CN112877453B

CN112877453B - Method for identifying authenticity of pepper variety

Info

Publication number: CN112877453B
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: 2022-11-22
Anticipated expiration: 2041-01-26
Also published as: CN112877453A

Abstract

The invention discloses a method for identifying the authenticity of pepper varieties. 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, the number of registered pepper varieties is 2464 in China, but the number of 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 an improved variety production base is not strict, the phenomena of stealing breeding, purchasing varieties and homonymy heterogeneity are serious, and the production quality accidents of seeds sometimes occur. 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, according to the requirements of the non-main crop variety registration guide, DNA detection results can be directly submitted if the related traits involved in the variety characteristic description and the variety DUS test report are clearly related 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 invention firstly protects a primer combination which comprises a primer group 1 for amplifying CaSNP01 locus, a primer group 2 for amplifying CaSNP02 locus, a primer group 3 for amplifying CaSNP03 locus, a primer group 4 for amplifying CaSNP04 locus, a primer group 5 for amplifying CaSNP05 locus, a primer group 6 for amplifying CaSNP06 locus, a primer group 7 for amplifying CaSNP07 locus, a primer group 8 for amplifying CaSNP08 locus, a primer group 9 for amplifying CaSNP09 locus, a primer group 10 for amplifying CaSNP10 locus, a primer group 11 for amplifying CaSNP11 locus, a primer group 12 for amplifying CaSNP12 locus, a primer group 13 for amplifying CaSNP13 locus, a primer group 14 for amplifying CaSNP14 locus, a primer group 15 for amplifying Ca15 locus, a primer group 16 for amplifying Ca16 locus, a primer group 17 for amplifying CaSNP17 locus, a primer group 18 for amplifying CaSNP18 locus, a primer group 19 for amplifying CaSNP19 locus, a primer group 20 for amplifying CaSNP 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, a primer group 29 for amplifying a CaSNP29 locus, a primer group 30 for amplifying a CaSNP30 locus, a primer group 31 for amplifying a CaSNP31 locus, a primer group 32 for amplifying a CaSNP32 locus, a primer group 33 for amplifying a CaSNP33 locus, a primer group 34 for amplifying a CaSNP34 locus, a primer group 35 for amplifying a CaSNP35 locus, a primer group 36 for amplifying a Ca36 locus, a primer group 37 for amplifying a CaSNP37 locus, a primer group 38 for amplifying a CaSNP38 locus, a primer group 38 for amplifying a CaSNP39 locus, and a primer group 40 for amplifying a CaSNP39 locus.

