CN111793708A

CN111793708A - Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof

Info

Publication number: CN111793708A
Application number: CN202010625401.1A
Authority: CN
Inventors: 张凤兰; 于拴仓; 温常龙; 张建; 罗江; 苏同兵; 李佩荣
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2019-10-11
Filing date: 2020-07-02
Publication date: 2020-10-20
Anticipated expiration: 2040-07-02
Also published as: CN111793708B

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 Chinese cabbage variety and a special SSR primer combination thereof. The SSR 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, ensuring the authenticity of the variety, practically protecting the rights and interests of producers and breeders, and providing technical support for protecting Chinese cabbage germplasm resources and new varieties. The method of the invention can identify that: whether the Chinese cabbage variety to be detected belongs to one of the standard Chinese cabbage varieties or not and which type is determined specifically. Therefore, the method can be used for identifying the unknown Chinese cabbage variety and also can be used for identifying the authenticity of the known variety. The invention has the advantages of high flux, accuracy, low cost, simple operation, manpower and material resource saving and the like, and has very wide application prospect.

Description

Method for identifying authenticity of Chinese cabbage variety 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 Chinese cabbage variety and a special SSR primer combination thereof.

Background

Chinese cabbage belongs to cruciferae and biennial herbaceous plants, and has become the widest vegetable crop in the current 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. Because the varieties of the Chinese cabbages are various, the management of the varieties is not effectively followed up, and cases of counterfeit and shoddy seeds and the phenomena of same variety and different names frequently occur. According to statistics, 2653 Chinese cabbage varieties are totally applied for new variety registration in 2019 since a non-main crop variety registration system is implemented in 2017 and 4 months, and only 1565 Chinese cabbage varieties obtain new variety registration authorization. The explosive growth of the number of the Chinese cabbage varieties provides new challenges for intellectual property protection and variety management of the Chinese cabbage varieties. Therefore, how to quickly 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 environment, 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, SSR molecular markers are widely applied to the identification of variety authenticity by virtue of the advantages of large quantity, rich variation, stable heredity and the like. At present, the current Chinese cabbage variety DNA identification SSR molecular marker method (NTY 2476-. Moreover, the traditional SSR locus screening lacks the support of big data of Chinese cabbage variation groups, the number of detected varieties is small, and the detection mode easily causes unreal, false positive and false negative results.

Therefore, in the work of identifying the varieties of the Chinese cabbage by using the SSR molecular markers, a stable and efficient SSR locus and a determination method based on large-scale Chinese cabbage re-sequencing and Chinese cabbage variation group information analysis are needed.

Recently, the inventor subjects group can clearly grasp the sequence variation in SSR Motif and the real situation of PCR amplified fragments by analyzing the big data of 194 Chinese cabbage genome variation groups. By adopting a multiple PCR amplification method, the authenticity SSR identification of the Chinese cabbage variety can be performed stably and efficiently with high flux.

Disclosure of Invention

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

The invention is realized by the following technical scheme:

an SSR locus for identifying the authenticity of a Chinese cabbage variety, which is selected from any 1 to 18 of the following first SSR locus to eighteenth SSR locus: the first SSR locus is positioned at the 15735196-15735240 th chromosome 3 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the second SSR locus is positioned at the No. 991401-991412 locus of the 7 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome; a third SSR locus which is positioned at the 11395449-11395488 th site of the 7 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; the fourth SSR locus is positioned at the 19600720-19600729 th chromosome 9 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the fifth SSR locus is positioned at the 126746-126765 th chromosome of the 2 nd chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome; the sixth SSR locus is located at the No. 4 chromosome position 9033544 and 9033555 of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof; the seventh SSR locus is positioned at the 5 th chromosome 1804454-1804465 of the Chinese cabbage reference genome or the homologous genome segment among varieties thereof; the eighth SSR locus is positioned at the 8241771-8241805 position of the 6 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome; the ninth SSR locus is positioned at the No. 9523723 and No. 9523746 chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the ninth SSR locus; the tenth SSR locus is positioned at the 872029-872052 th chromosome 10 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the eleventh SSR locus is positioned at the 8273753-8273794 th chromosome 6 of the Chinese cabbage reference genome or a homologous genome fragment among varieties thereof; the twelfth SSR locus is positioned at the No. 3531906 and No. 3531913 position of the 4 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome; the thirteenth SSR locus is positioned at the 8 th chromosome 15216555-15216601 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof; a fourteenth SSR locus which is positioned at the 1017146-1017173 locus of the 1 st chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof; a fifteenth SSR locus which is positioned at the 2535156-2535179 th chromosome 3 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof; the sixteenth SSR locus is positioned at the No. 2606495-2606533 of the No. 2 chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof; the seventeenth SSR locus is positioned at the 28975700-28975721 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome; the eighteenth SSR locus is positioned at the 10162405-10162432 th chromosome of the Chinese cabbage reference genome 10 or a homologous genome segment among varieties thereof; the Chinese cabbage reference genome is a Chinese cabbage Chiifu-401-42 reference genome V1 edition.

