CN112877453A

CN112877453A - Method for identifying authenticity of pepper variety

Info

Publication number: CN112877453A
Application number: CN202110103476.8A
Authority: CN
Inventors: 温常龙; 张建; 杜和山; 杨静静; 罗江; 张晓飞
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-06-01
Anticipated expiration: 2041-01-26
Also published as: CN112877453B

Abstract

The invention discloses a method for identifying the authenticity of a pepper variety. The method comprises the following steps: respectively detecting genotypes of the pepper to be detected and 286 pepper varieties based on 40 SNP loci, and then judging as follows: and if the genotypes of the to-be-detected pepper based on the 40 SNP sites are completely consistent with a certain variety of the 286 pepper varieties, the to-be-detected pepper and the pepper variety belong to the same variety. The method provided by the invention can be used for early identification of the pepper 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 pepper germplasm resources and new varieties. The method provided by the invention can be used for identifying unknown pepper varieties and also can be used for identifying the authenticity of known varieties. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

Method for identifying authenticity of pepper variety

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a method for identifying the authenticity of pepper varieties.

Background

Variety identification is an important method for agricultural production, crop breeding and seed inspection, and is also an important means for protecting crop varieties and preventing counterfeit and shoddy varieties from entering the market. The pepper is the vegetable crop with the largest planting area in China, and the planting area in 2019 reaches 178.42 ten thousand hectares. According to the "registration of non-staple crop varieties", pepper belongs to the first non-staple crop registration crop. Only between 2017 and 2019, 2464 pepper varieties pass through registration nationwide, but the number of the pepper varieties on the market far exceeds the number. Because the variety approval among provinces and cities is inconsistent, the management of the variety of the hot pepper cannot be effectively followed; in addition, the management of the fine seed production base is not strict, the phenomena of stealing breeding, purchasing varieties and homonymy heterogeneity are serious, and the production quality accidents of seeds occur occasionally. The quality supervision of pepper varieties is an important link in pepper production. However, the traditional pepper seed variety identification method has field character identification, grain shape identification and the like, but the identification period is long, and a large amount of manpower, material resources and financial resources are consumed. With the rapid development of molecular biology technology, molecular marker technology is mature continuously, and a way is opened up from the identification of variety genome level. In addition, DNA detection results can be directly submitted according to the requirements of the registration guidelines for non-major crop varieties, and the related traits involved in the variety characterization and variety DUS test reports, such as clear associated genes. Therefore, establishing a set of standards for effectively performing the authenticity of pepper variety resources based on DNA fingerprints is very important.

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 high-throughput sequencing technology and the reduction of sequencing cost, a large amount of pepper resequencing data is obtained. Based on analysis of pepper variation group information, more reliable and efficient SNP sites can be mined. By adopting an allele competitive specific PCR method, specific primers can be developed, and finally the genotype of the sample at the SNP site is obtained.

At present, no SNP molecular marking method special for identifying pepper varieties exists in China. At present, other crops mostly adopt SSR molecular markers, but an SSR detection mode easily causes unreal, false positive and false negative results; can not meet the requirements of automation, high flux and large scale. SNPs have several advantages over SSR markers: the variation is clear, stable and easy to detect, and the authenticity is high in accuracy; millions of SNPs are available for selection per crop; the method is suitable for high-throughput, low-cost and automatic rapid detection; SNP typing does not need to contrast varieties, results are presented by accurate bases, and human errors can be reduced.

Disclosure of Invention

The invention aims to identify pepper varieties, in particular 286 pepper varieties.

The primer combination is firstly protected, and the primer combination can comprise a primer group 1 for amplifying a CaSNP01 locus, a primer group 2 for amplifying a CaSNP02 locus, a primer group 3 for amplifying a CaSNP03 locus, a primer group 4 for amplifying a CaSNP04 locus, a primer group 5 for amplifying a CaSNP05 locus, a primer group 6 for amplifying a CaSNP06 locus, a primer group 7 for amplifying a CaSNP07 locus, a primer group 8 for amplifying a CaSNP08 locus, a primer group 9 for amplifying a CaSNP09 locus, a primer group 10 for amplifying a CaSNP10 locus, a primer group 11 for amplifying a CaSNP11 locus, a primer group 12 for amplifying a CaSNP12 locus, a primer group 13 for amplifying a CaSNP13 locus, a primer group 14 for amplifying a CaSNP29 locus, a primer group 15 for amplifying a CaSNP2 locus, a primer group 638 for amplifying a CaSNP 638 locus, a primer group 16 for amplifying a CaSNP17 locus, a CaSNP 6319 for amplifying a CaSNP 638 locus, and a primer group 638 for amplifying a CaSNP18 locus, A primer group 20 for amplifying a CaSNP20 locus, a primer group 21 for amplifying a CaSNP21 locus, a primer group 22 for amplifying a CaSNP22 locus, a primer group 23 for amplifying a CaSNP23 locus, a primer group 24 for amplifying a CaSNP24 locus, a primer group 25 for amplifying a CaSNP25 locus, a primer group 26 for amplifying a CaSNP26 locus, a primer group 27 for amplifying a CaSNP27 locus, a primer group 28 for amplifying a CaSNP28 locus, and a primer group 29 for amplifying a CaSNP29 locus, the primer group 30 for amplifying the CaSNP30 locus, the primer group 31 for amplifying the CaSNP31 locus, the primer group 32 for amplifying the CaSNP32 locus, the primer group 33 for amplifying the CaSNP33 locus, the primer group 34 for amplifying the CaSNP34 locus, the primer group 35 for amplifying the CaSNP35 locus, the primer group 36 for amplifying the CaSNP36 locus, the primer group 37 for amplifying the CaSNP37 locus, the primer group 38 for amplifying the CaSNP38 locus, the primer group 39 for amplifying the CaSNP39 locus and the primer group 40 for amplifying the CaSNP40 locus.