The CaSNP01 site-the CaSNP40 site is 40 SNP sites of a pepper genome. The CaSNP01 locus is 46784687 th nucleotide on chromosome 01. The CaSNP02 locus is nucleotide 64927233 on chromosome 01. The CaSNP03 site is 128314154 th nucleotide on chromosome 01. The CaSNP04 site is 293847360 th nucleotide on a No. 01 chromosome. The CaSNP05 site is 75886898 th nucleotide on the 02 th chromosome. The CaSNP06 site is the 93209608 th nucleotide on the 02 th chromosome. The CaSNP07 locus is nucleotide 140042546 on chromosome 02. The CaSNP08 site is the 158395138 th nucleotide on the 02 th chromosome. The CaSNP09 site is the 99284562 th nucleotide on the No. 03 chromosome. The CaSNP10 site is 251295225 th nucleotide on chromosome 03. The CaSNP11 site is 1205997 th nucleotide on the number 04 chromosome. The CaSNP12 site is the 12579985 th nucleotide on the 04 th chromosome. The CaSNP13 locus is a 30522616 th nucleotide on a chromosome 04. The CaSNP14 locus is the 42814994 th nucleotide on the 04 th chromosome. The CaSNP15 site is 29437076 th nucleotide on the 05 th chromosome. The CaSNP16 site is 85520600 th nucleotide on a chromosome 05. The CaSNP17 site is the nucleotide at the 198838826 th site on the chromosome 05. The CaSNP18 site is 388508 th nucleotide on chromosome 06. The CaSNP19 site is 58719488 nucleotide on chromosome 06. The CaSNP20 site is 95206292 th nucleotide on chromosome 06. The CaSNP21 site is the 101332262 nucleotide on the number 07 chromosome. The CaSNP22 site is the 110735031 th nucleotide on the number 07 chromosome. The CaSNP23 site is 204576291 th nucleotide on chromosome number 07. 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 126315482 th nucleotide on the No. 08 chromosome. The CaSNP27 site is 147458068 th nucleotide on the No. 08 chromosome. The CaSNP28 site is nucleotide 159969 on chromosome 09. 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 494205 th nucleotide on chromosome 10. The CaSNP32 site is the 1684877 site nucleotide on the chromosome 10. The CaSNP33 site is the 49575961 th nucleotide on the chromosome 10. The CaSNP34 site is 133308217 th nucleotide on chromosome 10. The CaSNP35 site is 7376328 th nucleotide on chromosome 11. The CaSNP36 site is the 20394748 nucleotide on the 11 th chromosome. The CaSNP37 site is 83162468 th nucleotide on 11 th chromosome. The CaSNP38 site is 204038344 th nucleotide on chromosome 11. The CaSNP39 site is the 40630040 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 by SEQ ID NO. 1, a forward primer 01F2 shown by SEQ ID NO. 2 and a reverse primer 01R shown by 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 by SEQ ID NO. 7, a forward primer 03F2 shown by SEQ ID NO. 8 and a reverse primer 03R shown by 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 group 5 can be composed of a forward primer 05F1 shown by SEQ ID NO. 13, a forward primer 05F2 shown by SEQ ID NO. 14 and a reverse primer 05R shown by SEQ ID NO. 15. The primer group 6 can be composed 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 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 may be composed of a forward primer 09F1 shown by SEQ ID NO. 25, a forward primer 09F2 shown by SEQ ID NO. 26, and a reverse primer 09R shown by SEQ ID NO. 27. The primer group 10 can be composed 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 set 11 can be composed 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 set 12 can be composed 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 set 13 may be composed of a forward primer 13F1 shown by SEQ ID NO. 37, a forward primer 13F2 shown by SEQ ID NO. 38, and a reverse primer 13R shown by SEQ ID NO. 39. The primer set 14 may be composed 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 set 15 can be composed of a forward primer 15F1 shown by SEQ ID NO. 43, a forward primer 15F2 shown by SEQ ID NO. 44, and a reverse primer 15R shown by SEQ ID NO. 45. The primer set 16 may be composed 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 set 17 may be composed 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 set 18 can be composed 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 set 19 can be composed 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 set 20 may be composed 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 set 21 may be composed 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 set 22 may be composed 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 can be composed 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 set 24 may be composed 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 set 25 may be composed 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 set 26 may be composed 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 set 27 may be composed 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 set 28 may be composed of a forward primer 28F1 shown by SEQ ID NO. 82, a forward primer 28F2 shown by SEQ ID NO. 83, and a reverse primer 28R shown by SEQ ID NO. 84. The primer group 29 may 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 be composed 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 set 31 may be composed 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 may be composed 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 set 33 may be composed 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 set 34 can be composed of a forward primer 34F1 shown by SEQ ID NO. 100, a forward primer 34F2 shown by SEQ ID NO. 101, and a reverse primer 34R shown by SEQ ID NO. 102. The primer set 35 may be composed 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 set 36 can be composed 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 set 37 may be composed 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 set 38 can be composed 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 set 39 may be composed 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 set 40 can be composed 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.