An SSR primer group for identifying the authenticity of Chinese cabbage varieties is used for respectively amplifying the SSR loci, and comprises: a first SSR primer pair for amplifying said first SSR site; a second SSR primer pair for amplifying said second SSR site; a third SSR primer pair for amplifying said third SSR site; a fourth SSR primer pair for amplifying said fourth SSR site; a fifth SSR primer pair for amplifying said fifth SSR site; a sixth SSR primer pair for amplifying said sixth SSR site; a seventh SSR primer pair for amplifying said seventh SSR locus; an eighth SSR primer pair for amplifying said eighth SSR locus; a ninth SSR primer pair for amplifying said ninth SSR locus; a tenth SSR primer pair for amplifying said tenth SSR site; an eleventh SSR primer pair for amplifying said eleventh SSR site; a twelfth SSR primer pair for amplifying said twelfth SSR site; a thirteenth SSR primer pair for amplifying said thirteenth SSR site; a fourteenth SSR primer pair for amplifying said fourteenth SSR site; a fifteenth SSR primer pair for amplifying said fifteenth SSR site; a sixteenth SSR primer pair for amplifying the sixteenth SSR site; a seventeenth SSR primer pair for amplifying the seventeenth SSR site; and the eighteenth SSR primer pair is used for amplifying the eighteenth SSR locus.

In some embodiments, the SSR primer sets, the first SSR primer pairs, are each identical to seq id NOs: 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched 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%; and the twelfth SSR primer pair is respectively matched with 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 respectively matched with 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%; and the fourteenth SSR primer pair is respectively matched with 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%; and the fifteenth SSR primer pair is respectively matched with SEQID 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 respectively matched with 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 respectively matched 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%; and the eighteenth SSR primer pair is respectively matched with 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%; 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.

An SSR kit for identifying the authenticity of Chinese cabbage varieties, wherein reagents of the SSR kit are used for preparing a PCR reaction system; the PCR reaction system comprises: the SSR primer group is preferably characterized in that the concentration ratio of the upstream primer to the downstream primer of each pair in the SSR primer group in the system is 1: 1; more preferably, 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 Chinese cabbage varieties comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of the Chinese cabbage to be detected; step two: the variety judgment of the Chinese cabbage to be detected comprises the following steps: if the number of the difference loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in the Chinese cabbage standard variety library based on the 18 SSR loci is 0, the Chinese cabbage to be detected and the standard variety in the Chinese cabbage standard variety library belong to the same variety; if the number of the difference loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in the Chinese cabbage standard variety library based on the 18 SSR loci is 1, the Chinese cabbage to be detected and the standard variety in the Chinese cabbage standard variety library belong to similar varieties; and if the number of the different loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of each variety in the Chinese cabbage standard variety library based on the 18 SSR loci is more than or equal to 2, the varieties of the Chinese cabbage to be detected and each of the Chinese cabbage standard varieties in the Chinese cabbage standard variety library are different.

In some embodiments, the step of detecting the SSR locus genotype of the Chinese cabbage to be detected comprises the following sub-steps: the method comprises the following steps: respectively taking the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of the Chinese cabbage standard variety 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 carrying out length detection on the PCR amplification product to obtain genotypes of the Chinese cabbage to be detected and the Chinese cabbage standard variety based on 18 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 Chinese cabbage and the standard Chinese cabbage variety based on the 18 SSR loci; or: detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the Chinese cabbage to be detected and the standard Chinese cabbage variety based on the 18 SSR loci.

In some embodiments, in the second step, the determination result is obtained according to a clustering analysis.

In some embodiments, the standard chinese cabbage variety bank is selected from any one or more of the following 216 chinese cabbage varieties: beijing Xin No. 1, Beijing Xiaoza 50, Tianwa, Dahuahuang, Qinqiao 60, Zhengbai 65, Tianzheng orange red 62, Tianzheng orange red 65, Jihong 308, Lichun, Qingyangqinbai No. 1, Beijing Xiaoza 67, Beijing Xinxin No. 2, Beijing orange red heart, Zhongbai 65, Zhongbai 81, Jingchuzao, Jingxiawang, Jingluo 60, Xiaoza 56, Zhongbai 50, Zhongbai 60, Delong No. 1, Jingxian No. 1, Beijing Xinxin No. 4, Yangchun, Chunbao, Chunqiu 54, Qiangqing, Beijing orange No. 2, Jingchunbai, Jingchun 988, Beijing improved 67, Beijing great ox heart, Beijing, Chunxun, Chunquan, Shichun 50, Beijing 80, Wangchun, Guang 58, Cuiyoubao, Beijing yellow 70, Beijing Xiaodeqinqiu No. 60, Jingdeqinqiu No. 60, Beijing white Beijing, Beijing Baichun No. 2, Beijing orange No. 2, Beijing Baichun, Beijing Baichun No. 60, Beijing Baichun, Beijing Baichun No. 2, Beijing Baichun, Beijing Xinchun No, Mini star, degao 16, jin qiu 606, zhong bai 62, wei bai 4, jing qiu 75, jin qiu 78, huichun, jing qiu baby vegetable, jin qiu 90, jing chun huang, jing chun wa 2, jin zao 9, jin zao 58, jing qiu 3, xiao 60, jing qiu 70, jin hua, you 3, jin qiu 1, jiu jian 85, jing qiu 4, jing qiu 76, jin xiu 1, beijing xiao 51, zhen green 55, zhen green 80, jiao bai 7, jing chun huang 2, jing chun 3, zhong bai 61, jing qiu 701, jing cui 75, ji hong hua, SW701, lai 38, jin green 75, jing hao green 2, ji cai yi 30, zhen hao jian hao cui 30, zhen hao cui, jing kuan 70, zhong nong qing hao qing huan jian hua 78, SW 356, qing bai 303, qing hua jian qing CR1, qing hua 58, zhen qing hao, zhen hao CR1, zhen hao 55, zhen hao, zhen huan hao 7, zhen hao 2, zhe, Middle bracket, Yi and 1410, Fushan walnut grain, Tian Bai 65, jin doll No. 1, Chun Yu Huang, jin Jian Kuo Hao Green No. 1, SGK1008, Xiao Yi and Qiu, 87-114, YHBC1511, YHBC1516, Shen Bai GMS02, Qing nong No. 45, Yuxin No. 1, Shi Green 85, Huanai CC001, Huanai CC002, Yi and No. 1, Zhen Lu 60, Jing Chun CR3, mountain king No. 2, Jing Chun Wa 4, Ling Huang, CR Tian Bai 15, De Gao CR117, Jing Qiu No. 635, Jihong 65, Lu Jian 60, Juxin No. one, Lichun Yi No. 70, Lu Bai No. 50, Zhongbai No. 61, Zhongbai No. 81 selected, Zhongbai No. 83, Zhongbai No. 85, Zhongbai 58 Zhongbai No. 76, Qiu Green 78, jin Green 80, Qiu Green 75, Qiu Green 60, Qiu Green No. 55, Qiu Green bud No. 6, Zao Huang No. 14, Xin Zan Shen No. 3, Xin Shen No. 5, Zhengbai Shen, Preferably 56 small miscellaneous crops, 68 middle packet, 89-8 Xinzao, four Zhengbai crops, three Xinzao crops, 007 Chunfeng, 5 Xibai crops, four Xibai crops, 3 Xichunbai crops, 50 Taijing, 78 Zhongbai crops, 3 Xibai crops, 10 Xibai crops, one Lebai crops, five precocious crops, two Tianzhengzhengchaobai crops, ten Liaobai crops, 4 Xibai crops, LI-1, 19 Jinhuayuan crops, 2 Qinbai crops, 2 Guanchun crops, spring crops, 58 Xinzao crops, 16 Lubai crops, 78 Qiyu 78, four Qingyan crops, F115-93 Fengnan crops, 5 Shandong crops, GMS01, Jingcui 60 crops, 9 Qiuchao crops, 60 Qingzhuang spring crops, one Luchunbun crops, 6 Zhejiang white crops, 75 Zhengbai crops, 23 Xinyunlong crops, 6 Zhenglv small size crops, five Shandong crops, 62 Beixiang spring crops, 62 Zhengchun spring crops, 1102 Zhengchun white flowers and 1 winter flowers.

