CN109811075B - Method for identifying authenticity of Chinese cabbage variety and special SNP primer combination thereof - Google Patents

Abstract

The invention discloses a method for identifying the authenticity of a Chinese cabbage variety and a special SNP primer combination thereof. The SNP primer combination provided by the invention consists of 32 primer groups. Each primer set consists of 3 primer sequences and is used for amplifying one SNP site. The nucleotide sequences of the primers in the 32 primer groups are sequentially shown as a sequence 1 to a sequence 96 in a sequence table. The SNP primer combination provided by the invention can be used for carrying out early identification on the Chinese cabbage 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 Chinese cabbage germplasm resources and new varieties. The method provided by the invention can be used for identifying the unknown Chinese cabbage variety and also identifying the authenticity of the known variety. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

Method for identifying authenticity of Chinese cabbage variety and special SNP primer combination thereof

Technical Field

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

Background

Chinese cabbage belongs to cruciferae and biennial herbaceous plants, and has become the widest vegetable crop in the current vegetable planting area of China due to balanced nutrition and unlimited planting area, the planting area is about 260 ten thousand hectares, and the annual yield is about 11300 ten thousand tons. The Chinese cabbage is native to the northern China, has long cultivation history and various varieties. Therefore, how to rapidly and effectively identify the authenticity of the Chinese cabbage variety is very important. The most effective method for identifying the authenticity of the conventional Chinese cabbage variety is field plot planting, but the method is easily interfered by the outside, has a long period and consumes time and labor. According to the requirements of the registration guidelines for non-major crop varieties, DNA detection results can be directly submitted for the variety trait specifications and related traits involved in the variety DUS test reports, such as clear associated genes. Therefore, a DNA technical system for rapidly, conveniently and effectively identifying the authenticity of the Chinese cabbage variety is urgently needed to be established.

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 continuous development of high-throughput sequencing technology and the continuous reduction of sequencing cost, a large amount of Chinese cabbage re-sequencing data is obtained. Based on analysis of variation group information of Chinese cabbage, stable 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, DNA molecular detection for identifying the authenticity of Chinese cabbage varieties is mainly based on an SSR molecular marking method, but the research and development of Chinese cabbage SSR primers do not refer to genome variation group information, and unreal variation conditions exist; the SSR primers are small in screening variety quantity and cannot represent the sales varieties in the current market in China; in addition, the method is limited by that unreal, false positive and false negative results are easily caused by an SSR detection mode; can not meet the requirements of automation, high flux and large-scale detection. Compared to SSR markers, SNP markers have several advantages: 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 the Chinese cabbage variety.

The invention firstly protects the SNP locus combination, which can comprise 32 SNP loci of the Chinese cabbage genome; the 32 SNP sites are as follows: the BrSNP01 site is the 1795429 th nucleotide on the chromosome 1; the BrSNP02 site is the 4668251 th nucleotide on the chromosome 1; the BrSNP03 site is the 8659686 th nucleotide on the chromosome 1; the BrSNP04 site is the 22523311 th nucleotide on the chromosome 1; the BrSNP05 site is the 157034 th nucleotide on chromosome 2; the BrSNP06 site is the 1758653 th nucleotide on chromosome 2; the BrSNP07 site is the 2421548 th nucleotide on chromosome 2; the BrSNP08 site is the 9141887 th nucleotide on chromosome 2; the BrSNP09 site is the 11181828 th nucleotide on chromosome 2; the BrSNP10 site is the 19966721 th nucleotide on chromosome 2; the BrSNP11 site is the 26263824 th nucleotide on chromosome 2; the BrSNP12 site is the 3067264 th nucleotide on the 3 rd chromosome; the BrSNP13 site is the 15431305 th nucleotide on the 3 rd chromosome; the BrSNP14 site is the 28161015 th nucleotide on the 3 rd chromosome; the BrSNP15 site is the 28629888 th nucleotide on the 3 rd chromosome; the BrSNP16 site is the 1833604 th nucleotide on chromosome 4; the BrSNP17 site is the 13764622 th nucleotide on chromosome 4; the BrSNP18 site is the 1283352 th nucleotide on chromosome 5; the BrSNP19 site is the 4556351 th nucleotide on chromosome 5; the BrSNP20 site is the 24583581 th nucleotide on chromosome 5; the BrSNP21 site is the 9159584 th nucleotide on chromosome 6; the BrSNP22 site is the 16193195 th nucleotide on chromosome 6; the BrSNP23 site is the 23521839 th nucleotide on the 7 th chromosome; the BrSNP24 site is the 2374634 th nucleotide on chromosome 8; the BrSNP25 site is the 3289888 th nucleotide on chromosome 8; the BrSNP26 site is the 12382048 th nucleotide on chromosome 8; the BrSNP27 site is the 14112886 th nucleotide on chromosome 8; the BrSNP28 site is the 1379762 th nucleotide on the 9 th chromosome; the BrSNP29 site is the 3019969 th nucleotide on the 9 th chromosome; the BrSNP30 site is the 277083 th nucleotide on the 10 th chromosome; the BrSNP31 site is the 6477381 th nucleotide on the 10 th chromosome; the BrSNP32 site is the 15835648 th nucleotide on chromosome 10.