In the primer combination, the primer set 1 can 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 represented by positions 22 to 42 from the 5 'end of SEQ ID NO. 4, a forward primer 02F2 represented by positions 22 to 43 from the 5' end of SEQ ID NO. 5, and a reverse primer 02R represented by SEQ ID NO. 6. The primer set 3 may be composed of a forward primer 03F1 represented by SEQ ID NO. 7 from position 22 to 40 from the 5 'terminus, a forward primer 03F2 represented by SEQ ID NO. 8 from position 22 to 39 from the 5' terminus, and a reverse primer 03R represented by 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 'end of SEQ ID NO. 10, a forward primer 04F2 shown in positions 22 to 49 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 by positions 22 to 51 from the 5 'terminus of SEQ ID NO. 13, a forward primer 05F2 shown by positions 22 to 52 from the 5' terminus of SEQ ID NO. 14, and a reverse primer 05R shown by SEQ ID NO. 15. The primer set 6 may be composed of a forward primer 06F1 shown in the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 16, a forward primer 06F2 shown in the 22 nd 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 a forward primer 09F1 represented by SEQ ID NO. 25 from position 22 to 49 from the 5 'terminus, a forward primer 09F2 represented by SEQ ID NO. 26 from position 22 to 49 from the 5' terminus, and a reverse primer 09R represented by SEQ ID NO. 27. The primer set 10 may be composed of a forward primer 10F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 28, a forward primer 10F2 shown in the 22 nd 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 set 11 may be composed of a forward primer 11F1 represented by positions 22 to 39 from the 5 'terminus of SEQ ID NO. 31, a forward primer 11F2 represented by positions 22 to 40 from the 5' terminus of SEQ ID NO. 32, and a reverse primer 11R represented by 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 'end of SEQ ID NO. 37, a forward primer 13F2 shown in positions 22 to 45 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 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 represented by positions 22 to 50 from the 5 'terminus of SEQ ID NO. 46, a forward primer 16F2 represented by positions 22 to 48 from the 5' terminus of SEQ ID NO. 47, and a reverse primer 16R represented by SEQ ID NO. 32. The primer set 17 may be composed 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 the 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 'terminus of SEQ ID NO. 52, a forward primer 18F2 shown in positions 22 to 44 from the 5' terminus 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 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 set 21 may be composed of a forward primer 21F1 represented by positions 22 to 43 from the 5 'terminus of SEQ ID NO. 61, a forward primer 21F2 represented by positions 22 to 44 from the 5' terminus of SEQ ID NO. 62, and a reverse primer 21R represented by SEQ ID NO. 63. The primer set 22 may be composed 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 set 23 may be composed of a forward primer 23F1 shown in the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 67, a forward primer 23F2 shown in the 22 nd 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 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 the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown in the 22 nd to 46 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 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 'terminus of SEQ ID NO. 79, a forward primer 27F2 shown in positions 22 to 48 from the 5' terminus 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 49 from the 5 'terminus of SEQ ID NO. 82, a forward primer 28F2 shown in positions 22 to 48 from the 5' terminus 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 'terminus of SEQ ID NO. 85, a forward primer 29F2 shown in positions 22 to 51 from the 5' terminus 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 represented by positions 22 to 46 from the 5 'terminus of SEQ ID NO. 88, a forward primer 30F2 represented by positions 22 to 45 from the 5' terminus of SEQ ID NO. 89, and a reverse primer 30R represented by SEQ ID NO. 90. The primer group 31 is composed of a forward primer 31F1 shown by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown by 22 th to 47 th positions from the 5' end of SEQ ID NO. 92, and a reverse primer 31R shown by 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 'end of SEQ ID NO. 97, a forward primer 33F2 shown in positions 22 to 48 from the 5' end 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 the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown in the 22 nd to 48 th positions from the 5' end of SEQ ID NO. 116, and a reverse primer 39R shown in the 117 th position of 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" to the primer named as "F2" to the primer named as "R" may be specifically 2.

Any one of the primer combinations described above 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 particular. 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 separately packaging each primer in any one primer group.

The application of the kit also belongs to the protection scope of the invention. The application of the kit can be x 3) or x 4): x 3) identifying 286 pepper varieties; x 4) identifying the authenticity of 286 pepper varieties.

The invention also protects the application of any one of the primer combinations, which can be any one of x 1) to x 4): x 1) preparing a kit for identifying 286 pepper varieties; x 2) preparing a kit for identifying the authenticity of 286 pepper varieties; x 3) identifying 286 pepper varieties; x 4) 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 loci, and then judging as follows: if the genotypes of the 40 SNP sites of the hot pepper to be detected and the 40 SNP sites of a certain variety of the 286 hot pepper to be detected are completely consistent, the hot pepper to be detected and the hot 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 pepper to be detected and 286 pepper varieties based on 40 SNP sites may be as follows:

(1) Respectively taking the genome DNA of the hot pepper to be detected and the genome DNA of 286 hot 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) After the step (1) is finished, detecting a fluorescent signal of a PCR amplification product by using an instrument, and obtaining genotypes of the to-be-detected pepper and 286 pepper varieties based on the 40 SNP sites according to the color of the fluorescent signal.

