse primer 31R shown in the sequence 93; the primer set 32 is composed of a forward primer 32F1 shown in 22 nd to 56 th positions from the 5 'end of the sequence 94, a forward primer 32F2 shown in 22 nd to 56 th positions from the 5' end of the sequence 95, and a reverse primer 32R shown in 96.

3. The primer combination A of claim 1 or the primer combination B of claim 2, which is any one of x1) to x 4):

x1) preparing a kit for identifying 241 cucumber varieties;

x2) preparing a kit for identifying the authenticity of 241 cucumber varieties;

x3) identifying 241 cucumber varieties;

x4) identifying the authenticity of 241 cucumber varieties;

the 241 cucumber varieties are emerald green, Jiza 16, Zhongnong 20, Lu cucumber 9, Zhongnong 29, Dianna, Zhongyanmao 8F1, Beisha, Zhongyan 23F1, Jinbo 99-3F1, Zhongyan 17F1, Jinbo 99-2F1, Jinbo 99F1, Jinbo 99-1F1, Jinbo 98F1, Jinbo 98-1F1, Zhongyan 19F1, silver embryo 88F1, 128-generation hybrid, Zhongnong 6, Zhongnong 10, Beinong Jia Xiu, Jing Yan 106, surpass cucumber F1, Ningxing Chunyun F1, Jingyameite, AMATA, Jinjinshan cucumber, Jingshan mini Qinshan Minqiu No. 9, Jingyan Jia Xian No. 3, Jingyan Jia Zhenghua 5, Cucumis 4, Jing Ming Zheng Hui Bao, Min Zhen Hao Bao, Min Hua Hao, Jing Hao, Jing Hui Li Shi, Fine Baiye San, Tangchun 100, hu melon, Aoguang, Manguan, almighty spring and autumn F1, Jing Yan Lu Linglong No. 2, Jing Yanhanbao No. 5, Youmiangzhongwang, Jing Yanxiamei, Jing Yanyousheng, Jing Feng 298, Jing you No. 35, Jing Yanqimei, Jing Yanchunmei, Luboheng No. 2, Luboqi No. 403, Beijing, Lvxiu, Beijing 204, Xinyansihao No. four, Jinyansihao, Mici king CUCUMBER, Xibai not Bailu, Longquan-Longquan Luguan, XSBN4, XSBN6, XSBN8, XSBN11, XSBN 6348, XSBN2, XSBN3, XSBN5, XSBN7, XSBN9, XSBN6, XSBN13, XSBN14, XSBN 29, XSBN # 84, XSBN # 3, JUN No. 2, XSBN 4672, JUN No. 2, XSBN # 19, JUN, JUN No. 2, JUN, XSBN 4672, JUN No. 2, XSBN # 3, XSBN 4672, XSBN # 19, JUN, XSBN # 3, XSBN 4611, XSBN #, Jingyan mini No. 1, liangliang, Dongnong 804, Ningjia No. 3, Yougan Quansha 09, He dryland, Yunv, Jinyou No. 12, Jinchun No. 4, Huza No. 6, Baohe No. 2, Zi Si, Jinyou No. 2, Dongli, Jinyou No. 36, MC2065, Zhongnong No. 15, North Japan, Zhongnong No. 26, Jinyou No. 38, Jinyou No. 31, Bomei No. 11, Bomei No. 74, Jinchun No. three, Jinlv No. 3, Jizaojiu No. eight, Shufeng No. eight, Sifeng Wang, Cucumis No. 1, Lu Cucumis No. 10, Bomei No. 6913, Qiande 117, Jinchun Chun No. 3, Jinyou No. 315, AK No. 308, P02, Wannong No. three, 9930, white horse seed, Cucumis, Kuihe Ruidezi No. 008, Beijing Yide No. 32, Beijing Yijie Jie Wan No. 32, Beijing Mao Wan Jie Ruojia, Beijing Mao No. 52, Beijing Mao Wan Shi No. 4, Beijing Mao Wan Shi, Dride F16, Dride Y8, Dinghao Youang replant, Black oil Bright 999, Jinzheng A207, Lvbaozhu No. 8, Dride 727, Dride 7876, Dride, Deler LD-3, oil Green prince, Jingyan 118, Huiyin 15, Cyanin big cucumber, Mini No. 3, Jinwang 203, Cucumis sativus, Ldelbrunbao, Shuojiu, Tianyi, Qijian No. 1, Lvyou 188, Chunxi F1, Qianglianjie knot, Xingyuan No. four, Xingyuan No. two, Black oil Bright 507, Qijian No. 2, Xinjin Chunrou No. 409, Guyumailixin, 83-ms702, Jingyan Mini No. 2, Duchangjixing, Lvjingling No. 2, CU No. 003, Weijiya, CU 9006, Sakulin No. 5, Lvjingling No. 3, Lvjingling No. 66, Cucumis No. 88, Jici No. 2, Mijizhi No. 701, Mijizhi No. 2, Mijizhi-136, Mijizhi No. 2, Mijizhi Ji Shi, Jingbaiyu super white leaf III, specially selected Jizai No. four, Jingyan green exquisite, Cuiguan, Tianjiao No. 7 south China, Ruiguang No. two, Berseoul green excellent cucumber, carefully selected leaf three, Guyu Jiali, Guyu drought cucumber, Tong 52, De Ruite cucumber L14-2, De Ruite cucumber GZ1601, De Ruite cucumber L14-5, De Ruite cucumber, Korean cucumber 2-06-97-164, Shengfeng 908, De Ruite 15-10 Mici cucumber, Jindong Rui 99 Mici cucumber, Zhongnong No. 106, Zhongnong No. 27, Jinyou No. 4, Jinyou No. 11, Jinza No. 2, Zhongnong No. 16, Jinyou 38, PI197088, H9, WI2757, true lemon, 13642014.11 and Z2982013.11.