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

The invention also protects a SNP primer combination, which can comprise a primer set 1 for amplifying the BrSNP01, a primer set 2 for amplifying the BrSNP02, a primer set 3 for amplifying the BrSNP03, a primer set 4 for amplifying the BrSNP04, a primer set 5 for amplifying the BrSNP05, a primer set 6 for amplifying the BrSNP06, a primer set 7 for amplifying the BrSNP07, a primer set 8 for amplifying the BrSNP08, a primer set 9 for amplifying the BrSNP09, a primer set 10 for amplifying the BrSNP, a primer set 11 for amplifying the BrSNP11, a primer set 12 for amplifying the BrSNP12, a primer set 13 for amplifying the BrSNP 6334, a primer set 14 for amplifying the BrSNP14, a primer set 15 for amplifying the BrSNP15, a primer set 16 for amplifying the BrSNP16, a primer set for amplifying the BrSNP17, a primer set for amplifying the BrSNP 59617, a primer set for amplifying the BrSNP 59618, and a primer set 15 for amplifying the BrSNP 3919, A primer set 20 for amplifying the BrSNP20, a primer set 21 for amplifying the BrSNP21, a primer set 22 for amplifying the BrSNP22, a primer set 23 for amplifying the BrSNP23, a primer set 24 for amplifying the BrSNP24, a primer set 25 for amplifying the BrSNP25, a primer set 26 for amplifying the BrSNP26, a primer set 27 for amplifying the BrSNP27, a primer set 28 for amplifying the BrSNP28, a primer set 29 for amplifying the BrSNP29, a primer set 30 for amplifying the BrSNP30, a primer set 31 for amplifying the BrSNP31, and a primer set 32 for amplifying the BrSNP 32.

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

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

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

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

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

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

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

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

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

The invention also discloses a method for identifying the variety of the Chinese cabbage to be detected, which belongs to 236 Chinese cabbage varieties, which comprises the following steps: and respectively detecting the genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on the 32 SNP loci, and then judging as follows: if the genotypes of the Chinese cabbage to be detected based on the 32 SNP loci are completely consistent with the genotypes of a certain variety of the 236 Chinese cabbage varieties based on the 32 SNP loci, the Chinese cabbage to be detected and the Chinese cabbage variety belong to the same variety; if the genotypes of the Chinese cabbage to be detected based on the 32 SNP sites are different from the genotypes of all the varieties in the 236 Chinese cabbage varieties based on the 32 SNP sites, the varieties of the Chinese cabbage to be detected are different from those of the 236 Chinese cabbage varieties.

In the above method, the step of detecting the genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on the 32 SNP loci may be as follows:

(1) respectively taking the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of 236 Chinese cabbage varieties as templates, and respectively carrying out PCR amplification by adopting the primer groups in any one of the SNP primer combinations 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 Chinese cabbage to be detected and 236 Chinese cabbage varieties based on the 32 SNP sites according to the color of the fluorescent signal.

In the above method, the step of detecting the genotypes of the detected Chinese cabbage and 236 Chinese cabbage varieties based on the 32 SNP loci may be as follows:

(1) respectively taking the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of 236 Chinese cabbage varieties as templates, and respectively carrying out PCR amplification by adopting the primer groups in any one of the SNP primer combinations to obtain PCR amplification products;

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

(3) and (3) obtaining genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on the 32 SNP loci according to the sequencing result obtained in the step (2).

The invention also discloses a method for identifying the variety of the Chinese cabbage to be detected, which belongs to 236 Chinese cabbage varieties, which comprises the following steps:

(1) taking the genome DNA of the Chinese cabbage to be detected as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the SNP primer combinations to obtain PCR amplification products; taking the genome DNA of each Chinese cabbage variety in a standard Chinese cabbage population as a template, and respectively carrying out PCR amplification by adopting the primer group in any one of the SNP primer combinations to obtain PCR amplification products; the standard Chinese cabbage variety group consists of 236 Chinese cabbage varieties;