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:

(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 the to-be-detected pepper among 286 pepper varieties, 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 15min; denaturation at 94 ℃ for 20s and 61-55 ℃ (touch down program is selected, the temperature is reduced by 0.6 ℃ per cycle), 1min, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. If the fluorescence signal is weak after PCR amplification is finished and data analysis is influenced, cycles (denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min and 5 cycles) can be added until the result is satisfactory.

The 286 capsicum varieties can be LW1-1, LW1-2, QINGTIANHONGSANJIA, LW1-4, LW1-5, LW1-6, LW1-7, LW1-8, LW1-9, LW1-10, LW1-11, LW1-12, LW1-13, LW1-14, LW1-15, and Yb 1-4 LW1-16, LW1-17, LW1-18, LW1-19, LW1-20, LW1-21, LW1-22, LW1-23, LW1-24, peak, green-light early-growing, xiang La Qiu Xiang, HONGQIU No. 1, SV8233HD, HONGFENGTEZHENG Thick beef Horn, YANG JIAO No. 2, YONG JIAO Donghuang No. 1, seed pepper 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 No. ten, top-quality line pepper, baosheng, fulong, kai Jun, yu Dazao pepper, zhongbo No. I, forster 9109, DY10986, YP863, guxi 121, gufu 308, LW1-36, kgla No. 4, guxi 105, LW1-37, guxi 805, gufu 901, gufu Fu K901, gufu K863, guxi 121, gufu 308, and WU 1-36908. LW1-38, LW1-39, LW1-40, national tower 109, LW1-41, guofu 208, guofu 305, guofu 115, guoshan No. 1, national tower 106, LW1-42, LW1-43, LW1-44, lvxing No. 1, chocolate sweet pepper, guofu 210, LW1-45, LW1-46, guofu 316, guofu 903, guofu 413, guofu 910, gaoshan 750, gaoshan, G25, DHF1, A11-11, walter, jin 601, HL4, li research No. 6, G17, haifeng 1055, xiou, 10-71, foster 960, A11-147, A11-13, radical tower, ji, wojun # 1, wojiao, 6, li research No. 6, G17, ji # 748, xiujin

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 pepper

Dragon flying, G26, chinese pepper 105, chinese pepper 0808, 217, azure 169, ji grind No. 12, 11-19, tianxi jade, aoye 119, rongyang rich, huangshuai, huanglong, G21, A11-46, guirenxiang, youmei thin-skin pickled pepper, forster 5110, ANS #4, zhuang pepper 2005, gold pepper eight, aoyan 218, gold pepper 2313, spicy 129, qiangfeng 7301, ANS #9, 364, LW1-47, A11-512, improved Huaguan, G19, gold screw, jufeng screw No. 5, A11-165, 732, G18, hongbao 202, A11-104, xiangfei line pepper, spicy No. 7, radical root-root

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, aojin, guangxi 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, jiaozhuang, 12T-272, ATX-3-IMP, 11-G13, youtan No. 6021, jinhui 13-F, zb1208, L02. MS-004, jinhui 21, juanquan, zb1214, 608, aoyxue dry pepper king, WSP-2322, zanthoxylum piperitum, jinhui 24, weiton 5, WSP-2329, 9566 dry pepper, rizhao 11, WSP-RDO, zb1202, hua green hot line pepper No. 8, jufeng green arrow, LJ104, zhongla No. 1, jiaolong No. 7, zb1203, 221, SPE-104, LJ06002, jingte 2018, zb2, LJ102, su color pepper No. 1, SPE-102, zb1222, 11-G35, DTSP-104, hybrid F1, zb122B, xifeng 601, MS-002, 12, zb1211, hancheng Tianjiao, zb1205, liangjian, zb1206, zb 2010-11, zb1209, tiannu 004, DY1164, L03, jinla Changsheng, forster 1135, sy10-314, jinguan, WSP-2318, 1001, lexing 601, yumeiren, super star, ATX02B, jinla Qingguanwang No. 2, jingte 1107, LW1-48, jinhui 30, A03, L05, X11-3, midline 109, longjiao No. 3, WSP-2330, hualin 301, WSP-CB, haizha No. 3, zb1201, WSP-BB, zanthoxylum piperitum No. 2, zhongla No. 4, weidun No. 4, red pepper, zb1224, BLW, 09-Q111, LJ1118, su No. 14, 4192, hangzhou No. two 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 a 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, simple 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 intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto.