The CaSNP01 site-the CaSNP40 site is 40 SNP sites of the pepper genome. The CaSNP01 site is the 46784687 th nucleotide on the chromosome 01. The CaSNP02 site is the 64927233 th nucleotide on the chromosome 01. The CaSNP03 site is the 128314154 th nucleotide on the chromosome 01. The CaSNP04 site is the 293847360 th nucleotide on the chromosome 01. The CaSNP05 site is the 75886898 th nucleotide on the 02 th chromosome. The CaSNP06 site is the 93209608 th nucleotide on the 02 th chromosome. The CaSNP07 site is the 140042546 th nucleotide on the 02 th chromosome. The CaSNP08 site is the 158395138 th nucleotide on the 02 th chromosome. The CaSNP09 site is the 99284562 th nucleotide on the 03 th chromosome. The CaSNP10 site is the 251295225 th nucleotide on the 03 th chromosome. The CaSNP11 site is the 1205997 th nucleotide on the 04 th chromosome. The CaSNP12 site is the 12579985 th nucleotide on the 04 th chromosome. The CaSNP13 site is the 30522616 th nucleotide on the 04 th chromosome. The CaSNP14 site is the 42814994 th nucleotide on the 04 th chromosome. The CaSNP15 site is the 29437076 th nucleotide on the 05 th chromosome. The CaSNP16 site is the 85520600 th nucleotide on the 05 th chromosome. The CaSNP17 site is the 198838826 th nucleotide on the 05 th chromosome. The CaSNP18 site is the 388508 th nucleotide on chromosome 06. The CaSNP19 site is the 58719488 th nucleotide on chromosome 06. The CaSNP20 site is the 95206292 th nucleotide on chromosome 06. The CaSNP21 site is the 101332262 th nucleotide on the number 07 chromosome. The CaSNP22 site is the 110735031 th nucleotide on the number 07 chromosome. The CaSNP23 site is the 204576291 th nucleotide on the number 07 chromosome. The CaSNP24 site is the 119957941 th nucleotide on the 08 th chromosome. The CaSNP25 site is the 122468853 th nucleotide on the 08 th chromosome. The CaSNP26 site is the 126315482 th nucleotide on the 08 th chromosome. The CaSNP27 site is the 147458068 th nucleotide on the 08 th chromosome. The CaSNP28 site is the 159969 th nucleotide on the No. 09 chromosome. The CaSNP29 site is the 151594116 th nucleotide on the No. 09 chromosome. The CaSNP30 site is the 236428132 th nucleotide on the No. 09 chromosome. The CaSNP31 site is the 494205 th nucleotide on the 10 th chromosome. The CaSNP32 site is the 1684877 th nucleotide on the 10 th chromosome. The CaSNP33 site is the 49575961 th nucleotide on the 10 th chromosome. The CaSNP34 site is the 133308217 th nucleotide on the 10 th chromosome. The CaSNP35 site is the 7376328 th nucleotide on the 11 th chromosome. The CaSNP36 site is the 20394748 th nucleotide on the 11 th chromosome. The CaSNP37 site is the 83162468 th nucleotide on the 11 th chromosome. The CaSNP38 site is the 204038344 th nucleotide on the 11 th chromosome. The CaSNP39 site is the 40630040 th nucleotide on the 12 th chromosome. The CaSNP40 site is the 157503920 th nucleotide on the 12 th chromosome.

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. 32. 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.

In the primer combination, the primer set 1 may be composed of a forward primer 01F1 shown in the 22 nd to 51 th positions from the 5 'end of SEQ ID NO. 1, a forward primer 01F2 shown in the 22 nd to 53 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 positions 22 to 42 from the 5 'end of SEQ ID NO. 4, a forward primer 02F2 shown in positions 22 to 43 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 40 from the 5 'end of SEQ ID NO. 7, a forward primer 03F2 shown in positions 22 to 39 from the 5' end of SEQ ID NO. 8, and a reverse primer 03R shown in SEQ ID NO. 9. The primer set 4 may be composed of a forward primer 04F1 shown in positions 22 to 48 from the 5 'terminus of SEQ ID NO. 10, a forward primer 04F2 shown in positions 22 to 49 from the 5' terminus 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 51 from the 5 'end of SEQ ID NO. 13, a forward primer 05F2 shown in positions 22 to 52 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 45 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 43 th positions from the 5 'end of SEQ ID NO. 19, a forward primer 07F2 shown in 22 th to 42 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 48 th positions from the 5 'end of SEQ ID NO. 22, a forward primer 08F2 shown in the 22 nd to 50 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 49 from the 5 'terminus of SEQ ID NO. 25, the forward primer 09F2 shown in positions 22 to 49 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 46 from the 5 'end of SEQ ID NO. 28, a forward primer 10F2 shown in positions 22 to 44 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 39 from the 5 'terminus of SEQ ID NO. 31, a forward primer 11F2 shown in positions 22 to 40 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 positions 22 to 48 from the 5 'end of SEQ ID NO. 34, a forward primer 12F2 shown in positions 22 to 47 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 'terminus of SEQ ID NO. 37, a forward primer 13F2 shown in positions 22 to 45 from the 5' terminus 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 the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 40, a forward primer 14F2 shown in the 22 nd to 46 th positions from the 5' end of SEQ ID NO. 41, and a reverse primer 14R shown in the SEQ ID NO. 42. The primer set 15 may be composed of a forward primer 15F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 43, a forward primer 15F2 shown in the 22 nd to 47 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 the 22 nd to 50 th positions from the 5 'end of SEQ ID NO. 46, a forward primer 16F2 shown in the 22 nd to 48 th positions from the 5' end of SEQ ID NO. 47, and a reverse primer 16R shown in SEQ ID NO. 32. The primer set 17 may be composed of a forward primer 17F1 shown in positions 22 to 46 from the 5 'end of SEQ ID NO. 49, a forward primer 17F2 shown in positions 22 to 45 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 positions 22 to 46 from the 5 'end of SEQ ID NO. 52, a forward primer 18F2 shown in positions 22 to 44 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 51 from the 5 'end of SEQ ID NO. 55, a forward primer 19F2 shown in positions 22 to 49 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 41 from the 5 'end of SEQ ID NO. 58, a forward primer 20F2 shown in positions 22 to 42 from the 5' end 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 52 from the 5 'end of SEQ ID NO. 64, a forward primer 22F2 shown in positions 22 to 54 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 44 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 48 from the 5 'end of SEQ ID NO. 70, a forward primer 24F2 shown in positions 22 to 49 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 positions 22 to 45 from the 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown in positions 22 to 46 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 46 from the 5 'end of SEQ ID NO. 76, a forward primer 26F2 shown in positions 22 to 45 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 49 from the 5 'end of SEQ ID NO. 79, a forward primer 27F2 shown in positions 22 to 48 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 consist of a forward primer 28F1 shown in positions 22 to 49 from the 5 'end of SEQ ID NO. 82, a forward primer 28F2 shown in positions 22 to 48 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 49 from the 5 'end of SEQ ID NO. 85, a forward primer 29F2 shown in positions 22 to 51 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 46 from the 5 'end of SEQ ID NO. 88, a forward primer 30F2 shown in positions 22 to 45 from the 5' end of SEQ ID NO. 89, and a reverse primer 30R shown in SEQ ID NO. 90. The primer set 31 is composed of a forward primer 31F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown in 22 th to 47 th positions 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 46 from the 5 'end of SEQ ID NO. 94, a forward primer 32F2 shown in positions 22 to 43 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 47 from the 5 'terminus of SEQ ID NO. 97, a forward primer 33F2 shown in positions 22 to 48 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 positions 22 to 39 from the 5 'end of SEQ ID NO. 100, a forward primer 34F2 shown in positions 22 to 41 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 43 from the 5 'end of SEQ ID NO. 103, a forward primer 35F2 shown in positions 22 to 44 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 54 from the 5 'end of SEQ ID NO 106, a forward primer 36F2 shown in positions 22 to 52 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 48 from the 5 'terminus of SEQ ID NO. 109, a forward primer 37F2 shown in positions 22 to 47 from the 5' terminus 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 50 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 22 th to 48 th positions from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown in 22 th to 48 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 may be composed of a forward primer 40F1 shown in positions 22 to 44 from the 5 'end of SEQ ID NO. 118, a forward primer 40F2 shown in positions 22 to 43 from the 5' end of SEQ ID NO. 119, and a reverse primer 40R shown in SEQ ID NO. 120.

