CN111235299B

CN111235299B - Method for identifying authenticity of cabbage heart varieties and special SSR primer combination thereof

Info

Publication number: CN111235299B
Application number: CN202010209250.1A
Authority: CN
Inventors: 温常龙; 张建; 张德双; 杨静静; 罗江; 刘慧�
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2021-04-27
Anticipated expiration: 2040-03-23
Also published as: CN111235299A

Abstract

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a method for identifying the authenticity of a cabbage variety and a special SSR primer combination thereof. Identifying SSR sites of the authenticity of the cabbage variety, and performing data mining according to Brassica _ rapa V1.5 of a reference genome of the cabbage and 20 parts of germplasm resource whole genome sequencing data to obtain the SSR sites; the SSR primer combination is selected from: the first SSR primer pair to the twentieth SSR primer pair are respectively used for PCR amplification of the first SSR locus to the twentieth SSR locus, and are preferably the same as SEQ ID NO: 1-40 is 85% -100% homologous to the nucleotide sequence. The method can be used for identifying the authenticity of the full life cycle of the cabbage variety from the seeds and provides technical support for the protection of the cabbage germplasm resources and new varieties.

Description

Method for identifying authenticity of cabbage heart varieties and special SSR primer combination thereof

Technical Field

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a method for identifying the authenticity of a cabbage variety and a special SSR primer combination thereof.

Background

The flowering cabbage is one of the largest-scale leaf vegetable crops planted in south China, and is gradually spread to all over the country in recent years, as well as to the coastal areas of southeast Asia. The cabbage heart is a brassica crop of cruciferae, can be regarded as a variety of a special edible flower bolt of Chinese cabbage, is suitable for being sowed in warm areas in the south, is one of special vegetables in the south of China, can be sowed all the year round, and is introduced and cultivated all the world at present.

In recent years, with the continuous progress of breeding work and the continuous introduction of new breeding technologies, the number of varieties of cabbage heart is increasing and even the well-spraying type is increased, the variety identification should be completed by adopting a DUS test method according to the identification scheme of the current standard, but the DUS identification needs a large amount of manpower and material resources, especially the experience of identification personnel is high, and the existing manpower cannot complete the large-scale identification task. At the moment, a more advanced molecular identification technology should be introduced, the SSR molecular marker technology is the current molecular identification standard for authenticity identification of most crops at the present stage, the identification result is stable and reliable, the identification mode is simple and efficient, and the identification cost is lower compared with that of a DUS test.

However, the molecular research basis of the cabbage heart is weak, and the existing technology related to molecular identification of the cabbage heart cannot meet the requirements at all, so that no method for identifying the authenticity of the cabbage heart variety by utilizing an SSR molecular marker exists in the prior art.

Disclosure of Invention

The invention provides a method for identifying the authenticity of a cabbage heart variety and a special SSR primer combination thereof, which can obtain a stable and efficient identification result: whether the variety of the Chinese flowering cabbage to be detected belongs to one of the standard Chinese flowering cabbage varieties or not, and specifically which one of the standard Chinese flowering cabbage varieties belongs to.

The invention is realized by the following technical scheme:

SSR loci for identifying the authenticity of a variety of flowering cabbage, said SSR loci being selected from any of 1 to 20 of the following first to twentieth SSR loci: a first SSR locus which is positioned at the 16508732-16508752 th chromosome 6 of a Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; a second SSR locus which is positioned at the No. 3 chromosome 29633824 and 29633849 of the Chinese cabbage reference genome or an interspecific homologous genome segment thereof; a third SSR locus which is positioned at the 228915 th and 22898932 th chromosome 5 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof; a fourth SSR locus which is located at the position 20048333-20048347 of the 7 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; a fifth SSR locus which is positioned at the 5 th chromosome 3713470-3713479 position of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; a sixth SSR locus which is positioned at the No. 8 chromosome 3292094-3292113 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; a seventh SSR locus which is positioned at the 24843934-24843945 th chromosome 1 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the eighth SSR locus is positioned at the 24887368-24887377 th chromosome 1 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; a ninth SSR locus located at the 7545410-7545424 th chromosome 10 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof; the tenth SSR locus is positioned at the No. 6297691-6297705 chromosome of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the eleventh SSR locus is positioned at 17713412-17713441 of the 4 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; the twelfth SSR locus is positioned at the No. 10 chromosome 6277936-6277951 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; the thirteenth SSR locus is located at the No. 16090886-16090906 position of the 4 th chromosome of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof; a fourteenth SSR locus which is located at the 14318215-14318228 th chromosome 9 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; a fifteenth SSR locus which is positioned at the 11405907-; a sixteenth SSR locus located at chromosome 9 of the Chinese cabbage reference genome at position 37194484-37194495 or an interspecies homologous genome fragment thereof; the seventeenth SSR locus is positioned at the No. 7 chromosome 22746168-22746179 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof; an eighteenth SSR locus is positioned at the 23245100-23245115 th chromosome 2 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the nineteenth SSR locus is positioned at the 3 rd chromosome 27883995-27884012 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the twentieth SSR locus is positioned at the 6 th chromosome 9752435 and 9752446 positions of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the reference genome is a Chinese cabbage reference genome Brassica oleracea V2.1.

An SSR primer group for identifying the authenticity of the variety of the flowering cabbage, wherein the SSR primer group is used for amplifying the SSR loci respectively, and comprises: a first SSR primer pair to a twentieth SSR primer pair for amplifying the first SSR site to the twentieth SSR site, respectively.

In some embodiments, the SSR primer set wherein said first SSR primer pair is identical to SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the second SSR primer pair is similar to the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the third SSR primer pair is similar to the primer pair shown in SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fourth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fifth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the sixth SSR primer pair is similar to the sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the seventh SSR primer pair is similar to SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the eighth SSR primer pair is similar to SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the ninth SSR primer pair is similar to SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the tenth SSR primer pair is similar to SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SSR primer pair is similar to SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the twelfth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the thirteenth SSR primer pair is similar to SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the fifteenth SSR primer pair is similar to SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and the sixteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SSR primer pair is similar to SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighteenth SSR primer pair is similar to SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the nineteenth SSR primer pair is similar to SEQ ID NO: 37 and SEQ ID NO: 38 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twentieth SSR primer pair is similar to SEQ ID NO: 39 and SEQ ID NO: 40 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, one primer of each pair of said primers is linked to a fluorescent molecule, more preferably said fluorescent molecule is selected from the group consisting of ROX, TAMRA, FAM, HEX.

The SSR kit for identifying the authenticity of the variety of the flowering Chinese cabbage is prepared into a PCR reaction system; the PCR reaction system comprises:

the SSR primer group; preferably, the concentration ratio of the upstream primer and the downstream primer of each pair in the SSR primer group in the system is 1: 1; the final concentration of the upstream primer and the final concentration of the downstream primer in the system are both preferably 0.25 mu mol/L; preferably, the system further comprises: dNTPs: final concentration in the system was 0.15mmol/L each, magnesium chloride: the final concentration in the system is 2.5mmol/L, DNA polymerase: the final concentration in the system was 0.05U/. mu.L, PCR buffer: is prepared from potassium chloride with final concentration of 10-50mmol/L in the system and Tris-HCL (pH7.5-9.0) with final concentration of 1-10mmol/L in the system.

A detection method for identifying the authenticity of a cabbage variety comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of the flowering Chinese cabbage to be detected; step two: and (3) judging the variety of the to-be-detected flowering cabbage:

if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is 0-2, judging the to-be-detected Chinese flowering cabbage and the specified variety of the Chinese flowering cabbage standard variety as a similar variety; if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is more than 2, judging the specified variety of the Chinese flowering cabbage to be different from the specified variety of the Chinese flowering cabbage standard variety; preferably, the result of the determination is obtained from a cluster analysis.

In some embodiments, the step of detecting the SSR locus genotype of the flowering cabbage to be detected comprises the following sub-steps: the method comprises the following steps: respectively taking the genome DNA of the cabbage heart to be detected and the genome DNA of the standard variety of the cabbage heart as templates, and respectively adopting the primer groups in the SSR primer combination to carry out PCR amplification to obtain PCR amplification products; step two is carried out: and detecting the PCR amplification product to obtain the genotypes of the to-be-detected cabbage heart and the standard variety of the cabbage heart based on 20 SSR loci.

In some embodiments, the detection method of the second step comprises: and (3) fluorescent signal detection: detecting a fluorescent signal of the PCR amplification product to obtain genotypes of the to-be-detected cabbage heart and the standard cabbage heart varieties based on the 20 SSR sites; or: detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the cabbage heart to be detected and the standard cabbage heart variety based on the 20 SSR sites.