The SSR locus, or the SSR primer combination, or the SSR kit, or the detection method, in the following X1 or X2: x1: identifying whether the variety of the Chinese cabbage to be detected belongs to one of standard Chinese cabbage varieties; x2: and identifying the variety of the Chinese cabbage to be detected to be the standard variety of the Chinese cabbage.

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

1. the SSR 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, ensuring the authenticity of the variety, practically protecting the rights and interests of producers and breeders, and providing technical support for protecting Chinese cabbage germplasm resources and new varieties.

2. The method provided by the invention can be used for identifying that: whether the Chinese cabbage variety to be detected belongs to one of the standard Chinese cabbage varieties or not and which type is determined specifically. Therefore, the method can be used for identifying the unknown Chinese cabbage variety and also can be used for identifying the authenticity of the known variety.

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.

Drawings

FIG. 1 is a cluster plot of 216 tested Brassica napus varieties established on 18 primer sets in example 1.

FIG. 2 is a difference marker chart of example 2, which shows the number of SSR markers (i.e., the number of SSR loci) and 216 varieties of Chinese cabbage to be tested.

FIGS. 3-20 are graphs showing SSR typing effects of the 18 primer sets of example 2 on part of tested varieties of Chinese cabbage. Wherein, fig. 3: SSR typing effect profile using BrSSr004 primer, fig. 4: SSR typing effect profile using BrSSr010 primer, fig. 5: SSR-typing effect profile using BrSSr011 primer, fig. 6: SSR-typing effect profile using BrSSr017 primer, fig. 7: SSR typing effect profile using BrSSr023 primer, fig. 8: SSR typing effect profile using BrSSr026 primer, fig. 9: SSR typing effect profile using BrSSr027 primer, fig. 10: SSR typing effect profile using BrSSR028 primer, fig. 11: SSR typing effect profile using BrSSr044 primer, fig. 12: SSR typing effect profile using BrSSr049 primer, fig. 13: SSR typing effect profile using BrSSr059 primer, fig. 14: SSR typing effect profile using BrSSr060 primers, fig. 15: SSR typing effect profile using BrSSr064 primer, fig. 16: SSR typing effect profile using BrSSr065 primer, fig. 17: b SSR typing effect profile using rSSr067 primer, fig. 18: SSR typing effect profile using BrSSr068 primer, fig. 19: SSR typing effect profile using BrSSr069 primer, fig. 20: SSR typing effect map using BrSSr071 primer.

Detailed Description

The definition is as follows:

authenticity of Chinese cabbage varieties: the method refers to the real correspondence between a Chinese cabbage variety and the genetic background of the Chinese cabbage variety.

Interspecies homologous genomic fragments: the gene group fragment is homologous with the specific gene group fragment sequence in the Chinese cabbage Chiifu-401-42 reference gene group sequence, wherein the version number of the reference gene group sequence of the Chinese cabbage Chiifu-401-42 is V1, and the version number of the reference gene group sequence of the Chinese cabbage Chiifu-401-42 is V1. For example, among the varieties in Table 3 of the present invention, the genome fragment having homology with the version number V1 of the reference genome sequence of Chinese cabbage Chiifu-401-42 is the specific genome fragment.