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 40.

As described above, the nucleotide sequence shown in the 1 st to 21 st positions from the 5' end of SEQ ID NO. 1 is a fluorescent tag sequence (i.e., FAM fluorescent tag sequence), and the fluorescent signal is blue in color. The nucleotide sequence shown in the 1 st to 21 st positions from the 5' end of SEQ ID NO. 2 is also a fluorescent tag sequence (namely, HEX fluorescent tag sequence), and the fluorescent signal is red in particular.

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 286 pepper varieties; x4) identifying the authenticity of 286 pepper varieties.

The invention also protects the application of any primer combination, which can be any one of x1) to x 4): x1) preparing a kit for identifying 286 pepper varieties; x2) preparing a kit for identifying the authenticity of 286 pepper varieties; x3) identifying 286 pepper varieties; x4) identifying the authenticity of 286 pepper varieties.

The invention also provides a method for identifying the variety of 286 capsicum to which the capsicum to be detected belongs, which comprises the following steps: respectively detecting genotypes of the pepper to be detected and 286 pepper varieties based on the 40 SNP sites, and then judging as follows: if the genotypes of the pepper to be detected based on the 40 SNP sites are completely consistent with the genotypes of a certain variety of 286 pepper varieties based on the 40 SNP sites, the pepper to be detected and the pepper variety belong to the same variety; and if the genotypes of the pepper to be detected based on the 40 SNP sites are different from the genotypes of all varieties in the 286 pepper varieties based on the 40 SNP sites, the varieties of the pepper to be detected are different from those of the 286 pepper varieties.

In the above method, the step of detecting the genotypes of the to-be-detected pepper and the 286 pepper varieties based on the 40 SNP sites may be as follows:

(1) respectively taking the genome DNA of the pepper to be detected and the genome DNA of 286 pepper 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 to-be-detected pepper and the 286 pepper varieties based on the 40 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 pepper to be detected and the 286 pepper varieties based on the 40 SNP sites according to the sequencing result obtained in the step (2).

The invention also provides a method for identifying the variety of 286 capsicum to which the capsicum to be detected belongs, which comprises the following steps:

(1) taking the genome DNA of the pepper 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 pepper variety in the standard pepper species population as a template, and respectively adopting the primer groups in any one of the primer combinations to carry out PCR amplification to obtain PCR amplification products; the standard pepper species population consists of 286 pepper species;

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

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 286 above pepper varieties can be LW1-1, LW1-2, Wangtianhong No. three, LW1-4, LW1-5, LW1-6, LW1-7, LW1-8, LW1-9, LW1-10, LW1-11, LW1-12, LW1-13, LW1-14, LW1-15, LW1-16, LW1-17, LW1-18, LW1-19, LW1-20, LW1-21, LW1-22, L1-16, LW1-17, LW1-18, LW1-19, LW1-20, LW 539 1-21, LW1-22, LW1-23, LW1-24, spike, green early growth, Xiang La Qiu Xiang, Hongbao No. 1, SV8233HD, Hongfeng specially selected thick beef cattle horn, Yang jiao No. 2, Donghuang No. 1, Zanthoxylum piperitum No. 1, LW1-25, HNX14470065, LW1-26, LW1-27, LW1-28, LW1-29, LW1-30, LW1-31, LW1-32, LW1-33, LW1-34, LW1-35, high-yield ten-size, top quality line pepper, Lansheng Sheng, Fulong Jun, Yunan super early season pepper, Zhongbo No. I, Foster 09, DY10986, LW 863, LW 121, LW1-36, Fu Guo 464, 36105-24, Xiang Han Guo 27-27, Fu Guo 4642, Fu Si Tu-33, Wo-33, LW-33, Wo Fu-33, Wo 4641, LW-33, LW 6-33, LW mille-33, LW Hao 3, LW 4641, LW-33, LW 3, LW 6-33, LW 3, LW1-43, LW1-44, Luxing No. 1, chocolate sweet pepper, Guofu 210, LW1-45, LW1-46, Guofu 316, Guofu 903, Guofu 413, Guofu 910, Shenghan 750, Shenghan 748, G25, DHF1, A11-11, Walter, Jinli 601, HL4, Ji research No. 6, G17, Haifeng 1055, Jingu, 10-71, Foster 960, A11-147, A11-13, and fundamentally

12-03, G16, Haifeng 1022, all

12-07, 12-28, 329, Haifeng 0854, Yangtai pepper No. 11, Hongfeng big long pepper, 8-98, Zungzheng, Zaishen, HL7, Jixing big sweet pepper, CR fragrance, Nongyoujiufa, No. 1, Jinyi big sweet pepper

Dragon flying, G26, Chinese red pepper 105, Chinese red pepper 0808, 217, azure 169, Ji ground No. 12, 11-19, Tianxi jade, Aoyxue 119, Rongyang rich, Huangshuai, Huanglong, G21, A11-46, Guirenxiang, Youmei thin-skin pickled pepper, Foster 5110, ANS #4, Zhuang pepper 2005, Jinjiao eighth, Aodan 218, Jinjiao 2313, Xiang La 129, Qiang Feng 7301, ANS #9, 364, LW1-47, A11-512, improved Hua crown, G19, gold screw, Jufeng screw No. 5, A11-165, 732, G18, Hongbao 202, A11-104, Xiangfei linear pepper, Xiang La 7, root-bark pepper, Yongzhuang 3538, Jufeng screw No. 5, A11-165, G18, Hongbao 202, A

12-05, 405, century red, 2011-A27, Fuxing No. 2, Fushun 23, ATX-67, Shenjian, Shenglong, 261, Zb1218, LJ06020, Zhuang jiao 2008, Top-quality big fruit 201, Ojin, Guangshi 1009, Hualv HL-13, ANS #2, Forster 289, 9562 dried pepper, 1003, super 158, Youyang 1048, WSP-2320, 2011-A19, Youyang No. 7, Jinshan big tip pepper, LJ103, Zb1212, Jinhui 11-F, Jinhui 22, 165, L04, Youyang No. 8, Hangjiao, 12T-272, ATX-3-IMP, 11-G13, Sanhao No. 6021, Jinhui-F, Zb1208, L02, MS-004, Jinhui 1214, super crown, Zb, Zao, Aoyudong pepper, Aoqiong dry pepper, No. 2322, No. 2325, Woo No. 2, WSP-959, Woo No. 11-Woo No. 11, Woodfish green Zb No. 11, Woodfish green pepper, WSO-1202, Woodfish stick No. 8, Woodfish stick No. 2, Woodfish stick, Jufeng green arrow, LJ104, Zhongla No. 1, Jiaolong No. 7, Zb1203, 221, SPE-104, LJ06002, Jingte 2018, Zb2, LJ102, Sucai pepper No. 1, SPE-102, Zb1222, 11-G35, DTSP-104, hybrid F1, Zb122B, Xifeng 601, MS-002, HL12, Zb1211, Hancheng Tianjiao, Zb1205, Liangjian, Zb1206, 2010-11, Zb1209, Tiannu 004, DY 4, L03, Jinchang peppery, Foster 1135, Sy10-314, Jinguan, WSP-2318, 1001, Lexing 601, Yumeiren, Su star, ATX02 Wang 4, Jinla Qingguan No. 8292, LW 5-48, Jinhuiton 38730, A03, L05, L583-1118, Mianmein No. 3, Shuangwu Wo No. 3, Zb No. 11, Zb No. 111, No. 11-K No. 11, No., 4192. Hangzhou pepper II and X11-6.