In some embodiments, the standard flowering cabbage variety is selected from the following 179 flowering cabbage varieties: jinmantian, Hongliang No. 1, Zhuhai dwarf, emerald green No. 1, Cuimei 888, late 80 days, 06 Qiubao No. 1, Lilong dwarf, Liji willow leaf, Dongguan sharp leaf, Changji 70, famous sharp leaf, Jinmiao Meilv 702, Changji, Jiaxin, Huangxin 701, Jinshui, Hongliang No. 2, Hongliang No. 3, Guangxian vegetable field, Jinhan, nongpu's Lbao, Guanlv 60, Shengda New Zealand, Xinnong 50, Xinnong's best heart king, Hongliang 50, Hongliang super, Vanji 50, Japanese tame crisp, nonglong dwarf, Lilong dwarf foot 45, Yigbai, Kunjin, Guangyu vegetable heart, Guangxin No. 3, Dingkun vegetable heart king, vegetable heart No. 4, vegetable heart No. 19, vegetable heart of Zao, Xintian Rough spring No. 6, Jinhan spring plum, Jinhan Han Hao, 8, Konjia japonica, Shuangjia Hao, Shuichun green flower, Shuichong green flower 31, Shuichun green flower Shiwo green tea-flavored oil, Shuichang Shih green flower 31, Shuichong flower, guangliang oil green, Guangliang early oil green, Guangdong Youxian No. 1, Jinhanqiju, New century No. 20, nongxin anti-disease oil green, 09A Bao, Japan oil Mei No. 1, Zhou 008 petiole, Guangdong 1 flower, 288 sweet cabbage heart, Wangzhongwang heat-resistant, Baofeng short foot, 06 autumn Bao-A, Weixing Huangye forty-nine, Jinlida No. 19, nongyue 49, pure Chinese peduncle heart, nongxin Xiagun green, Xintian oil green 25, Weixiong A-3, Green crown 608, New seedling 002, New seedling T28, super Xinxin Huang, Green garden crisp, Xinyuao, Minan Japan, 12 Bao, 13A 9-Bao, 12 Caxin No. 1, 12 Caxin No. 3, Qing A, Qing stemming B, 14A 9-22, 45 Tian Bao Xin Bao, Bai Jian Xin, 13A 9-Bao, 12 Cai No. 1, 12 Cai-Cai, Tai Cai-Tian Cao, Tai Cao Yun Cao, Tai Cao, shennong thick strips, xiang vegetable crisp and tender sijiu, uncover and grind No. 31 heart of green beets, 14 Guangzhou heart of beets, Guangfu No. 2, 06 Qiubao-B, forty-nine heart of yellow beets, Guangmei green, Tianpai, Henlida Aoqiao Haoshao heart of sugar beets, Nonpu oil green 701, Jinhan 70 day heart of vegetables, Jinhan Kong 70 day oil green, Lilong oil green dwarf foot of 70 days, Nanshu super product of 70 day heart of beets, sloping head of 70 day heart of oil green vegetables, Weixing Sanli, Qingcui 701, oil green 702 heart of vegetables, Liji Baisha, specially selected 80 day heart of green vegetables, Weixing 80 day leaf of Shayeqing, Aipu super late heart 808, Aipu super late heart of agricultural, Pilv 80 day heart of oil green beets, emerald green, Severn super 80 day heart of vegetables, Guanhai Jianjian 80 days, Lijian 802, Liguan Ji 805, Yiguan Qing super Xin, Yiwang Xin Yihao 801, Guang Xin Yihao 801, Guangxi Wang Xin Yihao, red jadeite flowering cabbage heart, gold korean red flowering cabbage heart, plump flowering cabbage heart 2, maoxing flowering cabbage, 15 guangzhou flowering cabbage-10, 15, 80, 62036x62013 raac0.5xc2030, PI175054Aaa-1 BAREPOP 15376, Aaa-1,1976FAST flower PO, White Stemmed Taitsai (germany), central lichen, エウサィタイ, chu brand new red moss, early White moss, red autumn flowering cabbage, hong jinqinbao 49 flowering cabbage, hong kong cabbage heart, xiang bao cabbage 60 days cabbage heart, late cabbage 2, eighty day rape heart, baoqing 50 days cabbage, jinqiong red second number, late cabbage moss, chi cabbage heart 019 (ping) cabbage, 20 cabbage (ping) x cabbage, 14A-2 x mustard cabbage, mustard heart (si), swamp mustard cabbage (cmi heart, gachc 4), swamp cabbage heart (cmi heart) (cmi heart, 4) mustard heart (cmi heart), swamp cabbage heart (cmi) 3), CMS8. flowering cabbage (purple), (07-882 × abc3-1)2, 019 flowering cabbage, little cauliflower, 16E 9-16- (C-24), 17E 9-22- (C-16E 9-16- (C-24 × 16E 9-13) - (C-21), 16E 9-49- (C-24 × 16E 9-22) - (C-21), Pistacia oleracea 77-44 (Japan), Qianjin Jingcai (Japan), Kadsura japonica (Japan), Undaria natans (Japan), and Pinus palustris (Japan).

A detection method for identifying whether the types of Chinese flowering cabbage are the same or not is disclosed, wherein the Chinese flowering cabbage to be detected is Chinese flowering cabbage of two unknown types; the detection method comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of the flowering cabbage to be detected; step two: and judging whether the varieties of the to-be-detected flowering Chinese cabbage are the same:

if the number of the different loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci is 0-2, judging the to-be-detected Chinese flowering cabbage to be a similar variety; and if the difference locus of the cabbage heart to be detected based on the genotypes of the 20 SSR loci is more than 2, judging the cabbage heart to be detected to be different varieties.

The SSR locus, or the SSR primer combination, or the SSR kit of 4, or the detection method, is applied to the following X1 or X2 or X3: x1: identifying whether the variety of the cabbage heart to be detected belongs to one of standard cabbage heart varieties; x2: identifying the variety of the to-be-detected Chinese flowering cabbage as any one of standard Chinese flowering cabbage varieties; x3: and identifying whether the cabbage heart samples to be detected are the same varieties.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides an SSR molecular marker identification method which can quickly, accurately and stably detect variety authenticity. According to the invention, data mining is carried out according to the reference genome of the Chinese cabbage and a large amount of re-sequencing data, thousands of candidate SSR markers are selected, and further optimized screening is carried out according to various conditions, so that a set of molecular markers which are stable and reliable in identification result, and rapid and efficient in identification means are finally obtained. The SSR primer combination can be used for carrying out early identification on the cabbage variety in the seed or seedling stage, and also can carry out authenticity identification of the full life cycle from the seed, thereby ensuring the authenticity of the variety, solving the problems of homonymy and heteronymy in the seed and seedling market in China, practically protecting the rights and interests of producers and breeders, and providing powerful technical support for protecting the cabbage germplasm resources and new varieties.

2. The method provided by the invention can be used for identifying that: whether the variety of the Chinese flowering cabbage to be detected belongs to one of the standard Chinese flowering cabbage varieties or not, and specifically which one of the standard Chinese flowering cabbage varieties belongs to. Therefore, the method can be used for identifying the authenticity of the known variety and also can be used for identifying the authenticity of the unknown cabbage variety with the standard sample; it is also possible to identify whether two unknown varieties belong to similar varieties.

3. 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.

4. Compared with other related technical schemes of SSR in the prior art, the invention has the following differences: firstly, plant large-scale sequencing has been in the rise for few years, most of the prior SSR related technologies do not have reference genome as data base, so that the SSR related technologies can only be selected blindly and randomly, the selected markers are completely healthy, and the potential identification capability of the SSR related technologies cannot be mentioned at all. The method is essentially different from the prior art that marks used by others in literature are cited or marks are selected from some free databases. The invention adopts the variety provided by the breeding expert as the verification material, so that the identification capability of the SSR marker is fully guaranteed. Because of the data mining method, the selected SSR marker can very effectively represent whole genome information, and the adopted cabbage heart variety materials cover all types of commercially available varieties, the selected SSR marker has very strong potential capability of identifying unknown cabbage heart varieties.

5. The invention also has the following characteristics: the method is rapid, when the variety identification is carried out by the method, the DNA directly extracted from seedlings and even seeds can be used for detection, and the method is different from the method that the identification is carried out only by mature plants in DUS identification, and the identification time is shortened from several months to several hours. Compared with a phenotype identification method, the identification method based on DNA detection is not influenced by external environment, cannot change due to the change of environmental conditions, and has stable and reliable result. And thirdly, the identification is carried out by the method of the invention in each phenological period of the heart of the cabbage, and the results are stable and consistent. And fourthly, the 20 SSR loci of the invention are detected by Target-seq sequencing, 3730 fluorescent capillary detection and polyacrylamide gel electrophoresis, and the multi-platform detection results are highly consistent, thereby fully proving the reliability of each SSR locus of the invention. Operation of the invention does not require long experience accumulation as compared to the DUS test.

Drawings

FIG. 1 is a cluster plot of 179 tested flowering cabbage varieties established on 20 primer sets in example 1.

FIGS. 2-21 are graphs showing the SSR typing effect of 20 primer sets in example 2 in part of the tested cabbage heart varieties.