4. A method for identifying to which of the 241 cucumber varieties described in claim 3 the cucumber to be tested belongs, comprising the steps of: the genotypes of cucumber to be detected and 241 cucumber varieties based on 32 SNP loci are respectively detected, and then the following judgments are carried out: if the genotypes of the cucumber to be detected based on the 32 SNP loci are completely consistent with the genotypes of a certain variety of 241 cucumber varieties based on the 32 SNP loci, the cucumber to be detected and the cucumber variety belong to the same variety; if the genotypes of the cucumber to be detected based on the 32 SNP sites are different from the genotypes of the 241 cucumber varieties based on the 32 SNP sites, the varieties of the cucumber to be detected are different from the varieties of the 241 cucumber varieties;

the 32 SNP sites are as follows: the CuSNP01 site is the 1976800 th nucleotide on the No. 1 chromosome; the CuSNP02 site is the 8433330 th nucleotide on the No. 1 chromosome; the CuSNP03 site is the 13502481 th nucleotide on the No. 1 chromosome; the CuSNP04 site is the 17508118 th nucleotide on the No. 1 chromosome; the CuSNP05 site is the 25296763 th nucleotide on the No. 1 chromosome; the CuSNP06 site is the 1200640 th nucleotide on chromosome 2; the CuSNP07 site is the 2563528 th nucleotide on chromosome 2; the CuSNP08 site is the 5891488 th nucleotide on chromosome 2; the CuSNP09 site is the 6045153 th nucleotide on chromosome 2; the CuSNP10 site is the 11947823 th nucleotide on chromosome 2; the CuSNP11 site is 40726 th nucleotide on chromosome 3; the CuSNP12 site is the 1001975 th nucleotide on the 3 rd chromosome; the CuSNP13 site is the 10713052 th nucleotide on the 3 rd chromosome; the CuSNP14 site is the 14161884 th nucleotide on the 3 rd chromosome; the CuSNP15 site is the 18678235 th nucleotide on the 3 rd chromosome; the CuSNP16 site is the 27913661 th nucleotide on the 3 rd chromosome; the CuSNP17 site is the 16975775 th nucleotide on the 4 th chromosome; the CuSNP18 site is the 21068185 th nucleotide on the 4 th chromosome; the CuSNP19 site is the 2133319 th nucleotide on the chromosome 5; the CuSNP20 site is the 4054461 th nucleotide on the chromosome 5; the CuSNP21 site is the 8103814 th nucleotide on the chromosome 5; the CuSNP22 site is the 9582207 th nucleotide on the chromosome 5; the CuSNP23 site is the 17245080 th nucleotide on the chromosome 5; the CuSNP24 site is the 195377 th nucleotide on the 6 th chromosome; the CuSNP25 site is the 2397556 th nucleotide on the 6 th chromosome; the CuSNP26 site is the 18079846 th nucleotide on the 6 th chromosome; the CuSNP27 site is the 20929474 th nucleotide on the 6 th chromosome; the CuSNP28 site is the 24439780 th nucleotide on the 6 th chromosome; the CuSNP29 site is the 2085399 th nucleotide on the 7 th chromosome; the CuSNP30 site is the 4866176 th nucleotide on the 7 th chromosome; the CuSNP31 site is the 6739101 th nucleotide on the 7 th chromosome; the CuSNP32 site is the 15415870 th nucleotide on the 7 th chromosome;

wherein the position of the SNP locus on the chromosome is determined based on the alignment of cucumber 9930 reference genome sequences; the version number of the cucumber 9930 reference genome sequence is V2.

5. The method of claim 4, wherein: the method comprises the following steps of detecting 32 SNP locus-based genotypes of cucumber to be detected and 241 cucumber varieties:

(1) respectively taking the genome DNA of cucumber to be detected and the genome DNA of 241 cucumber varieties as templates, and respectively adopting the primer group in the primer combination A in claim 1 to carry out PCR amplification to obtain PCR amplification products;

(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 genotypes of the cucumber to be detected and 241 cucumber varieties based on 32 SNP loci according to the color of the fluorescent signal.

6. The method of claim 4, wherein: the method comprises the following steps of detecting 32 SNP locus-based genotypes of cucumber to be detected and 241 cucumber varieties:

(1) respectively carrying out PCR amplification by using the genome DNA of cucumber to be detected and the genome DNA of 241 cucumber varieties as templates and respectively adopting the primer group in the primer combination B of claim 2 to obtain PCR amplification products;

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

(3) and (3) obtaining the 32 SNP locus-based genotypes of the cucumber to be detected and 241 cucumber varieties according to the sequencing result obtained in the step (2).

Images