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

In any of the above methods, 236 varieties of Chinese cabbage may be West Bai No. 10, 87-114, CR117, CR Tianbai No. 15, J405, LI-1, SGK1008, SW701, YHBC1511, YHBC1516, Beijing 75, Beijing 80, Beijing Daniu heart, Beijing improved 67, Beijing orange Red No. 2, Beijing orange Red Heart, Beijing Xiaoza 50, Beijing Xiaoza 51, Beijing Xiaoza 61, Beijing Xiaoza 67, Beijing Xin No. 1, Beijing New No. 2, Beijing New No. 3, Beijing New No. 4, Beijing New No. three, Beijing New No. 9, Chunbao Huang, Chunbao, Chunfeng, Chunbeng, Chunbao, Chunfeng 54, Chunwang, Chunxang, Chunwang No. 1, Degao No. 16, Gao No. 8, oriental crown, Huangguan, Huanghua, Huang, Ching Huachun No. 70, Huachun No. 5, Huazhong Ying Feng-Fu-5, Huang-Fu-Hua No. 93, anti-Gui-Ying No. 5, anti-Miao-, Yellow bud No. 14, Heichun, Jihong 308, Jihong 65, Jihong 82, Jihong doll, Jiaobei No. 7, Jiaobei Xiaxing, jin Guang No. 2, jin Huayuan No. 19, jin Ling, jin Qiu 68, jin Qiu 70, jin Qiu 90, jin Zao 58, jin Bai 56, jin Lu 75, jin Lu 80, jin Qiu 606, jin Qiu 78, jin doll No. 1, jin Xiu No. 1, jin Shukuai vegetable No. 30, jin Shukuai Kuai Quai green No. 1, jin Jiankuai green No. 2, jin Qiu No. 1, Jingchun 99, Jingchun CR3, Jingchun white No. 2, Jingchun Huang No. 2, Jingchun Chun Wawa No. 2, Jingchun Wawa No. 3, Jingchun Chun Wawa No. 4, Jingchun Chun Wai baby vegetable, Jingchun Mao 60, Jingchun Cui 70, Cuo No. 75, Jingchun No. 8, Jingchun No. 1, Jingchun Ju 70, Jingchun No. 70, Jingchun No. 70, Jingchun No. 1, Jingchun Wu 70, Jingchun Qiu Wa 70, Jingchun Xin, Jingyankuaicai, Juxin I, Lai 6014, Lai Bai I, Li Chun I, Liao Bai Ten, Linglong Huang, Lu Bai 15, Lu Bai 16, Lu Bai II, Lu Chun Bai No. 1, Lu Chun Bai I, Lu Jian 60, Lv Jian 85, Lv bamboo shoot 70, Qian Bai 4, Qin Bai II, Qin hetero 60, Qing Yan Chun No. 3, Qing Chun Bai I, Qing Jiang No. 1, Qing Jiang Bai, Qing nong 45 days, Qiu Green 55, Qiu Green 60, Qiu Green 75, Qiu Green 78, Qiu Yu 78, Qiu Zao 9, Huo 7, Shandi 2, Shandong No. 5, Shandong five No. 5201, Shen Qing Shen Yun Bao, Shen white GMS01, Bai GMS02, Shi Lu 85, Shi Di Ding Shen Shi Wan Shi No. 65, Jing Zhen Wu Zhen No. 65, Jing Zhen Hao Hongtao No, Tianzheng tangerine 62, Tianzheng tangerine 65, Tianzheng Yiyouyi No. 1, Tianwa, mophuang, Wannong No. two, Wannong No. six, Wangchun, Weibai No. 4, Xibai No. 3, Xibai No. 4, Xibai No. 5, Xibai No. 6, Xibai No. 9, Xibai No. four, Xichunbai No. 3, Xishuan, Xiguan, Xiyangyang 303, Xiaoyi and Qiu, Xiaoza 56, Xiaoza 60, Xinxiang Xiaoza 23, Xinzai 56, Xinzai 58, Xinzai 89-8, Xinzhong 78, Yanchun, Yi and 0906, Yi and No. 1, preferably Xiaoza 56, preferably precocious No. 5F1, YOULV No. 3, Xinxin No. 1, NXINXIN No. 2, YuXIN No. 3, YuXINXIN No. 9, Zhengzai Zhengzhen Zheng Zanbai No. 2, Zhengchun Xinchun No. 5, Zhengqing No. 6, Zhengqing Xinjiang No. 5, Zhengqing No. 6, Zhengqing No. 5, Zhengqing, Zhengbai 78, Zhengbai NO. four, Zhengzhengzhu 23, Zhengzao NO. 2, Zhengzao NO. two, Zhengzao 55, Zhengzao 60, Zhongbai 50, Zhongbai 58, Zhongbai 60, Zhongbai 61, Zhongbai 62, Zhongbai 65, Zhongbai 66, Zhongbai 76, Zhongbai 78, Zhongbai 81, Zhongbai 83, Zhongbai 85 and Zhongbao 68.

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

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

Drawings

FIG. 1 is a cluster diagram of 236 tested Chinese cabbage varieties established on 32 SNP primer sets.

FIG. 2 is a graph showing the relationship between the number of SNP markers (i.e., the number of SNP loci) and the discrimination of 236 varieties of Chinese cabbage under test.

FIG. 3 shows the SNP typing effect of 32 primer sets in some tested varieties of Chinese cabbage.