For a particular SSR sequence of chinese cabbage, the interspecies homologous genomic fragments refer to: the version number of the specific Chinese cabbage variety and the Chinese cabbage Chiifu-401-42 reference genome sequence is the same or similar with the SSR sequence flanking sequence in V1, and the SSR sequences in the specific Chinese cabbage variety and the Chinese cabbage Chiifu-401-42 reference genome sequence are the same or similar with each other.

In a first aspect, the invention provides SSR loci for identifying the authenticity of a chinese cabbage variety, each of which is located in the genome of the chinese cabbage, the number of the SSR loci is 18, and 1 or more of the SSR loci can be selected, and specific information is shown in table 1.

The SSR site is selected from any 1 to 18 of the following first to eighteenth SSR sites:

the first SSR locus is positioned at the 15735196-15735240 th chromosome 3 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the second SSR locus is positioned at the No. 991401-991412 locus of the 7 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome;

a third SSR locus which is positioned at the 11395449-11395488 th site of the 7 th chromosome of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

the fourth SSR locus is positioned at the 19600720-19600729 th chromosome 9 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the fifth SSR locus is positioned at the 126746-126765 th chromosome of the 2 nd chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome;

the sixth SSR locus is located at the No. 4 chromosome position 9033544 and 9033555 of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof;

the seventh SSR locus is positioned at the 5 th chromosome 1804454-1804465 of the Chinese cabbage reference genome or the homologous genome segment among varieties thereof;

the eighth SSR locus is positioned at the 8241771-8241805 position of the 6 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome;

the ninth SSR locus is positioned at the No. 9523723 and No. 9523746 chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the ninth SSR locus;

the tenth SSR locus is positioned at the 872029-872052 th chromosome 10 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the eleventh SSR locus is positioned at the 8273753-8273794 th chromosome 6 of the Chinese cabbage reference genome or a homologous genome fragment among varieties thereof;

the twelfth SSR locus is positioned at the No. 3531906 and No. 3531913 position of the 4 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome;

the thirteenth SSR locus is positioned at the 8 th chromosome 15216555-15216601 of the Chinese cabbage reference genome or an interspecies homologous genome segment thereof;

a fourteenth SSR locus which is positioned at the 1017146-1017173 locus of the 1 st chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof;

a fifteenth SSR locus which is positioned at the 2535156-2535179 th chromosome 3 of the Chinese cabbage reference genome or an interspecies homologous genome fragment thereof;

the sixteenth SSR locus is positioned at the No. 2606495-2606533 of the No. 2 chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties thereof;

the seventeenth SSR locus is positioned at the 28975700-28975721 th chromosome of the Chinese cabbage reference genome or a homologous genome segment among varieties of the Chinese cabbage reference genome;

the eighteenth SSR locus is located at the 10162405-10162432 th chromosome 10 of the Chinese cabbage reference genome or the homologous genome segment among varieties thereof.

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

The SSR primer combination is selected from:

the first SSR primer pair is respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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 respectively matched with 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%;

and the eleventh SSR primer pair is respectively matched 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%;

and the twelfth SSR primer pair is respectively matched with 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 respectively matched with 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%;

and the fourteenth SSR primer pair is respectively matched with 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%;

and the fifteenth SSR primer pair is respectively matched 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%;

and the sixteenth SSR primer pair is respectively matched with 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%;

and the seventeenth SSR primer pair is respectively matched 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 respectively matched with 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%;

one primer of each of the above primer pairs is linked to a fluorescent molecule, more preferably, the fluorescent molecule is selected from the group consisting of ROX, TAMRA, FAM, HEX.

In a preferred embodiment, the SSR primer combination is selected from one or more of primer sets 01-18; the DNA sequence information of the primer groups 01-18 is shown in a sequence table SEQ ID: 1-36, see table 2.

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

In a third aspect, the invention provides an SSR kit for identifying the authenticity of a chinese cabbage variety, the SSR reagent is formulated as a PCR reaction system, and the system preferably comprises:

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

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

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

The method comprises the following steps: respectively taking the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of the Chinese cabbage standard variety 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 (3) carrying out length detection on the PCR amplification product to obtain genotypes of the Chinese cabbage to be detected and the Chinese cabbage standard variety based on 18 SSR loci.

The detection may be a fluorescence signal detection: detecting the fluorescent signal of the PCR amplification product to obtain genotypes of the Chinese cabbage to be detected and the standard Chinese cabbage variety based on the 18 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 using capillary electrophoresis to obtain the genotypes of the Chinese cabbage to be detected and the standard Chinese cabbage variety based on the 18 SSR loci.

Step two: judging varieties of Chinese cabbages to be detected:

the following results are obtained by clustering analysis of genotypes of the Chinese cabbage to be detected and the standard Chinese cabbage variety based on the 18 SSR loci:

if the number of the difference loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in the Chinese cabbage standard variety library based on the 18 SSR loci is 0, the Chinese cabbage to be detected and the standard variety in the Chinese cabbage standard variety library belong to the same variety;

if the number of the difference loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in the Chinese cabbage standard variety library based on the 18 SSR loci is 1, the Chinese cabbage to be detected and the standard variety in the Chinese cabbage standard variety library belong to similar varieties;

and if the number of the difference loci of the genotypes of the Chinese cabbage to be detected based on the 18 SSR loci and the genotypes of all varieties in the Chinese cabbage standard substance seed bank based on the 18 SSR loci is more than or equal to 2, the varieties of each Chinese cabbage standard variety in the Chinese cabbage standard substance seed bank to be detected are different.