The invention establishes the DNA fingerprint database for identifying the authenticity of the pepper variety based on the allele competitive specificity PCR method, can be used for carrying out early identification on the pepper 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 pepper germplasm resources and new variety protection. The method provided by the invention can be used for identifying unknown pepper varieties and also can be used for identifying the authenticity of known varieties. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect. The invention has important application value.

Drawings

FIG. 1 shows the SNP typing effect of 40 primer sets in a part of pepper varieties tested.

FIG. 2 is a cluster plot of 286 pepper species tested on 40 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 among 286 pepper 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 obtaining of primer combination for authenticating the genuineness of Capsicum species

Discovery of one or 40 SNP sites

The method is based on 30 parts of re-sequencing data of pepper representative resources to obtain 40 SNP loci. The 30 parts of pepper are rich in resource types, cover the linear pepper (13 parts), the horn pepper (7 parts), the screw pepper (4 parts) and the square pepper (6 parts), basically comprise the main ecological type of the pepper, have higher genetic diversity in the aspects of agronomic characters such as growth period, fruit shape, fruit color, quality and the like, 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 40 SNP sites is detailed in columns 1 to 4 of Table 1. Wherein the position of the SNP site on the chromosome is determined based on … reference genome sequence alignment (download address: ftp:// ftp. solgenomics. net/genes/Capsicum _ annuum).

TABLE 1.40 basic information of SNP sites

Secondly, obtaining a primer combination for identifying the authenticity of the pepper variety

According to the 40 SNP loci found 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 pepper variety by using an allele competitive specific PCR method is developed.

The primer combination consisted of 40 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 respective primers in the 40 primer sets are shown in column 4 of Table 2.

TABLE 2.40 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

286 pepper species tested were randomly selected and tested for validity against the primer combination developed in example 1.

The basic information of 286 pepper varieties tested is shown in table 3. The 286 tested pepper varieties are common fine varieties or partially foreign introduced varieties.

Table 3.286 basic information on pepper varieties tested

1. Acquisition of genomic DNA of test Capsicum species

The genomic DNA of the leaves (mixed leaves of 30 seeds) of 286 tested pepper varieties is extracted by a CTAB method respectively to obtain the genomic DNA of the tested pepper varieties.

The quality and concentration of the genome DNA of the pepper variety to be tested meet the PCR requirement, and the standard of 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 pepper variety is 10-30 ng/. mu.L.

2. And respectively adopting the genome DNA of 286 tested pepper varieties as templates and respectively adopting 40 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 ℃, the fluorescence value is read through FAM and HEX light beam scanning of a microplate reader (the reading value is observed when the FAM fluorescent label sequence is at 485nm of exciting light and 520nm of emitting light, the reading value is observed when the HEX fluorescent label sequence is at 528nm of exciting light and 560nm of emitting light), and the genotype of 286 tested pepper varieties based on each SNP site is judged according to the color of the fluorescence signal. The specific judgment principle is as follows: if a certain test pepper variety shows a blue fluorescent signal based on a certain SNP site, the test pepper variety is homozygotic based on the genotype of the SNP site, namely the complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP site and contains F1 in the name; if a certain test pepper variety shows a red fluorescent signal based on a certain SNP locus, the test pepper variety is homozygotic based on the genotype of the SNP locus, namely the complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP locus and contains F2 in the name; when a certain test pepper variety shows a green fluorescent signal based on a certain SNP site, the test pepper 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 amplifying the SNP site and containing" F1 "in the name", and the other base is "a complementary base of the 1 st base at the 3' end of the primer amplifying the SNP site and containing" 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 results show that each primer group can obtain good typing effect in the tested pepper varieties.

4. Cluster analysis

And (3) performing clustering analysis on the 286 test pepper varieties by using MiniMarker and MEGA7 software according to the genotypes of the 286 test pepper varieties based on 40 SNP loci.

The cluster map of 286 tested pepper varieties established on the 40 primer sets is shown in fig. 2. The results show that 40 primer sets can completely distinguish 286 tested pepper species in table 3. Therefore, the primer combination developed in the example 1 can be applied to the construction of the DNA fingerprint database of the pepper variety and the identification of variety authenticity.

5. Evaluation of efficiency

The variety authenticity identification can reduce the workload by adopting a sequential analysis mode. The inventors of the present invention compared the relationship between the number of SNP markers (i.e., the number of primer sets) and the discrimination rate for discriminating 286 test pepper varieties.

The experimental result shows (figure 3), the discrimination rate of 40 primer groups (namely 40 SNP markers) in 286 tested pepper varieties reaches 100%.

Embodiment 3, method for establishing and detecting to which variety of 286 capsicums in embodiment 2 the capsicum to be detected belongs

Method for establishing and detecting variety of 286 capsicums in example 2 to which capsicum to be detected belongs

1. Obtaining genomic DNA of pepper to be tested

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

2. And (3) performing PCR amplification by respectively adopting 40 primer groups by taking the genome DNA of the pepper to be detected as a template 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. After the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading value is observed when the FAM fluorescent label sequence is at 485nm of exciting light and at 520nm of emitting light, reading value is observed when the HEX fluorescent label sequence is at 528nm of exciting light and at 560nm of emitting light), and judging the genotype of the pepper to be detected based on each SNP site 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 genotypes of the 40 SNP sites of the hot pepper to be detected are completely consistent with the genotypes of the corresponding SNP sites of a certain variety of 286 hot pepper varieties, the hot pepper to be detected and the hot pepper variety belong to the same variety; if the genotypes of the pepper to be detected based on the 40 SNP sites are different from the genotypes of the corresponding SNP sites of the 286 pepper varieties, the pepper to be detected is different from the varieties of the 286 pepper varieties to be detected.

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

The varieties of the hot pepper to be detected 1-5 are LJ1118, Su pepper No. 14, 4192, Hangzhou pepper No. two and X11-6 in sequence. The leaves of the hot pepper to be tested 1-5 are all taken from the test base of the vegetable research center of agriculture and forestry academy of sciences of Beijing.

1. And (3) obtaining the genotypes of the hot pepper to be detected 1-5 based on 40 SNP sites according to the method 1-3 in the step one.

2. And comparing the genotypes of the pepper to be detected 1-5 based on 40 SNP sites with the genotypes of 286 pepper varieties based on 40 SNP sites.