Wherein, fig. 2: the primer is BcSSr067, and the adopted variety is Weixing Sanlin; FIG. 3: the primer is BcSSR039, and the adopted variety is Guangfu 2; FIG. 4: the primer is BcSSR057, and the adopted variety is uncovered and researched No. 31 green beet heart; FIG. 5: the primer is BcSSR083, and the adopted variety is Changhe No. 1; FIG. 6: the primer is BcSSR051, and the adopted variety is CMS, cabbage heart (purple) No. 1; FIG. 7: the primer is BcSSR090, and the adopted variety is red-leaf emerald green cauliflower heart; FIG. 8: the primer is BcSSR007, and the adopted variety is Guangmuimei green; FIG. 9: the primer is BcSSR008, and the adopted variety is crispy, tender and forty-nine of Xiang vegetables; FIG. 10: the primer is BcSSR113, and the adopted variety is Cochinchong Dongguan sloping head; FIG. 11: the primer is BcSSR016, and the adopted variety is verdant 701; FIG. 12: the primer is BcSSR045, and the adopted variety is Jinmantian; FIG. 13: the primer is BcSSR111, and the adopted variety is a south vegetable top-grade 70-day beet heart; FIG. 14: the primer is BcSSR044, and the adopted variety is Weixing Sanlin; FIG. 15: the primer is BcSSR099, and the adopted variety is ChangHe No. 2; FIG. 16: the primer is BcSSR092, and the adopted variety is emerald 80-day flowering cabbage; FIG. 17: the primer is BcSSR107, and the adopted variety is New century No. 20; FIG. 18: the primer is BcSSR085, and the adopted variety is Weixing late flower; FIG. 19: the primer is BcSSR024, and the adopted variety is green flowering cabbage; FIG. 20: the primer is BcSSR127, and the adopted variety is 15 large 80; FIG. 21: the primer is BcSSR066, and the adopted variety is 70 days oil green of Jinhanhong variety. FIG. 22 is a difference labeling chart of SSR marker number (i.e., SSR locus number) and the discrimination of 179 varieties of Chinese cabbage to be tested in example 2.

Detailed Description

The definition is as follows:

authenticity of the variety of the flowering cabbage: essentially refers to the real correspondence of a cabbage variety to its genetic background; in actual work, whether a certain variety to be tested has authenticity means whether the variety to be tested conforms to a file record (such as a variety specification, a label and the like).

Interspecies homologous genomic fragments: the gene group is a gene group fragment which is homologous with the Chinese cabbage reference genome Brassica _ rapa V1.5 in the Chinese cabbage of other varieties except the Chinese cabbage reference genome Brassica _ rapa V1.5. For example, for a specific genome segment, the same genes exist in 179 standard varieties of the invention as in Brassica _ rapa V1.5, the chinese cabbage reference genome.

In a first aspect, the invention provides SSR loci for identifying the authenticity of a cabbage variety, wherein the SSR loci are respectively located in the genome of a cabbage, the number of the SSR loci is 20, and 1 or more SSR loci can be selected, and specific information is shown in table 1.

The SSR site is selected from any 1 to 20 of the following first to twentieth SSR sites: a first SSR locus which is positioned at the 16508732-16508752 th chromosome 6 of a Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

a second SSR locus which is positioned at the No. 3 chromosome 29633824 and 29633849 of the Chinese cabbage reference genome or an interspecific homologous genome segment thereof;

a third SSR locus which is positioned at the 228915 th and 22898932 th chromosome 5 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof;

a fourth SSR locus which is located at the position 20048333-20048347 of the 7 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

a fifth SSR locus which is positioned at the 5 th chromosome 3713470-3713479 position of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

a sixth SSR locus which is positioned at the No. 8 chromosome 3292094-3292113 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

a seventh SSR locus which is positioned at the 24843934-24843945 th chromosome 1 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the eighth SSR locus is positioned at the 24887368-24887377 th chromosome 1 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

a ninth SSR locus located at the 7545410-7545424 th chromosome 10 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof;

the tenth SSR locus is positioned at the No. 6297691-6297705 chromosome of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the eleventh SSR locus is positioned at 17713412-17713441 of the 4 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

the twelfth SSR locus is positioned at the No. 10 chromosome 6277936-6277951 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

the thirteenth SSR locus is located at the No. 16090886-16090906 position of the 4 th chromosome of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof;

a fourteenth SSR locus which is located at the 14318215-14318228 th chromosome 9 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

a fifteenth SSR locus which is positioned at the 11405907-;

a sixteenth SSR locus located at chromosome 9 of the Chinese cabbage reference genome at position 37194484-37194495 or an interspecies homologous genome fragment thereof;

the seventeenth SSR locus is positioned at the No. 7 chromosome 22746168-22746179 of the Chinese cabbage reference genome or an interspecific homologous genome fragment thereof;

an eighteenth SSR locus is positioned at the 23245100-23245115 th chromosome 2 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the nineteenth SSR locus is positioned at the 3 rd chromosome 27883995-27884012 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the twentieth SSR locus is positioned at the 6 th chromosome 9752435 and 9752446 positions of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the Chinese cabbage reference genome Brassica _ rapa V1.5.

In a second aspect, the invention provides an SSR primer group for identifying the authenticity of the variety of Chinese flowering cabbage, and a PCR amplification product based on the SSR locus can be obtained through PCR amplification reaction.

The SSR primer combination is selected from: and the first SSR primer pair to the twentieth SSR primer pair are respectively used for PCR amplification of the first SSR locus to the twentieth SSR locus. The first SSR primer pair is similar to SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second SSR primer pair is similar to SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the third SSR primer pair is similar to SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourth SSR primer pair is similar to SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fifth SSR primer pair is similar to SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the sixth SSR primer pair is similar to SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the seventh SSR primer pair is similar to SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighth SSR primer pair is similar to SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the ninth SSR primer pair is similar to SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the tenth SSR primer pair is similar to SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SSR primer pair, together with SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twelfth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirteenth SSR primer pair, as described above, is identical to SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourteenth SSR primer pair is a primer pair that binds to SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the fifteenth SSR primer pair, together with SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the sixteenth SSR primer pair is similar to SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SSR primer pair, in combination with SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighteenth SSR primer pair is similar to SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the nineteenth SSR primer pair is similar to SEQ ID NO: 37 and SEQ ID NO: 38 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twentieth SSR primer pair is similar to SEQ ID NO: 39 and SEQ ID NO: 40 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%.

In a preferred embodiment, the SSR primer combination is selected from one or more of primer sets 01-20; the DNA sequence information of the primer group 01-20 is shown in a sequence table SEQ ID: 1-40, see table 2.

In the primer set, the 5' end of the upstream primer can be provided with a fluorescent label sequence for fluorescent PCR detection, for example, the fluorescent signal of the FAM fluorescent label sequence is blue, and the fluorescent signal of the HEX fluorescent label sequence is green.

In a third aspect, the invention provides an SSR kit for identifying the authenticity of the variety of the flowering cabbage, wherein an SSR reagent is prepared by PCR

A reaction system, preferably comprising:

in the SSR primer group, the ratio of the final concentration of the upstream primer to the final concentration of the downstream primer is 1: 1.

In a fourth aspect, the invention provides a detection method for identifying the authenticity of a cabbage heart variety, which comprises the following steps:

the method comprises the following steps: and detecting the SSR locus genotype of the flowering cabbage to be detected.

The method comprises the following steps: respectively taking the genome DNA of the cabbage heart to be detected and the genome DNA of the standard variety of the cabbage heart as templates, and respectively adopting the primer groups in the SSR primer combination to perform PCR amplification reaction to obtain PCR amplification products;

step two is carried out: and detecting the PCR amplification product to obtain the genotypes of the cabbage heart and the cabbage heart standard variety to be detected based on 20 SSR sites.

The detection may be a fluorescence signal detection: detecting the fluorescent signal of the PCR amplification product to obtain genotypes of the cabbage heart to be detected and the standard cabbage heart variety based on the 20 SSR loci;

the detection can also be the detection of amplified product fragments: and (3) detecting the fragment size of the PCR amplification product by utilizing capillary electrophoresis to obtain the genotypes of the to-be-detected Chinese flowering cabbage and the standard Chinese flowering cabbage variety based on the 20 SSR sites.

Step two: and (3) judging the variety of the flowering cabbage to be detected:

the following results are obtained by clustering analysis of the genotypes of the to-be-detected Chinese flowering cabbage and the standard Chinese flowering cabbage variety based on the 20 SSR loci:

if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is 0-2, the Chinese flowering cabbage to be detected and the variety of the Chinese flowering cabbage standard variety belong to similar varieties;

and if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is more than 2, judging the specified variety of the Chinese flowering cabbage to be different from the specified variety of the Chinese flowering cabbage standard variety.

The procedure of the PCR amplification reaction is preferably:

pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and reducing the temperature by 0.8 ℃ per cycle for 12 cycles; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 25 cycles; final extension at 72 ℃ for 10 min. The amplification products were stored at-20 ℃ or on ice prior to electrophoresis.

