CN111979350A

CN111979350A - Method for identifying authenticity of cucurbita pepo variety

Info

Publication number: CN111979350A
Application number: CN202011048135.7A
Authority: CN
Inventors: 温常龙; 李海真; 张建; 杨静静; 罗江; 张晓飞; 李向晶; 赵泓
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2020-11-24
Anticipated expiration: 2040-09-29
Also published as: CN111979350B

Abstract

The invention discloses a method for identifying the authenticity of a cucurbita pepo variety. The method provided by the invention can be used for early identification of the zucchini variety in the seed or seedling stage, the authenticity of the variety is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for the protection of zucchini germplasm resources and new varieties. The method provided by the invention can be used for identifying the unknown variety of the zucchini and also can be used for identifying the authenticity of the known variety. 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 cucurbita pepo variety

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a method for identifying the authenticity of a cucurbita pepo variety, which can be applied to biotechnology popularization service and provides technical support for the protection of cucurbita pepo germplasm resources and varieties.

Background

Cucurbita pepo, also called American pumpkin, is an important cultivated vegetable of Cucurbitaceae Cucurbita, is rich in nutrition, contains an inducer of interferon, and can improve immunity. The pumpkin has the advantages of short growing period, high yield, storage and transportation resistance, suitability for intercropping and the like, and becomes a melon vegetable with the cultivation area second to that of the cucumber. The planting area of the summer squash in China is more than 500 ten thousand mu, the yield of the summer squash in Shandong is 7.5-10 ten thousand kilograms per day, and the method has wide application prospect and great economic potential. In some regions, cucurbita pepo is brought into a non-main crop registration catalogue, but due to the fact that variety approval is inconsistent between provinces and cities, the management of cucurbita pepo varieties in the market cannot be effectively followed. The intellectual property protection of the cucurbita pepo variety is not perfect, so that the phenomenon that the cucurbita pepo is counterfeited with seeds, the parents are indiscriminately used and F1 exists, the enthusiasm of breeding workers is seriously attacked, and huge economic loss is brought to vast cucurbita pepo growers. For example, judicial identification of seeds of a pumpkin threshed by "Top-quality Ruifeng 9" by the center for agricultural, forestry and pasturing in Xinjiang, 4 months in 2016 shows that the seed lot of the pumpkin purchased by farmers is seriously mixed and degenerated, which causes great loss to the farmers. How to efficiently monitor the variety of the zucchini and protect the economic interests of breeders and vegetable farmers becomes one of the main problems facing the development of the current zucchini industry. According to the requirements of the registration guidelines for non-major crop varieties, DNA detection results can be directly submitted for the variety trait specifications and related traits involved in the variety DUS test reports, such as clear associated genes. Therefore, establishing a set of standards for effectively identifying the authenticity of the cucurbita pepo variety based on the DNA fingerprint is very important.

The method is used for identifying varieties based on a molecular marker technology, and has the advantages of no influence of environmental factors, high accuracy and the like. At present, no DNA molecular detection method for identifying the variety of the cucurbita pepo exists in China. In recent years, SNPs have been widely regarded as third-generation molecular markers because of their advantages such as large number, wide distribution, and genetic stability. With the development of the second generation high-throughput sequencing technology and the reduction of the sequencing cost, more stable and efficient SNP sites can be mined by utilizing the zucchini variant group information. And finally obtaining the genotype of the cucurbita pepo variety at the SNP locus by adopting an allele competitive specific PCR method and designing a specific primer thereof. The SNP typing detection technology has the advantages of high throughput, low cost, no need of species comparison for SNP typing and the like, results are presented by accurate bases, the molecular genetic difference of the cucurbita pepo variety can be directly reflected, and the SNP typing detection technology is an ideal marker for identifying the authenticity of the cucurbita pepo variety.

Disclosure of Invention

The invention aims to identify cucurbita pepo varieties, in particular 265 cucurbita pepo varieties.

The invention firstly protects the SNP locus combination, which can comprise 48 SNP loci of the pumpkin genome; the 48 SNP sites are as follows: the CupopSNP 01 site is 2807880 th nucleotide on the chromosome of Chr 01; the CupopSNP 02 site is 9791465 th nucleotide on the chromosome of Chr 01; the CupopSNP 03 site is 19069993 th nucleotide on the chromosome of Chr 01; the CupopSNP 04 site is 1687667 th nucleotide on the chromosome of Chr 02; the CupopSNP 05 site is 794938 th nucleotide on the chromosome of Chr 03; the CupopSNP 06 site is 799507 th nucleotide on the chromosome of Chr 03; the CupopSNP 07 site is 4509600 th nucleotide on the chromosome of Chr 04; the CupopSNP 08 site is 7015999 th nucleotide on the chromosome of Chr 04; the CupopSNP 09 site is 11177727 th nucleotide on the chromosome of Chr 04; the CupopSNP 10 site is 4771908 th nucleotide on the chromosome of Chr 05; the CupopSNP 11 site is 9266170 th nucleotide on the chromosome of Chr 05; the CupopSNP 12 site is 3551450 th nucleotide on the chromosome of Chr 06; the CupopSNP 13 site is 1076750 th nucleotide on the chromosome of Chr 07; the CupopSNP 14 site is 7776963 th nucleotide on the chromosome of Chr 07; the CupopSNP 15 site is 8150946 th nucleotide on the chromosome of Chr 07; the CupopSNP 16 site is 419732 th nucleotide on the chromosome of Chr 08; the CupopSNP 17 site is 7173914 th nucleotide on the chromosome of Chr 08; the CupopSNP 18 site is 8024934 th nucleotide on the chromosome of Chr 08; the CupopSNP 19 site is 1524784 th nucleotide on the chromosome of Chr 09; the CupopSNP 20 site is 1823037 th nucleotide on the chromosome of Chr 09; the CupopSNP 21 site is 2169965 th nucleotide on the chromosome of Chr 09; the CupopSNP 22 site is 2352404 th nucleotide on the chromosome of Chr 10; the CupopSNP 23 site is 4943533 th nucleotide on the chromosome of Chr 10; the CupopSNP 24 site is 7237965 th nucleotide on the chromosome of Chr 11; the CupopSNP 25 site is 9818902 th nucleotide on the chromosome of Chr 11; the CupopSNP 26 site is 4936646 th nucleotide on the chromosome of Chr 12; the CupopSNP 27 site is 6034493 th nucleotide on the chromosome of Chr 12; the CupopSNP 28 site is 8635520 th nucleotide on the chromosome of Chr 12; the CupopSNP 29 site is 6704302 th nucleotide on the chromosome of Chr 13; the CupopSNP 30 site is 6770190 th nucleotide on the chromosome of Chr 13; the CupopSNP 31 site is 7984331 th nucleotide on the chromosome of Chr 13; the CupopSNP 32 site is 8283301 th nucleotide on the chromosome of Chr 13; the CupopSNP 33 site is 5675208 th nucleotide on the chromosome of Chr 14; the CupopSNP 34 site is 8439875 th nucleotide on the chromosome of Chr 14; the CupopSNP 35 site is 107674 th nucleotide on the chromosome of Chr 15; the CupopSNP 36 site is 997997 th nucleotide on the chromosome of Chr 15; the CupopSNP 37 site is 8052733 th nucleotide on the chromosome of Chr 15; the CupopSNP 38 site is 441092 th nucleotide on the chromosome of Chr 16; the CupopSNP 39 site is 4936150 th nucleotide on the chromosome of Chr 16; the CupopSNP 40 site is 8226807 th nucleotide on the chromosome of Chr 16; the CupopSNP 41 site is 365539 th nucleotide on the chromosome of Chr 17; the CupopSNP 42 site is 3071924 th nucleotide on the chromosome of Chr 17; the CupopSNP 43 site is 5566093 th nucleotide on the chromosome of Chr 18; the CupopSNP 44 site is 105081 th nucleotide on the chromosome of Chr 19; the CupopSNP 45 site is 1475103 th nucleotide on the chromosome of Chr 19; the CupopSNP 46 site is 6337473 th nucleotide on the chromosome of Chr 19; the CupopSNP 47 site is 1391021 th nucleotide on the chromosome of Chr 20; the CupopSNP 48 site is nucleotide 7417753 on chromosome Chr 20.

The SNP site combination can specifically consist of the 48 SNP sites.

The invention also protects a primer combination, which can comprise a primer group 1 for amplifying the CupopSNP 01 locus, a primer group 2 for amplifying the CupopSNP 02 locus, a primer group 3 for amplifying the CupopSNP 03 locus, a primer group 4 for amplifying the CupopSNP 04 locus, a primer group 5 for amplifying the CupopSNP 05 locus, a primer group 6 for amplifying the CupopSNP 06 locus, a primer group 7 for amplifying the CupopSNP 07 locus, a primer group 8 for amplifying the CupopSNP 08 locus, a primer group 9 for amplifying the CupopSNP 09 locus, a primer group 10 for amplifying the CupopSNP 10 locus, a primer group 11 for amplifying the CupopSNP 73745 locus, a primer group 12 for amplifying the CupopSNP 12 locus, a primer group 13 for amplifying the CupopSNP 13 locus, a primer group 14 for amplifying the CupopSNP 14 locus, a primer group 15 locus, a primer group for amplifying the CupopSNP 828238 locus, and a primer group 16 for amplifying the CupopSNP 398936 locus, A primer set 18 for amplifying the CupopSNP 18 site, a primer set 19 for amplifying the CupopSNP 19 site, a primer set 20 for amplifying the CupopSNP 20 site, a primer set 21 for amplifying the CupopSNP 21 site, a primer set 22 for amplifying the CupopSNP 22 site, a primer set 23 for amplifying the CupopSNP 23 site, a primer set 24 for amplifying the CupopSNP 24 site, a primer set 25 for amplifying the CupopSNP 25 site, a primer set 26 for amplifying the CupopSNP 26 site, a primer set 27 for amplifying the CupopSNP 27 site, a primer set 28 for amplifying the CupopSNP 28 site, a primer set 29 for amplifying the CupopSNP 29 site, a primer set 30 for amplifying the CupopSNP 30 site, a primer set 31 for amplifying the CupopSNP 31 site, a primer set 32 for amplifying the CupopSNP 32 site, a primer set 5832 for amplifying the CupopSNP 35 6 site, a primer set for amplifying the CupopSNP 59635 site, and a primer set 22 for amplifying the CupopSNP 382 site, A primer set 36 for amplifying the CuPoSNP36 site, a primer set 37 for amplifying the CuPoSNP37 site, a primer set 38 for amplifying the CuPoSNP38 site, a primer set 39 for amplifying the CuPoSNP39 site, a primer set 40 for amplifying the CuPoSNP40 site, a primer set 41 for amplifying the CuPoSNP41 site, a primer set 42 for amplifying the CuPoSNP42 site, a primer set 43 for amplifying the CuPoSNP43 site, a primer set 44 for amplifying the CuPoSNP44 site, a primer set 45 for amplifying the CuPoSNP45 site, a primer set 46 for amplifying the CuPoSNP46 site, a primer set 47 for amplifying the CuPoSNP47 site, and a primer set 48 for amplifying the CuPoSNP48 site.