The result shows that the genotype of the pepper 1 to be detected based on 40 SNP sites is completely consistent with the genotype of the SNP site corresponding to the LJ1118, and the pepper 1 to be detected belongs to the LJ 1118; the genotype of the pepper 2 to be detected based on 40 SNP loci is completely consistent with the genotype of the corresponding SNP locus of Su pepper No. 14, and the pepper 2 to be detected belongs to Su pepper No. 14; the genotype of the pepper 3 to be detected based on the 40 SNP sites is completely consistent with the genotype of the corresponding SNP site 4192, and the pepper 3 to be detected belongs to 4192; the genotype of the pepper 4 to be detected based on the 40 SNP loci is completely consistent with the genotype of the corresponding SNP locus of Hangzhou pepper II, and the pepper 4 to be detected belongs to Hangzhou pepper II; the genotype of the pepper 5 to be detected based on the 40 SNP sites is completely consistent with the genotype of the corresponding SNP site X11-6, and the pepper 5 to be detected belongs to X11-6. 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> method for identifying authenticity of pepper variety

<160>120

<170> PatentIn version 3.5

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<213> Artificial sequence

<400> 58

gaaggtgacc aagttcatgc tgacggtttg acggaccgtc g 41

<210> 59

<211> 42

<212> DNA

<213> Artificial sequence

<400> 59

gaaggtcgga gtcaacggat tggacggttt gacggaccgt ca 42

<210> 60

<211> 22

<212> DNA

<213> Artificial sequence

<400> 60

cttgggagac ggtccgttgc ta 22

<210> 61

<211> 43

<212> DNA

<213> Artificial sequence

<400> 61

gaaggtgacc aagttcatgc ttggtctaat cctagccaac ccg 43

<210> 62

<211> 44

<212> DNA

<213> Artificial sequence

<400> 62

gaaggtcgga gtcaacggat tgtggtctaa tcctagccaa ccca 44

<210> 63

<211> 24

<212> DNA

<213> Artificial sequence

<400> 63

cgtagtcttg acggtcagtc ggaa 24

<210> 64

<211> 52

<212> DNA

<213> Artificial sequence

<400> 64

gaaggtgacc aagttcatgc tcaaacaatt aaaactgtaa aagttacata cc 52

<210> 65

<211> 54

<212> DNA

<213> Artificial sequence

<400> 65

gaaggtcgga gtcaacggat taacaaacaa ttaaaactgt aaaagttaca taca 54

<210> 66

<211> 27

<212> DNA

<213> Artificial sequence

<400> 66

gggtatcaaa agtgtaattg ttgtggg 27

<210> 67

<211> 46

<212> DNA

<213> Artificial sequence

<400> 67

gaaggtgacc aagttcatgc tacctagtgg ttctatgcag gagtta 46

<210> 68

<211> 44

<212> DNA

<213> Artificial sequence

<400> 68

gaaggtcgga gtcaacggat tctagtggtt ctatgcagga gttc 44

<210> 69

<211>30

<212> DNA

<213> Artificial sequence

<400> 69

cactttttcc cacttccaag taggaatatt 30 <210> 70

<211>48

<212> DNA

<213> Artificial sequence

<400> 70

gaaggtgacc aagttcatgc tgtgatgttt atgtattgct taatcccg 48

<210> 71

<211>49

<212> DNA

<213> Artificial sequence

<400> 71

gaaggtcgga gtcaacggat tcgtgatgtt tatgtattgc ttaatccct 49

<210> 72

<211>29

<212> DNA

<213> Artificial sequence

<400> 72

ccacataagg ggtcacataa gataaagta 29

<210> 73

<211>45

<212> DNA

<213> Artificial sequence

<400> 73

gaaggtgacc aagttcatgc tggaagcaaa gaggtggttg aatac 45

<210> 74

<211>46

<212> DNA

<213> Artificial sequence

<400> 74

gaaggtcgga gtcaacggat tgggaagcaa agaggtggtt gaatat 46

<210> 75

<211>30

<212> DNA

<213> Artificial sequence

<400> 75

gggtaattca tcccatgttg tgattgaatt 30

<210> 76

<211>46

<212> DNA

<213> Artificial sequence

<400> 76

gaaggtgacc aagttcatgc tcgatgttgt acctgttcca ttgtga 46

<210> 77

<211>45

<212> DNA

<213> Artificial sequence

<400> 77

gaaggtcgga gtcaacggat tgatgttgta cctgttccat tgtgg 45

<210> 78

<211>28

<212> DNA

<213> Artificial sequence

<400> 78

ccgcgattag ccagtattgt atcaaaaa 28

<210> 79

<211>49

<212> DNA

<213> Artificial sequence

<400> 79

gaaggtgacc aagttcatgc tctcacaatt tcggtttcat caacaactt 49

<210> 80

<211>48

<212> DNA

<213> Artificial sequence

<400> 80

gaaggtcgga gtcaacggat ttcacaattt cggtttcatc aacaactc 48

<210> 81

<211>25

<212> DNA

<213> Artificial sequence

<400> 81

tggggggaag aatccaaaag aggta 25

<210> 82

<211>49

<212> DNA

<213> Artificial sequence

<400> 82

gaaggtgacc aagttcatgc tcttggacat aatagctgaa ttgcctaaa 49

<210> 83

<211>48

<212> DNA

<213> Artificial sequence

<400> 83

gaaggtcgga gtcaacggat tttggacata atagctgaat tgcctaag 48 <210> 84

<211>28

<212> DNA

<213> Artificial sequence

<400> 84

cataagcagc ataatccatc agcttcaa 28

<210> 85

<211> 49

<212> DNA

<213> Artificial sequence

<400> 85

gaaggtgacc aagttcatgc ttgccattat acctagatgg ttttatgag 49

<210> 86

<211> 51

<212> DNA

<213> Artificial sequence

<400> 86

gaaggtcgga gtcaacggat taatgccatt atacctagat ggttttatga a 51

<210> 87

<211>30

<212> DNA

<213> Artificial sequence

<400> 87

ggaagaattt ctttaattcc tccgaagcat 30

<210> 88

<211>46

<212> DNA

<213> Artificial sequence

<400> 88

gaaggtgacc aagttcatgc tggttttcat ggtgggctca aatgtt 46

<210> 89

<211>45

<212> DNA

<213> Artificial sequence

<400> 89

gaaggtcgga gtcaacggat tgttttcatg gtgggctcaa atgtc 45

<210> 90

<211>29

<212> DNA

<213> Artificial sequence

<400> 90

gggaagcttg gttgattttt gaccaaaat 29

<210> 91

<211>48

<212> DNA

<213> Artificial sequence

<400> 91

gaaggtgacc aagttcatgc taggagagag aaaattgata ccgacaat 48

<210> 92

<211> 47

<212> DNA

<213> Artificial sequence

<400> 92

gaaggtcgga gtcaacggat tggagagaga aaattgatac cgacaac 47