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

The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 obtaining of primer combination for authenticating variety of Capsicum annuum

1. Discovery of 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, include 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 types of the pepper, have higher genetic diversity in the aspects of agronomic traits 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 \8230: (download address: ftp:// ftp. Solgenomics. Net/genes/Capsicum _ annuum) by reference to genome sequence alignment.

TABLE 1.40 basic information of SNP sites

2. Obtaining of primer combination for identifying authenticity of pepper variety

According to the 40 SNP sites 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 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 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.

Validity test of the primer combinations developed in example 2 and example 1

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

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

TABLE 3.286 basic information on the 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; the ratio of A260/A280 is about 1.8 when the ultraviolet spectrophotometer Nanodrop2000 (Thermo) detects, and the ratio of A260/A230 is more than 1.8; 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 containing "F1" in the name, the primer containing "F2" in the name, and the primer containing "R" in the name was 2.

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

3. After the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, reading fluorescence values (reading values are observed by FAM and HEX light beams of a microplate reader when the FAM fluorescent label sequence is 485nm in exciting light and 520nm in emitting light, reading values are observed by HEX fluorescent label sequence at 528nm in exciting light and 560nm in emitting light) through scanning of FAM and HEX light beams of the microplate reader, and determining the genotypes of the 286 tested pepper varieties based on each SNP site according to the colors of fluorescence signals. 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 the F1 in the name; if a certain tested pepper variety shows a red fluorescent signal based on a certain SNP locus, the tested pepper variety is homozygotic based on the genotype of the SNP locus, namely the complementary base of the 1 st base at the 3' tail end of a primer which amplifies the SNP locus and contains F2 in the name of the SNP locus; if a certain test pepper variety shows a green fluorescent signal based on a certain SNP site, the test pepper variety is a heterozygous type based on the genotype of the SNP site, one base is a "complementary base amplifying the 1 st base at the 3 'end of the primer of the SNP site and containing the" F1 "in the name", and the other base is a "complementary base amplifying the 1 st base at the 3' end of the primer of the SNP site and containing the" F2 "in the name".

If the fluorescence signal is weak after the PCR amplification, which affects the data analysis, cycles (94 ℃ denaturation 20s,55 ℃ renaturation and elongation 1min,5 cycles) may be added until the results are 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 tested pepper varieties by utilizing MiniMarker and MEGA7 software according to the genotypes of the 286 tested 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 authenticity identification of the variety.

5. Evaluation of efficiency

The variety authenticity identification can adopt a sequential analysis mode to reduce the workload. 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 (fig. 3), the discrimination of 40 primer sets (i.e. 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

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

1. Obtaining genomic DNA of pepper to be tested

The method of step 1 in example 2 was followed to replace the "leaf of the pepper variety to be tested" with the "leaf of the pepper to be tested", and the genomic DNA of the pepper to be tested was obtained without changing the other steps.

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 containing "F1" in the name, the primer containing "F2" in the name, and the primer containing "R" in the name was 2.

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, which affects the data analysis, cycles (94 ℃ denaturation 20s,55 ℃ renaturation and elongation 1min,5 cycles) may be added until the results are satisfactory.

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

4. After the step 3 is finished, 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.

2. Accuracy identification of the method established in step one

The varieties of the 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 to 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 LJ1118; 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 of 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, forestry, and scientific colleges in Beijing

<120> method for identifying authenticity of pepper variety

<160>120

<170> PatentIn version 3.5

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gaaggtcgga gtcaacggat tgggaaagga aaagttttga tgctaatag 49

<210> 57

<211> 31

<212> DNA

<213> Artificial sequence

<400> 57

gtatatttcc ttatcaggtg cttgtttcta t 31

<210> 58

<211> 41

<212> DNA

<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. The primer combination A consists of a primer group 1-a primer group 40;

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

the primer group 2 consists 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 consists of a forward primer 03F1 shown by SEQ ID NO. 7, a forward primer 03F2 shown by SEQ ID NO. 8 and a reverse primer 03R shown by 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 by SEQ ID NO. 13, a forward primer 05F2 shown by SEQ ID NO. 14 and a reverse primer 05R shown by 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 by SEQ ID NO. 19, a forward primer 07F2 shown by SEQ ID NO. 20 and a reverse primer 07R shown by 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 by SEQ ID NO. 25, a forward primer 09F2 shown by SEQ ID NO. 26 and a reverse primer 09R shown by 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 by SEQ ID NO. 37, a forward primer 13F2 shown by SEQ ID NO. 38 and a reverse primer 13R shown by 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 by SEQ ID NO. 43, a forward primer 15F2 shown by SEQ ID NO. 44 and a reverse primer 15R shown by 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 by SEQ ID NO. 82, a forward primer 28F2 shown by SEQ ID NO. 83 and a reverse primer 28R shown by 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 by SEQ ID NO. 100, a forward primer 34F2 shown by SEQ ID NO. 101 and a reverse primer 34R shown by 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.