In the primer combination, the primer group 1 can be composed of a forward primer 01F1 shown in SEQ ID NO. 1, a forward primer 01F2 shown in SEQ ID NO. 2 and a reverse primer 01R shown in SEQ ID NO. 3. The primer group 2 can be composed of a forward primer 02F1 shown by SEQ ID NO. 4, a forward primer 02F2 shown by SEQ ID NO. 5 and a reverse primer 02R shown by SEQ ID NO. 6. The primer group 3 can be composed of a forward primer 03F1 shown in SEQ ID NO. 7, a forward primer 03F2 shown in SEQ ID NO. 8 and a reverse primer 03R shown in SEQ ID NO. 9. The primer group 4 can be composed of a forward primer 04F1 shown by SEQ ID NO. 10, a forward primer 04F2 shown by SEQ ID NO. 11 and a reverse primer 04R shown by SEQ ID NO. 12. The primer set 5 can be composed of a forward primer 05F1 shown in SEQ ID NO. 13, a forward primer 05F2 shown in SEQ ID NO. 14, and a reverse primer 05R shown in SEQ ID NO. 15. The primer group 6 can be composed of a forward primer 06F1 shown in SEQ ID NO. 16, a forward primer 06F2 shown in SEQ ID NO. 17 and a reverse primer 06R shown in SEQ ID NO. 18. The primer group 7 can be composed of a forward primer 07F1 shown in SEQ ID NO. 19, a forward primer 07F2 shown in SEQ ID NO. 20, and a reverse primer 07R shown in SEQ ID NO. 21. The primer group 8 can be composed of a forward primer 08F1 shown by SEQ ID NO. 22, a forward primer 08F2 shown by SEQ ID NO. 23, and a reverse primer 08R shown by SEQ ID NO. 24. The primer set 9 can be composed of a forward primer 09F1 shown in SEQ ID NO. 25, a forward primer 09F2 shown in SEQ ID NO. 26, and a reverse primer 09R shown in SEQ ID NO. 27. The primer set 10 can be composed of a forward primer 10F1 shown in SEQ ID NO. 28, a forward primer 10F2 shown in SEQ ID NO. 29, and a reverse primer 10R shown in SEQ ID NO. 30. The primer set 11 may be composed of a forward primer 11F1 shown in SEQ ID NO. 31, a forward primer 11F2 shown in SEQ ID NO. 32, and a reverse primer 11R shown in SEQ ID NO. 33. The primer set 12 can be composed of a forward primer 12F1 shown in SEQ ID NO. 34, a forward primer 12F2 shown in SEQ ID NO. 35, and a reverse primer 12R shown in SEQ ID NO. 36. The primer set 13 may be composed of a forward primer 13F1 shown in SEQ ID NO. 37, a forward primer 13F2 shown in SEQ ID NO. 38, and a reverse primer 13R shown in SEQ ID NO. 39. The primer set 14 may be composed of a forward primer 14F1 shown in SEQ ID NO. 40, a forward primer 14F2 shown in SEQ ID NO. 41, and a reverse primer 14R shown in SEQ ID NO. 42. The primer set 15 may be composed of a forward primer 15F1 shown in SEQ ID NO. 43, a forward primer 15F2 shown in SEQ ID NO. 44, and a reverse primer 15R shown in SEQ ID NO. 45. The primer set 16 may consist of a forward primer 16F1 shown in SEQ ID NO. 46, a forward primer 16F2 shown in SEQ ID NO. 47, and a reverse primer 16R shown in SEQ ID NO. 48. The primer set 17 may be composed of a forward primer 17F1 shown in SEQ ID NO. 49, a forward primer 17F2 shown in SEQ ID NO. 50, and a reverse primer 17R shown in SEQ ID NO. 51. The primer set 18 can be composed of a forward primer 18F1 shown in SEQ ID NO. 52, a forward primer 18F2 shown in SEQ ID NO. 53, and a reverse primer 18R shown in SEQ ID NO. 54. The primer set 19 may be composed of a forward primer 19F1 shown in SEQ ID NO. 55, a forward primer 19F2 shown in SEQ ID NO. 56, and a reverse primer 19R shown in SEQ ID NO. 57. The primer set 20 may consist of the forward primer 20F1 shown in SEQ ID NO. 58, the forward primer 20F2 shown in SEQ ID NO. 59, and the reverse primer 20R shown in SEQ ID NO. 60. The primer set 21 may be composed of a forward primer 21F1 shown in SEQ ID NO. 61, a forward primer 21F2 shown in SEQ ID NO. 62, and a reverse primer 21R shown in SEQ ID NO. 63. The primer set 22 may be composed of a forward primer 22F1 shown in SEQ ID NO. 64, a forward primer 22F2 shown in SEQ ID NO. 65, and a reverse primer 22R shown in SEQ ID NO. 66. The primer set 23 may consist of a forward primer 23F1 shown in SEQ ID NO. 67, a forward primer 23F2 shown in SEQ ID NO. 68, and a reverse primer 23R shown in SEQ ID NO. 69. The primer set 24 can be composed of a forward primer 24F1 shown in SEQ ID NO. 70, a forward primer 24F2 shown in SEQ ID NO. 71, and a reverse primer 24R shown in SEQ ID NO. 72. The primer set 25 may consist of the forward primer 25F1 shown in SEQ ID NO. 73, the forward primer 25F2 shown in SEQ ID NO. 74, and the reverse primer 25R shown in SEQ ID NO. 75. The primer set 26 may consist of a forward primer 26F1 shown in SEQ ID NO. 76, a forward primer 26F2 shown in SEQ ID NO. 77, and a reverse primer 26R shown in SEQ ID NO. 78. The primer set 27 may consist of a forward primer 27F1 shown in SEQ ID NO. 79, a forward primer 27F2 shown in SEQ ID NO. 80, and a reverse primer 27R shown in SEQ ID NO. 81. The primer set 28 may consist of a forward primer 28F1 shown in SEQ ID NO. 82, a forward primer 28F2 shown in SEQ ID NO. 83, and a reverse primer 28R shown in SEQ ID NO. 84. The primer group 29 can be composed of a forward primer 29F1 shown by SEQ ID NO. 85, a forward primer 29F2 shown by SEQ ID NO. 86, and a reverse primer 29R shown by SEQ ID NO. 87. The primer set 30 may consist of a forward primer 30F1 shown in SEQ ID NO. 88, a forward primer 30F2 shown in SEQ ID NO. 89, and a reverse primer 30R shown in SEQ ID NO. 90. The primer set 31 may be composed of a forward primer 31F1 shown in SEQ ID NO. 91, a forward primer 31F2 shown in SEQ ID NO. 92, and a reverse primer 31R shown in SEQ ID NO. 93. The primer set 32 may consist of a forward primer 32F1 shown in SEQ ID NO. 94, a forward primer 32F2 shown in SEQ ID NO. 95, and a reverse primer 32R shown in SEQ ID NO. 96. The primer set 33 may be composed of a forward primer 33F1 shown in SEQ ID NO. 97, a forward primer 33F2 shown in SEQ ID NO. 98, and a reverse primer 33R shown in SEQ ID NO. 99. The primer set 34 can be composed of a forward primer 34F1 shown in SEQ ID NO. 100, a forward primer 34F2 shown in SEQ ID NO. 101, and a reverse primer 34R shown in SEQ ID NO. 102. The primer set 35 can be composed of a forward primer 35F1 shown in SEQ ID NO. 103, a forward primer 35F2 shown in SEQ ID NO. 104, and a reverse primer 35R shown in SEQ ID NO. 105. The primer set 36 can be composed of a forward primer 36F1 shown in SEQ ID NO. 106, a forward primer 36F2 shown in SEQ ID NO. 107, and a reverse primer 36R shown in SEQ ID NO. 108. The primer set 37 may be composed of a forward primer 37F1 shown in SEQ ID NO. 109, a forward primer 37F2 shown in SEQ ID NO. 110, and a reverse primer 37R shown in SEQ ID NO. 111. The primer set 38 can be composed of a forward primer 38F1 shown in SEQ ID NO. 112, a forward primer 38F2 shown in SEQ ID NO. 113, and a reverse primer 38R shown in SEQ ID NO. 114. The primer set 39 may consist of a forward primer 39F1 shown in SEQ ID NO. 115, a forward primer 39F2 shown in SEQ ID NO. 116, and a reverse primer 39R shown in SEQ ID NO. 117. The primer set 40 may consist of the forward primer 40F1 shown in SEQ ID NO. 118, the forward primer 40F2 shown in SEQ ID NO. 119, and the reverse primer 40R shown in SEQ ID NO. 120. The primer set 41 may be composed of a forward primer 41F1 shown in SEQ ID NO. 121, a forward primer 41F2 shown in SEQ ID NO. 122, and a reverse primer 41R shown in SEQ ID NO. 123. The primer set 42 may consist of a forward primer 42F1 shown in SEQ ID NO. 124, a forward primer 42F2 shown in SEQ ID NO. 125, and a reverse primer 42R shown in SEQ ID NO. 126. The primer set 43 may consist of a forward primer 43F1 shown in SEQ ID NO. 127, a forward primer 43F2 shown in SEQ ID NO. 128, and a reverse primer 43R shown in SEQ ID NO. 129. The primer set 44 may consist of forward primer 44F1 shown in SEQ ID NO. 130, forward primer 44F2 shown in SEQ ID NO. 131, and reverse primer 44R shown in SEQ ID NO. 132. The primer set 45 may consist of a forward primer 45F1 shown in SEQ ID NO. 133, a forward primer 45F2 shown in SEQ ID NO. 134, and a reverse primer 45R shown in SEQ ID NO. 135. The primer set 46 may consist of the forward primer 46F1 shown in SEQ ID NO. 136, the forward primer 46F2 shown in SEQ ID NO. 137, and the reverse primer 46R shown in SEQ ID NO. 138. The primer set 47 may consist of a forward primer 47F1 shown in SEQ ID NO. 139, a forward primer 47F2 shown in SEQ ID NO. 140, and a reverse primer 47R shown in SEQ ID NO. 141. The primer set 48 may be composed of a forward primer 48F1 shown in SEQ ID NO:142, a forward primer 48F2 shown in SEQ ID NO:143, and a reverse primer 48R shown in SEQ ID NO: 144.

In the 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 SEQ ID NO. 1, a forward primer 01F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 2, and a reverse primer 01R shown in SEQ ID NO. 3. The primer set 2 may be composed of a forward primer 02F1 shown in the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 4, a forward primer 02F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 5, and a reverse primer 02R shown in SEQ ID NO. 6. The primer set 3 may be composed of a forward primer 03F1 shown in positions 22 to 45 from the 5 'end of SEQ ID NO. 7, a forward primer 03F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 8, and a reverse primer 03R shown in SEQ ID NO. 9. The primer group 4 consists of a forward primer 04F1 shown in the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 10, a forward primer 04F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 11, and a reverse primer 04R shown in SEQ ID NO. 12. The primer set 5 may be composed of a forward primer 05F1 shown in positions 22 to 48 from the 5 'end of SEQ ID NO. 13, a forward primer 05F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO. 14, and a reverse primer 05R shown in SEQ ID NO. 15. The primer set 6 may be composed of a forward primer 06F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 16, a forward primer 06F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 17, and a reverse primer 06R shown in SEQ ID NO. 18. 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 SEQ ID NO. 19, a forward primer 07F2 shown in 22 th to 47 th positions from the 5' end of SEQ ID NO. 20, and a reverse primer 07R shown in SEQ ID NO. 21. The primer set 8 may be composed of a forward primer 08F1 shown in the 22 nd to 54 th positions from the 5 'end of SEQ ID NO. 22, a forward primer 08F2 shown in the 22 nd to 52 th positions from the 5' end of SEQ ID NO. 23, and a reverse primer 08R shown in SEQ ID NO. 24. The primer set 9 may be composed of the forward primer 09F1 shown in positions 22 to 52 from the 5 'terminus of SEQ ID NO. 25, the forward primer 09F2 shown in positions 22 to 52 from the 5' terminus of SEQ ID NO. 26, and the reverse primer 09R shown in SEQ ID NO. 27. The primer set 10 may be composed of a forward primer 10F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 28, a forward primer 10F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 29, and a reverse primer 10R shown in SEQ ID NO. 30. The primer set 11 may be composed of a forward primer 11F1 shown in positions 22 to 49 from the 5 'terminus of SEQ ID NO. 31, a forward primer 11F2 shown in positions 22 to 48 from the 5' terminus of SEQ ID NO. 32, and a reverse primer 11R shown in SEQ ID NO. 33. The primer set 12 may be composed of a forward primer 12F1 shown in 22 nd to 54 th positions from the 5 'end of SEQ ID NO. 34, a forward primer 12F2 shown in 22 nd to 53 th positions from the 5' end of SEQ ID NO. 35, and a reverse primer 12R shown in SEQ ID NO. 36. The primer set 13 may be composed of a forward primer 13F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 37, a forward primer 13F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 38, and a reverse primer 13R shown in SEQ ID NO. 39. The primer set 14 may be composed of a forward primer 14F1 shown in positions 22 to 48 from the 5 'end of SEQ ID NO. 40, a forward primer 14F2 shown in positions 22 to 49 from the 5' end of SEQ ID NO. 41, and a reverse primer 14R shown in SEQ ID NO. 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 SEQ ID NO. 43, a forward primer 15F2 shown in the 22 nd to 49 th positions from the 5' end of SEQ ID NO. 44, and a reverse primer 15R shown in SEQ ID NO. 45. The primer set 16 may be composed of a forward primer 16F1 shown in positions 22 to 48 from the 5 'terminus of SEQ ID NO. 46, a forward primer 16F2 shown in positions 22 to 49 from the 5' terminus of SEQ ID NO. 47, and a reverse primer 16R shown in SEQ ID NO. 48. The primer set 17 may be composed of a forward primer 17F1 shown in positions 22 to 49 from the 5 'end of SEQ ID NO. 49, a forward primer 17F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO. 50, and a reverse primer 17R shown in SEQ ID NO. 51. The primer set 18 may be composed of a forward primer 18F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 52, a forward primer 18F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 53, and a reverse primer 18R shown in SEQ ID NO. 54. The primer set 19 may be composed of a forward primer 19F1 shown in positions 22 to 49 from the 5 'end of SEQ ID NO. 55, a forward primer 19F2 shown in positions 22 to 51 from the 5' end of SEQ ID NO. 56, and a reverse primer 19R shown in SEQ ID NO. 57. The primer set 20 may be composed of a forward primer 20F1 shown in positions 22 to 43 from the 5 'terminus of SEQ ID NO. 58, a forward primer 20F2 shown in positions 22 to 46 from the 5' terminus of SEQ ID NO. 59, and a reverse primer 20R shown in SEQ ID NO. 60. The primer set 21 may be composed of a forward primer 21F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 61, a forward primer 21F2 shown in positions 22 to 44 from the 5' end of SEQ ID NO. 62, and a reverse primer 21R shown in SEQ ID NO. 63. The primer set 22 may be composed of a forward primer 22F1 shown in positions 22 to 41 from the 5 'end of SEQ ID NO. 64, a forward primer 22F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 65, and a reverse primer 22R shown in SEQ ID NO. 66. The primer set 23 may be composed of a forward primer 23F1 shown in positions 22 to 46 from the 5 'end of SEQ ID NO. 67, a forward primer 23F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 68, and a reverse primer 23R shown in SEQ ID NO. 69. The primer set 24 may be composed of a forward primer 24F1 shown in positions 22 to 49 from the 5 'end of SEQ ID NO. 70, a forward primer 24F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO. 71, and a reverse primer 24R shown in SEQ ID NO. 72. The primer set 25 may be composed of a forward primer 25F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown in the 22 nd to 47 th positions from the 5' end of SEQ ID NO. 74, and a reverse primer 25R shown in SEQ ID NO. 75. The primer set 26 may be composed of a forward primer 26F1 shown in positions 22 to 48 from the 5 'end of SEQ ID NO. 76, a forward primer 26F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 77, and a reverse primer 26R shown in SEQ ID NO. 78. The primer set 27 may be composed of a forward primer 27F1 shown in positions 22 to 51 from the 5 'end of SEQ ID NO. 79, a forward primer 27F2 shown in positions 22 to 49 from the 5' end of SEQ ID NO. 80, and a reverse primer 27R shown in SEQ ID NO. 81. The primer set 28 may be composed of a forward primer 28F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 82, a forward primer 28F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 83, and a reverse primer 28R shown in SEQ ID NO. 84. The primer set 29 may be composed of a forward primer 29F1 shown in positions 22 to 44 from the 5 'end of SEQ ID NO. 85, a forward primer 29F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 86, and a reverse primer 29R shown in SEQ ID NO. 87. The primer set 30 may be composed of a forward primer 30F1 shown in positions 22 to 51 from the 5 'end of SEQ ID NO. 88, a forward primer 30F2 shown in positions 22 to 51 from the 5' end of SEQ ID NO. 89, and a reverse primer 30R shown in SEQ ID NO. 90. The primer set 31 may be composed of a forward primer 31F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 92, and a reverse primer 31R shown in SEQ ID NO. 93. The primer set 32 may be composed of a forward primer 32F1 shown in positions 22 to 48 from the 5 'end of SEQ ID NO. 94, a forward primer 32F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 95, and a reverse primer 32R shown in SEQ ID NO. 96. The primer set 33 may be composed of a forward primer 33F1 shown in positions 22 to 48 from the 5 'terminus of SEQ ID NO. 97, a forward primer 33F2 shown in positions 22 to 50 from the 5' terminus of SEQ ID NO. 98, and a reverse primer 33R shown in SEQ ID NO. 99. The primer set 34 may be composed of a forward primer 34F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 100, a forward primer 34F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 101, and a reverse primer 34R shown in SEQ ID NO. 102. The primer set 35 may be composed of a forward primer 35F1 shown in positions 22 to 47 from the 5 'end of SEQ ID NO. 103, a forward primer 35F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 104, and a reverse primer 35R shown in SEQ ID NO. 105. The primer set 36 may be composed of a forward primer 36F1 shown in positions 22 to 50 from the 5 'end of SEQ ID NO 106, a forward primer 36F2 shown in positions 22 to 51 from the 5' end of SEQ ID NO 107, and a reverse primer 36R shown in SEQ ID NO 108. The primer set 37 may be composed of a forward primer 37F1 shown in positions 22 to 43 from the 5 'end of SEQ ID NO. 109, a forward primer 37F2 shown in positions 22 to 45 from the 5' end of SEQ ID NO. 110, and a reverse primer 37R shown in SEQ ID NO. 111. The primer set 38 may be composed of a forward primer 38F1 shown in positions 22 to 51 from the 5 'end of SEQ ID NO. 112, a forward primer 38F2 shown in positions 22 to 52 from the 5' end of SEQ ID NO. 113, and a reverse primer 38R shown in SEQ ID NO. 114. The primer set 39 may be composed of a forward primer 39F1 shown in positions 22 to 49 from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown in positions 22 to 51 from the 5' end of SEQ ID NO. 116, and a reverse primer 39R shown in SEQ ID NO. 117. The primer set 40 may be composed of a forward primer 40F1 shown in positions 22 to 49 from the 5 'terminus of SEQ ID NO. 118, a forward primer 40F2 shown in positions 22 to 48 from the 5' terminus of SEQ ID NO. 119, and a reverse primer 40R shown in SEQ ID NO. 120. The primer set 41 may be composed of a forward primer 41F1 shown in the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 121, a forward primer 41F2 shown in the 22 nd to 47 th positions from the 5' end of SEQ ID NO. 122, and a reverse primer 41R shown in the SEQ ID NO. 123. The primer set 42 may be composed of a forward primer 42F1 shown in positions 22 to 47 from the 5 'end of SEQ ID NO. 124, a forward primer 42F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO. 125, and a reverse primer 42R shown in SEQ ID NO. 126. The primer set 43 may be composed of a forward primer 43F1 shown in positions 22 to 49 from the 5 'terminus of SEQ ID NO. 127, a forward primer 43F2 shown in positions 22 to 48 from the 5' terminus of SEQ ID NO. 128, and a reverse primer 43R shown in SEQ ID NO. 129. The primer set 44 may be composed of a forward primer 44F1 shown in 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 130, a forward primer 44F2 shown in 22 nd to 43 th positions from the 5' end of SEQ ID NO. 131, and a reverse primer 44R shown in SEQ ID NO. 132. The primer set 45 may be composed of a forward primer 45F1 shown in 22 nd to 51 nd positions from the 5 'end of SEQ ID NO. 133, a forward primer 45F2 shown in 22 nd to 49 nd positions from the 5' end of SEQ ID NO. 134, and a reverse primer 45R shown in SEQ ID NO. 135. The primer set 46 may be composed of a forward primer 46F1 shown in positions 22 to 51 from the 5 'end of SEQ ID NO. 136, a forward primer 46F2 shown in positions 22 to 51 from the 5' end of SEQ ID NO. 137, and a reverse primer 46R shown in SEQ ID NO. 138. The primer set 47 may be composed of a forward primer 47F1 shown in positions 22 to 48 from the 5 'terminus of SEQ ID NO. 139, a forward primer 47F2 shown in positions 22 to 46 from the 5' terminus of SEQ ID NO. 140, and a reverse primer 47R shown in SEQ ID NO. 141. The primer set 48 may be composed of a forward primer 48F1 shown in positions 22 to 39 from the 5 'end of SEQ ID NO 142, a forward primer 48F2 shown in positions 22 to 40 from the 5' end of SEQ ID NO 143, and a reverse primer 48R shown in SEQ ID NO 144.