<210> 93

<211>23

<212> DNA

<213> Artificial sequence

<400> 93

cggtgatgtt ctcccttgcg ctt 23

<210> 94

<211> 46

<212> DNA

<213> Artificial sequence

<400> 94

gaaggtgacc aagttcatgc tttagctggt caaactagcg aaacga 46

<210> 95

<211> 43

<212> DNA

<213> Artificial sequence

<400> 95

gaaggtcgga gtcaacggat tgctggtcaa actagcgaaa cgg 43

<210> 96

<211>27

<212> DNA

<213> Artificial sequence

<400> 96

cgatttggcc taaaaatgcc ccttcat 27

<210>97

<211>47

<212>DNA

<213>Artificial sequence

<400>97

gaaggtgacc aagttcatgc tggtaaggca taaaaccaat tttggcg 47

<210>98

<211>48

<212>DNA

<213>Artificial sequence

<400>98

gaaggtcgga gtcaacggat ttggtaaggc ataaaaccaa ttttggca 48

<210>99

<211>26

<212>DNA

<213>Artificial sequence

<400>99

catcatccag tattggtggt ggtcaa 26

<210>100

<211>39

<212>DNA

<213>Artificial sequence

<400>100

gaaggtgacc aagttcatgc tggacgcaag gccaggcag 39

<210>101

<211>41

<212>DNA

<213>Artificial sequence

<400>101

gaaggtcgga gtcaacggat tgaggacgca aggccaggca a 41

<210>102

<211>32

<212>DNA

<213>Artificial sequence

<400>102

ggtccaggtc ggaaaatttt tgtaaattta aa 32

<210>103

<211>43

<212>DNA

<213>Artificial sequence

<400>103

gaaggtgacc aagttcatgc tgcctcctag tgttgtccgt ttg 43

<210>104

<211>44

<212>DNA

<213>Artificial sequence

<400>104

gaaggtcgga gtcaacggat tagcctccta gtgttgtccg ttta 44

<210>105

<211>27

<212>DNA

<213>Artificial sequence

<400>105

ccgttttggt tgtttgacca cccaaat 27

<210>106

<211>54

<212>DNA

<213>Artificial sequence

<400>106

gaaggtgacc aagttcatgc tatttgagat atggatcttt ctagattgat aatt 54

<210>107

<211>52

<212>DNA

<213>Artificial sequence

<400>107

gaaggtcgga gtcaacggat tttgagatat ggatctttct agattgataa tg 52

<210>108

<211>31

<212>DNA

<213>Artificial sequence

<400>108

tttaacttct ctttctcaat ctgttgagca t 31

<210>109

<211>48

<212>DNA

<213>Artificial sequence

<400>109

gaaggtgacc aagttcatgc tctctcaatg acaatccatt cgatagat 48

<210>110

<211>47

<212>DNA

<213>Artificial sequence

<400>110

gaaggtcgga gtcaacggat ttctcaatga caatccattc gatagac 47

<210>111

<211>24

<212>DNA

<213>Artificial sequence

<400>111

catcaatggc ccgttggtag gcta 24

<210>112

<211>51

<212>DNA

<213>Artificial sequence

<400>112

gaaggtgacc aagttcatgc tggaaattag taatttttta gtagtgaccc a 51

<210>113

<211>50

<212>DNA

<213>Artificial sequence

<400>113

gaaggtcgga gtcaacggat tgaaattagt aattttttag tagtgacccg 50

<210>114

<211>28

<212>DNA

<213>Artificial sequence

<400>114

tgacagggac aaagttgaaa agggatat 28

<210>115

<211>48

<212>DNA

<213>Artificial sequence

<400>115

gaaggtgacc aagttcatgc tcattccgat gaaaattctg aatcctca 48

<210>116

<211>48

<212>DNA

<213>Artificial sequence

<400>116

gaaggtcgga gtcaacggat tcattccgat gaaaattctg aatcctct 48

<210>117

<211>28

<212>DNA

<213>Artificial sequence

<400>117

aaggtgcttc cccaattatt gttgctta 28

<210>118

<211>44

<212>DNA

<213>Artificial sequence

<400>118

gaaggtgacc aagttcatgc tgttgcgatt gctggagacc gaaa 44

<210>119

<211>43

<212>DNA

<213>Artificial sequence

<400>119

gaaggtcgga gtcaacggat tttgcgattg ctggagaccg aag 43

<210>120

<211>26

<212>DNA

<213>Artificial sequence

<400>120

ccgcagcacc caaataatga ccattt 26

Claims

1. A primer combination comprising

Primer set 1 for amplifying CaSNP01 site,

A primer group 2 for amplifying the CaSNP02 site,

A primer group 3 for amplifying the CaSNP03 locus,

A primer group 4 for amplifying the CaSNP04 locus,

A primer group 5 for amplifying the CaSNP05 site,

A primer group 6 for amplifying the CaSNP06 site,

A primer group 7 for amplifying the CaSNP07 site,

A primer group 8 for amplifying the CaSNP08 locus,

A primer group 9 for amplifying the CaSNP09 site,

A primer group 10 for amplifying the CaSNP10 site,

A primer group 11 for amplifying the CaSNP11 site,

A primer group 12 for amplifying the CaSNP12 site,

A primer group 13 for amplifying the CaSNP13 site,

A primer group 14 for amplifying the CaSNP14 site,

A primer group 15 for amplifying the CaSNP15 site,

A primer group 16 for amplifying the CaSNP16 site,

A primer group 17 for amplifying the CaSNP17 site,

A primer group 18 for amplifying the CaSNP18 site,

A primer group 19 for amplifying the CaSNP19 site,

A primer group 20 for amplifying the CaSNP20 site,

A primer group 21 for amplifying the CaSNP21 site,

A primer group 22 for amplifying the CaSNP22 site,

A primer group 23 for amplifying the CaSNP23 site,

A primer group 24 for amplifying the CaSNP24 site,

A primer group 25 for amplifying the CaSNP25 site,

A primer group 26 for amplifying the CaSNP26 site,

A primer group 27 for amplifying the CaSNP27 site,

A primer group 28 for amplifying the CaSNP28 site,

A primer group 29 for amplifying the CaSNP29 site,

A primer group 30 for amplifying the CaSNP30 site,

A primer group 31 for amplifying the CaSNP31 site,

A primer group 32 for amplifying the CaSNP32 site,

A primer group 33 for amplifying the CaSNP33 site,

A primer group 34 for amplifying the CaSNP34 site,

A primer group 35 for amplifying the CaSNP35 site,

A primer group 36 for amplifying the CaSNP36 site,

A primer group 37 for amplifying the CaSNP37 site,

A primer group 38 for amplifying the CaSNP38 site,

Primer set 39 for amplifying CaSNP39 site and method for preparing the same

A primer set 40 for amplifying the CaSNP40 site;

CaSNP01 site-CaSNP 40 site is 40 SNP sites of the pepper genome;

the CaSNP01 site is the 46784687 th nucleotide on the 01 st chromosome;

the CaSNP02 site is the 64927233 th nucleotide on the 01 st chromosome;

the CaSNP03 site is the 128314154 th nucleotide on the 01 st chromosome;

the CaSNP04 site is the 293847360 th nucleotide on the 01 st chromosome;

the CaSNP05 site is the 75886898 th nucleotide on the 02 th chromosome;

the CaSNP06 site is the 93209608 th nucleotide on the 02 th chromosome;

the CaSNP07 site is the 140042546 th nucleotide on the 02 th chromosome;

the CaSNP08 site is the 158395138 th nucleotide on the 02 th chromosome;

the CaSNP09 site is the 99284562 th nucleotide on the 03 th chromosome;

the CaSNP10 site is the 251295225 th nucleotide on the 03 th chromosome;

the CaSNP11 site is the 1205997 th nucleotide on the 04 th chromosome;

the CaSNP12 site is the 12579985 th nucleotide on the 04 th chromosome;

the CaSNP13 site is the 30522616 th nucleotide on the 04 th chromosome;

the CaSNP14 site is the 42814994 th nucleotide on the 04 th chromosome;

the CaSNP15 site is the 29437076 th nucleotide on the 05 th chromosome;

the CaSNP16 site is the 85520600 th nucleotide on the 05 th chromosome;

the CaSNP17 site is the 198838826 th nucleotide on the 05 th chromosome;

the CaSNP18 site is the 388508 th nucleotide on the 06 th chromosome;

the CaSNP19 site is the 58719488 th nucleotide on the 06 th chromosome;

the CaSNP20 site is the 95206292 th nucleotide on the 06 th chromosome;

the CaSNP21 site is the 101332262 th nucleotide on the number 07 chromosome;

the CaSNP22 site is the 110735031 th nucleotide on the number 07 chromosome;

the CaSNP23 site is the 204576291 th nucleotide on the number 07 chromosome;

the CaSNP24 site is the 119957941 th nucleotide on the 08 th chromosome;

the CaSNP25 site is the 122468853 th nucleotide on the 08 th chromosome;

the CaSNP26 site is the 126315482 th nucleotide on the 08 th chromosome;

the CaSNP27 site is the 147458068 th nucleotide on the 08 th chromosome;

the CaSNP28 site is the 159969 th nucleotide on the No. 09 chromosome;

the CaSNP29 site is the 151594116 th nucleotide on the No. 09 chromosome;

the CaSNP30 site is the 236428132 th nucleotide on the No. 09 chromosome;

the CaSNP31 site is the 494205 th nucleotide on the 10 th chromosome;

the CaSNP32 site is the 1684877 th nucleotide on the 10 th chromosome;

the CaSNP33 site is the 49575961 th nucleotide on the 10 th chromosome;

the CaSNP34 site is the 133308217 th nucleotide on the 10 th chromosome;

the CaSNP35 site is the 7376328 th nucleotide on the 11 th chromosome;

the CaSNP36 site is the 20394748 th nucleotide on the 11 th chromosome;

the CaSNP37 site is the 83162468 th nucleotide on the 11 th chromosome;

the CaSNP38 site is the 204038344 th nucleotide on the 11 th chromosome;

the CaSNP39 site is the 40630040 th nucleotide on the 12 th chromosome;

the CaSNP40 locus is the 157503920 th nucleotide on chromosome 12.

2. The primer combination of claim 1, 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. 32;

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.

3. The primer combination of claim 1, wherein:

the primer group 1 consists of a forward primer 01F1 shown in 22 th to 51 th positions from the 5 'end of SEQ ID NO. 1, a forward primer 01F2 shown in 22 th to 53 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 42 th positions from 5 'end of SEQ ID NO. 4, a forward primer 02F2 shown in 22 th to 43 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 40 th positions from the 5 'end of SEQ ID NO. 7, a forward primer 03F2 shown in the 22 nd to 39 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 48 th positions from the 5 'end of SEQ ID NO. 10, a forward primer 04F2 shown in 22 th to 49 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 the 22 nd to 51 th positions from the 5 'end of SEQ ID NO. 13, a forward primer 05F2 shown in the 22 nd to 52 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 45 th positions from 5 'end of SEQ ID NO. 16, a forward primer 06F2 shown in 22 th to 46 th positions from 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 43 th positions from the 5 'end of SEQ ID NO. 19, a forward primer 07F2 shown in 22 th to 42 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 48 th positions from the 5 'end of SEQ ID NO. 22, a forward primer 08F2 shown in 22 th to 50 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 49 th positions from the 5 'end of SEQ ID NO. 25, a forward primer 09F2 shown in the 22 nd to 49 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 46 th positions from the 5 'end of SEQ ID NO. 28, a forward primer 10F2 shown in 22 th to 44 th positions from the 5' end of SEQ ID NO. 29, and a reverse primer 10R shown in SEQ ID NO. 30;

the primer group 11 consists of a forward primer 11F1 shown in 22 th to 39 th positions from the 5 'end of SEQ ID NO. 31, a forward primer 11F2 shown in 22 th to 40 th positions from the 5' end of SEQ ID NO. 32, and a reverse primer 11R shown in SEQ ID NO. 33;

the primer group 12 consists of a forward primer 12F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 34, a forward primer 12F2 shown in 22 th to 47 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 the 5 'end of SEQ ID NO. 37, a forward primer 13F2 shown in 22 th to 45 th positions from the 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 46 th positions from the 5 'end of SEQ ID NO. 40, a forward primer 14F2 shown in the 22 nd to 46 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 46 th positions from the 5 'end of SEQ ID NO. 43, a forward primer 15F2 shown in the 22 nd to 47 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 50 th positions from the 5 'end of SEQ ID NO. 46, a forward primer 16F2 shown in the 22 nd to 48 th positions from the 5' end of SEQ ID NO. 47, and a reverse primer 16R shown in SEQ ID NO. 32;

the primer set 17 consists of a forward primer 17F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 49, a forward primer 17F2 shown in the 22 nd to 45 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 44 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 51 th positions from the 5 'end of SEQ ID NO. 55, a forward primer 19F2 shown in the 22 nd to 49 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 41 th positions from the 5 'end of SEQ ID NO. 58, a forward primer 20F2 shown in the 22 nd to 42 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 52 th positions from the 5 'end of SEQ ID NO. 64, a forward primer 22F2 shown in the 22 nd to 54 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 44 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 the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 70, a forward primer 24F2 shown in the 22 nd to 49 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 22 th to 45 th positions from 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown in 22 th to 46 th positions from 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 46 th positions from the 5 'end of SEQ ID NO. 76, a forward primer 26F2 shown in the 22 nd to 45 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 49 th positions from the 5 'end of SEQ ID NO. 79, a forward primer 27F2 shown in 22 th to 48 th positions from the 5' end of SEQ ID NO. 80, and a reverse primer 27R shown in SEQ ID NO. 81;

the primer group 28 consists of a forward primer 28F1 shown in the 22 nd to 49 th positions from the 5 'end of SEQ ID NO. 82, a forward primer 28F2 shown in the 22 nd to 48 th positions from the 5' end of 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 in 22 th to 49 th positions from the 5 'end of SEQ ID NO. 85, a forward primer 29F2 shown in 22 th to 51 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 46 th positions from the 5 'end of SEQ ID NO. 88, a forward primer 30F2 shown in 22 th to 45 th positions from the 5' end of SEQ ID NO. 89, and a reverse primer 30R shown in SEQ ID NO. 90;

the primer group 31 consists of a forward primer 31F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown in 22 th to 47 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 the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 94, a forward primer 32F2 shown in the 22 nd to 43 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 47 th positions from 5 'end of SEQ ID NO. 97, a forward primer 33F2 shown in 22 th to 48 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 22 th to 39 th positions from the 5 'end of SEQ ID NO. 100, a forward primer 34F2 shown in 22 th to 41 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 43 th positions from the 5 'end of SEQ ID NO. 103, a forward primer 35F2 shown in the 22 nd to 44 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 54 th positions from the 5 'end of SEQ ID NO. 106, a forward primer 36F2 shown in 22 th to 52 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 48 th positions from the 5 'end of SEQ ID NO. 109, a forward primer 37F2 shown in the 22 nd to 47 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 50 th positions from the 5' end of SEQ ID NO. 113, and a reverse primer 38R shown in SEQ ID NO. 114;

the primer group 39 consists of a forward primer 39F1 shown in 22 th to 48 th positions from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown in 22 th to 48 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 is composed of a forward primer 40F1 shown in the 22 nd to 44 th positions from the 5 'end of SEQ ID NO. 118, a forward primer 40F2 shown in the 22 nd to 43 th positions from the 5' end of SEQ ID NO. 119, and a reverse primer 40R shown in SEQ ID NO. 120.

4. Use of the primer combination of any one of claims 1 to 3, being any one of x1) to x 4): x1) preparing a kit for identifying 286 pepper varieties; x2) preparing a kit for identifying the authenticity of 286 pepper varieties; x3) identifying 286 pepper varieties; x4) identifying the authenticity of 286 pepper varieties;

the 286 pepper varieties are LW-1, LW-2, Wangtianhong No. three, LW-4, LW-5, LW-6, LW-7, LW-8, LW-9, LW-10, LW-11, LW-12, LW-13, LW-14, LW-15, LW-16, LW-17, LW-18, LW-19, LW-20, LW-21, LW-22, LW-23, LW-24, spike, green-early-growing, Xiangchu-qiu, Hongyou No. 1, SV8233, HONGFENGTECHROU, HONGJIAO No. 2, DONGHUANG No. 1, LW-25, HNX14470065, LW-26, LW-27, LW-28, LW-29, LW-30, LW-31, LW-32, LW-33, LW-34, LW-34-24, LW-30, LW-31, LW-13, LW-14, LW-, LW1-35, high-yield ten-grade, fine line pepper, Shengsheng, Fulong, Kawasi Jun, Yuyuan super early pepper, Zhongbo I, Foster 9109, DY10986, YP863, Guoxi 121, Guoxi 308, LW 685 1-36, Jing La No. 4, Guoxi 105, LW1-37, Guoxi 805, Guoxi 901, Guoxi 908 Fu, LW1-38, LW1-39, LW1-40, Golgi tower 109, LW1-41, Guoxi 208, Guoxi 305, Guoxi 115, Guoxi No. 1, LW1-42, LW1-43, LW1-44, Lvxinxing 1, chocolate sweet pepper, LW1-45, LW1-46, LW 36316, Guoxi 903, Guoxi Fu 413, Cold Hei 910, Huoxi 11, DH 11-8, Haoxi 27-48, Haifeng Haifei No. 3, DH 27-8, Haifeng 27, Haifeng 48, Haifeng 27, Haifeng 48, Haifeng 27, DHe, Haifeng 27, A11-13,

12-03, G16, Haifeng 1022,

12-07, 12-28, 329, Haifeng 0854, Yangtai pepper No. 11, Hongfeng big long pepper, 8-98, Zungzheng, Zaishen, HL7, Jixing big sweet pepper, CR fragrance, Nongyoujiufa, No. 1 pepper, Kangkuan, Ka,

Dragon flying, G26, Chinese prickly ash 105, Chinese prickly ash 0808, 217, azure 169, Ji grind No. 12, 11-19, Tianxi jade, Aoyxue 119, Rongyang rich, Huangshuai, Huanglong, G21, A11-46, Guirenxiang, Youmei thin-skin pickled pepper, Fusite 5110, ANS #4, Zhuang pepper 2005, Jinjiao No. eight, Aodan 218, Jinjiao 2313, spicy 129, Qiangfeng 7301, ANS #9, 364, 129, and 129,LW1-47, A11-512, modified Huaguan, G19, Jinju, Jufeng screw No. 5, A11-165, 732, G18, Hongbao 202, A11-104, Xiangfei cayenne pepper, Xiang La jiao No. 7, and,

5. A method for identifying which of the 286 pepper varieties of claim 4 the pepper to be tested belongs to, comprising the steps of: respectively detecting the genotypes of the to-be-detected pepper and 286 pepper varieties based on the 40 SNP sites of claim 1, and then judging as follows: if the genotypes of the pepper to be detected based on the 40 SNP sites are completely consistent with the genotypes of a certain variety of 286 pepper varieties based on the 40 SNP sites, the pepper to be detected and the pepper variety belong to the same variety; and if the genotypes of the pepper to be detected based on the 40 SNP sites are different from the genotypes of all varieties in the 286 pepper varieties based on the 40 SNP sites, the varieties of the pepper to be detected are different from those of the 286 pepper varieties.

6. The method of claim 5, wherein: the steps for detecting the genotypes of the pepper to be detected and the 286 pepper varieties based on 40 SNP loci are as follows:

(1) respectively carrying out PCR amplification by using the genomic DNA of the pepper to be detected and the genomic DNAs of 286 pepper varieties as templates and respectively adopting the primer groups in the primer combination according to claim 2 to obtain PCR amplification products;

7. The method of claim 5, wherein: the steps for detecting the genotypes of the pepper to be detected and the 286 pepper varieties based on 40 SNP loci are as follows:

(1) respectively carrying out PCR amplification by using the genomic DNA of the pepper to be detected and the genomic DNAs of 286 pepper varieties as templates and respectively adopting the primer groups in the primer combination according to claim 3 to obtain PCR amplification products;

8. A method for identifying which of the 286 pepper varieties of claim 4 the pepper to be tested belongs to, comprising the steps of:

(1) performing PCR amplification by respectively adopting the primer groups in the primer combination of claim 2 by taking the genome DNA of the pepper to be detected as a template to obtain PCR amplification products; performing PCR amplification by respectively adopting the primer groups in the primer combination according to claim 2 by taking the genome DNA of each pepper variety in the standard pepper species population as a template to obtain PCR amplification products; the standard pepper species population consists of 286 pepper species;

9. A kit comprising a primer combination according to any one of claims 1 to 3.

10. The use of the kit of claim 9, being x3) or x 4): x3) identifying the 286 pepper varieties of claim 4; x4) identifying the authenticity of the 286 pepper varieties as claimed in claim 4.