Detailed Description

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

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

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

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

Example 1 acquisition of primer combination for authenticating variety of Chinese cabbage

Discovery of one, 32 SNP sites

The invention obtains 32 SNP loci based on the re-sequencing data of 194 parts of Chinese cabbage inbred lines. The 194 parts of Chinese cabbage are rich in resource types, comprise autumn Chinese cabbage (117 parts), summer Chinese cabbage (37 parts) and spring Chinese cabbage (40 parts), basically comprise the main ecological type and agronomic characters of the Chinese cabbage, have representativeness of Chinese cabbage germplasm, and have higher genetic diversity. Specifically, the screening criteria for SNP sites are as follows: SNP loci with uniform positions, good polymorphism, small heterozygosity, MAF >0.3, good population distinguishing effect, obvious PCA clustering and two-wing 50bp sequence conservation (no InDel, no SSR and no other SNP) are selected in the whole genome range. The basic information of the 32 SNP sites is detailed in columns 1 to 4 of Table 1. Wherein the position of the SNP site on the chromosome is determined based on the alignment of the Chiifu-401-42 reference genome sequence, and the version number of the Chiifu-401-42 reference genome sequence is V1.5 (download address: https:// www.plantcyc.org/databases/breppacapsyc/1.5).

TABLE 1.32 basic information of SNP sites

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

According to the 32 SNP loci discovered in the first step, a primer combination which has higher polymorphism information content (PIC value) (see the 5 th column in the table 1) and is suitable for identifying the authenticity of the Chinese cabbage variety by using an allele competitive specific PCR method is developed.

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

TABLE 2

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

Example 2 and example 1 validation of the primer combinations developed

The basic information of 236 tested Chinese cabbage varieties in this example is shown in Table 3.236 tested Chinese cabbage varieties are all common fine varieties or partially foreign introduced varieties.

Table 3.236 basic information of tested Chinese cabbage varieties

1. Acquisition of genomic DNA of the tested Brassica rapa variety

And respectively extracting the genome DNA of 236 leaves (true leaves of 30 mixed seeds) of the Chinese cabbage variety to be tested by adopting a CTAB method to obtain the genome DNA of the Chinese cabbage variety to be tested.

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

2. And respectively taking the genome DNA of 236 varieties of the Chinese cabbages to be tested as templates, and respectively adopting 32 primer groups to carry out PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer named "F1", the primer named "F2" and the primer named "R" was 2:2: 5.

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

3. After the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, scanning and reading fluorescence values through FAM and HEX light beams of a microplate reader (reading values are observed when the FAM fluorescent label sequence is at 485nm of exciting light and at 520nm of emitting light, reading values are observed when the HEX fluorescent label sequence is at 528nm of exciting light and at 560nm of emitting light), and judging the genotypes of 236 tested Chinese cabbage varieties based on each SNP site according to the colors of fluorescence signals. The specific judgment principle is as follows: if a certain test Chinese cabbage variety shows a blue fluorescent signal based on a certain SNP locus, the test Chinese cabbage 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 F1 in the name; if a certain test Chinese cabbage variety shows a red fluorescent signal based on a certain SNP locus, the test Chinese cabbage 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; if a certain test cabbage variety shows a green fluorescent signal based on a certain SNP site, the test cabbage variety is heterozygous based on the genotype of the SNP site, one base is a complementary base "amplifying the SNP site and containing the 1 st base at the 3 'end of the primer of" F1 "in the name", and the other base is a complementary base "amplifying the SNP site and containing the 1 st base at the 3' end of the primer of" F2 "in the name".

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

Partial results are shown in FIG. 3. The results show that each primer group can obtain good typing effect in the tested Chinese cabbage variety.

4. Cluster analysis

And (3) carrying out cluster analysis on 236 varieties of the Chinese cabbages to be tested by utilizing MiniMarker and MEGA7 software according to the genotypes of the 236 varieties of the Chinese cabbages to be tested based on 32 SNP loci.

The clustering chart of 236 tested brassica rapa varieties established on 32 primer sets is shown in figure 1. The results show that 236 tested Chinese cabbage varieties in the table 3 can be completely distinguished by 32 primer groups. Therefore, the SNP primer combination developed in the embodiment 1 can be applied to the construction of a DNA fingerprint database of the Chinese cabbage 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 level of 236 varieties of Chinese cabbage to be tested.

The experimental result shows (figure 2) that the discrimination rate of the 32 primer groups (namely the number of the 32 SNP markers) in 236 varieties of the Chinese cabbages to be tested reaches 100 percent.