2. The primer combination B consists of a primer group 1-a primer group 40;

the primer group 1 consists of a forward primer 01F1 shown by 22 th to 51 th positions from the 5 'end of SEQ ID NO. 1, a forward primer 01F2 shown by 22 th to 53 th positions from the 5' end of SEQ ID NO. 2 and a reverse primer 01R shown by SEQ ID NO. 3;

the primer group 2 consists of a forward primer 02F1 shown in 22 th to 42 th positions from the 5 'end of SEQ ID NO. 4, a forward primer 02F2 shown in 22 th to 43 th positions from the 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 by 22 th to 40 th positions from the 5 'end of SEQ ID NO. 7, a forward primer 03F2 shown by 22 th to 39 th positions from the 5' end of SEQ ID NO. 8 and a reverse primer 03R shown by SEQ ID NO. 9;

the primer group 4 consists of a forward primer 04F1 shown by the 22 nd to 48 th positions from the 5 'end of SEQ ID NO. 10, a forward primer 04F2 shown by the 22 nd to 49 th positions from the 5' end of 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 22 th to 51 th positions from the 5 'end of SEQ ID NO. 13, a forward primer 05F2 shown in 22 th 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 by the 22 nd to 45 th positions from the 5 'end of SEQ ID NO. 16, a forward primer 06F2 shown by the 22 nd to 46 th positions from the 5' end of 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 as 22 th to 43 th positions from the 5 'end of SEQ ID NO. 19, a forward primer 07F2 shown as 22 th to 42 th positions from the 5' end of SEQ ID NO. 20, and a reverse primer 07R shown as SEQ ID NO. 21;

the primer group 8 consists of a forward primer 08F1 shown by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 22, a forward primer 08F2 shown by 22 th to 50 th positions from the 5' end of SEQ ID NO. 23, and a reverse primer 08R shown by 24 SEQ ID NO. 24;

the primer group 9 consists of a forward primer 09F1 shown as the 22 nd to 49 th positions from the 5 'end of SEQ ID NO. 25, a forward primer 09F2 shown as the 22 nd to 49 th positions from the 5' end of SEQ ID NO. 26, and a reverse primer 09R shown as 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 by 22 th to 39 th positions from the 5 'end of SEQ ID NO. 31, a forward primer 11F2 shown by 22 th to 40 th positions from the 5' end of 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 22 th to 48 th positions from the 5 'end of SEQ ID NO. 34, a forward primer 12F2 shown by 22 th to 47 th positions from the 5' end of 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 by 22 th to 43 th positions from the 5 'end of SEQ ID NO. 37, a forward primer 13F2 shown by 22 th to 45 th positions from the 5' end of SEQ ID NO. 38, and a reverse primer 13R shown by SEQ ID NO. 39;

the primer group 14 consists of a forward primer 14F1 shown by 22 th to 46 th positions from the 5 'end of SEQ ID NO. 40, a forward primer 14F2 shown by 22 th to 46 th positions from the 5' end of SEQ ID NO. 41, and a reverse primer 14R shown by 42 SEQ ID NO. 42;

the primer set 15 consists of a forward primer 15F1 shown by 22 th to 46 th positions from the 5 'end of SEQ ID NO. 43, a forward primer 15F2 shown by 22 th to 47 th positions from the 5' end of SEQ ID NO. 44, and a reverse primer 15R shown by SEQ ID NO. 45;