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 cabbage variety bank comprises any one variety 1-216 of the following 216 Chinese cabbage varieties:

beijing Xin No. 1, Beijing Xiaoza 50, Tianwa, Dahuahuang, Qinqiao 60, Zhengbai 65, Tianzheng orange red 62, Tianzheng orange red 65, Jihong 308, Lichun, Qingyangqinbai No. 1, Beijing Xiaoza 67, Beijing Xinxin No. 2, Beijing orange red heart, Zhongbai 65, Zhongbai 81, Jingchuzao, Jingxiawang, Jingluo 60, Xiaoza 56, Zhongbai 50, Zhongbai 60, Delong No. 1, Jingxian No. 1, Beijing Xinxin No. 4, Yangchun, Chunbao, Chunqiu 54, Qiangqing, Beijing orange No. 2, Jingchunbai, Jingchun 988, Beijing improved 67, Beijing great ox heart, Beijing, Chunxun, Chunquan, Shichun 50, Beijing 80, Wangchun, Guang 58, Cuiyoubao, Beijing yellow 70, Beijing Xiaodeqinqiu No. 60, Jingdeqinqiu No. 60, Beijing white Beijing, Beijing Baichun No. 2, Beijing orange No. 2, Beijing Baichun, Beijing Baichun No. 60, Beijing Baichun, Beijing Baichun No. 2, Beijing Baichun, Beijing Xinchun No, Mini star, degao 16, jin qiu 606, zhong bai 62, wei bai 4, jing qiu 75, jin qiu 78, hui chun, jing qiu baby vegetable, jin qiu 90, jing chun huang, jing chun wa 2, jin zao 9, jin zao 58, jing qiu 3, xiao 60, jing qiu 70, jin hua, you 3, jin qiu 1, lv jian 85, jing qiu 4, jing qiu 76, jin xiu 1, beijing xiao 51, zhen green 55, zhen green 80, jiao bai 7, jing chun huang 2, jing chun 3, zhong bai 61, jing qiu 701, jing cui 75, ji hong hua, SW701, lai 38, jin green 75, jing hao green 2, ji cai yi 30, zhen hao jian cui 30, zhen hao cui, zihao 70, zhong nong qing hao 78, zhong bai jian qing 303, SW 21, qing yang CR1, jinzao CR 32, zhen qing hua hao 92, zhen qing hao CR1, zhen hao 80, zhen hao 2, zhen hao 7, zhen hao 2, zhen hao, Yi and 0906, Zhongparen, Yi and 1410, Fushan walnut vein, Tianbai 65, jin doll No. 1, Chunyhuang, jin Jian Kuai Hao No. 1, SGK1008, Xiaoyi and Qiu, 87-114, YHBC1511, YHBC1516, Shenbai GMS02, Qingnong 45 days, Yuxin No. 1, Shilv 85, Huanai CC001, Huanai CC002, Yi and No. 1, Zhen Lu 60, Jingchun CR3, mountain No. 2, Jingchun Wao No. 4, Linglong yellow, CR Tianbai 15, Degao CR117, Jingqiu No. 5, Jihong 65, Lujian 60, Juxin No. 1, Lichun No. 70, Zhongbai No. 50, Zhongbai No. 61, Zhongbai No. 81, Zhongbai No. 83, Zhongbai No. 85, Zhongbai No. 58, Zhongbai Shenbai 76, Qiu Green 6378, jin Green 80, Qiu Green 75, Qiu Green, Zao Lu 60, Zhongbai Lu No. 6, Zhongbai Shao No. 5, Zhongbai Ji Shen, Zhengza No. two, preferably Xiaoza No. 56, Zhongbao No. 68, Xinzao No. 89-8, Zhengbai No. four, Xinxiao No. three, Chunfeng No. 007, Xibai No. 5, Xibai No. four, Xichunbai No. 3, Taijing No. 50, Zhongbai No. 78, Xibai No. 3, Xibai No. 10, Laibai No. one, precocious No. five, Tianzhengchaobai No. two, Liaobai No. ten, Xibai No. 4, LI-1, Jinhuayuan No. 19, Qinbai No. 2, Guanchun, Chunrao, Xinzao No. 58, Lubai No. 16, Qiuyu No. 78, Qingyan No. four, Fennan No. F115-93, Shandong No. 5, Shenbai GMS01, Jingcui No. 60, Sicui Cui No. 9, Fenggong No. 60, Qingchun Bai No. one, Luchunbai No. 6, Zhejiang white Zheng No. 6, Yun Baizheng No. 75, Xinxiang pao No. 23, Luxiang No. 6, Shandong No. 6, Shandon.

In a fifth aspect, the invention provides the above SSR locus, SSR primer combination, SSR kit, detection method, use in x1 or x 2:

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

x2: and identifying the variety of the Chinese cabbage to be detected to be the standard variety of the Chinese cabbage.

Both X1 and X2 belong to the application of identifying the authenticity of Chinese cabbage varieties.

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 Chinese cabbage variety

Discovery of 18 SSR loci

The invention obtains 18 SSR loci based on the re-sequencing data (NCBI: SRP158243) 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 agricultural variety of Chinese cabbage, have representativeness of Chinese cabbage germplasm, and have high genetic diversity.

Specifically, the screening criteria for SSR sites are as follows: the SSR sites of each chromosome are uniform in quantity, the spacing is more than 1Mb, the minimum allele frequency MAF is more than 0.1, the heterozygosity He is less than 0.1, PIC is more than 0.3, and the two wings of the SSR sites are 50bp sequence conservative (no InDel, no SSR, no SNP and no other SSR).