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

<120> method for identifying authenticity of Chinese cabbage variety and special SNP primer combination thereof

<160> 96

<170> PatentIn version 3.5

<210> 1

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

gaaggtgacc aagttcatgc tagactgttc aaagctactt acccaaat 48

<210> 2

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

gaaggtcgga gtcaacggat tgactgttca aagctactta cccaaac 47

<210> 3

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

catgtctcca ctagagtcat gggaa 25

<210> 4

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 4

gaaggtgacc aagttcatgc tgctttgaga gagtatcatt aggcatg 47

<210> 5

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 5

gaaggtcgga gtcaacggat ttgctttgag agagtatcat taggcata 48

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 6

gctgaaggaa atgagagact gttggta 27

<210> 7

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 7

gaaggtgacc aagttcatgc tgttacccat cagaatttgc atcatatttc 50

<210> 8

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 8

gaaggtcgga gtcaacggat tgttacccat cagaatttgc atcatatttt 50

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 9

gcgtagtctg gtttatcatc aaaacgaa 28

<210> 10

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 10

gaaggtgacc aagttcatgc taatcgtgat tctagcactc catcca 46

<210> 11

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 11

gaaggtcgga gtcaacggat tcgtgattct agcactccat ccg 43

<210> 12

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 12

cataaactgg cacttagcca ctgacta 27

<210> 13

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 13

gaaggtgacc aagttcatgc tggtttctcc caaacccaac acc 43

<210> 14

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 14

gaaggtcgga gtcaacggat tataggtttc tcccaaaccc aacact 46

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 15

ggaagtcgag ttcgggagct gat 23

<210> 16

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 16

gaaggtgacc aagttcatgc tagaccaaaa aaacaatgtc aagagatgat a 51

<210> 17

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 17

gaaggtcgga gtcaacggat tgaccaaaaa aacaatgtca agagatgatg 50

<210> 18

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 18

gtagcaagat catcaagatc agtctcat 28

<210> 19

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 19

gaaggtgacc aagttcatgc taaacatgat acaaagagat gtttacttac ata 53

<210> 20

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 20

gaaggtcgga gtcaacggat tacatgatac aaagagatgt ttacttacat c 51

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 21

ggtgccggac ttgcatgctt cta 23

<210> 22

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 22

gaaggtgacc aagttcatgc tgctggatga gattaaccag gttttc 46

<210> 23

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 23

gaaggtcgga gtcaacggat tgctggatga gattaaccag gttttg 46

<210> 24

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 24

gacatttgaa tacacatggt agaataagga aa 32

<210> 25

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 25

gaaggtgacc aagttcatgc tgcgaatgtt gacatttacg gatgct 46

<210> 26

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 26

gaaggtcgga gtcaacggat tgcgaatgtt gacatttacg gatgca 46

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 27

cggccacaag tgactcgtct tcta 24

<210> 28

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 28

gaaggtgacc aagttcatgc tggttcaaca aagttgcatc tccact 46

<210> 29

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 29

gaaggtcgga gtcaacggat tggttcaaca aagttgcatc tccaca 46

<210> 30

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 30

ccactgtttg tcatttcctc attgtgaat 29

<210> 31

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 31

gaaggtgacc aagttcatgc tccgaccgat ccgaagagcc t 41

<210> 32

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 32

gaaggtcgga gtcaacggat tcgaccgatc cgaagagccc 40

<210> 33

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 33

cggagccttc tgcgagcaaa cat 23

<210> 34

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 34

gaaggtgacc aagttcatgc taaatgttgt tcgattagtg aagtgtctt 49

<210> 35

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 35

gaaggtcgga gtcaacggat ttgttgttcg attagtgaag tgtctc 46

<210> 36

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 36

gttggaaaat cgtttaaatg tgtgcgaca 29

<210> 37

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 37

gaaggtgacc aagttcatgc tgaggttaca atgatctatc aaatgctg 48

<210> 38

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 38

gaaggtcgga gtcaacggat tgaggttaca atgatctatc aaatgctc 48

<210> 39

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 39

tgctatactt gactccttgt ttaccgtt 28

<210> 40

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 40

gaaggtgacc aagttcatgc tgcagtgaat tccagcagtc ctg 43

<210> 41

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 41

gaaggtcgga gtcaacggat tgcagtgaat tccagcagtc ctc 43

<210> 42

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 42

gttaatagct taaggcatga ctgcacaa 28

<210> 43

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 43

gaaggtgacc aagttcatgc tgagtattcc ctatttatag taaaaggaaa ga 52

<210> 44

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 44

gaaggtcgga gtcaacggat tagtattccc tatttatagt aaaaggaaag c 51

<210> 45

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 45

gccatgcact cctccttcat ttttgta 27

<210> 46

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 46

gaaggtgacc aagttcatgc tatacaggag attaagattg gtacctga 48

<210> 47

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 47

gaaggtcgga gtcaacggat tacaggagat taagattggt acctgc 46

<210> 48

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 48