the primer group 16 consists of a forward primer 16F1 shown by 22 th to 50 th positions from the 5 'end of SEQ ID NO. 46, a forward primer 16F2 shown by 22 th to 48 th positions from the 5' end of 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 the 22 nd to 46 th positions from the 5 'end of SEQ ID NO. 49, a forward primer 17F2 shown by the 22 nd to 45 th positions from the 5' end of 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 in 22 th to 46 th positions from the 5 'end of SEQ ID NO. 52, a forward primer 18F2 shown in 22 th 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 by 22 th to 51 th positions from the 5 'end of SEQ ID NO. 55, a forward primer 19F2 shown by 22 th to 49 th positions from the 5' end of SEQ ID NO. 56, and a reverse primer 19R shown by SEQ ID NO. 57;

the primer set 20 consists of a forward primer 20F1 represented by the 22 nd to 41 th positions from the 5 'terminus of SEQ ID NO. 58, a forward primer 20F2 represented by the 22 nd to 42 th positions from the 5' terminus of SEQ ID NO. 59, and a reverse primer 20R represented by SEQ ID NO. 60;

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

the primer set 22 consists of a forward primer 22F1 shown by 22 th to 52 th positions from the 5 'end of SEQ ID NO. 64, a forward primer 22F2 shown by 22 nd to 54 th positions from the 5' end of SEQ ID NO. 65, and a reverse primer 22R shown by 66 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 by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 70, a forward primer 24F2 shown by 22 th to 49 th positions from the 5' end of SEQ ID NO. 71 and a reverse primer 24R shown by 72 SEQ ID NO. 72;

the primer group 25 consists of a forward primer 25F1 shown by 22 th to 45 th positions from the 5 'end of SEQ ID NO. 73, a forward primer 25F2 shown by 22 th to 46 th positions from the 5' end of SEQ ID NO. 74, and a reverse primer 25R shown by 75 SEQ ID NO;

the primer group 26 consists of a forward primer 26F1 shown by 22 th to 46 th positions from the 5 'end of SEQ ID NO. 76, a forward primer 26F2 shown by 22 th to 45 th positions from the 5' end of SEQ ID NO. 77, and a reverse primer 26R shown by 78 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 22 th to 49 th positions from the 5 'end of SEQ ID NO. 82, a forward primer 28F2 shown in 22 th 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 by 22 th to 49 th positions from the 5 'end of SEQ ID NO. 85, a forward primer 29F2 shown by 22 th to 51 th positions from the 5' end of 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 represented by 22 th to 46 th positions from the 5 'end of SEQ ID NO. 88, a forward primer 30F2 represented by 22 th to 45 th positions from the 5' end of SEQ ID NO. 89, and a reverse primer 30R represented by SEQ ID NO. 90;

the primer group 31 consists of a forward primer 31F1 shown by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 91, a forward primer 31F2 shown by 22 th to 47 th positions from the 5' end of 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 22 th to 46 th positions from the 5 'end of SEQ ID NO. 94, a forward primer 32F2 shown by 22 th to 43 th positions from the 5' end of SEQ ID NO. 95 and a reverse primer 32R shown by 96 SEQ ID NO. 96;

the primer group 33 consists of a forward primer 33F1 shown by the 22 nd to 47 th positions from the 5 'end of SEQ ID NO. 97, a forward primer 33F2 shown by the 22 nd to 48 th positions from the 5' end of 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 the 22 nd to 39 th positions from the 5 'end of SEQ ID NO. 100, a forward primer 34F2 shown in the 22 nd 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 represented by the 22 nd to 43 th positions from the 5 'end of SEQ ID NO. 103, a forward primer 35F2 represented by the 22 nd to 44 th positions from the 5' end of SEQ ID NO. 104, and a reverse primer 35R represented by SEQ ID NO. 105;

the primer group 36 consists of a forward primer 36F1 shown by 22 th to 54 th positions from the 5 'end of SEQ ID NO. 106, a forward primer 36F2 shown by 22 th to 52 th positions from the 5' end of SEQ ID NO. 107 and a reverse primer 36R shown by 108 SEQ ID NO. 108;

the primer group 37 consists of a forward primer 37F1 shown by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 109, a forward primer 37F2 shown by 22 th to 47 th positions from the 5' end of 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 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 set 39 consists of a forward primer 39F1 shown by 22 th to 48 th positions from the 5 'end of SEQ ID NO. 115, a forward primer 39F2 shown by 22 th to 48 th positions from the 5' end of SEQ ID NO. 116, and a reverse primer 39R shown by SEQ ID NO. 117;

the primer set 40 is composed of a forward primer 40F1 represented by positions 22 to 44 from the 5 'terminus of SEQ ID NO. 118, a forward primer 40F2 represented by positions 22 to 43 from the 5' terminus of SEQ ID NO. 119, and a reverse primer 40R represented by SEQ ID NO. 120.