Table 1 shows that 18 kinds of SSR information relative to a Chinese cabbage Chiifu-401-42 reference genome sequence are determined based on basic data determined by alignment of the Chinese cabbage Chiifu-401-42 reference genome sequence, wherein the version number of the Chinese cabbage Chiifu-401-42 reference genome sequence is V1 (download address: https:// www.ncbi.nlm.nih.gov/genome/.

Table 1: basic information of 18 SSR loci

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

Based on the 18 SSR loci found in step one, the inventors of the present invention developed SSR primer combinations 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 a 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; see column 5 in Table 4). Primers are designed on the basis that the SSR locus is in an upstream sequence and a downstream sequence of a Chinese cabbage Chiifu-401-42 reference genome sequence, the SSR primer combination consists of 18 primer groups, and the name of each primer group is shown in a2 nd column in a 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 18 primer sets are shown in column 4 of Table 2.

Table 2: 18 pairs of primer sequences of Chinese cabbage SSR sites and fluorescent modifying groups

Example 2

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

The basic information of 216 tested brassica rapa pekinensis varieties in the embodiment is shown in table 3. The 216 tested Chinese cabbage varieties are common fine varieties or partially foreign introduced varieties.

Table 3: basic information of 216 tested Chinese cabbage varieties

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

The genome DNA of 216 leaves of the tested Chinese cabbage variety (true leaves of 30 seeds are mixed, namely the true leaves of 30 seeds of each variety are mixed, namely the true leaves of 30 different plants of the same variety are mixed) is respectively extracted by adopting a CTAB method, so that the genome DNA of the tested Chinese cabbage variety is obtained.

The CTAB method is specifically operated as follows:

respectively picking up the leaves of the 216 varieties in the seedling stage, and dehydrating in a freeze dryer (CoolSafe 55-4); then grinding with high fluxThe leaves were broken with an instrument (Geno/Grind6875), 20. mu.g 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 65 ℃ water bath for 30min, added with chloroform/isoamyl alcohol (24: 1) of equal volume, centrifuged at 10000rpm/min for 10min, the supernatant was taken and transferred to a new centrifuge tube, 0.8 times the volume of precooled isopropanol was added, mixed well by gentle inversion, left at-20 ℃ for 30min and centrifuged at 4 ℃ and 12,000r/min for 10 min. Discarding supernatant, washing with 70% ethanol solution for 2 times, naturally drying, and adding 100 μ L ddH₂And dissolving DNA to obtain the genomic DNA of the tested Chinese cabbage variety, and detecting the concentration for later use at 4 ℃.

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: 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. Respectively taking the genome DNA of 216 tested Chinese cabbage varieties as templates, and respectively adopting 18 primer pairs 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.

Each 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 primer, the amplification products of different fluorescence labels of the same combined primer with the same volume are respectively taken, the TAMRA fluorescence product is diluted by 50 times, and other fluorescence products are fully and uniformly mixed after being diluted by 100 times. Pipette 2. 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 DNA analyzer is turned on, and the working state and the reagent state of the instrument 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 reference instrument default values (the maximum excitation wavelength of FAM is 494nm, the maximum excitation wavelength of HEX is 535nm, the maximum excitation wavelength of TAMRA is 560nm, and the maximum excitation wavelength of ROX is 587nm) for detecting the fluorescent primers need to perform spectrum correction on the capillary electrophoresis equipment regularly.

Some of the results are shown in FIGS. 3-20. The results show that each primer group can obtain good typing effect in the tested Chinese cabbage variety (Beijing New No. three).

4. Cluster analysis

According to the genotypes of the 216 tested Chinese cabbage varieties based on the 18 SSR loci, the MEGA7 software is used for carrying out cluster analysis on the 216 tested Chinese cabbage varieties.

The clustering chart of 216 tested brassica rapa varieties established on 18 primer sets is shown in figure 1. The results show that the 18 primer sets can completely distinguish 216 tested Chinese cabbage varieties in the table 3. Therefore, the SSR primer combination developed in the embodiment 1 can be applied to the construction of a Chinese cabbage variety DNA fingerprint database 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 inventor of the invention compares the relationship between the number of SSR markers (namely the number of primer groups) and the distinguishing rate of 216 varieties of Chinese cabbages to be tested.

The number of the difference marks compared and counted between 216 varieties is shown in figure 2, wherein the number of the results compared between two varieties is

A plurality of; in 23220, the number of differential sites was about 22% of the total 12, 15% of the total 13, 5% of the total 14, 1% of the total 15, and 0% of the total 16-18, so that the results for more than 12 differential sites accounted for about 43%, indicating that the polymorphisms were good in 216 varieties using these markers; the discrimination rate of 18 primer groups (namely 18 SSR markers) in 216 tested Chinese cabbage varieties reaches 100 percent.

Based on the SSR genotype data of 18 SSR loci in 216 parts of Chinese cabbage varieties, main allelic variation and frequency (shown in table 4) of each marker are counted, and the 18 SSRs are high in polymorphism in 216 parts of Chinese cabbage varieties and suitable for authenticity identification of the Chinese cabbage varieties.

Table 4: the typing frequency of 216 varieties at 18 SSR sites was statistically as follows:

wherein, the main allelic variation is obtained according to the statistics of 216 varieties of the Chinese cabbages to be tested in the table 3. For example: if a sample only has 1 allelic variation at a certain locus and the size is 282bp, the genotype of the main allelic variation at the locus is written as 282/282; if a sample has two allelic variations at a site, the sizes of which are 114bp and 122bp, respectively, the genotype of the major allelic variation at the site is written as 114/122.