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.

Any of the above primer combinations may specifically consist of the primer set 1 to the primer set 48.

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 of the 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 x3) or x 4): x3) identifying the variety of the zucchini; x4) identifying the authenticity of the cucurbita pepo variety.

The invention also protects the application of any one of the SNP site combinations or any one of the primer combinations, which can be any one of x1) to x 4): x1) preparing a kit for identifying the variety of the cucurbita pepo; x2) preparing a kit for identifying the authenticity of the cucurbita pepo variety; x3) identifying the variety of the zucchini; x4) identifying the authenticity of the cucurbita pepo variety.

The invention also discloses a method for identifying the variety of the pumpkin to be detected, which belongs to 265 varieties of pumpkin, which comprises the following steps: and (3) respectively detecting the genotypes of the cucurbita pepo variety to be detected and the 265 cucurbita pepo variety based on the 48 SNP sites, and then judging as follows: if the genotypes of the pumpkin to be detected based on the 48 SNP sites are completely consistent with the genotypes of some variety of the 265 pumpkin varieties based on the 48 SNP sites, the pumpkin to be detected and the pumpkin variety belong to the same variety; and if the genotypes of the pumpkin to be detected based on the 48 SNP sites are different from the genotypes of all varieties in the 265 pumpkin varieties based on the 48 SNP sites, the varieties of the pumpkin to be detected are different from the varieties of the 265 pumpkin varieties.

In the above method, the step of detecting the genotypes of the cucurbita pepo and 265 cucurbita pepo varieties to be detected based on the 48 SNP sites may be as follows:

(1) respectively taking the genome DNA of the cucurbita pepo to be detected and the genome DNA of 265 cucurbita pepo varieties as templates, and respectively adopting the primer groups in any one of the 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 cucurbita pepo variety to be detected and 265 cucurbita pepo varieties based on the 48 SNP sites according to the color of the fluorescent signal.

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

(3) and (3) obtaining genotypes of the cucurbita pepo and 265 cucurbita pepo varieties to be detected based on the 48 SNP loci according to the sequencing result obtained in the step (2).

The invention also discloses a method for identifying the variety of the pumpkin to be detected, which belongs to 265 varieties of pumpkin, which comprises the following steps:

(1) taking genome DNA of the cucurbita pepo to be detected as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the primer combinations to obtain PCR amplification products; taking the genome DNA of each cucurbita pepo variety in a standard cucurbita pepo variety population as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the primer combinations to obtain a PCR amplification product; the standard zucchini product population consists of 265 zucchini varieties;

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

The 265 Cucurbita pepo varieties can be the Beijing Feng 131, the Beijing Feng No. 13, the Beijing Feng No. 12, the Beijing Feng No. 11, the Zifeng No. 9, the Zifeng No. 4, the Zifeng No. 3, the Zifeng No. 2, the Zifeng No. 1, the sheng Hu No. 11, the sheng Hu No. 7, the sheng Hu No. 5, the Cucumis pellucida 336, the Cucumis pepo No. 22, the Cucumis 8, the Jingbi, the Yuzhu No. 2, the Xinjing Zhu No. 2, the Xinzhu, the Jingzhu No. 66, the Jinghu 42, the Jinghu 38, the Jinghu 37, the Jinghu 36, the Jinghu 35, the Jinghu 33, the Jinghu No. 35-2, the Jinghu CRV3-3, the Jinghu CRV4, the Jinghu No. 3, the Jinghu No. 2, the Jinghu No. 539 No. 3, the Jinghu No. 3 No. 2, the Jinghu No. 3-No. 2, the Jinghu No. 6, the Jinghu No. 3, the Jinghu No. 2, the Jinghu No. 3, the Jinghu No. 7, the Jinghu No. 3, the Jinghu No. 6, 201-LSQ236, 205-8G504 Cucurbita pepo, 206-8G506, 210-ZG522, 202-LSQ237 Cucurbita pepo, XHL1476, XHL1485, Lilang No. 3, Dongfeng No. 5, XHL14106, XHL1471, XHL14109, XHL14104, Meijian 8018, XH01587, LZ-4, XHL14103, Xixingxi Cucurbita No. 10, Xinrui 1212, 209-8G521 Cucurbita pepo, TRQ-18, TRL 14108, XHL1472, XHL1474, XHL1449, XHL14107, L14101, Lhuang 38, Lhuangguan 29, Lhuangguan 40, xiduo, Xiyu, DTZU-42, Chunri No. 1430, TRQ-15, S-1482 Cucurbita, ATX S34, ATX S4656, ATX S04, XHS 1445, XHX No. 2002, XHX No. 46, XH-S-94 ATX No. 2002, XH-6, ATX No. 2002, XH-94, XH-S-6, ATX No. 2002, XH-6, XH-5, XH-6-S-6, ATX-94-6, ATX-S-94, ATX No. 2002, XH-6, XH-29, XH-6-1, XH-8-6-X-, XHL1497, XHL14101, XHL14102, XHL14103, XHL14104, XHL14107, XHL14108, No. 132, XHL14109, 14108, Lvshui, sq217218, sq219220, XH01587, XH01588 pumpkin, XH01589 pumpkin, XH01590 pumpkin, XH01591 pumpkin, XH01592 pumpkin, XH01594 pumpkin, XH01595 pumpkin, Saina, XH01446, XH01447, Jade, Guanyu, Jinhu 36, Hanhu, Dongguan No. 16, Donggu 1712, Donggou 3329, Donggou No. 22, Donggou No. 33, Donggu No. 28, Qiupi 04002, XH04003, XH04004, XH04005, 01412, XH01414, Xiwakamikaowu, XH Rou bao (Rou de), XH Rou de Rou No. 375, XH 03, XH Rou de la, XH-Rou de 2, XH-0135, Hwa Rou de la-802, Hga-Rou de, Hga-802, XH Rou de, XH # 35, Hga-mo-2, Hga-mo-2, Hga Hao Rou de, Hga-2, Hga-mo-802, Hga-K # 35, Hga-2, Hga-K # XH # 35, Hga-2, Hga-K2, Hga, Yue Lang Yuan Jade, Xiyu, Xiduozi, Ximei, Xizhiyu, Yuyu-Dongxu, Xili, France Shengna, France Lisa, Luhuang Guang 24, Luhuang Guang 27, Luhuang Guang 29, Luhuang Guang 38, Luhuang Guang 34, Luhuang Guang 40, Luhuang Guang 41, Lufeng 55F1, Tianfeng, France Lubei 7810, Meihu 38, Lubaowang oil-green Sihuo 889, Fabao, Cuibao, Mozuo Andoni, Xihusq-17, Milao Xiao Dada, Xinfu No. 9, Feitian No. 8, Jinfeng No. eight, Dongguan Xihuo F45, S-3 Xihuo, ATX S03, ATX S04, ATX S05, ATX S07, ATX S08, ATX S09, ATX 10, ATX 11, ATX 12, ATX S3527, ATX S898, ATX 368, ATX 1, ATX 368, Tailiang Luo Yuan Luo, Tailiang Luo, Yuan, Yuan, Fengchun No. 2, Fengchun No. 3, Fengchun No. 4, Fengchun No. 5, Fengchun No. 6, Fengchun No. 7, Black gourd No. 3, Zhao Zhen X3, Zhao Zhen No. 4, Qisheng Xihuo, SQLH7, SQLH10, SQLH11, SQLH12, Ruifeng Jiu No. F1, gold 318F1, gold 618F1, WS 4112F 1, 2088G517, 2068G506, 2058G504 Cucurbita pepo, Rui 1212, 201LSQ236, early spring 202LSQ237 Cucurbita pepo, green jade, Nanao No. 26, Haimei No. 1207, W1501F 1, W368F 1 Alexander, Lide Lneng Lanzhong, Huasheng 31 and 327.

In any of the above methods, the reaction procedure for performing PCR amplification using the primer sets in any of the above 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 at 55 ℃ and extension for 1min, and amplification is continued 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 invention establishes the DNA fingerprint database for identifying the authenticity of the cucurbita pepo variety based on the allele competitive specificity PCR method, can be used for carrying out early identification on the cucurbita pepo variety in the seed or seedling stage, ensures the authenticity of the variety, practically protects the rights and interests of producers and breeders, and provides technical support for the protection of cucurbita pepo germplasm resources and new varieties. The method provided by the invention can be used for identifying the unknown variety of the zucchini and also can be used for identifying the authenticity of the known variety. 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. The invention has important application value.

Drawings

FIG. 1 shows the SNP typing effect of 48 primer sets in a part of the Cucurbita pepo varieties tested.

FIG. 2 is a cluster map of 265 Cucurbita pepo varieties tested, established on 48 SNP primer sets.

FIG. 3 is a graph showing the relationship between the number of SNP markers (i.e., the number of SNP sites) and the discrimination of 265 cucurbita pepo varieties to be 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 conventional biochemicals, unless otherwise specified.

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

Example 1 acquisition of primer combinations for identifying the authenticity of Cucurbita pepo varieties

Discovery of one, 48 SNP sites

The invention obtains 48 SNP loci based on 30 parts of re-sequencing data of cucurbita pepo representative resources. The 30 parts of zucchini are rich in representative resource types, cover 8 parts of zucchini, 7 parts of seedcoat-free zucchini, 8 parts of green-peel zucchini and 7 parts of long-vine zucchini, basically comprise the main ecological types of the zucchini, have higher genetic diversity in the aspect of agronomic traits and embody germplasm representativeness as much as possible.

Specifically, the screening criteria for SNP sites are as follows: SNP sites 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 48 SNP sites is detailed in Table 1, columns 1 to 4. Wherein the position of the SNP site on the chromosome is determined based on Zucchini reference genome sequence alignment (download address: ftp:// curbitangenomics. org/pub/curbitat/genome/curbita _ pepo /).

TABLE 1.48 essential information of SNP sites

Secondly, obtaining of primer combination for identifying authenticity of cucurbita pepo variety

According to the 48 SNP loci discovered in the first step, a primer combination which has higher polymorphism information content (PIC value) (see the 5 th column in the table 1) and is suitable for identifying the authenticity of the cucurbita pepo variety by using an allele competitive specific PCR method is developed.

The primer combination consisted of 48 primer sets. The name of each primer set is shown in column 2 of 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 48 primer sets are shown in column 4 of Table 2.

TABLE 2.48 primer sets and nucleotide sequences of the primers thereof

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

Example 2 and example 1 validation of the primer combinations developed

The validity of the primer combination developed in example 1 was tested by randomly selecting 265 varieties of zucchini to be tested.

The basic information of 265 cucurbita pepo varieties tested is shown in table 3. 265 cucurbita pepo varieties to be tested are common fine varieties or partially foreign introduced varieties.

TABLE 3.265 names and sources of the varieties of Cucurbita pepo L.for testing

1. Acquisition of genomic DNA of the Cucurbita pepo variety tested

And respectively extracting the genome DNA of the leaves (mixing true leaves of 30 seeds) of 265 Cucurbita pepo varieties to be tested by a CTAB method to obtain the genome DNA of the Cucurbita pepo varieties to be tested.

The quality and concentration of the genome DNA of the Cucurbita pepo 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 genome DNA of the tested cucurbita pepo variety is 10-30 ng/mu L.

2. And respectively adopting genome DNAs of 265 cucurbita pepo varieties to be tested as templates and respectively adopting 48 primer groups to carry out PCR amplification so as 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 fluorescence values through FAM and HEX light beams of a microplate reader (reading values are observed when the FAM fluorescent label sequence is at 485nm of exciting light and at 520nm of emitting light, reading values are observed when the HEX fluorescent label sequence is at 528nm of exciting light and at 560nm of emitting light), and judging the genotypes of 265 zucchini varieties to be tested based on each SNP locus according to the colors of fluorescence signals. The specific judgment principle is as follows: if a certain test Cucurbita pepo variety shows a blue fluorescent signal based on a certain SNP locus, the test Cucurbita pepo variety is homozygotic based on the genotype of the SNP locus, wherein the complementary base is ' the complementary base of the 1 st base at the 3 ' end of a primer which amplifies the SNP locus and contains ' F1 ' in the name '; if a certain test Cucurbita pepo variety shows a red fluorescent signal based on a certain SNP locus, the test Cucurbita pepo variety is homozygotic based on the genotype of the SNP locus, wherein the complementary base is ' the complementary base of the 1 st base at the 3 ' end of a primer which amplifies the SNP locus and contains ' F2 ' in the name '; if a certain test Cucurbita pepo variety shows a green fluorescent signal based on a certain SNP site, the test Cucurbita pepo 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 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. 1. The result shows that each primer group can obtain good typing effect in the cucurbita pepo variety to be tested.

4. Cluster analysis

And (3) carrying out cluster analysis on the 265 zucchini varieties to be tested by utilizing MiniMarker and MEGA7 software according to the genotypes of the 265 zucchini varieties to be tested based on 48 SNP loci.

The cluster map of 265 cucurbita pepo varieties tested, built on the 48 primer sets, is shown in figure 2. The results show that the 48 primer sets can completely distinguish 265 cucurbita pepo varieties to be tested in the table 3. Therefore, the primer combination developed in the embodiment 1 can be applied to the construction of the DNA fingerprint database of the cucurbita pepo variety and the authenticity identification of the variety.

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 265 varieties of Cucurbita pepo L.to be tested.

The experimental result shows (figure 3), the discrimination of 48 primer groups (namely the number of 48 SNP markers) in 265 cucurbita pepo varieties to be tested reaches 100%.

Embodiment 3, method for establishing and detecting which variety of 265 cucurbita pepo varieties to be detected belongs to in embodiment 2

Method for establishing and detecting variety of 265 cucurbita pepo varieties to be detected in embodiment 2

1. Obtaining of genome DNA of Cucurbita pepo to be tested

According to the method of the step 1 in the embodiment 2, the leaves of the zucchini variety to be tested are replaced by the leaves of the zucchini to be tested, and the other steps are not changed, so that the genome DNA of the zucchini to be tested is obtained.

2. And (3) taking the genome DNA of the cucurbita pepo to be detected as a template, and respectively adopting 48 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.

3. And (3) after the step (2) is completed, when the temperature of the 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 the reading value of the FAM fluorescence label sequence is observed under the wavelength of 485nm of exciting light and 520nm of emitted light, reading the reading value of the HEX fluorescence label sequence is observed under the wavelength of 528nm of exciting light and 560nm of emitted light), and judging the genotype of the zucchini to be detected based on each SNP locus according to the color of a fluorescence signal. 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.

The specific judgment principle is the same as that of step 3 in example 2.

4. After the step 3 is completed, the following judgment is carried out: if the genotype of the zucchini to be detected based on 48 SNP loci is completely consistent with the genotype of the corresponding SNP locus of a certain variety of 265 zucchini varieties, the zucchini to be detected and the zucchini variety belong to the same variety; if the genotypes of the 48 SNP sites of the Cucurbita pepo to be detected are different from the genotypes of the corresponding SNP sites of the 265 Cucurbita pepo varieties, the Cucurbita pepo to be detected is different from the varieties of the 265 Cucurbita pepo varieties to be detected.

Secondly, identifying the accuracy of the method established in the first step

The varieties of the zucchini 1 to be tested and the zucchini 5 to be tested are respectively No. 131, No. 13, No. 12, No. 11 and No. 9. The leaves of the summer squash 1 to be tested and the summer squash 5 to be tested are all taken from the test base of the vegetable research center of the academy of agriculture and forestry, Beijing.

1. And (3) obtaining the genotypes of the pumpkin to be detected 1-pumpkin to be detected 5 based on 48 SNP loci according to the method 1-3 in the step one.

2. And comparing the genotypes of the pumpkin 1 to be detected and the pumpkin 5 to be detected based on 48 SNP loci with the genotypes of 265 pumpkin varieties based on 48 SNP loci.

The result shows that the genotype of the pumpkin 1 to be detected based on 48 SNP loci is completely consistent with the genotype of the SNP locus corresponding to the seed abundance 131, and the pumpkin 1 to be detected belongs to the seed abundance 131; the genotype of the to-be-detected zucchini 2 based on 48 SNP loci is completely consistent with the genotype of the corresponding SNP locus of the Fengfeng No. 13, and the to-be-detected zucchini 2 belongs to the Fengfeng No. 13; the genotype of the to-be-detected zucchini 3 based on 48 SNP loci is completely consistent with the genotype of the corresponding SNP locus of the Bianfeng No. 12, and the to-be-detected zucchini 3 belongs to the Bianfeng No. 12; the genotype of the to-be-detected zucchini 4 based on 48 SNP loci is completely consistent with the genotype of the corresponding SNP locus of the Bianfeng No. 11, and the to-be-detected zucchini 4 belongs to the Bianfeng No. 11; the genotype of the to-be-detected zucchini 5 based on 48 SNP loci is completely consistent with the genotype of the corresponding SNP locus of the Bingfeng No. 9, and the to-be-detected zucchini 5 belongs to the Bingfeng No. 9. The detection result is completely consistent with the expected result.

Therefore, the method established in the step one has higher accuracy.

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

<120> a method for identifying the authenticity of cucurbita pepo varieties

<160> 144

<170> PatentIn version 3.5

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<210> 13

<211> 48

<212> DNA

<213> Artificial sequence

<400> 13

gaaggtgacc aagttcatgc tactatctct gagttgctat caagtttc 48

<210> 14

<211> 48

<212> DNA

<213> Artificial sequence

<400> 14

gaaggtcgga gtcaacggat tactatctct gagttgctat caagtttg 48

<210> 15

<211> 34

<212> DNA

<213> Artificial sequence

<400> 15

gacatcagat tcgttatcta taattcttgt caat 34

<210> 16

<211> 48

<212> DNA

<213> Artificial sequence

<400> 16

gaaggtgacc aagttcatgc taaagggcat gaatccataa gttcctta 48

<210> 17

<211> 46

<212> DNA

<213> Artificial sequence

<400> 17

gaaggtcgga gtcaacggat tagggcatga atccataagt tccttg 46

<210> 18

<211> 27

<212> DNA

<213> Artificial sequence

<400> 18

cttttctgct gtttctatcg gtggcat 27

<210> 19

<211> 46

<212> DNA

<213> Artificial sequence

<400> 19

gaaggtgacc aagttcatgc tagctcattt gagattcctc ttccag 46

<210> 20

<211> 47

<212> DNA

<213> Artificial sequence

<400> 20

gaaggtcgga gtcaacggat tgagctcatt tgagattcct cttccaa 47

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<400> 21

ggtaggcact acattgtgga tgcta 25

<210> 22

<211> 54

<212> DNA

<213> Artificial sequence

<400> 22

gaaggtgacc aagttcatgc ttataatttt tggagttttg ctaatgaact ctta 54

<210> 23

<211> 52

<212> DNA

<213> Artificial sequence

<400> 23

gaaggtcgga gtcaacggat ttaatttttg gagttttgct aatgaactct tg 52

<210> 24

<211> 28

<212> DNA

<213> Artificial sequence

<400> 24

aaatgtggga gagagggaag agaatatt 28

<210> 25

<211> 52

<212> DNA

<213> Artificial sequence

<400> 25

gaaggtgacc aagttcatgc tgaatttttg aagaaatgcc tgaaaagaat gt 52

<210> 26

<211> 52

<212> DNA

<213> Artificial sequence

<400> 26

gaaggtcgga gtcaacggat tgaatttttg aagaaatgcc tgaaaagaat ga 52

<210> 27

<211> 26

<212> DNA

<213> Artificial sequence

<400> 27

ccctgcaatc atcgcattcc atgaaa 26

<210> 28

<211> 43

<212> DNA

<213> Artificial sequence

<400> 28

gaaggtgacc aagttcatgc tataatcccc ctccaccaac tcg 43

<210> 29

<211> 43

<212> DNA

<213> Artificial sequence

<400> 29

gaaggtcgga gtcaacggat tataatcccc ctccaccaac tcc 43

<210> 30

<211> 29

<212> DNA

<213> Artificial sequence

<400> 30

gttgaagatg atgttgtggt cgaatgttt 29

<210> 31

<211> 49

<212> DNA

<213> Artificial sequence

<400> 31

gaaggtgacc aagttcatgc tgcacttgat ccaacttgtt tattttggt 49

<210> 32

<211> 48

<212> DNA

<213> Artificial sequence

<400> 32

gaaggtcgga gtcaacggat tcacttgatc caacttgttt attttggg 48

<210> 33

<211> 31

<212> DNA

<213> Artificial sequence

<400> 33

agaatcaatg aagggatttg aaccatttgt t 31

<210> 34

<211> 54

<212> DNA

<213> Artificial sequence

<400> 34

gaaggtgacc aagttcatgc ttgtttggta atcatttata attcataagt gcta 54

<210> 35

<211> 53

<212> DNA

<213> Artificial sequence

<400> 35

gaaggtcgga gtcaacggat tgtttggtaa tcatttataa ttcataagtg ctc 53

<210> 36

<211> 34

<212> DNA

<213> Artificial sequence

<400> 36

aaaccataga aaggagttag ttaaatccaa ttat 34

<210> 37

<211> 43

<212> DNA

<213> Artificial sequence

<400> 37

gaaggtgacc aagttcatgc ttgcttacct taccggttgc agc 43

<210> 38

<211> 46

<212> DNA

<213> Artificial sequence

<400> 38

gaaggtcgga gtcaacggat tatttgctta ccttaccggt tgcagt 46

<210> 39

<211> 27

<212> DNA

<213> Artificial sequence

<400> 39

gcagcatatc ctcaactgag tcatcaa 27

<210> 40

<211> 48

<212> DNA

<213> Artificial sequence

<400> 40

gaaggtgacc aagttcatgc tatgtccatt cttctttgat tcccaaac 48

<210> 41

<211> 49

<212> DNA

<213> Artificial sequence

<400> 41

gaaggtcgga gtcaacggat tcatgtccat tcttctttga ttcccaaat 49

<210> 42

<211> 28

<212> DNA

<213> Artificial sequence

<400> 42

ccgacactgg aatctataca tgtttgat 28

<210> 43

<211> 48

<212> DNA

<213> Artificial sequence

<400> 43

gaaggtgacc aagttcatgc tatcaaagag aagatcactt caactacg 48

<210> 44

<211> 49

<212> DNA

<213> Artificial sequence

<400> 44

gaaggtcgga gtcaacggat tgatcaaaga gaagatcact tcaactaca 49

<210> 45

<211> 31

<212> DNA

<213> Artificial sequence

<400> 45

aatagatacc ttctggctgc aagtattaaa a 31

<210> 46

<211> 48

<212> DNA

<213> Artificial sequence

<400> 46

gaaggtgacc aagttcatgc tcaggccttc aaggttaata aagataag 48

<210> 47

<211> 49

<212> DNA

<213> Artificial sequence

<400> 47

gaaggtcgga gtcaacggat tgcaggcctt caaggttaat aaagataat 49

<210> 48

<211> 28

<212> DNA

<213> Artificial sequence

<400> 48

aattctgact gcaggaacca agtttctt 28

<210> 49

<211> 49

<212> DNA

<213> Artificial sequence

<400> 49

gaaggtgacc aagttcatgc tgttgaacaa gaagtcgttt agagaatca 49

<210> 50

<211> 48

<212> DNA

<213> Artificial sequence

<400> 50

gaaggtcgga gtcaacggat tttgaacaag aagtcgttta gagaatcg 48

<210> 51

<211> 24

<212> DNA

<213> Artificial sequence

<400> 51

gagctcctca ccagtcttcg actt 24

<210> 52

<211> 46

<212> DNA

<213> Artificial sequence

<400> 52

gaaggtgacc aagttcatgc ttaacaccaa tcccaagaag tgcaag 46

<210> 53

<211> 46

<212> DNA

<213> Artificial sequence

<400> 53

gaaggtcgga gtcaacggat ttaacaccaa tcccaagaag tgcaac 46

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence

<400> 54

aggtctactc tctcttctgg tgctt 25

<210> 55

<211> 49

<212> DNA

<213> Artificial sequence

<400> 55

gaaggtgacc aagttcatgc tgaacacatc tctgtattta agttccttc 49

<210> 56

<211> 51

<212> DNA

<213> Artificial sequence

<400> 56

gaaggtcgga gtcaacggat taagaacaca tctctgtatt taagttcctt t 51

<210> 57

<211> 34

<212> DNA

<213> Artificial sequence

<400> 57

actcgaataa attacagctt cattttagag ttat 34

<210> 58

<211> 43

<212> DNA

<213> Artificial sequence

<400> 58

gaaggtgacc aagttcatgc tctcgcttaa ctcgacggct atg 43

<210> 59

<211> 46

<212> DNA

<213> Artificial sequence

<400> 59

gaaggtcgga gtcaacggat tttactcgct taactcgacg gctata 46

<210> 60

<211> 28

<212> DNA

<213> Artificial sequence

<400> 60

ttcgagagat tggcttagtt tgtagcat 28

<210> 61

<211> 43

<212> DNA

<213> Artificial sequence

<400> 61

gaaggtgacc aagttcatgc tacatggtga ggcactggac tag 43

<210> 62

<211> 44

<212> DNA

<213> Artificial sequence

<400> 62

gaaggtcgga gtcaacggat tgacatggtg aggcactgga ctat 44

<210> 63

<211> 26

<212> DNA

<213> Artificial sequence

<400> 63

gctgatgctg atgggaaact tgtgat 26

<210> 64

<211> 41

<212> DNA

<213> Artificial sequence

<400> 64

gaaggtgacc aagttcatgc tcatcggacg gtcctgaatc g 41

<210> 65

<211> 43

<212> DNA

<213> Artificial sequence

<400> 65

gaaggtcgga gtcaacggat tatcatcgga cggtcctgaa tca 43

<210> 66

<211> 22

<212> DNA

<213> Artificial sequence

<400> 66

agagctagcg gtttggccag tg 22

<210> 67

<211> 46

<212> DNA

<213> Artificial sequence

<400> 67

gaaggtgacc aagttcatgc taggaaagtt tgaaggagcg cttact 46

<210> 68

<211> 46

<212> DNA

<213> Artificial sequence

<400> 68

gaaggtcgga gtcaacggat taggaaagtt tgaaggagcg cttaca 46

<210> 69

<211> 27

<212> DNA

<213> Artificial sequence

<400> 69

cgcgcttcag tttcttactt ccagaaa 27

<210> 70

<211> 49

<212> DNA

<213> Artificial sequence

<400> 70

gaaggtgacc aagttcatgc tcaatatgtg ctagctgtca acttacatt 49

<210> 71

<211> 48

<212> DNA

<213> Artificial sequence

<400> 71

gaaggtcgga gtcaacggat taatatgtgc tagctgtcaa cttacatc 48

<210> 72

<211> 25

<212> DNA

<213> Artificial sequence

<400> 72

gggccagtgt ctttaacctc tagta 25

<210> 73

<211> 46

<212> DNA

<213> Artificial sequence

<400> 73

gaaggtgacc aagttcatgc ttgtcccaaa aatcgaccat gttcag 46

<210> 74

<211> 47

<212> DNA

<213> Artificial sequence

<400> 74

gaaggtcgga gtcaacggat tatgtcccaa aaatcgacca tgttcat 47

<210> 75

<211> 27

<212> DNA

<213> Artificial sequence

<400> 75

gctggcaaca atgcccttga atttcaa 27

<210> 76

<211> 48

<212> DNA

<213> Artificial sequence

<400> 76

gaaggtgacc aagttcatgc ttatgccctc gatatacctt tgttagtt 48

<210> 77

<211> 46

<212> DNA

<213> Artificial sequence

<400> 77

gaaggtcgga gtcaacggat ttgccctcga tatacctttg ttagtc 46

<210> 78

<211> 25

<212> DNA

<213> Artificial sequence

<400> 78

tgtgtttgtc actccccgtt ggttt 25

<210> 79

<211> 51

<212> DNA

<213> Artificial sequence

<400> 79

gaaggtgacc aagttcatgc taatgacaag ttgttcttgc gtataaactt a 51

<210> 80

<211> 49

<212> DNA

<213> Artificial sequence

<400> 80

gaaggtcgga gtcaacggat ttgacaagtt gttcttgcgt ataaacttg 49

<210> 81

<211> 29

<212> DNA

<213> Artificial sequence

<400> 81

ccttacgatc tacgactcca ttttgataa 29

<210> 82

<211> 43

<212> DNA

<213> Artificial sequence

<400> 82

gaaggtgacc aagttcatgc tcaagaagaa ggcgaaggaa gcg 43

<210> 83

<211> 43

<212> DNA

<213> Artificial sequence

<400> 83

gaaggtcgga gtcaacggat tcaagaagaa ggcgaaggaa gcc 43

<210> 84

<211> 26

<212> DNA

<213> Artificial sequence

<400> 84

ctcttcccct tgaagcttag cttctt 26

<210> 85

<211> 44

<212> DNA

<213> Artificial sequence

<400> 85

gaaggtgacc aagttcatgc tcgattcgca ccggcaagta ttct 44

<210> 86

<211> 43

<212> DNA

<213> Artificial sequence

<400> 86

gaaggtcgga gtcaacggat tgattcgcac cggcaagtat tcg 43

<210> 87

<211> 27

<212> DNA

<213> Artificial sequence

<400> 87

cagattcctg ggcagaaaag tgagaat 27

<210> 88

<211> 51

<212> DNA

<213> Artificial sequence

<400> 88

gaaggtgacc aagttcatgc tttatcactt tattttccaa cattgtaacg c 51

<210> 89

<211> 51

<212> DNA

<213> Artificial sequence

<400> 89

gaaggtcgga gtcaacggat tttatcactt tattttccaa cattgtaacg g 51

<210> 90

<211> 28

<212> DNA

<213> Artificial sequence

<400> 90

cgtggatagt cacgtcgata tttagtaa 28

<210> 91

<211> 43

<212> DNA

<213> Artificial sequence

<400> 91

gaaggtgacc aagttcatgc tcttaattgg gccgccagga act 43

<210> 92

<211> 43

<212> DNA

<213> Artificial sequence

<400> 92

gaaggtcgga gtcaacggat tcttaattgg gccgccagga aca 43

<210> 93

<211> 26

<212> DNA

<213> Artificial sequence

<400> 93

gcaatggctt tagccaacaa ggtctt 26

<210> 94

<211> 48

<212> DNA

<213> Artificial sequence

<400> 94

gaaggtgacc aagttcatgc taatagcaca gccccaaaac aatacaaa 48

<210> 95

<211> 46

<212> DNA

<213> Artificial sequence

<400> 95

gaaggtcgga gtcaacggat ttagcacagc cccaaaacaa tacaag 46

<210> 96

<211> 25

<212> DNA

<213> Artificial sequence

<400> 96

gttaggaagg tgagcctttc tgctt 25

<210>97

<211>48

<212>DNA

<213>Artificial sequence

<400>97

gaaggtgacc aagttcatgc ttaccaacag gagaatctat actatcac 48

<210>98

<211>50

<212>DNA

<213>Artificial sequence

<400>98

gaaggtcgga gtcaacggat tgataccaac aggagaatct atactatcat 50

<210>99

<211>27

<212>DNA

<213>Artificial sequence

<400>99

cctgattgga cagaacctga accaaat 27

<210>100

<211>43

<212>DNA

<213>Artificial sequence

<400>100

gaaggtgacc aagttcatgc ttccctatgg gcaaatcgca tcg 43

<210>101

<211>46

<212>DNA

<213>Artificial sequence

<400>101

gaaggtcgga gtcaacggat tttttcccta tgggcaaatc gcatca 46

<210>102

<211>30

<212>DNA

<213>Artificial sequence

<400>102

ctctctctta gtcgcttttt tggatttctt 30

<210>103

<211>47

<212>DNA

<213>Artificial sequence

<400>103

gaaggtgacc aagttcatgc tgtgtccgtt tgtgatgagt tgctaat 47

<210>104

<211>46

<212>DNA

<213>Artificial sequence

<400>104

gaaggtcgga gtcaacggat ttgtccgttt gtgatgagtt gctaac 46

<210>105

<211>26

<212>DNA

<213>Artificial sequence

<400>105

ggacgaatcg tccacattct cagaaa 26

<210>106

<211>50

<212>DNA

<213>Artificial sequence

<400>106

gaaggtgacc aagttcatgc tgacatacaa tgaaaactag gttaagaacg 50

<210>107

<211>51

<212>DNA

<213>Artificial sequence

<400>107

gaaggtcgga gtcaacggat ttgacataca atgaaaacta ggttaagaac a 51

<210>108

<211>30

<212>DNA

<213>Artificial sequence

<400>108

caagatacag ccttttaggt aaatggtctt 30

<210>109

<211>43

<212>DNA

<213>Artificial sequence

<400>109

gaaggtgacc aagttcatgc tctgcagaag atgaagtttg gcc 43

<210>110

<211>45

<212>DNA

<213>Artificial sequence

<400>110

gaaggtcgga gtcaacggat tttctgcaga agatgaagtt tggct 45

<210>111

<211>22

<212>DNA

<213>Artificial sequence

<400>111

agacaagcaa gagctcgccg ag 22

<210>112

<211>51

<212>DNA

<213>Artificial sequence

<400>112

gaaggtgacc aagttcatgc tgaggaaaga aagatttgag atgattaaat g 51

<210>113

<211>52

<212>DNA

<213>Artificial sequence

<400>113

gaaggtcgga gtcaacggat tggaggaaag aaagatttga gatgattaaa ta 52

<210>114

<211>34

<212>DNA

<213>Artificial sequence

<400>114

aattcttaaa ttcttcggaa taatacacca agaa 34

<210>115

<211>49

<212>DNA

<213>Artificial sequence

<400>115

gaaggtgacc aagttcatgc tttaaaacca gcctgctata cataagttc 49

<210>116

<211>51

<212>DNA

<213>Artificial sequence

<400>116

gaaggtcgga gtcaacggat ttattaaaac cagcctgcta tacataagtt t 51

<210>117

<211>34

<212>DNA

<213>Artificial sequence

<400>117

cctagccacc aaaagactaa aataactatt ataa 34

<210>118

<211>49

<212>DNA

<213>Artificial sequence

<400>118

gaaggtgacc aagttcatgc tgacaataga attggagaag gatcagatt 49

<210>119

<211>48

<212>DNA

<213>Artificial sequence

<400>119

gaaggtcgga gtcaacggat tacaatagaa ttggagaagg atcagatg 48

<210>120

<211>30

<212>DNA

<213>Artificial sequence

<400>120

ctgaacctcc atccttttca ttggatatat 30

<210>121

<211>48

<212>DNA

<213>Artificial sequence

<400>121

gaaggtgacc aagttcatgc tccatcataa ccattcatag atgcaaga 48

<210>122

<211>47

<212>DNA

<213>Artificial sequence

<400>122

gaaggtcgga gtcaacggat tcatcataac cattcataga tgcaagg 47

<210>123

<211>27

<212>DNA

<213>Artificial sequence

<400>123

ctcatcaaag aaccctctgg gatacat 27

<210>124

<211>47

<212>DNA

<213>Artificial sequence

<400>124

gaaggtgacc aagttcatgc tggacttgct ataacttcta ccagtct 47

<210>125

<211>46

<212>DNA

<213>Artificial sequence

<400>125

gaaggtcgga gtcaacggat tgacttgcta taacttctac cagtcc 46

<210>126

<211>25

<212>DNA

<213>Artificial sequence

<400>126

atagttctcc ccagccaact tcgta 25

<210>127

<211>49

<212>DNA

<213>Artificial sequence

<400>127

gaaggtgacc aagttcatgc tcctaaaagt aagttacact tggctactt 49

<210>128

<211>48

<212>DNA

<213>Artificial sequence

<400>128

gaaggtcgga gtcaacggat tctaaaagta agttacactt ggctactg 48

<210>129

<211>30

<212>DNA

<213>Artificial sequence

<400>129

cacattagaa aatttcagcc aggagaactt 30

<210>130

<211>46

<212>DNA

<213>Artificial sequence

<400>130

gaaggtgacc aagttcatgc taattcctac gcctggcatg tcaaat 46

<210>131

<211>43

<212>DNA

<213>Artificial sequence

<400>131

gaaggtcgga gtcaacggat ttcctacgcc tggcatgtca aac 43

<210>132

<211>31

<212>DNA

<213>Artificial sequence

<400>132

gaacatcatg ttaatgctga tggatagata t 31

<210>133

<211>51

<212>DNA

<213>Artificial sequence

<400>133

gaaggtgacc aagttcatgc ttatcctaaa gagagaatta aaaaggtctg t 51

<210>134

<211>49

<212>DNA

<213>Artificial sequence

<400>134

gaaggtcgga gtcaacggat ttcctaaaga gagaattaaa aaggtctgg 49

<210>135

<211>35

<212>DNA

<213>Artificial sequence

<400>135

acattctatt gataaatgac agaaacaata atctt 35

<210>136

<211>51

<212>DNA

<213>Artificial sequence

<400>136

gaaggtgacc aagttcatgc tttttcatgt cgcaaataat taaggaagca t 51

<210>137

<211>51

<212>DNA

<213>Artificial sequence

<400>137

gaaggtcgga gtcaacggat tttttcatgt cgcaaataat taaggaagca a 51

<210>138

<211>28

<212>DNA

<213>Artificial sequence

<400>138

aggtgaaaga aagcaagtgg ctagttaa 28

<210>139

<211>48

<212>DNA

<213>Artificial sequence

<400>139

gaaggtgacc aagttcatgc tattcggagc agtgaaaagc agtaaaat 48

<210>140

<211>46

<212>DNA

<213>Artificial sequence

<400>140

gaaggtcgga gtcaacggat ttcggagcag tgaaaagcag taaaac 46

<210>141

<211>26

<212>DNA

<213>Artificial sequence

<400>141

cttgcccgga aatcaattct gggaaa 26

<210>142

<211>39

<212>DNA

<213>Artificial sequence

<400>142

gaaggtgacc aagttcatgc tccctctcct cgacgccgg 39

<210>143

<211>40

<212>DNA

<213>Artificial sequence

<400>143

gaaggtcgga gtcaacggat taccctctcc tcgacgccga 40

<210>144

<211>19

<212>DNA

<213>Artificial sequence

<400>144

gggcggcgga ggagtcgat 19

Claims

SNP locus combinations comprising 48 SNP loci of a Cucurbita pepo genome; the 48 SNP sites are as follows:

the CupopSNP 01 site is 2807880 th nucleotide on the chromosome of Chr 01;

the CupopSNP 02 site is 9791465 th nucleotide on the chromosome of Chr 01;

the CupopSNP 03 site is 19069993 th nucleotide on the chromosome of Chr 01;

the CupopSNP 04 site is 1687667 th nucleotide on the chromosome of Chr 02;

the CupopSNP 05 site is 794938 th nucleotide on the chromosome of Chr 03;

the CupopSNP 06 site is 799507 th nucleotide on the chromosome of Chr 03;

the CupopSNP 07 site is 4509600 th nucleotide on the chromosome of Chr 04;

the CupopSNP 08 site is 7015999 th nucleotide on the chromosome of Chr 04;

the CupopSNP 09 site is 11177727 th nucleotide on the chromosome of Chr 04;

the CupopSNP 10 site is 4771908 th nucleotide on the chromosome of Chr 05;

the CupopSNP 11 site is 9266170 th nucleotide on the chromosome of Chr 05;

the CupopSNP 12 site is 3551450 th nucleotide on the chromosome of Chr 06;

the CupopSNP 13 site is 1076750 th nucleotide on the chromosome of Chr 07;

the CupopSNP 14 site is 7776963 th nucleotide on the chromosome of Chr 07;

the CupopSNP 15 site is 8150946 th nucleotide on the chromosome of Chr 07;

the CupopSNP 16 site is 419732 th nucleotide on the chromosome of Chr 08;

the CupopSNP 17 site is 7173914 th nucleotide on the chromosome of Chr 08;

the CupopSNP 18 site is 8024934 th nucleotide on the chromosome of Chr 08;

the CupopSNP 19 site is 1524784 th nucleotide on the chromosome of Chr 09;

the CupopSNP 20 site is 1823037 th nucleotide on the chromosome of Chr 09;

the CupopSNP 21 site is 2169965 th nucleotide on the chromosome of Chr 09;

the CupopSNP 22 site is 2352404 th nucleotide on the chromosome of Chr 10;

the CupopSNP 23 site is 4943533 th nucleotide on the chromosome of Chr 10;

the CupopSNP 24 site is 7237965 th nucleotide on the chromosome of Chr 11;

the CupopSNP 25 site is 9818902 th nucleotide on the chromosome of Chr 11;

the CupopSNP 26 site is 4936646 th nucleotide on the chromosome of Chr 12;

the CupopSNP 27 site is 6034493 th nucleotide on the chromosome of Chr 12;

the CupopSNP 28 site is 8635520 th nucleotide on the chromosome of Chr 12;

the CupopSNP 29 site is 6704302 th nucleotide on the chromosome of Chr 13;

the CupopSNP 30 site is 6770190 th nucleotide on the chromosome of Chr 13;

the CupopSNP 31 site is 7984331 th nucleotide on the chromosome of Chr 13;

the CupopSNP 32 site is 8283301 th nucleotide on the chromosome of Chr 13;

the CupopSNP 33 site is 5675208 th nucleotide on the chromosome of Chr 14;

the CupopSNP 34 site is 8439875 th nucleotide on the chromosome of Chr 14;

the CupopSNP 35 site is 107674 th nucleotide on the chromosome of Chr 15;

the CupopSNP 36 site is 997997 th nucleotide on the chromosome of Chr 15;

the CupopSNP 37 site is 8052733 th nucleotide on the chromosome of Chr 15;

the CupopSNP 38 site is 441092 th nucleotide on the chromosome of Chr 16;

the CupopSNP 39 site is 4936150 th nucleotide on the chromosome of Chr 16;

the CupopSNP 40 site is 8226807 th nucleotide on the chromosome of Chr 16;

the CupopSNP 41 site is 365539 th nucleotide on the chromosome of Chr 17;

the CupopSNP 42 site is 3071924 th nucleotide on the chromosome of Chr 17;

the CupopSNP 43 site is 5566093 th nucleotide on the chromosome of Chr 18;

the CupopSNP 44 site is 105081 th nucleotide on the chromosome of Chr 19;

the CupopSNP 45 site is 1475103 th nucleotide on the chromosome of Chr 19;

the CupopSNP 46 site is 6337473 th nucleotide on the chromosome of Chr 19;

the CupopSNP 47 site is 1391021 th nucleotide on the chromosome of Chr 20;

the CupopSNP 48 site is nucleotide 7417753 on chromosome Chr 20.
2. A primer combination comprising

Primer set 1 for amplifying CupopSNP 01 site defined in claim 1,

Primer set 2 for amplifying CupopSNP 02 site defined in claim 1,

Primer set 3 for amplifying CupopSNP 03 site defined in claim 1,

Primer set 4 for amplifying CupopSNP 04 site defined in claim 1,

Primer set 5 for amplifying CupopSNP 05 site defined in claim 1,

Primer set 6 for amplifying CupopSNP 06 site defined in claim 1,

Primer set 7 for amplifying CupopSNP 07 site defined in claim 1,

The primer set 8 for amplifying the CupopSNP 08 site defined in claim 1,

The primer set 9 for amplifying the CupopSNP 09 site defined in claim 1,

A primer set 10 for amplifying the CupopSNP 10 site defined in claim 1,

The primer set 11 for amplifying the CupopSNP 11 site defined in claim 1,

A primer set 12 for amplifying the CupopSNP 12 site defined in claim 1,

The primer set 13 for amplifying the CupopSNP 13 site defined in claim 1,

The primer set 14 for amplifying the CupopSNP 14 site defined in claim 1,

A primer set 15 for amplifying the CupopSNP 15 site defined in claim 1,

A primer set 16 for amplifying the CupopSNP 16 site defined in claim 1,

A primer set 17 for amplifying the CupopSNP 17 site defined in claim 1,

The primer set 18 for amplifying the CupopSNP 18 site defined in claim 1,

A primer set 19 for amplifying the CupopSNP 19 site defined in claim 1,

A primer set 20 for amplifying the CupopSNP 20 site defined in claim 1,

A primer set 21 for amplifying the CupopSNP 21 site defined in claim 1,

A primer set 22 for amplifying the CupopSNP 22 site defined in claim 1,

The primer set 23 for amplifying the CupopSNP 23 site defined in claim 1,

The primer set 24 for amplifying the CupopSNP 24 site defined in claim 1,

A primer set 25 for amplifying the CupopSNP 25 site defined in claim 1,

The primer set 26 for amplifying the CupopSNP 26 site defined in claim 1,

A primer set 27 for amplifying the CupopSNP 27 site defined in claim 1,

A primer set 28 for amplifying the CupopSNP 28 site defined in claim 1,

A primer set 29 for amplifying the CupopSNP 29 site defined in claim 1,

A primer set 30 for amplifying the CupopSNP 30 site defined in claim 1,

A primer set 31 for amplifying the CupopSNP 31 site defined in claim 1,

The primer set 32 for amplifying the CupopSNP 32 site defined in claim 1,

A primer set 33 for amplifying the CupopSNP 33 site defined in claim 1,

A primer set 34 for amplifying the CupopSNP 34 site defined in claim 1,

The primer set 35 for amplifying the CupopSNP 35 site defined in claim 1,

The primer set 36 for amplifying the CupopSNP 36 site defined in claim 1,

A primer set 37 for amplifying the CupopSNP 37 site defined in claim 1,

A primer set 38 for amplifying the CupopSNP 38 site defined in claim 1,

A primer set 39 for amplifying the CupopSNP 39 site defined in claim 1,

A primer set 40 for amplifying the CupopSNP 40 site defined in claim 1,

A primer set 41 for amplifying the CupopSNP 41 site defined in claim 1,

A primer set 42 for amplifying the CupopSNP 42 site defined in claim 1,

A primer set 43 for amplifying the CupopSNP 43 site defined in claim 1,

A primer set 44 for amplifying the CupopSNP 44 site defined in claim 1,

A primer set 45 for amplifying the CupopSNP 45 site defined in claim 1,

A primer set 46 for amplifying the CupopSNP 46 site defined in claim 1,

Primer set 47 and primer set for amplifying CupopSNP 47 site described in claim 1

A primer set 48 for amplifying the CupopSNP 48 site as set forth in claim 1.
3. The primer combination of claim 2, wherein:

the primer group 1 consists of a forward primer 01F1 shown in SEQ ID NO. 1, a forward primer 01F2 shown in SEQ ID NO. 2 and a reverse primer 01R shown in SEQ ID NO. 3;

the primer group 2 consists of a forward primer 02F1 shown in SEQ ID NO. 4, a forward primer 02F2 shown in SEQ ID NO. 5 and a reverse primer 02R shown in SEQ ID NO. 6;

the primer group 3 consists of a forward primer 03F1 shown in SEQ ID NO. 7, a forward primer 03F2 shown in SEQ ID NO. 8 and a reverse primer 03R shown in SEQ ID NO. 9;

the primer group 4 consists of a forward primer 04F1 shown by SEQ ID NO. 10, a forward primer 04F2 shown by SEQ ID NO. 11 and a reverse primer 04R shown by SEQ ID NO. 12;

the primer group 5 consists of a forward primer 05F1 shown in SEQ ID NO. 13, a forward primer 05F2 shown in SEQ ID NO. 14 and a reverse primer 05R shown in SEQ ID NO. 15;

the primer group 6 consists of a forward primer 06F1 shown by SEQ ID NO. 16, a forward primer 06F2 shown by SEQ ID NO. 17 and a reverse primer 06R shown by SEQ ID NO. 18;

the primer group 7 consists of a forward primer 07F1 shown in SEQ ID NO. 19, a forward primer 07F2 shown in SEQ ID NO. 20 and a reverse primer 07R shown in SEQ ID NO. 21;

the primer group 8 consists of a forward primer 08F1 shown by SEQ ID NO. 22, a forward primer 08F2 shown by SEQ ID NO. 23 and a reverse primer 08R shown by SEQ ID NO. 24;

the primer group 9 consists of a forward primer 09F1 shown in SEQ ID NO. 25, a forward primer 09F2 shown in SEQ ID NO. 26 and a reverse primer 09R shown in SEQ ID NO. 27;

the primer group 10 consists of a forward primer 10F1 shown by SEQ ID NO. 28, a forward primer 10F2 shown by SEQ ID NO. 29 and a reverse primer 10R shown by SEQ ID NO. 30;

the primer group 11 consists of a forward primer 11F1 shown by SEQ ID NO. 31, a forward primer 11F2 shown by SEQ ID NO. 32 and a reverse primer 11R shown by SEQ ID NO. 33;

the primer group 12 consists of a forward primer 12F1 shown by SEQ ID NO. 34, a forward primer 12F2 shown by SEQ ID NO. 35 and a reverse primer 12R shown by SEQ ID NO. 36;

the primer group 13 consists of a forward primer 13F1 shown in SEQ ID NO. 37, a forward primer 13F2 shown in SEQ ID NO. 38 and a reverse primer 13R shown in SEQ ID NO. 39;

the primer group 14 consists of a forward primer 14F1 shown by SEQ ID NO. 40, a forward primer 14F2 shown by SEQ ID NO. 41 and a reverse primer 14R shown by SEQ ID NO. 42;

the primer group 15 consists of a forward primer 15F1 shown in SEQ ID NO. 43, a forward primer 15F2 shown in SEQ ID NO. 44 and a reverse primer 15R shown in SEQ ID NO. 45;

the primer group 16 consists of a forward primer 16F1 shown by SEQ ID NO. 46, a forward primer 16F2 shown by SEQ ID NO. 47 and a reverse primer 16R shown by SEQ ID NO. 48;

the primer group 17 consists of a forward primer 17F1 shown by SEQ ID NO. 49, a forward primer 17F2 shown by SEQ ID NO. 50 and a reverse primer 17R shown by SEQ ID NO. 51;

the primer group 18 consists of a forward primer 18F1 shown by SEQ ID NO. 52, a forward primer 18F2 shown by SEQ ID NO. 53 and a reverse primer 18R shown by SEQ ID NO. 54;

the primer group 19 consists of a forward primer 19F1 shown by SEQ ID NO. 55, a forward primer 19F2 shown by SEQ ID NO. 56 and a reverse primer 19R shown by SEQ ID NO. 57;

the primer group 20 consists of a forward primer 20F1 shown by SEQ ID NO. 58, a forward primer 20F2 shown by SEQ ID NO. 59 and a reverse primer 20R shown by SEQ ID NO. 60;

the primer group 21 consists of a forward primer 21F1 shown by SEQ ID NO. 61, a forward primer 21F2 shown by SEQ ID NO. 62 and a reverse primer 21R shown by SEQ ID NO. 63;

the primer group 22 consists of a forward primer 22F1 shown by SEQ ID NO. 64, a forward primer 22F2 shown by SEQ ID NO. 65 and a reverse primer 22R shown by SEQ ID NO. 66;

the primer group 23 consists of a forward primer 23F1 shown by SEQ ID NO. 67, a forward primer 23F2 shown by SEQ ID NO. 68 and a reverse primer 23R shown by SEQ ID NO. 69;

the primer group 24 consists of a forward primer 24F1 shown by SEQ ID NO. 70, a forward primer 24F2 shown by SEQ ID NO. 71 and a reverse primer 24R shown by SEQ ID NO. 72;

the primer group 25 consists of a forward primer 25F1 shown by SEQ ID NO. 73, a forward primer 25F2 shown by SEQ ID NO. 74 and a reverse primer 25R shown by SEQ ID NO. 75;

the primer group 26 consists of a forward primer 26F1 shown by SEQ ID NO. 76, a forward primer 26F2 shown by SEQ ID NO. 77 and a reverse primer 26R shown by SEQ ID NO. 78;

the primer group 27 consists of a forward primer 27F1 shown by SEQ ID NO. 79, a forward primer 27F2 shown by SEQ ID NO. 80 and a reverse primer 27R shown by SEQ ID NO. 81;

the primer group 28 consists of a forward primer 28F1 shown in SEQ ID NO. 82, a forward primer 28F2 shown in SEQ ID NO. 83 and a reverse primer 28R shown in SEQ ID NO. 84;

the primer group 29 consists of a forward primer 29F1 shown by SEQ ID NO. 85, a forward primer 29F2 shown by SEQ ID NO. 86 and a reverse primer 29R shown by SEQ ID NO. 87;

the primer group 30 consists of a forward primer 30F1 shown by SEQ ID NO. 88, a forward primer 30F2 shown by SEQ ID NO. 89 and a reverse primer 30R shown by SEQ ID NO. 90;

the primer group 31 consists of a forward primer 31F1 shown by SEQ ID NO. 91, a forward primer 31F2 shown by SEQ ID NO. 92 and a reverse primer 31R shown by SEQ ID NO. 93;

the primer group 32 consists of a forward primer 32F1 shown by SEQ ID NO. 94, a forward primer 32F2 shown by SEQ ID NO. 95 and a reverse primer 32R shown by SEQ ID NO. 96;

the primer group 33 consists of a forward primer 33F1 shown by SEQ ID NO. 97, a forward primer 33F2 shown by SEQ ID NO. 98 and a reverse primer 33R shown by SEQ ID NO. 99;

the primer group 34 consists of a forward primer 34F1 shown in SEQ ID NO. 100, a forward primer 34F2 shown in SEQ ID NO. 101 and a reverse primer 34R shown in SEQ ID NO. 102;

the primer group 35 consists of a forward primer 35F1 shown by SEQ ID NO. 103, a forward primer 35F2 shown by SEQ ID NO. 104 and a reverse primer 35R shown by SEQ ID NO. 105;

the primer group 36 consists of a forward primer 36F1 shown by SEQ ID NO. 106, a forward primer 36F2 shown by SEQ ID NO. 107 and a reverse primer 36R shown by SEQ ID NO. 108;

the primer group 37 consists of a forward primer 37F1 shown by SEQ ID NO. 109, a forward primer 37F2 shown by SEQ ID NO. 110 and a reverse primer 37R shown by SEQ ID NO. 111;

the primer group 38 consists of a forward primer 38F1 shown by SEQ ID NO. 112, a forward primer 38F2 shown by SEQ ID NO. 113 and a reverse primer 38R shown by SEQ ID NO. 114;

the primer group 39 consists of a forward primer 39F1 shown by SEQ ID NO. 115, a forward primer 39F2 shown by SEQ ID NO. 116 and a reverse primer 39R shown by SEQ ID NO. 117;

the primer group 40 consists of a forward primer 40F1 shown by SEQ ID NO. 118, a forward primer 40F2 shown by SEQ ID NO. 119 and a reverse primer 40R shown by SEQ ID NO. 120;

the primer group 41 consists of a forward primer 41F1 shown by SEQ ID NO. 121, a forward primer 41F2 shown by SEQ ID NO. 122 and a reverse primer 41R shown by SEQ ID NO. 123;

the primer group 42 consists of a forward primer 42F1 shown by SEQ ID NO. 124, a forward primer 42F2 shown by SEQ ID NO. 125 and a reverse primer 42R shown by SEQ ID NO. 126;

the primer group 43 consists of a forward primer 43F1 shown by SEQ ID NO. 127, a forward primer 43F2 shown by SEQ ID NO. 128 and a reverse primer 43R shown by SEQ ID NO. 129;

the primer group 44 consists of a forward primer 44F1 shown in SEQ ID NO. 130, a forward primer 44F2 shown in SEQ ID NO. 131 and a reverse primer 44R shown in SEQ ID NO. 132;

the primer group 45 consists of a forward primer 45F1 shown by SEQ ID NO. 133, a forward primer 45F2 shown by SEQ ID NO. 134 and a reverse primer 45R shown by SEQ ID NO. 135;

the primer group 46 consists of a forward primer 46F1 shown by SEQ ID NO. 136, a forward primer 46F2 shown by SEQ ID NO. 137 and a reverse primer 46R shown by SEQ ID NO. 138;

the primer group 47 consists of a forward primer 47F1 shown by SEQ ID NO. 139, a forward primer 47F2 shown by SEQ ID NO. 140 and a reverse primer 47R shown by SEQ ID NO. 141;

the primer set 48 consists of a forward primer 48F1 shown in SEQ ID NO:142, a forward primer 48F2 shown in SEQ ID NO:143 and a reverse primer 48R shown in SEQ ID NO: 144.
4. The primer combination of claim 2, wherein:

the primer group 1 consists of a forward primer 01F1 shown in 22 th to 46 th positions from the 5 'end of SEQ ID NO. 1, a forward primer 01F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 2 and a reverse primer 01R shown in SEQ ID NO. 3;

the primer group 2 consists of a forward primer 02F1 shown in 22 th to 45 th positions from 5 'end of SEQ ID NO. 4, a forward primer 02F2 shown in 22 th to 46 th positions from 5' end of SEQ ID NO. 5, and a reverse primer 02R shown in SEQ ID NO. 6;

the primer group 3 consists of a forward primer 03F1 shown in the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 7, a forward primer 03F2 shown in the 22 nd to 43 th positions from the 5' end of SEQ ID NO. 8, and a reverse primer 03R shown in SEQ ID NO. 9;

the primer group 4 consists of a forward primer 04F1 shown in 22 th to 45 th positions from the 5 'end of SEQ ID NO. 10, a forward primer 04F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 11, and a reverse primer 04R shown in SEQ ID NO. 12;

the primer group 5 consists of a forward primer 05F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 13, a forward primer 05F2 shown in 22 th to 48 th positions from the 5' end of SEQ ID NO. 14, and a reverse primer 05R shown in SEQ ID NO. 15;

the primer group 6 consists of a forward primer 06F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 16, a forward primer 06F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 17, and a reverse primer 06R shown in SEQ ID NO. 18;

the primer group 7 consists of a forward primer 07F1 shown in 22 th to 46 th positions from the 5 'end of SEQ ID NO. 19, a forward primer 07F2 shown in 22 th to 47 th positions from the 5' end of SEQ ID NO. 20, and a reverse primer 07R shown in SEQ ID NO. 21;

the primer group 8 consists of a forward primer 08F1 shown in 22 th to 54 th positions from the 5 'end of SEQ ID NO. 22, a forward primer 08F2 shown in 22 th to 52 th positions from the 5' end of SEQ ID NO. 23, and a reverse primer 08R shown in SEQ ID NO. 24;

the primer group 9 consists of a forward primer 09F1 shown in the 22 nd to 52 th positions from the 5 'end of SEQ ID NO. 25, a forward primer 09F2 shown in the 22 nd to 52 th positions from the 5' end of SEQ ID NO. 26, and a reverse primer 09R shown in the SEQ ID NO. 27;

the primer group 10 consists of a forward primer 10F1 shown in 22 th to 43 th positions from 5 'end of SEQ ID NO. 28, a forward primer 10F2 shown in 22 th to 43 th positions from 5' end of SEQ ID NO. 29, and a reverse primer 10R shown in SEQ ID NO. 30;

the primer set 11 consists of a forward primer 11F1 shown in 22 th to 49 th positions from the 5 'end of SEQ ID NO. 31, a forward primer 11F2 shown in 22 th to 48 th positions from the 5' end of SEQ ID NO. 32, and a reverse primer 11R shown in SEQ ID NO. 33;

the primer set 12 consists of a forward primer 12F1 shown in 22 th to 54 th positions from the 5 'end of SEQ ID NO. 34, a forward primer 12F2 shown in 22 th to 53 th positions from the 5' end of SEQ ID NO. 35, and a reverse primer 12R shown in SEQ ID NO. 36;

the primer group 13 consists of a forward primer 13F1 shown in 22 th to 43 th positions from 5 'end of SEQ ID NO. 37, a forward primer 13F2 shown in 22 th to 46 th positions from 5' end of SEQ ID NO. 38, and a reverse primer 13R shown in SEQ ID NO. 39;

the primer set 14 consists of a forward primer 14F1 shown in the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 40, a forward primer 14F2 shown in the 22 nd to 49 th positions from the 5' end of SEQ ID NO. 41, and a reverse primer 14R shown in SEQ ID NO. 42;

the primer group 15 consists of a forward primer 15F1 shown in the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 43, a forward primer 15F2 shown in the 22 nd to 49 th positions from the 5' end of SEQ ID NO. 44, and a reverse primer 15R shown in SEQ ID NO. 45;

the primer set 16 consists of a forward primer 16F1 shown in the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 46, a forward primer 16F2 shown in the 22 nd to 49 th positions from the 5' end of SEQ ID NO. 47, and a reverse primer 16R shown in SEQ ID NO. 48;

the primer set 17 consists of a forward primer 17F1 shown in the 22 nd to 49 th positions from the 5 'end of SEQ ID NO. 49, a forward primer 17F2 shown in the 22 nd to 48 th positions from the 5' end of SEQ ID NO. 50, and a reverse primer 17R shown in SEQ ID NO. 51;

the primer group 18 consists of a forward primer 18F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 52, a forward primer 18F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 53, and a reverse primer 18R shown in SEQ ID NO. 54;

the primer group 19 consists of a forward primer 19F1 shown in the 22 nd to 49 th positions from the 5 'end of SEQ ID NO. 55, a forward primer 19F2 shown in the 22 nd to 51 th positions from the 5' end of SEQ ID NO. 56, and a reverse primer 19R shown in SEQ ID NO. 57;

the primer set 20 consists of a forward primer 20F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 58, a forward primer 20F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 59, and a reverse primer 20R shown in SEQ ID NO. 60;

the primer group 21 consists of a forward primer 21F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 61, a forward primer 21F2 shown in the 22 nd to 44 th positions from the 5' end of SEQ ID NO. 62, and a reverse primer 21R shown in SEQ ID NO. 63;

the primer set 22 consists of a forward primer 22F1 shown in the 22 nd to 41 th positions from the 5 'end of SEQ ID NO. 64, a forward primer 22F2 shown in the 22 nd to 43 th positions from the 5' end of SEQ ID NO. 65, and a reverse primer 22R shown in SEQ ID NO. 66;

the primer group 23 consists of a forward primer 23F1 shown in 22 th to 46 th positions from the 5 'end of SEQ ID NO. 67, a forward primer 23F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 68, and a reverse primer 23R shown in SEQ ID NO. 69;

the primer group 24 consists of a forward primer 24F1 shown in 22 th to 49 th positions from the 5 'end of SEQ ID NO. 70, a forward primer 24F2 shown in 22 th to 48 th positions from the 5' end of SEQ ID NO. 71 and a reverse primer 24R shown in SEQ ID NO. 72;

the primer set 25 consists of a forward primer 25F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown in the 22 nd to 47 th positions from the 5' end of SEQ ID NO. 74, and a reverse primer 25R shown in SEQ ID NO. 75;

the primer group 26 consists of a forward primer 26F1 shown in the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 76, a forward primer 26F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 77, and a reverse primer 26R shown in SEQ ID NO. 78;

the primer group 27 consists of a forward primer 27F1 shown in 22 th to 51 th positions from the 5 'end of SEQ ID NO. 79, a forward primer 27F2 shown in 22 th to 49 th positions from the 5' end of SEQ ID NO. 80, and a reverse primer 27R shown in SEQ ID NO. 81;

the primer set 28 consists of a forward primer 28F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 82, a forward primer 28F2 shown in the 22 nd to 43 th positions from the 5' end of SEQ ID NO. 83, and a reverse primer 28R shown in the SEQ ID NO. 84;