3. Use of the primer combination a of claim 1 or the primer combination b of claim 2, in any one of x 1) to x 4): x 1) preparing a kit for identifying 286 pepper varieties; x 2) preparing a kit for identifying the authenticity of 286 pepper varieties; x 3) identifying 286 pepper varieties; x 4) identifying the authenticity of 286 pepper varieties;

<xnotran> 286 LW1-1, LW1-2, , 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, LW1-23, LW1-24, , , , 1 , SV8233HD, , 2 , 1 , 1 , LW1-25, HNX14470065, LW1-26, LW1-27, LW1-28, LW1-29, LW1-30, LW1-31, LW1-32, LW1-33, LW1-34, LW1-35, , , , , , , , 9109, DY10986, YP863, 121, 308, LW1-36, 4 , 105, LW1-37, 805, 901, 908, LW1-38, LW1-39, LW1-40, 109, LW1-41, 208, 305, 115, 1 , 106, LW1-42, LW1-43, LW1-44, 1 , , 210, LW1-45, LW1-46, 316, 903, 413, 910, 750, 748, G25, DHF1, A11-11, , 601, HL4, 6 , G17, 1055, , 10-71, 960, A11-147, </xnotran> A11-13, at all

12-03, G16, haifeng 1022, all

12-07, 12-28, 329, haifeng 0854, yangtai pepper No. 11, hongfeng big long pepper, 8-98, zunzheng, zaisheng, HL7, jixing big sweet pepper, CR fragrance, nongyoujujujijujiufa, pepper No. 1, jinyi

Dragon flying, G26, chinese pepper 105, chinese pepper 0808, 217, azure 169, ji grind No. 12, 11-19, tianxi emerald, aoye 119, rongyang rich, huangshuai, huanglong dragonG21, A11-46, noble incense, beautiful thin-skin pickled pepper, forster 5110, ANS #4, zhuang Pepper 2005, golden Pepper eighth, aotai 218, golden Pepper 2313, spicy 129, qiangfeng 7301, ANS #9, 364, LW1-47, A11-512, improved Huaguan, G19, golden screw, jufeng screw No. 5, A11-165, 732, G18, hongbao 202, A11-104, xiangfei Ling Pepper, xiang La Langchao No. 7, radical

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, aujin, 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, kikui 22, and Yuanhe 165, L04, youyang No. 8, charming, 12T-272, ATX-3-IMP, 11-G13, hangzhou pepper No. three, 6021, jinhui 13-F, zb1208, L02, MS-004, jinhui 21, super crown, zb1214, 608, aoyxue dry pepper king, WSP-2322, zanthoxylum piperitum, jinhui 24, weidun No. 5, WSP-2329, 9566 dry pepper, rizhao No. 11, WSP-RDO, zb1202, hua green spicy line pepper No. 8, jufeng green arrow, LJ104, zb 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, DY1164, L03, jinla Changsheng, foster 1135, sy10-314, gold crown, yuan WSP-2318, 1001, lexing 601, yumeiren, suxiaxing, ATX02B, jinla Qingguanwang No. 2, jingte 1107, LW1-48, jinhui 30, A03, L05, X11-3, midline 109, longjiao No. 3, WSP-2330, hualin 301, WSP-CB, haizi No. 3, zb1201, WSP-BB, zanthoxylum piperitum No. 2, zhongla No. 4, weidun No. 4, hongdan, zb1224, BLW, 09-Q111, LJ1118, zanthoxylum piperitum No. 14, suyangjiang No. 14, yumeiren, super-star, ATX 11-3, longjiao No. 3, woo-Hualin 301, WSP-CB, haizhong-CB No. 3, zb1201, zb No. 4, zb-B1224, and Woo-Li-ZW, 4192. Hangzhou pepper II and X11-6.

4. A method of identifying which of the 286 pepper varieties of claim 3 the pepper to be tested belongs to, comprising the steps of:

(1) Performing PCR amplification by respectively adopting the primer group in the primer combination B 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 using the genome DNA of each pepper variety in the standard pepper species population as a template and respectively adopting the primer group in the primer combination B of claim 2 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 which 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.