The standard Chinese flowering cabbage variety comprises the following 179 Chinese flowering cabbage varieties:

jinmantian, Hongliang No. 1, Zhuhai dwarf, emerald green No. 1, Cuimei 888, late 80 days, 06 Qiubao No. 1, Lilong dwarf, Liji willow leaf, Dongguan sharp leaf, Changji 70, famous sharp leaf, Jinmiao Meilv 702, Changji, Jiaxin, Huangxin 701, Jinshui, Hongliang No. 2, Hongliang No. 3, Guangxian vegetable field, Jinhan, nongpu's Lbao, Guanlv 60, Shengda New Zealand, Xinnong 50, Xinnong's best heart king, Hongliang 50, Hongliang super, Vanji 50, Japanese tame crisp, nonglong dwarf, Lilong dwarf foot 45, Yigbai, Kunjin, Guangyu vegetable heart, Guangxin No. 3, Dingkun vegetable heart king, vegetable heart No. 4, vegetable heart No. 19, vegetable heart of Zao, Xintian Rough spring No. 6, Jinhan spring plum, Jinhan Han Hao, 8, Konjia japonica, Shuangjia Hao, Shuichun green flower, Shuichong green flower 31, Shuichun green flower Shiwo green tea-flavored oil, Shuichang Shih green flower 31, Shuichong flower, guangliang oil green, Guangliang early oil green, Guangdong Youxian No. 1, Jinhanqiju, New century No. 20, nongxin anti-disease oil green, 09A Bao, Japan oil Mei No. 1, Zhou 008 petiole, Guangdong 1 flower, 288 sweet cabbage heart, Wangzhongwang heat-resistant, Baofeng short foot, 06 autumn Bao-A, Weixing Huangye forty-nine, Jinlida No. 19, nongyue 49, pure Chinese peduncle heart, nongxin Xiagun green, Xintian oil green 25, Weixiong A-3, Green crown 608, New seedling 002, New seedling T28, super Xinxin Huang, Green garden crisp, Xinyuao, Minan Japan, 12 Bao, 13A 9-Bao, 12 Caxin No. 1, 12 Caxin No. 3, Qing A, Qing stemming B, 14A 9-22, 45 Tian Bao Xin Bao, Bai Jian Xin, 13A 9-Bao, 12 Cai No. 1, 12 Cai-Cai, Tai Cai-Tian Cao, Tai Cao Yun Cao, Tai Cao, shennong thick strips, xiang vegetable crisp and tender sijiu, uncover and grind No. 31 heart of green beets, 14 Guangzhou heart of beets, Guangfu No. 2, 06 Qiubao-B, forty-nine heart of yellow beets, Guangmei green, Tianpai, Henlida Aoqiao Haoshao heart of sugar beets, Nonpu oil green 701, Jinhan 70 day heart of vegetables, Jinhan Kong 70 day oil green, Lilong oil green dwarf foot of 70 days, Nanshu super product of 70 day heart of beets, sloping head of 70 day heart of oil green vegetables, Weixing Sanli, Qingcui 701, oil green 702 heart of vegetables, Liji Baisha, specially selected 80 day heart of green vegetables, Weixing 80 day leaf of Shayeqing, Aipu super late heart 808, Aipu super late heart of agricultural, Pilv 80 day heart of oil green beets, emerald green, Severn super 80 day heart of vegetables, Guanhai Jianjian 80 days, Lijian 802, Liguan Ji 805, Yiguan Qing super Xin, Yiwang Xin Yihao 801, Guang Xin Yihao 801, Guangxi Wang Xin Yihao, red jadeite flowering cabbage heart, gold korean red flowering cabbage heart, plump flowering cabbage heart 2, maoxing flowering cabbage, 15 guangzhou flowering cabbage-10, 15, 80, 62036x62013 raac0.5xc2030, PI175054Aaa-1 BAREPOP 15376, Aaa-1,1976FAST flower PO, White Stemmed Taitsai (germany), central lichen, エウサィタイ, chu brand new red moss, early White moss, red autumn flowering cabbage, hong jinqinbao 49 flowering cabbage, hong kong cabbage heart, xiang bao cabbage 60 days cabbage heart, late cabbage 2, eighty day rape heart, baoqing 50 days cabbage, jinqiong red second number, late cabbage moss, chi cabbage heart 019 (ping) cabbage, 20 cabbage (ping) x cabbage, 14A-2 x mustard cabbage, mustard heart (si), swamp mustard cabbage (cmi heart, gachc 4), swamp cabbage heart (cmi heart) (cmi heart, 4) mustard heart (cmi heart), swamp cabbage heart (cmi) 3), CMS8. flowering cabbage (purple), (07-882 × abc3-1)2, 019 flowering cabbage, little cauliflower, 16E 9-16- (C-24), 17E 9-22- (C-16E 9-16- (C-24 × 16E 9-13) - (C-21), 16E 9-49- (C-24 × 16E 9-22) - (C-21), Pistacia oleracea 77-44 (Japan), Qianjin Jingcai (Japan), Kadsura japonica (Japan), Undaria natans (Japan), and Pinus palustris (Japan).

In a fifth aspect, the invention provides a detection method for identifying whether the cabbage heart varieties are the same.

Wherein, the cabbage heart to be detected is the cabbage heart of two unknown varieties;

the detection method comprises the following steps:

the method comprises the following steps: detecting the genotypes of the 20 SSR loci of the flowering Chinese cabbage to be detected; the detection method is as described in the fourth aspect.

Step two: and (3) judging whether the varieties of the cabbage heart to be detected (two unknown varieties of the cabbage heart) are the same:

if the number of the difference loci of the two unknown cabbage heart varieties based on the genotypes of the 20 SSR loci is 0-2, judging the two unknown cabbage heart varieties as similar varieties;

and if the difference loci of the two unknown cabbage heart varieties based on the genotypes of the 20 SSR loci are more than 2, judging the two unknown cabbage heart varieties to be different varieties.

In a sixth aspect, the invention provides the above SSR locus, SSR primer combination, SSR kit, and the detection method, and uses thereof in the following X1 or X2 or X3:

x1: identifying whether the variety of the cabbage heart to be detected belongs to one of standard cabbage heart varieties;

x2: identifying the variety of the to-be-detected Chinese flowering cabbage as any one of standard Chinese flowering cabbage varieties;

x3: and identifying whether the cabbage heart samples to be detected are the same varieties.

X1, X2 and X3 all belong to the application of identifying the authenticity of the cabbage variety.

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.

Example 1

Acquisition of SSR primer combination for identifying authenticity of cabbage variety

Discovery of one, 20 SSR sites: the loci are obtained by carrying out data mining on Brassica _ rapa V1.5 serving as a reference genome and 20 parts of germplasm whole genome re-sequencing data of a closely related Chinese cabbage.

1. The invention is different from other molecular marker identification methods, and relates to a method for randomly and blindly selecting SSR markers. The method is characterized in that different detection means such as Target-Seq simplified sequencing technology, ABI3730 fluorescence capillary electrophoresis and the like are used for verification, and finally 20 SSR sites with the strongest identification ability are selected as the SSR sites used in the method by combining 20 parts of actual materials.

The invention discovers and obtains 20 SSR loci for the first time based on the re-sequencing data of 20 parts of cabbage heart representative resources. The 20 parts of Chinese cabbage have rich resource types, cover the main ecological types and the agronomic traits of the Chinese cabbage in the market, embody germplasm representativeness as much as possible and have higher genetic diversity.

2. The screening of SSR loci of the invention comprises the following steps:

firstly, according to the re-sequencing result of 20 parts of cabbage heart materials, 668325 SSR sites are found in total by comparing with a cabbage reference genome Brassica _ rapa V1.5 reference genome sequence, 84267 SSR sites are left after the base repetition times are filtered to be less than 5 times, 38435 SSR sites are left after the deletion proportion is more than 30% are filtered, 6777 SSR sites are left after the MAF is less than 0.1 are filtered, and 1254 SSR sites are left after sites containing indels and SNPs in a 150bp sequence of two wings are filtered. Then, data dimensionality reduction is carried out on the 1254 SSR loci, 117 most representative SSR loci are selected, and PCR primer design is carried out on the loci.

After the 117 SSR primers are used for amplifying 179 varieties (see example 2), the SSR sequence information of the varieties is obtained by a simplified sequencing technology, and data analysis is carried out, so that the residual 103 SSR loci with deletion ratio of more than 10% of SSR locus data are removed as alternative loci. And then selecting 20 SSR sites by using a minimarker algorithm, wherein the algorithm preferentially considers the identification capability of the SSR sites on 179 cabbage varieties, comprehensively considers the polymorphism of the SSR sites, and uniformly distributes the SSR sites on 10 pairs of chromosomes and other conditions.

In order to determine the reliability of the discrimination ability of the 20 SSR loci, the inventors redesign primers suitable for fluorescence capillary electrophoresis detection, perform PCR amplification on 179 cabbage heart varieties by using the 20 pairs of primers, and perform fragment length detection by using an AB 3730 fluorescence capillary electrophoresis system, and the results prove that the selected 20 SSR loci have very high discrimination ability and reliability.