the primer group 29 consists of a forward primer 29F1 shown in the 22 nd to 44 th positions from the 5 'end of SEQ ID NO. 85, a forward primer 29F2 shown in the 22 nd to 43 th positions from the 5' end of SEQ ID NO. 86, and a reverse primer 29R shown in SEQ ID NO. 87;

the primer group 30 consists of a forward primer 30F1 shown in 22 th to 51 th positions from the 5 'end of SEQ ID NO. 88, a forward primer 30F2 shown in 22 th to 51 th positions from the 5' end of SEQ ID NO. 89, and a reverse primer 30R shown in SEQ ID NO. 90;

the primer set 31 consists of a forward primer 31F1 shown in 22 th to 43 th positions from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown in 22 th to 43 th positions from the 5' end of SEQ ID NO. 92, and a reverse primer 31R shown in SEQ ID NO. 93;

the primer group 32 consists of a forward primer 32F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 94, a forward primer 32F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 95, and a reverse primer 32R shown in SEQ ID NO. 96;

the primer group 33 consists of a forward primer 33F1 shown in 22 th to 48 th positions from 5 'end of SEQ ID NO. 97, a forward primer 33F2 shown in 22 th to 50 th positions from 5' end of SEQ ID NO. 98, and a reverse primer 33R shown in SEQ ID NO. 99;

the primer group 34 consists of a forward primer 34F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 100, a forward primer 34F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 101, and a reverse primer 34R shown in SEQ ID NO. 102;

the primer group 35 consists of a forward primer 35F1 shown in the 22 nd to 47 th positions from the 5 'end of SEQ ID NO. 103, a forward primer 35F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 104, and a reverse primer 35R shown in SEQ ID NO. 105;

the primer group 36 consists of a forward primer 36F1 shown in 22 th to 50 th positions from the 5 'end of SEQ ID NO. 106, a forward primer 36F2 shown in 22 th to 51 th positions from the 5' end of SEQ ID NO. 107, and a reverse primer 36R shown in SEQ ID NO. 108;

the primer group 37 consists of a forward primer 37F1 shown in the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 109, a forward primer 37F2 shown in the 22 nd to 45 th positions from the 5' end of SEQ ID NO. 110, and a reverse primer 37R shown in SEQ ID NO. 111;

the primer set 38 consists of a forward primer 38F1 shown in the 22 nd to 51 th positions from the 5 'end of SEQ ID NO. 112, a forward primer 38F2 shown in the 22 nd to 52 th positions from the 5' end of SEQ ID NO. 113, and a reverse primer 38R shown in SEQ ID NO. 114;

the primer set 39 consists of a forward primer 39F1 shown in 22 th to 49 th positions from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown in 22 th to 51 th positions from the 5' end of SEQ ID NO. 116, and a reverse primer 39R shown in SEQ ID NO. 117;

the primer set 40 consists of a forward primer 40F1 shown in the 22 nd to 49 th positions from the 5 'end of SEQ ID NO. 118, a forward primer 40F2 shown in the 22 nd to 48 th positions from the 5' end of SEQ ID NO. 119, and a reverse primer 40R shown in SEQ ID NO. 120;

the primer group 41 consists of a forward primer 41F1 shown in 22 th to 48 th positions from 5 'end of SEQ ID NO. 121, a forward primer 41F2 shown in 22 th to 47 th positions from 5' end of SEQ ID NO. 122, and a reverse primer 41R shown in SEQ ID NO. 123;

the primer group 42 consists of a forward primer 42F1 shown in 22 nd to 47 th positions from the 5 'end of SEQ ID NO. 124, a forward primer 42F2 shown in 22 nd to 46 th positions from the 5' end of SEQ ID NO. 125, and a reverse primer 42R shown in SEQ ID NO. 126;

the primer set 43 consists of a forward primer 43F1 shown in 22 th to 49 th positions from the 5 'end of SEQ ID NO. 127, a forward primer 43F2 shown in 22 th to 48 th positions from the 5' end of SEQ ID NO. 128, and a reverse primer 43R shown in SEQ ID NO. 129;

the primer set 44 consists of a forward primer 44F1 shown in 22 th to 46 th positions from the 5 'end of SEQ ID NO. 130, a forward primer 44F2 shown in 22 th to 43 th positions from the 5' end of SEQ ID NO. 131, and a reverse primer 44R shown in SEQ ID NO. 132;

the primer group 45 consists of a forward primer 45F1 shown in 22 th to 51 th positions from the 5 'end of SEQ ID NO. 133, a forward primer 45F2 shown in 22 th to 49 th positions from the 5' end of SEQ ID NO. 134, and a reverse primer 45R shown in SEQ ID NO. 135;

the primer set 46 consists of a forward primer 46F1 shown in the 22 nd to 51 th positions from the 5 'end of SEQ ID NO. 136, a forward primer 46F2 shown in the 22 nd to 51 th positions from the 5' end of SEQ ID NO. 137, and a reverse primer 46R shown in the SEQ ID NO. 138;

the primer group 47 consists of a forward primer 47F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 139, a forward primer 47F2 shown in 22 th to 46 th positions from the 5' end of SEQ ID NO. 140, and a reverse primer 47R shown in SEQ ID NO. 141;

the primer set 48 is composed of a forward primer 48F1 shown in the 22 nd to 39 th positions from the 5 'end of SEQ ID NO 142, a forward primer 48F2 shown in the 22 nd to 40 th positions from the 5' end of SEQ ID NO 143, and a reverse primer 48R shown in SEQ ID NO 144.
5. The use of the combination of SNP sites according to claim 1 or the combination of primers according to any one of claims 2 to 4) in any one of x1) to x 4): x1) preparing a kit for identifying the variety of the cucurbita pepo; x2) preparing a kit for identifying the authenticity of the cucurbita pepo variety; x3) identifying the variety of the zucchini; x4) identifying the authenticity of the cucurbita pepo variety.
6. A method for identifying which variety of 265 cucurbita pepo varieties a cucurbita pepo to be detected belongs to comprises the following steps: respectively detecting genotypes of cucurbita pepo and 265 cucurbita pepo varieties to be detected based on the 48 SNP sites of claim 1, and then judging as follows: if the genotypes of the pumpkin to be detected based on the 48 SNP sites are completely consistent with the genotypes of some variety of the 265 pumpkin varieties based on the 48 SNP sites, the pumpkin to be detected and the pumpkin variety belong to the same variety; and if the genotypes of the pumpkin to be detected based on the 48 SNP sites are different from the genotypes of all varieties in the 265 pumpkin varieties based on the 48 SNP sites, the varieties of the pumpkin to be detected are different from the varieties of the 265 pumpkin varieties.
7. The method of claim 6, wherein: the steps for detecting the genotypes of the cucurbita pepo variety to be detected and 265 cucurbita pepo varieties based on the 48 SNP loci in the claim 1 are as follows:

(1) respectively carrying out PCR amplification by using the genome DNA of the cucurbita pepo to be detected and the genome DNA of 265 cucurbita pepo varieties as templates and respectively adopting the primer groups in the primer combination according to claim 3 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 cucurbita pepo variety to be detected and 265 cucurbita pepo varieties based on the 48 SNP sites according to the color of the fluorescent signal.
8. The method of claim 6, wherein: the steps for detecting the genotypes of the cucurbita pepo variety to be detected and 265 cucurbita pepo varieties based on the 48 SNP loci in the claim 1 are as follows:

(1) respectively carrying out PCR amplification by using the genome DNA of the Cucurbita pepo to be detected and the genome DNA of 265 Cucurbita pepo varieties as templates and respectively adopting the primer groups in the primer combination according to claim 4 to obtain PCR amplification products;

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

(3) and (3) obtaining genotypes of the cucurbita pepo and 265 cucurbita pepo varieties to be detected based on the 48 SNP loci according to the sequencing result obtained in the step (2).
9. A method for identifying which variety of 265 cucurbita pepo varieties a cucurbita pepo to be detected belongs to comprises the following steps:

(1) carrying out PCR amplification by respectively adopting the primer groups in the primer combination of claim 4 by taking the genome DNA of the cucurbita pepo to be detected as a template to obtain PCR amplification products; carrying out PCR amplification by respectively adopting the primer groups in the primer combination of claim 4 by taking the genome DNA of each cucurbita pepo variety in the standard cucurbita pepo variety population as a template to obtain PCR amplification products; the standard zucchini product population consists of 265 zucchini varieties;

(2) and performing cluster analysis on each PCR amplification product obtained from the Cucurbita pepo to be detected and the PCR amplification product corresponding to each standard Cucurbita pepo variety, wherein the Cucurbita pepo to be detected and which standard Cucurbita pepo variety are in the same class in the cluster analysis, and the Cucurbita pepo to be detected and the standard Cucurbita pepo variety belong to the same variety.
10. The method of any of claims 6 to 9, wherein: the 265 Cucurbita pepo varieties are the Cucurbita pepo 131, the Cucurbita pepo No. 13, the Cucurbita pepo No. 12, the Cucurbita pepo No. 11, the Cucurbita pepo No. 9, the Cucurbita pepo No. 4, the Cucurbita pepo No. 3, the Cucurbita pepo No. 2, the Cucurbita pepo No. 1, the Cucurbita pepo No. 11, the Cucurbita pepo No. 7, the Cucurbita pepo No. 5, the Cucurbita pepo No. 336, the Cucurbita pepo No. 22, the Cucurbita pepo No. 8, the Cucurbita pepo No. 2, the Yuzhu No. 2, the New Kyozhu, the Cucurbita pepo 66, the Cucurbita pepo No. 42, the Cucurbita pepo 38, the Cucurbita pepo 37, the Cucurbita pepo No. 36, the Cucurbita pepo No. 35, the Cucurbita pepo No. 33, the Cucurbita pepo CRV4-2, the Cucurbita pepo No. 3, the Beijing Cucurbita pepo No. 11, the Beijing Cucurbita CRV4-2, the Beijing Cucurbita pepo No. 3, the Beijing Cucurbita pepo No. 10, the Beijing No. 11, the Beijing Cucurbita pepo No. 11, the Beijing No. 11, the Bei, 205-8G504 Cucurbita pepo, 206-8G506, 210-ZG522, early spring 202-LSQ237 Cucurbita pepo, XHL1476, XHL1485, Lilang No. 3, Dongfeng No. 5, XHL14106, XHL1471, XHL14109, XHL14104, Meijian 8018, XH01587, LZ-4, XHL14103, Xixing Cucurbita pepo No. 10, Xinrui 1212, 209-8G521 Cucurbita pepo, TRQ-18, XHL14108, XHL1472, XHL1474, XHL1449, XHL14107, XHL14101, Green crown 38, Green crown 29, Green crown 40, Xiduo, West Jade, DTZU-42, Chunshen, Xinrui 1430, TRQ-15, S-2 Xixisi, ATX S06, ATX S4656, ATX S04, ATX S14125, ATX 10, XHX No. 2002, XHX No. 14911, XHL 1478, XHL1474, XHL 1478, XH-6, XH-7, XH-6, XH-S-2-6, ATX No. 2, ATX-94, ATX-6, ATX No. 2002, ATX-6, ATX No. 2, ATX-19, ATX-No. 2, XHL14103, XHL14104, XHL14107, XHL14108, No. 132, XHL14109, 14108, Lv Shuai, sq217218, sq219220, XH01587, XH01588 pumpkin, XH01589 pumpkin, XH01590 pumpkin, XH01591 pumpkin, XH01592 pumpkin, XH01594 pumpkin, XH01595 pumpkin, Saina, XH01446, XH01447, Jade, Guanyu, Jinhu 04036, Hanyu, Dong hu No. 16, Dong hu 1712, Dong hu 3329, Dong hu No. 22, Dong hu No. 33, Dong hu No. 28, XH04002, XH04003, XH04, Qiu 04005, XH014, XH01414, Wanbao, Roubabao (circular Xibao), Fuyu, Wuwanlu zui, Miao Rui Luo Rou 375, XH Rou, XH Shu Rou No. 377, XH No. 35, XH-01374, XH-Hwa-01, Zhao Rou-Mo Xihu, XH No. 373, Hga Yu-8, Hga Yu-2, Hga Yu Shu-2, Hga Yu-K # 35, Hga-Fu-2, Hga Yu Shu-Hga Yu-2, Hga Yu Shu-Fu-2, Hga Yu-2, West jade, west prince, west america, west jade, valley rain, winter beauty, xiali, france shenna, france lissa, green crown 24, green crown 27, green crown 29, green crown 38, green crown 34, green crown 40, green crown 41, lufeng 55F1, tianfeng, france green bei 7810, meihuhu 38, luba oil green zucchini 889, french treasure, cuibao, mouzuo antoni, zucchini SQ-17, zilao xiao, fuxin No. 9, feitian No. 8, jinfeng No. eight, dongguan zuhuo F1, S-3 zucchini, ATX S03, ATX S04, ATX S05, ATX S07, ATX S08, ATX S09, ATX S10, ATX 58s 26, ATX S12, ATX S13, ATX S3527, ATX S14, tai yun hui feng, tai yun feng 368, tai yun feng 368, yun feng 368, yun yu 368, yun feng 368, yu 368, yun feng 368, yun fen 368, fengchu No. 3, Fengchu No. 4, Fengchu No. 5, Fengchu No. 6, Fengchu No. 7, Heimai gourd No. 3, Chao Zhen X3, Chao Zhen No. 4, Qisheng Xigou, SQLH7, SQLH10, SQLH11, SQLH12, Ruifeng Jiu No. F1, gold 318F1, gold 618F1, WS 4112F 1, 2088G517, 2068G506, 2058G504 Cucurbita pepo, Xinrui 1212, 201LSQ236, early spring 202LSQ237 Cucurbita pepo, green jade, Naao No. 26, Haimei No. 1207, W1501F 1, W368F 1 Alexander, Lideluneng Lanzhong, Huasheng No. 31 and No. 327.