gttatcaagg acatttgttg agtgctacat 30

<210> 49

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 49

gaaggtgacc aagttcatgc tgtttggact ttgtcttgtt agatcaga 48

<210> 50

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 50

gaaggtcgga gtcaacggat ttttggactt tgtcttgtta gatcagg 47

<210> 51

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 51

gagaaaacac acctcaagaa tatcactgaa 30

<210> 52

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 52

gaaggtgacc aagttcatgc tagagacgtt ctaaggattg tgaaaatg 48

<210> 53

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 53

gaaggtcgga gtcaacggat taaagagacg ttctaaggat tgtgaaaata 50

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 54

ttccccgttt tcagctggat ggtta 25

<210> 55

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 55

gaaggtgacc aagttcatgc tctctaagtt taattcttgg tgctgtgat 49

<210> 56

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 56

gaaggtcgga gtcaacggat ttctaagttt aattcttggt gctgtgac 48

<210> 57

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 57

tgcggctctc ctggtggtcc at 22

<210> 58

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 58

gaaggtgacc aagttcatgc tcctttctca tttatcaact tcgccgt 47

<210> 59

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 59

gaaggtcgga gtcaacggat tctttctcat ttatcaactt cgccgc 46

<210> 60

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 60

gttttttagg gttttggaaa tgttggttct t 31

<210> 61

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 61

gaaggtgacc aagttcatgc tatagaggtg ggagataagt ttcataag 48

<210> 62

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 62

gaaggtcgga gtcaacggat tgatagaggt gggagataag tttcataaa 49

<210> 63

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 63

tcggatgaga tgtatcgcct acgta 25

<210> 64

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 64

gaaggtgacc aagttcatgc tattaaagga agtggcagaa ttgttaataa c 51

<210> 65

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 65

gaaggtcgga gtcaacggat tattaaagga agtggcagaa ttgttaataa g 51

<210> 66

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 66

cataaagaaa ccgtaaacgt aaatgcgcaa 30

<210> 67

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 67

gaaggtgacc aagttcatgc tcggtgaaaa ccagctcatt tctgtt 46

<210> 68

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 68

gaaggtcgga gtcaacggat tggtgaaaac cagctcattt ctgtg 45

<210> 69

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 69

gcaggagtca agtgttcctg acatt 25

<210> 70

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 70

gaaggtgacc aagttcatgc tgaatttttg gttcagcaga tgatcctta 49

<210> 71

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 71

gaaggtcgga gtcaacggat taatttttgg ttcagcagat gatccttg 48

<210> 72

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 72

cttcctcttc tctcagtcac ctcaa 25

<210> 73

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 73

gaaggtgacc aagttcatgc taaaacttat tttgaccaac acctctgc 48

<210> 74

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 74

gaaggtcgga gtcaacggat tcaaaactta ttttgaccaa cacctctga 49

<210> 75

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 75

catctatcat tgaaccaaca atcaataagg aa 32

<210> 76

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 76

gaaggtgacc aagttcatgc tgacaagagg gtacttggct cca 43

<210> 77

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 77

gaaggtcgga gtcaacggat tgacaagagg gtacttggct cct 43

<210> 78

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 78

cgagatcgcg tagtttgtga tccatt 26

<210> 79

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 79

gaaggtgacc aagttcatgc tgacgttgta cttttttgtg tagggca 47

<210> 80

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 80

gaaggtcgga gtcaacggat tacgttgtac ttttttgtgt agggcg 46

<210> 81

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 81

cgctgcaaca atgtaataaa gtttatttaa aaaaa 35

<210> 82

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 82

gaaggtgacc aagttcatgc ttctgacgat gaggtccctt tcg 43

<210> 83

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 83

gaaggtcgga gtcaacggat tatatctgac gatgaggtcc ctttca 46

<210> 84

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 84

cactttcgaa gacaccgtaa gaattaacat 30

<210> 85

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 85

gaaggtgacc aagttcatgc tgaggattgt aaggaagaag gttgct 46

<210> 86

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 86

gaaggtcgga gtcaacggat taggattgta aggaagaagg ttgcg 45

<210> 87

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 87

gacattaata aaggttccgt tggttatttc aa 32

<210> 88

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 88

gaaggtgacc aagttcatgc ttatatacag cctttcagag aatctagc 48

<210> 89

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 89

gaaggtcgga gtcaacggat ttatatacag cctttcagag aatctagg 48

<210> 90

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 90

cttcgccttg agcacttcta tgtcttt 27

<210> 91

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 91

gaaggtgacc aagttcatgc tagatgtcag aagcagatgg tattgc 46

<210> 92

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 92

gaaggtcgga gtcaacggat taaagatgtc agaagcagat ggtattga 48

<210> 93

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 93

ttctccaact ctataagtat ggtcccta 28

<210> 94

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 94

gaaggtgacc aagttcatgc tgccggtgtt tcacagatcc aac 43

<210> 95

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 95

gaaggtcgga gtcaacggat tgccggtgtt tcacagatcc aag 43

<210> 96

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 96

caaatcagcg gttcctccac ccaa 24

Claims (7)