Example 3

Method for detecting whether to-be-detected Chinese cabbage variety belongs to 216 Chinese cabbage varieties to be detected

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

The leaf of the Chinese cabbage variety to be tested (actually Jingjian 70) is taken from the test base of vegetable research center of agriculture and forestry academy of sciences of Beijing.

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

3. Fluorescence capillary electrophoresis detection

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

Comparing the fragment sizes of the 18 SSR amplification products of the Chinese cabbage variety to be detected with 18 SSR loci of 216 Chinese cabbage varieties to be detected (also standard Chinese cabbage varieties) (shown in Table 3), counting the number of difference loci of the Chinese cabbage variety to be detected and 216 standard Chinese cabbage varieties, and judging as follows:

if the number of the malposition points of the Chinese cabbage variety to be detected and a standard Chinese cabbage variety is 2 or more, the Chinese cabbage variety to be detected and the standard Chinese cabbage variety belong to different Chinese cabbage varieties; the greater the number of differential sites, the more distant the genetic relationship.

If the number of the malposition points of the Chinese cabbage variety to be detected and a standard Chinese cabbage variety is 1 or 0, the Chinese cabbage variety to be detected and the standard Chinese cabbage variety are or are suspected to be the same Chinese cabbage variety.

The result shows that the number of the difference sites of the Chinese cabbage variety to be detected and the 216 Chinese cabbage varieties to be tested on the 18 SSR sites is more than 4, so that the Chinese cabbage variety to be detected does not belong to any one of the 216 Chinese cabbage varieties to be tested, namely the Chinese cabbage variety to be detected is different from any one of the 216 Chinese cabbage varieties to be tested.

Example 4

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

In this case, the ABI 3730 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 primer, the amplification products of different fluorescence labels of the same combined primer with the same volume are respectively taken, the TAMRA labeled product is diluted by 50 times, and other fluorescence labeled products are fully and uniformly mixed after being diluted 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. The DNA was placed on a DNA analyzer after instantaneous centrifugation for 10 s.

S2: the ABI 3730 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: comparing the fragment sizes of the 18 SSR amplification products of the Chinese cabbage variety to be detected with 18 SSR loci of 216 Chinese cabbage varieties to be detected (shown in Table 3), counting the number of difference loci of the Chinese cabbage variety to be detected and 18 standard Chinese cabbage varieties, and judging as follows:

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.

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Claims

1. An SSR locus for identifying the authenticity of a Chinese cabbage variety, wherein the SSR locus is selected from any 1 to 18 of the following first SSR locus to eighteenth SSR locus:

the eighteenth SSR locus is positioned at the 10162405-10162432 th chromosome of the Chinese cabbage reference genome 10 or a homologous genome segment among varieties thereof;

the Chinese cabbage reference genome is a Chinese cabbage Chiifu-401-42 reference genome V1 edition.

2. An SSR primer group for identifying the authenticity of Chinese cabbage varieties, which is used for respectively amplifying SSR loci according to claim 1, and comprises:

a first SSR primer pair for amplifying said first SSR site;

a second SSR primer pair for amplifying said second SSR site;

a third SSR primer pair for amplifying said third SSR site;

a fourth SSR primer pair for amplifying said fourth SSR site;

a fifth SSR primer pair for amplifying said fifth SSR site;

a sixth SSR primer pair for amplifying said sixth SSR site;

a seventh SSR primer pair for amplifying said seventh SSR locus;

an eighth SSR primer pair for amplifying said eighth SSR locus;

a ninth SSR primer pair for amplifying said ninth SSR locus;

a tenth SSR primer pair for amplifying said tenth SSR site;

an eleventh SSR primer pair for amplifying said eleventh SSR site;

a twelfth SSR primer pair for amplifying said twelfth SSR site;

a thirteenth SSR primer pair for amplifying said thirteenth SSR site;

a fourteenth SSR primer pair for amplifying said fourteenth SSR site;

a fifteenth SSR primer pair for amplifying said fifteenth SSR site;

a sixteenth SSR primer pair for amplifying the sixteenth SSR site;

a seventeenth SSR primer pair for amplifying the seventeenth SSR site;

and the eighteenth SSR primer pair is used for amplifying the eighteenth SSR locus.

3. The SSR primer set according to claim 2 characterized in that:

and the second SSR primer pair is respectively matched with 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 eleventh SSR primer pair is respectively matched 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 seventeenth SSR primer pair is respectively matched 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%;

and the eighteenth SSR primer pair is respectively matched with 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%;

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.

4. An SSR kit for identifying the authenticity of Chinese cabbage varieties is characterized in that: the reagent in the SSR kit is used for preparing a PCR reaction system; the PCR reaction system comprises:

the SSR primer set according to claim 2 or 3,

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; more preferably, 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: 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 in the system and Tris-HCL (pH7.5-9.0) with final concentration of 1-10mmol/L in the system.

5. A detection method for identifying the authenticity of Chinese cabbage varieties 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 Chinese cabbage to be detected according to claim 1;

step two: the variety judgment of the Chinese cabbage to be detected comprises the following steps:

and if the number of the different loci of the genotype of the Chinese cabbage to be detected based on the 18 SSR loci and the genotype of each variety in the Chinese cabbage standard variety library based on the 18 SSR loci is more than or equal to 2, the varieties of the Chinese cabbage to be detected and each of the Chinese cabbage standard varieties in the Chinese cabbage standard variety library are different.