3. Specifically, the screening criteria for SSR sites are as follows: SSR sites which have uniform positions, good polymorphism, small heterozygosity, MAF >0.3, good PCA clustering effect and high discrimination and have 150bp sequence conservation (no InDel, no SNP and no other SSR) on both wings are selected in the whole genome range. The basic information of the 20 SSR sites is detailed in Table 1. The position of the SSR locus on the chromosome and the motif information are determined based on the Chinese cabbage reference genome sequence comparison, the version number of the Chinese cabbage Brassica _ rapa reference genome sequence is V1.5, and the website is referred to as: website address:

http://brassicadb.org/brad/datasets/pub/BrassicaceaeGenome/Brassica_rapa/V1.0/Bra_Chromoso me_V1.5/

the PIC values and the major allelic variant amplification lengths in Table 1 were obtained for 179 varieties according to example 2.

TABLE 1.20 basic information of SSR loci

Second, obtaining SSR primer combination for identifying authenticity of cabbage variety

Based on the 20 SSR sites found in step one, the inventors of the present invention developed an SSR primer combination for identifying the authenticity of a cabbage variety with a higher amount of polymorphism information (i.e., PIC value, which refers to the value of one marker for detecting polymorphisms in a population; PIC value depends on the number of detected alleles and their frequency distribution of alleles; PIC value is equal to 1 minus the sum of the squares of all allele frequencies). Primers are designed based on the upstream and downstream sequences of the SSR locus in the version number V1.5 of the Chinese cabbage Brassica _ rapa reference genome sequence, an SSR primer combination consists of 20 primer groups, and the name of each primer group is shown in the 2 nd column in the table 2. Each primer group consists of 2 primer sequences and is used for amplifying one SSR locus. The nucleotide sequences of the individual primers in the 20 primer sets are shown in Table 2.

TABLE 2.20 information on the primer sets

Example 2

This example is a validation test of the SSR primer combination developed in example 1.

The 179 tested flowering Chinese cabbage varieties in this example are all common fine varieties or partially foreign introduced varieties, and the sources are all vegetable center germplasm banks of agriculture and forestry academy of sciences in Beijing. The specific varieties are as follows:

1. acquisition of genomic DNA of the cabbage variety to be tested

Genome DNA of 179 leaves of the tested flowering cabbage variety (mixed true leaves of 30 seeds, namely true leaves of 30 seeds of each variety, namely that the true leaves of 30 different plants of the same variety are mixed) is extracted by adopting a CTAB method, so that the genome DNA of the tested flowering cabbage variety is obtained.

The CTAB method is specifically operated as follows:

respectively picking up the leaves of the 149 varieties in the seedling stage, and dehydrating in a freeze dryer (CoolSafe 55-4); then, the leaves were crushed with a high throughput grinder (Geno/Grind6875), 200mg of dry leaf powder was taken, 800. mu.L of CTAB extract (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1% PVP-40, 0.2% beta-mercaptoethanol) was added thereto, mixed well, washed in a water bath at 65 ℃ for 30min, added with chloroform/isoamyl alcohol (v: 24: 1) of equal volume, centrifuged at 10000rpm/min for 10min, the supernatant was aspirated and transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added, mixed well by gentle inversion, left at 20 ℃ for 30min and then centrifuged at 4 ℃ for 10min at 12,000 r/min. Discarding supernatant, washing with 70% ethanol solution for 2 times, naturally drying, and adding 100 μ L ddH₂And dissolving DNA by O to obtain the genomic DNA of the cabbage heart variety to be tested, and detecting the concentration for later use at 4 ℃.

The quality and the concentration of the genome DNA of the tested cabbage heart variety both need to meet the PCR requirement, and the standard of reaching the standard is as follows: 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 cabbage heart variety is 30-50 ng/. mu.L.

2. And respectively taking genome DNAs of 179 cabbage heart varieties to be tested as templates, and respectively adopting 20 primer groups to carry out PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name and the primer containing "R" in the name was 1: 1.

The reaction system comprises:

the ratio of the concentration of the forward primer (named as "F") to the concentration of the reverse primer (named as "R") in the system was 1: 1.

The reaction procedure is as follows: pre-denaturation: 5min at 94 ℃; amplification: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and reducing the temperature by 0.8 ℃ per cycle for 12 cycles; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 25 cycles; final extension: 10min at 72 ℃. The resulting amplification product was stored at 4 ℃ before electrophoresis.

3. Fluorescence capillary electrophoresis

After step 2 is completed, a plurality of primer combinations can be selected for electrophoresis according to different instruments according to different sizes of the SSR molecular marker amplified fragments. According to the predetermined combined primers, respectively taking different fluorescence-labeled amplification products of the same combined primer with the same volume, diluting TAMRA by 50 times, and fully and uniformly mixing other fluorescence products after diluting by 100 times. Pipette 1. mu.L of the mixture and add to a well dedicated to the sample loading plate of the DNA analyzer. Adding 0.1 μ L molecular weight internal standard and 8.9 μ L deionized formamide into each well, denaturing at 95 deg.C for 5min in PCR instrument, taking out, immediately placing in-20 deg.C refrigerator or ice, and cooling for 5 min. After being instantaneously centrifuged for 10s, the mixture was placed on a DNA analyzer. The test is started.

Partial results are shown in FIGS. 2-21. The result shows that each primer group can obtain good typing effect in the tested cabbage heart varieties.

4. Cluster analysis

And (3) carrying out cluster analysis on the 179 varieties of the cabbages to be tested by utilizing MEGA7 software according to the genotypes of the 179 varieties of the cabbages to be tested based on the 20 SSR loci.

The clustering plots for the 179 tested flowering cabbage varieties established on the 20 primer sets are shown in FIG. 1. The result shows that 20 primer groups can completely distinguish 179 varieties of flowering cabbage to be tested. Therefore, the SSR primer combination developed in the example 1 can be applied to the construction of the DNA fingerprint database of the cabbage heart variety and the identification of the 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 SSR markers (i.e., the number of primer sets) and the discrimination rate of 179 cabbage varieties to be tested.

The number of difference markers compared and counted between 179 varieties in pairs as shown in FIG. 22, wherein the number of results compared between pairs is C² ₁₇₉179 × 178 ÷ 2 ═ 15931; in these 15931 results, the number of differential sites was about 2% of the total 2, the number of differential sites was about 4% of the total 3, the number of differential sites was about 10% of the total 4, the number of differential sites was about 16% of the total 5, the number of differential sites was about 18.5% of the total 6, the number of differential sites was about 18% of the total 7, the number of differential sites was about 14% of the total 8, the number of differential sites was about 8.5% of the total 9, the number of differential sites was about 5% of the total 10, the number of differential sites was about 2.5% of the total 11, the number of differential sites was about 1% of the total 12, the number of differential sites was about 0.5% of the total 13, and the number of differential sites was about 0% of the total 14-20, which indicates that polymorphisms were good in 179 varieties using these markers; the discrimination rate of 20 primer groups (namely the number of 20 SSR markers) in 179 varieties of Chinese cabbages to be tested reaches 100 percent.

Example 3

The embodiment is a method for detecting whether a variety of a to-be-detected Chinese flowering cabbage belongs to one of 179 varieties of Chinese flowering cabbage to be detected, the variety of the to-be-detected Chinese flowering cabbage is unknown, and whether the variety of the to-be-detected Chinese flowering cabbage is one of the 179 varieties needs to be obtained through the detection method of the embodiment.

1. Obtaining genome DNA of variety of flowering cabbage to be detected

The leaf of the variety of the flowering Chinese cabbage to be detected is taken from the experimental base of the vegetable research center of the academy of agriculture and forestry, Beijing.

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

2. SSR primer and configuration of PCR reaction system

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

3. Fluorescence capillary electrophoresis detection

And taking a PCR product of the cabbage heart variety to be detected.

Respectively comparing the fragment sizes of 20 SSR amplification products of the cabbage heart varieties to be detected with 20 SSR sites of 179 cabbage heart varieties to be detected, counting the number of difference sites of the cabbage heart varieties to be detected and 20 standard cabbage heart varieties, and then judging as follows:

if the number of the different position points of the variety of the to-be-detected Chinese flowering cabbage and a standard Chinese flowering cabbage is more than 2, judging the variety of the to-be-detected Chinese flowering cabbage and the standard Chinese flowering cabbage to be different Chinese flowering cabbage; the greater the number of differential sites, the more distant the genetic relationship.

If the number of the different sites of the variety of the cabbage heart to be detected and a standard variety of the cabbage heart is 0-2, the variety of the cabbage heart to be detected and the standard variety of the cabbage heart are judged to be similar.

The result shows that the number of the different sites of the to-be-detected Chinese flowering cabbage variety and the 179 tested Chinese flowering cabbage variety is more than 2, so that the to-be-detected Chinese flowering cabbage variety does not belong to any one of the 179 tested Chinese flowering cabbage varieties, namely the to-be-detected Chinese flowering cabbage variety is not one of the 179 tested Chinese flowering cabbage varieties.

Example 4

In the embodiment, the variety of the flowering Chinese cabbage is judged by comparing the sizes of the fragments through capillary electrophoresis instead of adopting a fluorescent signal.

In this case, the ABI3730 fluorescent capillary detection platform is used as a reference, and if other platforms are used, corresponding adjustment is performed according to the operation requirements of the equipment.

According to the different sizes of the SSR molecular marker amplified fragments, a plurality of primer combinations can be selected for electrophoresis according to different instruments.

S1: according to the predetermined combined primers, respectively taking different fluorescence-labeled amplification products of the same combined primer with the same volume, diluting TAMRA by 50 times, and fully and uniformly mixing other fluorescence products after diluting by 100 times. Pipette 1. mu.L of the mixture and add to a well dedicated to the sample loading plate of the DNA analyzer. Adding 0.1 μ L molecular weight internal standard and 8.9 μ L deionized formamide into each well, denaturing at 95 deg.C for 1min in PCR instrument, taking out, immediately placing on ice, and cooling for 5 min. After being instantaneously centrifuged for 10s, the mixture was placed on a DNA analyzer.

S2: the ABI3730 DNA analyzer is opened and the instrument operating status and reagent status are checked. The loading plate with the sample is placed on the sample holder base, the buffer plate with the electrode buffer solution is placed on the buffer plate holder base, the data collection software is opened, and the operation is carried out according to the instruction manual of the DNA analyzer. The DNA analyzer will run the parameters automatically and save the raw data for electrophoresis. The excitation wavelength and color used by the fluorescent primers are detected by referring to default values of an instrument.

S3: exporting an electrophoresis original data file, and adopting data analysis software to perform data discrimination according to the following steps: presetting SSR primer names, fluorescence categories, molecular weight internal standards and amplification fragment sizes of corresponding primers in data analysis software; importing the electrophoresis original data file into analysis software, and selecting panel, molecular weight internal standard, Bin, quality control parameters and the like for analysis; the analysis software assigns a color mark to the detection quality for scoring, green indicates that the quality is reliable without intervention, red indicates that the quality is not over or does not fall within a specified segment size range, and yellow indicates that the original image needs to be checked for confirmation in question.

S4: the amplified fragment size was read after calibrating the data deviation between different electrophoresis plates by using a standard sample and a reference sample (a small amount of control was selected according to the primers) which were tested simultaneously. If the screened specific peak falls into the specified fragment size range, directly reading the size of the amplified fragment; if the peaks are not within the predetermined range, the data can be read by shifting the whole of the peak as far as possible within the peak setting range.

S5: respectively comparing the fragment sizes of 20 SSR amplification products of the cabbage heart varieties to be detected with 20 SSR sites of 179 cabbage heart varieties to be detected, counting the number of difference sites of the cabbage heart varieties to be detected and 20 standard cabbage heart varieties, and then judging as follows:

If the number of the difference sites between the variety of the cabbage heart to be detected and a standard variety of the cabbage heart is 0-2, judging the variety of the cabbage heart to be detected and the standard variety of the cabbage heart to be similar.

Example 5

In this embodiment, it is detected whether two unknown types of flowering cabbage are the same type, and the types of flowering cabbage to be detected in this embodiment are two unknown types of flowering cabbage.

1. Obtaining genome DNA of variety of flowering cabbage to be detected

And (3) respectively taking the leaves of two to-be-detected cabbage heart varieties, and respectively extracting the genomic DNAs of the two to-be-detected cabbage heart varieties by adopting a CTAB method, wherein the operation of the CTAB method is shown in example 2, so as to obtain the genomic DNAs of the to-be-detected cabbage heart varieties.

2. SSR primer and configuration of PCR reaction system

3. Fluorescence capillary electrophoresis detection and data recording

After the PCR reaction in step 2 is completed, performing grouping electrophoresis on the PCR product according to the difference of the main equal-length non-variant amplification lengths of different primers by combining detection equipment, detecting the fragment size of the PCR product and recording data, wherein the recording method comprises the following steps: if only 1 allelic variation occurs in a certain position of a sample, and the size is 150bp, the genotype of the main allelic variation at the position is written as 150/150; if a sample has two allelic variations at a site, the sizes of which are 141bp and 150bp, respectively, the genotype of the major allelic variation at the site is written as 141/150.

4. In step 3, 20 SSR site data of two unknown Chinese flowering cabbage varieties (Chinese flowering cabbage varieties to be detected) are respectively obtained, the data of the two unknown Chinese flowering cabbage varieties on each SSR site in the same 20 SSR sites are respectively compared, and the number of the different sites is recorded.

If the number of the ectopic difference points of the two unknown Chinese flowering cabbage varieties at the 20 SSR sites is more than 2, judging the two unknown Chinese flowering cabbage varieties as different Chinese flowering cabbage varieties; the more the number of the differential sites is, the farther the genetic relationship is;

and if the number of the ectopic points of the two unknown Chinese flowering cabbage varieties at the 20 SSR sites is 0-2, judging the two unknown Chinese flowering cabbage varieties as similar Chinese flowering cabbage varieties.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

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<120> method for identifying authenticity of cabbage heart varieties and special SSR primer combination thereof

<141> 2020-02-24

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<213> Artificial Sequence

<400> 2

catgagcggt ggtggtgg 18

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 3

gtagattcag agtagagatt ttggaagt 28

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 4

ggcgtttcac tctccttctc tc 22

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 5

gattttactc tcacatgtgc catg 24

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 6

ttccttgcat catcctaccc t 21

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 7

cattcctcgt tgcaatcaaa aca 23

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 8

gcttatggtt ttgccgtaca tttct 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

gaatttggag ctctctctag gtgat 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

tctgtaatac acaacaccaa atggt 25

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 11

gtaacggccg tcggtgac 18

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 12

taacataaca cgatcgcttt aaggc 25

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 13

aatggagtga tcgagaatgg agg 23

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 14

gctgtctaat ttgggcatac tagaa 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 15

tcgtttccaa atgcgacatt ctaac 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 16

gttcttgttt gctgtggaag ttttt 25

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 17

agagagagag tcagagacag ctatt 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 18

aatgcactat cttgcttcct ttctc 25

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 19

tctaaggtag agatagtagt agctgct 27

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 20

caaataggac atgtctttga agcca 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 21

ccgtttggaa ctgttattgc aaatg 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 22

tagaagcaac cgtgttacat gttgt 25

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 23

aaaccgtgaa tcttaattgg ccg 23

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 24

ttgattctct agttcagcta ctcgg 25

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 25

aacaatggcg tttgtccgaa at 22

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 26

tctgaaaagc tctttgaatg caact 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 27

aaagaagctt cgttcaaact ttcga 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 28

agtttgaagt tcatcctaca aagcc 25

<210> 29

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 29

aactacattg gcatagttgt tcaga 25

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 30

gctggtactt acagtggcaa ac 22

<210> 31

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 31

gacttgtaag agagatgacc tgtgt 25

<210> 32

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 32

tcacattcct cttacctgta ctctc 25

<210> 33

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 33

tggcgactct actaatgatg ttctt 25

<210> 34

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 34

gacatgatcc atcttcatcc cacc 24

<210> 35

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 35

aagcagagac attaagttct gaggt 25

<210> 36

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 36

ttttggtctt gttttgtctc tgtcg 25

<210> 37

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 37

ggattcagtt acagaggcaa aacaa 25

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 38

cacgtgtctc agacgataaa agaag 25

<210> 39

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 39

caaatcggct ccatagtgca taatt 25

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 40

cccttgattt gcaatgttga gtact 25

Claims

1. The SSR primer group for identifying the authenticity of the cabbage variety is characterized in that:

the SSR primer group is used for identifying the authenticity of the following 179 cabbage heart varieties: jinmantian, Hongliang No. 1, Zhuhai dwarf, emerald green No. 1, Cuimei 888, late 80 days, 06 Qiubao No. 1, Lilong dwarf, Liji willow leaf, Dongguan sharp leaf, Changji 70, famous sharp leaf, Jinmiao Meilv 702, Changji, Jiaxin, Huangxin 701, Jinshui, Hongliang No. 2, Hongliang No. 3, Guangxian vegetable field, Jinhan, nongpu's Lbao, Guanlv 60, Shengda New Zealand, Xinnong 50, Xinnong's best heart king, Hongliang 50, Hongliang super, Vanji 50, Japanese tame crisp, nonglong dwarf, Lilong dwarf foot 45, Yigbai, Kunjin, Guangyu vegetable heart, Guangxin No. 3, Dingkun vegetable heart king, vegetable heart No. 4, vegetable heart No. 19, vegetable heart of Zao, Xintian Rough spring No. 6, Jinhan spring plum, Jinhan Han Hao, 8, Konjia japonica, Shuangjia Hao, Shuichun green flower, Shuichong green flower 31, Shuichun green flower Shiwo green tea-flavored oil, Shuichang Shih green flower 31, Shuichong flower, guangliang oil green, Guangliang early oil green, Guangdong Youxian No. 1, Jinhanqiju, New century No. 20, nongxin anti-disease oil green, 09A Bao, Japan oil Mei No. 1, Zhou 008 petiole, Guangdong 1 flower, 288 sweet cabbage heart, Wangzhongwang heat-resistant, Baofeng short foot, 06 autumn Bao-A, Weixing Huangye forty-nine, Jinlida No. 19, nongyue 49, pure Chinese peduncle heart, nongxin Xiagun green, Xintian oil green 25, Weixiong A-3, Green crown 608, New seedling 002, New seedling T28, super Xinxin Huang, Green garden crisp, Xinyuao, Minan Japan, 12 Bao, 13A 9-Bao, 12 Caxin No. 1, 12 Caxin No. 3, Qing A, Qing stemming B, 14A 9-22, 45 Tian Bao Xin Bao, Bai Jian Xin, 13A 9-Bao, 12 Cai No. 1, 12 Cai-Cai, Tai Cai-Tian Cao, Tai Cao Yun Cao, Tai Cao, shennong thick strips, xiang vegetable crisp and tender sijiu, uncover and grind No. 31 heart of green beets, 14 Guangzhou heart of beets, Guangfu No. 2, 06 Qiubao-B, forty-nine heart of yellow beets, Guangmei green, Tianpai, Henlida Aoqiao Haoshao heart of sugar beets, Nonpu oil green 701, Jinhan 70 day heart of vegetables, Jinhan Kong 70 day oil green, Lilong oil green dwarf foot of 70 days, Nanshu super product of 70 day heart of beets, sloping head of 70 day heart of oil green vegetables, Weixing Sanli, Qingcui 701, oil green 702 heart of vegetables, Liji Baisha, specially selected 80 day heart of green vegetables, Weixing 80 day leaf of Shayeqing, Aipu super late heart 808, Aipu super late heart of agricultural, Pilv 80 day heart of oil green beets, emerald green, Severn super 80 day heart of vegetables, Guanhai Jianjian 80 days, Lijian 802, Liguan Ji 805, Yiguan Qing super Xin, Yiwang Xin Yihao 801, Guang Xin Yihao 801, Guangxi Wang Xin Yihao, red jadeite flowering cabbage heart, gold Korean red leaf flowering cabbage heart, plump flowering cabbage heart No. 2, Weixing flowering cabbage, 15 Guangzhou flowering cabbage-10, 15 large species 80, 62036x62013 RaaC0.5x2030, PI175054Aaa-1 BAROP 15376, Aaa-1, White Stemmed Taitsai, Carex meyeriana, エウサィタイ, Jingchu brand fresh red cabbage moss, Zaocai moss, red autumn flowering cabbage, Hongkong New 49 flowering cabbage, Hongkong cabbage 60, late cabbage No. 2, eighty day cabbage heart, Kingbao 50 day cabbage, Jinqihonghong No. two, tarda green cabbage, Tuyan cabbage X purple mustard 019, 20 cabbage X purple mustard, 14A-P2-21 flowering cabbage heart, SxS1, Sxcabbage X0192, S3, SxS3, Sbx 3, S7, CME heart 16, CME heart 16-35 CME heart, CMABE heart 882, 17E9-22, 16E9-16, 24X 16E9-13, 21, 16E9-49, 24X 16E9-22, 77-44 of Wanyeancheng, Qianjingjing vegetable, Guangdong vegetable, nonane vegetable and pinus xiao vegetable;

the SSR primer group consists of a first SSR primer pair, a second SSR primer pair, a third SSR primer pair, a fourth SSR primer pair, a fifth SSR primer pair, a sixth SSR primer pair, a seventh SSR primer pair, an eighth SSR primer pair, a ninth SSR primer pair, a tenth SSR primer pair, an eleventh SSR primer pair, a twelfth SSR primer pair, a thirteenth SSR primer pair, a fourteenth SSR primer pair, a fifteenth SSR primer pair, a sixteenth SSR primer pair, a seventeenth SSR primer pair, an eighteenth SSR primer pair, a nineteenth primer pair and a twentieth SSR primer pair;

the sequence of the first SSR primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

the sequence of the second SSR primer pair is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;

the sequence of the third SSR primer pair is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;

the sequence of the fourth SSR primer pair is shown as SEQ ID NO: 7 and SEQ ID NO: 8 is shown in the specification;

the sequence of the fifth SSR primer pair is shown as SEQ ID NO: 9 and SEQ ID NO: 10 is shown in the figure;

the sequence of the sixth SSR primer pair is shown as SEQ ID NO: 11 and SEQ ID NO: 12 is shown in the specification;

the sequence of the seventh SSR primer pair is shown as SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure;

the sequence of the eighth SSR primer pair is shown as SEQ ID NO: 15 and SEQ ID NO: 16 is shown in the figure;

the sequence of the ninth SSR primer pair is shown as SEQ ID NO: 17 and SEQ ID NO: 18 is shown in the figure;

the sequence of the tenth SSR primer pair is shown as SEQ ID NO: 19 and SEQ ID NO: 20 is shown in the figure;

the sequence of the eleventh SSR primer pair is shown as SEQ ID NO: 21 and SEQ ID NO: 22;

the sequence of the twelfth SSR primer pair is shown as SEQ ID NO: 23 and SEQ ID NO: shown at 24;

the sequence of the thirteenth SSR primer pair is shown as SEQ ID NO: 25 and SEQ ID NO: 26 is shown;

the sequence of the fourteenth SSR primer pair is shown as SEQ ID NO: 27 and SEQ ID NO: 28 is shown;

the sequence of the fifteenth SSR primer pair is shown as SEQ ID NO: 29 and SEQ ID NO: 30 is shown in the figure;

the sequence of the sixteenth SSR primer pair is shown as SEQ ID NO: 31 and SEQ ID NO: 32 is shown;

the sequence of the seventeenth SSR primer pair is shown as SEQ ID NO: 33 and SEQ ID NO: 34;

the sequence of the eighteenth SSR primer pair is shown as SEQ ID NO: 35 and SEQ ID NO: 36 is shown;

the sequence of the nineteenth SSR primer pair is shown as SEQ ID NO: 37 and SEQ ID NO: 38;

the sequence of the twentieth SSR primer pair is shown as SEQ ID NO: 39 and SEQ ID NO: 40 is shown in the figure;

one primer of each pair of primers is linked to a fluorescent molecule selected from the group consisting of ROX, TAMRA, FAM, HEX.

2. SSR kit of appraisal heart breed authenticity, its characterized in that: the SSR kit is prepared into a PCR reaction system; the PCR reaction system comprises:

the SSR primer set according to claim 1,

the concentration ratio of the upstream primer and the downstream primer of each pair in the SSR primer group in the system is 1: 1; the final concentration of the upstream primer and the final concentration of the downstream primer in the system are both 0.25 mu mol/L.

3. An SSR kit according to claim 2 characterized in that: the system further comprises:

dNTPs: the final concentration in the system was 0.15mmol/L each,

magnesium chloride: the final concentration in the system is 2.5mmol/L,

DNA polymerase: the final concentration in the system is 0.05U/. mu.L,

PCR buffer solution: is prepared from potassium chloride with final concentration of 10-50mmol/L and Tris-HCl with final concentration of 1-10mmol/L and pH of 7.5-9.0.

4. A detection method for identifying the authenticity of a cabbage variety is characterized by comprising the following steps: the detection method comprises the following steps:

the method comprises the following steps: detecting the genotype of the SSR locus of the flowering cabbage to be detected;

step two: and (3) judging the variety of the to-be-detected flowering cabbage:

if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is 0-2, judging the to-be-detected Chinese flowering cabbage and the specified variety of the Chinese flowering cabbage standard variety as a similar variety;

if the number of the difference loci of the to-be-detected Chinese flowering cabbage based on the genotypes of the 20 SSR loci and the genotypes of a certain specified variety in the Chinese flowering cabbage standard variety based on the 20 SSR loci is more than 2, judging the specified variety of the Chinese flowering cabbage to be different from the specified variety of the Chinese flowering cabbage standard variety;

the step of detecting the SSR locus genotype of the flowering cabbage to be detected comprises the following sub-steps:

the method comprises the following steps: respectively taking the genomic DNA of the cabbage heart to be detected and the genomic DNA of the standard variety of the cabbage heart as templates, and respectively adopting the primer group of claim 1 to carry out PCR amplification to obtain PCR amplification products;

step two is carried out: detecting the PCR amplification product to obtain genotypes of the to-be-detected cabbage heart and the standard variety of the cabbage heart based on 20 SSR loci;

the detection method is used for identifying the authenticity of the following 179 cabbage heart varieties:

jinmantian, Hongliang No. 1, Zhuhai dwarf, emerald green No. 1, Cuimei 888, late 80 days, 06 Qiubao No. 1, Lilong dwarf, Liji willow leaf, Dongguan sharp leaf, Changji 70, famous sharp leaf, Jinmiao Meilv 702, Changji, Jiaxin, Huangxin 701, Jinshui, Hongliang No. 2, Hongliang No. 3, Guangxian vegetable field, Jinhan, nongpu's Lbao, Guanlv 60, Shengda New Zealand, Xinnong 50, Xinnong's best heart king, Hongliang 50, Hongliang super, Vanji 50, Japanese tame crisp, nonglong dwarf, Lilong dwarf foot 45, Yigbai, Kunjin, Guangyu vegetable heart, Guangxin No. 3, Dingkun vegetable heart king, vegetable heart No. 4, vegetable heart No. 19, vegetable heart of Zao, Xintian Rough spring No. 6, Jinhan spring plum, Jinhan Han Hao, 8, Konjia japonica, Shuangjia Hao, Shuichun green flower, Shuichong green flower 31, Shuichun green flower Shiwo green tea-flavored oil, Shuichang Shih green flower 31, Shuichong flower, guangliang oil green, Guangliang early oil green, Guangdong Youxian No. 1, Jinhanqiju, New century No. 20, nongxin anti-disease oil green, 09A Bao, Japan oil Mei No. 1, Zhou 008 petiole, Guangdong 1 flower, 288 sweet cabbage heart, Wangzhongwang heat-resistant, Baofeng short foot, 06 autumn Bao-A, Weixing Huangye forty-nine, Jinlida No. 19, nongyue 49, pure Chinese peduncle heart, nongxin Xiagun green, Xintian oil green 25, Weixiong A-3, Green crown 608, New seedling 002, New seedling T28, super Xinxin Huang, Green garden crisp, Xinyuao, Minan Japan, 12 Bao, 13A 9-Bao, 12 Caxin No. 1, 12 Caxin No. 3, Qing A, Qing stemming B, 14A 9-22, 45 Tian Bao Xin Bao, Bai Jian Xin, 13A 9-Bao, 12 Cai No. 1, 12 Cai-Cai, Tai Cai-Tian Cao, Tai Cao Yun Cao, Tai Cao, shennong thick strips, xiang vegetable crisp and tender sijiu, uncover and grind No. 31 heart of green beets, 14 Guangzhou heart of beets, Guangfu No. 2, 06 Qiubao-B, forty-nine heart of yellow beets, Guangmei green, Tianpai, Henlida Aoqiao Haoshao heart of sugar beets, Nonpu oil green 701, Jinhan 70 day heart of vegetables, Jinhan Kong 70 day oil green, Lilong oil green dwarf foot of 70 days, Nanshu super product of 70 day heart of beets, sloping head of 70 day heart of oil green vegetables, Weixing Sanli, Qingcui 701, oil green 702 heart of vegetables, Liji Baisha, specially selected 80 day heart of green vegetables, Weixing 80 day leaf of Shayeqing, Aipu super late heart 808, Aipu super late heart of agricultural, Pilv 80 day heart of oil green beets, emerald green, Severn super 80 day heart of vegetables, Guanhai Jianjian 80 days, Lijian 802, Liguan Ji 805, Yiguan Qing super Xin, Yiwang Xin Yihao 801, Guang Xin Yihao 801, Guangxi Wang Xin Yihao, red jadeite flowering cabbage heart, gold Korean red leaf flowering cabbage heart, plump flowering cabbage heart No. 2, Weixing flowering cabbage, 15 Guangzhou flowering cabbage-10, 15 large species 80, 62036x62013 RaaC0.5x2030, PI175054Aaa-1 BAROP 15376, Aaa-1, White Stemmed Taitsai, Carex meyeriana, エウサィタイ, Jingchu brand fresh red cabbage moss, Zaocai moss, red autumn flowering cabbage, Hongkong New 49 flowering cabbage, Hongkong cabbage 60, late cabbage No. 2, eighty day cabbage heart, Kingbao 50 day cabbage, Jinqihonghong No. two, tarda green cabbage, Tuyan cabbage X purple mustard 019, 20 cabbage X purple mustard, 14A-P2-21 flowering cabbage heart, SxS1, Sxcabbage X0192, S3, SxS3, Sbx 3, S7, CME heart 16, CME heart 16-35 CME heart, CMABE heart 882, 17E9-22, 16E9-16, 24X 16E9-13, 21, 16E9-49, 24X 16E9-22, 77-44 of Wanyeancheng, Qianjingjing vegetable, Guangdong vegetable, nonane vegetable and pinus xiao vegetable.

5. The detection method of claim 4: the method is characterized in that:

the result of the determination is obtained from a cluster analysis.

6. The detection method of claim 4: the method is characterized in that:

the detection method of the substep two comprises the following steps:

and (3) fluorescent signal detection: detecting a fluorescent signal of the PCR amplification product to obtain genotypes of the to-be-detected cabbage heart and the standard cabbage heart varieties based on the 20 SSR sites; or:

detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the cabbage heart to be detected and the standard cabbage heart variety based on the 20 SSR sites.

7. The SSR primer set according to claim 1, or the SSR kit according to claim 2 or 3, or the detection method according to any of claims 4 to 6 for use in X1 or X2 or X3:

x3: identifying whether the cabbage heart samples to be detected are the same varieties or not;

the standard Chinese flowering cabbage variety is selected from the following 179 Chinese flowering cabbage varieties: jinmantian, Hongliang No. 1, Zhuhai dwarf, emerald green No. 1, Cuimei 888, late 80 days, 06 Qiubao No. 1, Lilong dwarf, Liji willow leaf, Dongguan sharp leaf, Changji 70, famous sharp leaf, Jinmiao Meilv 702, Changji, Jiaxin, Huangxin 701, Jinshui, Hongliang No. 2, Hongliang No. 3, Guangxian vegetable field, Jinhan, nongpu's Lbao, Guanlv 60, Shengda New Zealand, Xinnong 50, Xinnong's best heart king, Hongliang 50, Hongliang super, Vanji 50, Japanese tame crisp, nonglong dwarf, Lilong dwarf foot 45, Yigbai, Kunjin, Guangyu vegetable heart, Guangxin No. 3, Dingkun vegetable heart king, vegetable heart No. 4, vegetable heart No. 19, vegetable heart of Zao, Xintian Rough spring No. 6, Jinhan spring plum, Jinhan Han Hao, 8, Konjia japonica, Shuangjia Hao, Shuichun green flower, Shuichong green flower 31, Shuichun green flower Shiwo green tea-flavored oil, Shuichang Shih green flower 31, Shuichong flower, guangliang oil green, Guangliang early oil green, Guangdong Youxian No. 1, Jinhanqiju, New century No. 20, nongxin anti-disease oil green, 09A Bao, Japan oil Mei No. 1, Zhou 008 petiole, Guangdong 1 flower, 288 sweet cabbage heart, Wangzhongwang heat-resistant, Baofeng short foot, 06 autumn Bao-A, Weixing Huangye forty-nine, Jinlida No. 19, nongyue 49, pure Chinese peduncle heart, nongxin Xiagun green, Xintian oil green 25, Weixiong A-3, Green crown 608, New seedling 002, New seedling T28, super Xinxin Huang, Green garden crisp, Xinyuao, Minan Japan, 12 Bao, 13A 9-Bao, 12 Caxin No. 1, 12 Caxin No. 3, Qing A, Qing stemming B, 14A 9-22, 45 Tian Bao Xin Bao, Bai Jian Xin, 13A 9-Bao, 12 Cai No. 1, 12 Cai-Cai, Tai Cai-Tian Cao, Tai Cao Yun Cao, Tai Cao, shennong thick strips, xiang vegetable crisp and tender sijiu, uncover and grind No. 31 heart of green beets, 14 Guangzhou heart of beets, Guangfu No. 2, 06 Qiubao-B, forty-nine heart of yellow beets, Guangmei green, Tianpai, Henlida Aoqiao Haoshao heart of sugar beets, Nonpu oil green 701, Jinhan 70 day heart of vegetables, Jinhan Kong 70 day oil green, Lilong oil green dwarf foot of 70 days, Nanshu super product of 70 day heart of beets, sloping head of 70 day heart of oil green vegetables, Weixing Sanli, Qingcui 701, oil green 702 heart of vegetables, Liji Baisha, specially selected 80 day heart of green vegetables, Weixing 80 day leaf of Shayeqing, Aipu super late heart 808, Aipu super late heart of agricultural, Pilv 80 day heart of oil green beets, emerald green, Severn super 80 day heart of vegetables, Guanhai Jianjian 80 days, Lijian 802, Liguan Ji 805, Yiguan Qing super Xin, Yiwang Xin Yihao 801, Guang Xin Yihao 801, Guangxi Wang Xin Yihao, red jadeite flowering cabbage heart, gold Korean red leaf flowering cabbage heart, plump flowering cabbage heart No. 2, Weixing flowering cabbage, 15 Guangzhou flowering cabbage-10, 15 large species 80, 62036x62013 RaaC0.5x2030, PI175054Aaa-1 BAROP 15376, Aaa-1, White Stemmed Taitsai, Carex meyeriana, エウサィタイ, Jingchu brand fresh red cabbage moss, Zaocai moss, red autumn flowering cabbage, Hongkong New 49 flowering cabbage, Hongkong cabbage 60, late cabbage No. 2, eighty day cabbage heart, Kingbao 50 day cabbage, Jinqihonghong No. two, tarda green cabbage, Tuyan cabbage X purple mustard 019, 20 cabbage X purple mustard, 14A-P2-21 flowering cabbage heart, SxS1, Sxcabbage X0192, S3, SxS3, Sbx 3, S7, CME heart 16, CME heart 16-35 CME heart, CMABE heart 882, 17E9-22, 16E9-16, 24X 16E9-13, 21, 16E9-49, 24X 16E9-22, 77-44 of Wanyeancheng, Qianjingjing vegetable, Guangdong vegetable, nonane vegetable and pinus xiao vegetable.