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

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

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

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

3. The primer combination A of claim 1 or the primer combination B of claim 2, which is any one of x1) to x 4): x1) preparing a kit for identifying 236 Chinese cabbage varieties; x2) preparing a kit for identifying the authenticity of 236 Chinese cabbage varieties; x3) identifying 236 Chinese cabbage varieties; x4) identifying the authenticity of 236 Chinese cabbage varieties;

the 236 Chinese cabbage varieties are Xibai No. 10, 87-114, CR117, CR 15, J405, LI-1, SGK1008, SW701, YHBC1511, YHBC1516, Beijing 75, Beijing 80, Beijing great beef heart, Beijing improved 67, Beijing orange No. 2, Beijing orange red heart, Beijing Xiaoza 50, Beijing Xiaoza 51, Beijing Xiaoza 61, Beijing Xiaoza 67, Beijing Xinxin No. 1, Beijing New No. 2, Beijing New No. 3, Beijing New No. 4, Beijing New No. three, Beijing Zao No. 9, Chunbao, Chunbaohuang, Chunbaofeng, Chunbeng 007, Chunyao, Chunyou54, Chunyang, Chunwang, Chunwanfeng, Chunhao No. 1, Degao No. 16, Degao No. 8, Royaluan, Bao, Dongchunfeng, 58, Chunhuang Feng No. 70, Chunhao No. 70, Hua No. 1, Henyuan No. 70, Heiyuan No. 15, Heiyuan No. 3, Heiyuan-1, Heiyuan, He, Jihong 308, Jihong 65, Jihong 82, Jihong doll, Jiaobei No. 7, Jiaohuaxing, Jingguan No. 2, Jinhuayuan No. 19, Jinling, Jinqiu 68, Jinqiu 70, Jinqiu 90, Jinzao 58, Jinbai 56, Jinlv 75, Jinlv 80, Jinqiu 606, Jinqiu 78, Jinhua No. 1, Jinxiu No. 1, Jinjiakucai 30, Jinjiakukukukukulv No. 1, Jinjiangkulv No. 2, Jingqiu No. 1, Jingchun 99, Jingchun CR3, Jingchun white No. 2, Jingchun yellow No. 2, Jingchun child No. 3, Jingchun child No. 4, Jingchun chun child baby vegetable, Jingchun 60, Jingcui 70, Jingcuicui cui 75, Jingchun 988, Jingchun 1, Jingchun 4, Jingchun child No. 65, Jingchun 70, Jingchun child No. 70, Jingchun # 75, Jingchun # 701, Jingchun # 80, Jingchun Lai 6014, Lai Bai Yi, Li Chun I, Liao Bai Ten, Ling Huang, Lu Bai 15, Lu Bai 16, Lu Bai II, Lu Chun Bai 1, Lu Chun Bai I, Lv Jian 60, Lv Jian 85, Lv bamboo shoot 70, Qian Bai 4, Qin Bai II, Qin za 60, Qing Yan Chun Bai 3, Qing Yan Chun Bai I, Qing Qin Bai 1, Qing Yan Si No, Qing Jiang Bai 45 days, Qiu Green 55, Qiu Green 60, Qiu Green 75, Qiu Green 78, Qiu Yu 78, Qiu Zao 60, Qiu 9, Huo 7, Shandi Wang 2, Shandong 5, Shandong Wu No. shan, shan mountain shan 1, Shen Cuiyu Yu, Shen Qing Cao, Shen Qing Dong Bao, Bai Shen 01, Shen Bai 02, GMS Green 85, Shi Lu 50, Cuo Jia Fang, Cuo Hao Jia Kuai No. 1, Qin Kuai, Ji Hao 65, Ji Si Ji, Tianzheng tangerine 65, Tianzheng Yinyun Youyi No. 6, Tianwa, mopidan, Wannong No. two, Wannong No. six, Wangchun, Weibai No. 4, Xibai No. 3, Xibai No. 4, Xibai No. 5, Xibai No. 6, Xibai No. 9, Xibai No. four, Xichunbaibai No. 3, Xiyun iron root, Xiguan, Xiyang 303, Xiao and Qiu, Xiao and Xiao, Xiao za 56, Xiao za 60, Xinxiang Xiao Bao 23, Xin Zao 56, Xinzao 58, Xinzao 89-8, Xinzhong 78, Yanchun, Yi and 0906, Yi and No. 1, preferably Xiao za 56, Zaisheng 5 No. F1, YOu No. 3, Yuxin No. 1, Yuxin No. 2, Yuxin No. 3, Yuxin No. 9, Yuzao 0901, Yuchun Zheng white Zheng Zhen No. 5, Zheng Heng No. 5, Zao No. 6, Zhen Xin No. 6, Zhengzhengdan 23, Zhengza No. 2, Zhengza No. two, Zhengzaza No. 55, Zhengzaza No. 60, Zhongbai 50, Zhongbai 58, Zhongbai 60, Zhongbai 61, Zhongbai 62, Zhongbai 65, Zhongbai 66, Zhongbai No. 76, Zhongbai 78, Zhongbai No. 78, Zhongbai 81, Zhongbai No. 83, Zhongbai No. 85 and Zhongbao 68.

4. A method for identifying which variety of 236 Chinese cabbage varieties to be detected belongs to in claim 3, comprising the following steps: respectively detecting genotypes of Chinese cabbage to be detected and 236 Chinese cabbage varieties based on 32 SNP loci, and then judging as follows: if the genotypes of the Chinese cabbage to be detected based on the 32 SNP loci are completely consistent with the genotypes of a certain variety of the 236 Chinese cabbage varieties based on the 32 SNP loci, the Chinese cabbage to be detected and the Chinese cabbage variety belong to the same variety; if the genotypes of the Chinese cabbage to be detected based on the 32 SNP sites are different from the genotypes of each variety of the 236 Chinese cabbage varieties based on the 32 SNP sites, the varieties of the Chinese cabbage to be detected are different from the varieties of the 236 Chinese cabbage varieties;

the 32 SNP sites are as follows: the BrSNP01 site is the 1795429 th nucleotide on the chromosome 1; the BrSNP02 site is the 4668251 th nucleotide on the chromosome 1; the BrSNP03 site is the 8659686 th nucleotide on the chromosome 1; the BrSNP04 site is the 22523311 th nucleotide on the chromosome 1; the BrSNP05 site is the 157034 th nucleotide on chromosome 2; the BrSNP06 site is the 1758653 th nucleotide on chromosome 2; the BrSNP07 site is the 2421548 th nucleotide on chromosome 2; the BrSNP08 site is the 9141887 th nucleotide on chromosome 2; the BrSNP09 site is the 11181828 th nucleotide on chromosome 2; the BrSNP10 site is the 19966721 th nucleotide on chromosome 2; the BrSNP11 site is the 26263824 th nucleotide on chromosome 2; the BrSNP12 site is the 3067264 th nucleotide on the 3 rd chromosome; the BrSNP13 site is the 15431305 th nucleotide on the 3 rd chromosome; the BrSNP14 site is the 28161015 th nucleotide on the 3 rd chromosome; the BrSNP15 site is the 28629888 th nucleotide on the 3 rd chromosome; the BrSNP16 site is the 1833604 th nucleotide on chromosome 4; the BrSNP17 site is the 13764622 th nucleotide on chromosome 4; the BrSNP18 site is the 1283352 th nucleotide on chromosome 5; the BrSNP19 site is the 4556351 th nucleotide on chromosome 5; the BrSNP20 site is the 24583581 th nucleotide on chromosome 5; the BrSNP21 site is the 9159584 th nucleotide on chromosome 6; the BrSNP22 site is the 16193195 th nucleotide on chromosome 6; the BrSNP23 site is the 23521839 th nucleotide on the 7 th chromosome; the BrSNP24 site is the 2374634 th nucleotide on chromosome 8; the BrSNP25 site is the 3289888 th nucleotide on chromosome 8; the BrSNP26 site is the 12382048 th nucleotide on chromosome 8; the BrSNP27 site is the 14112886 th nucleotide on chromosome 8; the BrSNP28 site is the 1379762 th nucleotide on the 9 th chromosome; the BrSNP29 site is the 3019969 th nucleotide on the 9 th chromosome; the BrSNP30 site is the 277083 th nucleotide on the 10 th chromosome; the BrSNP31 site is the 6477381 th nucleotide on the 10 th chromosome; the BrSNP32 site is the 15835648 th nucleotide on chromosome 10.

5. The method of claim 4, wherein: the steps for detecting the genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on 32 SNP loci are as follows:

(1) respectively taking the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of 236 Chinese cabbage varieties as templates, and respectively adopting the primer group in the primer combination A1 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 Chinese cabbage to be detected and 236 Chinese cabbage varieties based on 32 SNP sites according to the color of the fluorescent signal.

6. The method of claim 4, wherein: the steps of detecting and detecting the genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on 32 SNP loci are as follows:

(1) respectively carrying out PCR amplification by using the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of 236 Chinese cabbage varieties as templates and respectively adopting the primer group in the primer combination B of claim 2 to obtain PCR amplification products;

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

(3) and (3) obtaining genotypes of the Chinese cabbage to be detected and 236 Chinese cabbage varieties based on 32 SNP loci according to the sequencing result obtained in the step (2).

7. A method for identifying which variety of 236 Chinese cabbage varieties to be detected belongs to in claim 3, comprising the following steps:

(1) taking the genomic DNA of the Chinese cabbage to be detected as a template, and respectively adopting the primer group in the primer combination A in the claim 1 to carry out PCR amplification to obtain PCR amplification products; respectively carrying out PCR amplification by using the primer group in the primer combination A in claim 1 by using the genome DNA of each Chinese cabbage variety in the standard Chinese cabbage population as a template to obtain a PCR amplification product; the standard Chinese cabbage variety group consists of 236 Chinese cabbage varieties;

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

Images