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

the step of detecting the SSR locus genotype of the Chinese cabbage to be detected comprises the following steps:

the method comprises the following steps: respectively carrying out PCR amplification by using the genomic DNA of the Chinese cabbage to be detected and the genomic DNA of the Chinese cabbage standard variety as templates and respectively adopting the primer group in the SSR primer combination according to claim 2 or 3 to obtain PCR amplification products;

step two is carried out: and carrying out length detection on the PCR amplification product to obtain genotypes of the Chinese cabbage to be detected and the Chinese cabbage standard variety based on 18 SSR loci.

7. The detection method of claim 6: 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 Chinese cabbage and the standard Chinese cabbage variety based on the 18 SSR loci; or:

detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the Chinese cabbage to be detected and the standard Chinese cabbage variety based on the 18 SSR loci.

8. The detection method of claim 5: the method is characterized in that: in the second step, the judgment result is obtained according to cluster analysis.

9. The detection method according to any one of claims 5 to 8, wherein: the standard Chinese cabbage variety bank is selected from any one or more of the following 216 Chinese cabbage varieties:

beijing Xin No. 1, Beijing Xiaoza 50, Tianwa, Dahuahuang, Qinqiao 60, Zhengbai 65, Tianzheng orange red 62, Tianzheng orange red 65, Jihong 308, Lichun, Qingyangqinbai No. 1, Beijing Xiaoza 67, Beijing Xinxin No. 2, Beijing orange red heart, Zhongbai 65, Zhongbai 81, Jingchuzao, Jingxiawang, Jingluo 60, Xiaoza 56, Zhongbai 50, Zhongbai 60, Delong No. 1, Jingxian No. 1, Beijing Xinxin No. 4, Yangchun, Chunbao, Chunqiu 54, Qiangqing, Beijing orange No. 2, Jingchunbai, Jingchun 988, Beijing improved 67, Beijing great ox heart, Beijing, Chunxun, Chunquan, Shichun 50, Beijing 80, Wangchun, Guang 58, Cuiyoubao, Beijing yellow 70, Beijing Xiaodeqinqiu No. 60, Jingdeqinqiu No. 60, Beijing white Beijing, Beijing Baichun No. 2, Beijing orange No. 2, Beijing Baichun, Beijing Baichun No. 60, Beijing Baichun, Beijing Baichun No. 2, Beijing Baichun, Beijing Xinchun No, Mini star, degao 16, jin qiu 606, zhong bai 62, wei bai 4, jing qiu 75, jin qiu 78, huichun, jing qiu baby vegetable, jin qiu 90, jing chun huang, jing chun wa 2, jin zao 9, jin zao 58, jing qiu 3, xiao 60, jing qiu 70, jin hua, you 3, jin qiu 1, jiu jian 85, jing qiu 4, jing qiu 76, jin xiu 1, beijing xiao 51, zhen green 55, zhen green 80, jiao bai 7, jing chun huang 2, jing chun 3, zhong bai 61, jing qiu 701, jing cui 75, ji hong hua, SW701, lai 38, jin green 75, jing hao green 2, ji cai yi 30, zhen hao jian hao cui 30, zhen hao cui, jing kuan 70, zhong nong qing hao qing huan jian hua 78, SW 356, qing bai 303, qing hua jian qing CR1, qing hua 58, zhen qing hao, zhen hao CR1, zhen hao 55, zhen hao, zhen huan hao 7, zhen hao 2, zhe, Middle bracket, Yi and 1410, Fushan walnut grain, Tian Bai 65, jin doll No. 1, Chun Yu Huang, jin Jian Kuo Hao Green No. 1, SGK1008, Xiao Yi and Qiu, 87-114, YHBC1511, YHBC1516, Shen Bai GMS02, Qing nong No. 45, Yuxin No. 1, Shi Green 85, Huanai CC001, Huanai CC002, Yi and No. 1, Zhen Lu 60, Jing Chun CR3, mountain king No. 2, Jing Chun Wa 4, Ling Huang, CR Tian Bai 15, De Gao CR117, Jing Qiu No. 635, Jihong 65, Lu Jian 60, Juxin No. one, Lichun Yi No. 70, Lu Bai No. 50, Zhongbai No. 61, Zhongbai No. 81 selected, Zhongbai No. 83, Zhongbai No. 85, Zhongbai 58 Zhongbai No. 76, Qiu Green 78, jin Green 80, Qiu Green 75, Qiu Green 60, Qiu Green bud Green No. 55, Qiu Green No. 6, Zao Huang No. 6, Zan Zao Shen No. 5, Zheng Bai Shen Huan Hao No. 5, chinese medicine 68, Xinzao 89-8, Zhengbai No. four, Xinsanhao, Chunfeng 007, Xibai No. 5, Xibai No. four, Xichunbai No. 3, Taijing No. 50, Zhongbai No. 78, Xibai No. 3, Xibai No. 10, Labai No. one, precocious five, Tianzhengzhengchaobai No. two, Liaobai No. ten, Xibai No. 4, LI-1, Jinhuayuan No. 19, Qinbai No. 2, Guanchun, Chunyao, Xinzao 58, Lubai No. 16, Qiuyu 78, Qingyan No. four, Fennan F115-93, Shandong No. 5, Shenbai GMS01, Jingcui 60, Sicui, Qiuchi No. 9, Fengcheng 60, Qingyangchun No. one, Luchunbai No. 6, Zhejiang white No. 75, Xinxiang 23, Zhendong green No. 6, Shandong Wu No. five, Beijing No. three, Zhenghuan hong No. 62, Zhengchun No. 2, Huachunbai No. 2, Beijing.

10. The SSR site of claim 1, or the SSR primer combination of claim 2 or 3, or the SSR kit of claim 4, or the detection method of any one of claims 5 to 9, for use in X1 or X2: