CN107988413B - Method for identifying authenticity of cucumber variety and special SSR primer group thereof - Google Patents
Method for identifying authenticity of cucumber variety and special SSR primer group thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying the authenticity of cucumber varieties and a special SSR primer group thereof. The SSR primer group provided by the invention consists of 32 primers, and the nucleotide sequences of the 32 primers are sequentially shown as a sequence 1 to a sequence 32 in a sequence table. The SSR primer group provided by the invention has the advantages of high polymorphism, good repeatability, stable and reliable marking, convenience in statistics and the like, and the DNA fingerprint database for identifying the authenticity of the cucumber variety based on high-throughput sequencing is also established, so that the SSR primer group can be used for carrying out early identification on the cucumber variety in the seed or seedling stage, ensuring the authenticity of the variety, practically protecting the rights and interests of producers and breeders, providing technical support for the protection of cucumber germplasm resources and new varieties, and having wide application prospect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for identifying the authenticity of cucumber varieties and a special SSR primer group thereof.
Background
China is the country with the largest cucumber cultivation area (about 139.6 million hectares) and the highest total yield (about 70 percent of the global yield) in the world. Since the reform was open, the cucumber industry has rapidly developed, and more than 300 varieties are approved and registered only by the country. Because the cucumber breeding enterprises are small and scattered, the variety management cannot follow effectively, and the variety approval among provinces and cities is mutually inconsistent. In addition, the management of an improved seed production base is disordered, the phenomena of stealing breeding and purchasing varieties are rampant, the phenomenon of same-species and different names is serious, the quality of seeds is not uniform, and the quality accidents of the seeds occur occasionally. At present, how to manage cucumber varieties efficiently and protect rights and interests of producers and breeders become one of the main problems facing the development of the current cucumber industry. 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 high-throughput DNA fingerprint technology system for effectively identifying the authenticity of cucumber varieties is urgently needed to be established.
SSR (simple Sequence repeats) is a series repeat Sequence which is composed of several nucleotides (generally 2-6) as repeat units and has the advantages of rich polymorphism, good repeatability, simple detection method and the like. However, the traditional cucumber SSR marker does not have reference genome variation group information at present, and unreal or variant situations exist; in addition, the SSR detection method is limited to easily cause unreal, false positive and false negative results, and the specific expression is as follows: 1) the SSR has simple sources, lacks the support of genome and genetic variation group, has poor polymorphism, and easily generates peak-cutting by the repeated SSR of two bases; 2) the SSR detects that the size of an amplified fragment is not changed (false negative) or is changed due to the interference of other SSRs, indels/SNPs (single nucleotide polymorphisms) in the amplified region and the motif region; 3) the traditional SSR detection method is characterized in that the length of a microsatellite marker is judged through gel electrophoresis, and a reference variety is needed for comparison, so that the detection workload is increased, and the result lacks reliable stability and reproducibility; 4) the requirements of automatic, high-throughput, low-cost detection cannot be met.
In recent years, with the continuous development of high-throughput sequencing technology and the continuous reduction of sequencing cost, the amplicon sequencing technology combining high-throughput sequencing and multiplex PCR brings a new solution for large-scale variety identification. By designing a specific SSR primer group for a site to be detected, different DNA samples are distinguished by different barcode sequences, and multiple PCR amplification is carried out in a single tube. And carrying out deep sequencing on the amplification product of the mixed sample, and finally obtaining SSR sequence information of different samples of each site according to a sequencing result. The method can solve the problems in the traditional SSR detection technology and has a very wide application prospect.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify cucumber varieties.
In order to solve the technical problems, the invention firstly provides an SSR primer group. The SSR primer group can comprise a primer pair M1, a primer pair M2, a primer pair M3, a primer pair M4, a primer pair M5, a primer pair M6, a primer pair M7, a primer pair M8, a primer pair M9, a primer pair M10, a primer pair M11, a primer pair M12, a primer pair M13, a primer pair M14, a primer pair M15 and a primer pair M16;
the primer pair M1 can be composed of a primer M1-F and a primer M1-R; the primer pair M2 can be composed of a primer M2-F and a primer M2-R; the primer pair M3 can be composed of a primer M3-F and a primer M3-R; the primer pair M4 can be composed of a primer M4-F and a primer M4-R; the primer pair M5 can be composed of a primer M5-F and a primer M5-R; the primer pair M6 can be composed of a primer M6-F and a primer M6-R; the primer pair M7 can be composed of a primer M7-F and a primer M7-R; the primer pair M8 can be composed of a primer M8-F and a primer M8-R; the primer pair M9 can be composed of a primer M9-F and a primer M9-R; the primer pair M10 can be composed of a primer M10-F and a primer M10-R; the primer pair M11 can be composed of a primer M11-F and a primer M11-R; the primer pair M12 can be composed of a primer M12-F and a primer M12-R; the primer pair M13 can be composed of a primer M13-F and a primer M13-R; the primer pair M14 can be composed of a primer M14-F and a primer M14-R; the primer pair M15 can be composed of a primer M15-F and a primer M15-R; the primer pair M16 can be composed of a primer M16-F and a primer M16-R.
The primer M1-F can be any one of the following single-stranded DNA molecules A1) or A2):
A1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
A2) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A1).
The primer M1-R can be any one of the following single-stranded DNA molecules A3) or A4):
A3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
A4) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A3).
The primer M2-F can be any one of the following single-stranded DNA molecules A5) or A6):
A5) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
A6) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A5).
The primer M2-R can be any one of the following single-stranded DNA molecules A7) or A8):
A7) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
A8) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A7).
The primer M3-F can be any one of the following single-stranded DNA molecules A9) or A10):
A9) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
A10) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A9).
The primer M3-R can be any one of the following single-stranded DNA molecules A11) or A12):
A11) a single-stranded DNA molecule shown in sequence 6 of the sequence table;
A12) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A11).
The primer M4-F can be any one of the following single-stranded DNA molecules A13) or A14):
A13) a single-stranded DNA molecule shown in sequence 7 of the sequence table;
A14) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A13).
The primer M4-R can be any one of the following single-stranded DNA molecules A15) or A16):
A15) a single-stranded DNA molecule shown in sequence 8 of the sequence table;
A16) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A15).
The primer M5-F can be any one of the following single-stranded DNA molecules A17) or A18):
A17) a single-stranded DNA molecule shown in sequence 9 of the sequence table;
A18) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A17).
The primer M5-R can be any one of the following single-stranded DNA molecules A19) or A20):
A19) a single-stranded DNA molecule shown in sequence 10 of the sequence table;
A20) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in A19).
The primer M6-F can be any one of the following single-stranded DNA molecules B1) or B2):
B1) a single-stranded DNA molecule shown in sequence 11 of the sequence table;
B2) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B1).
The primer M6-R can be any one of the following single-stranded DNA molecules B3) or B4):
B3) a single-stranded DNA molecule shown in sequence 12 of the sequence table;
B4) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B3).
The primer M7-F can be any one of the following single-stranded DNA molecules B5) or B6):
B5) a single-stranded DNA molecule shown in sequence 13 of the sequence table;
B6) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B5).
The primer M7-R can be any one of the following single-stranded DNA molecules B7) or B8):
B7) a single-stranded DNA molecule shown as a sequence 14 in a sequence table;
B8) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B7).
The primer M8-F can be any one of the following single-stranded DNA molecules B9) or B10):
B9) a single-stranded DNA molecule shown in sequence 15 of the sequence table;
B10) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B9).
The primer M8-R can be any one of the following single-stranded DNA molecules B11) or B12):
B11) a single-stranded DNA molecule shown as sequence 16 in the sequence table;
B12) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B11).
The primer M9-F can be any one of the following single-stranded DNA molecules B13) or B14):
B13) a single-stranded DNA molecule shown in sequence 17 of the sequence table;
B14) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B13).
The primer M9-R can be any one of the following single-stranded DNA molecules B15) or B16):
B15) a single-stranded DNA molecule shown in sequence 18 of the sequence table;
B16) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B15).
The primer M10-F can be any one of the following single-stranded DNA molecules B17) or B18):
B17) a single-stranded DNA molecule shown as sequence 19 in the sequence table;
B18) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B17).
The primer M10-R can be any one of the following single-stranded DNA molecules B19) or B20):
B19) a single-stranded DNA molecule shown in sequence 20 of the sequence table;
B20) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in B19).
The primer M11-F can be any one of the following single-stranded DNA molecules in C1) or C2):
C1) a single-stranded DNA molecule shown in sequence 21 of the sequence table;
C2) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C1).
The primer M11-R can be any one of the following single-stranded DNA molecules in C3) or C4):
C3) a single-stranded DNA molecule shown as a sequence 22 in a sequence table;
C4) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C3).
The primer M12-F can be any one of the following single-stranded DNA molecules in C5) or C6):
C5) a single-stranded DNA molecule shown as sequence 23 in the sequence table;
C6) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C5).
The primer M12-R can be any one of the following single-stranded DNA molecules in C7) or C8):
C7) a single-stranded DNA molecule shown in sequence 24 of the sequence table;
C8) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C7).
The primer M13-F can be any one of the following single-stranded DNA molecules in C9) or C10):
C9) a single-stranded DNA molecule shown as sequence 25 in the sequence table;
C10) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C9).
The primer M13-R can be any one of the following single-stranded DNA molecules in C11) or C12):
C11) a single-stranded DNA molecule shown as a sequence 26 in a sequence table;
C12) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C11).
The primer M14-F can be any one of the following single-stranded DNA molecules in C13) or C14):
C13) a single-stranded DNA molecule shown as sequence 27 in the sequence table;
C14) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C13).
The primer M14-R can be any one of the following single-stranded DNA molecules in C15) or C16):
C15) a single-stranded DNA molecule shown as sequence 28 in the sequence table;
C16) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C15).
The primer M15-F can be any one of the following single-stranded DNA molecules in C17) or C18):
C17) a single-stranded DNA molecule shown as sequence 29 in the sequence table;
C18) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C17).
The primer M15-R can be any one of the following single-stranded DNA molecules in C19) or C20):
C19) a single-stranded DNA molecule shown as a sequence 30 in a sequence table;
C20) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in C19).
The primer M16-F can be any one of the following single-stranded DNA molecules D1) or D2):
D1) a single-stranded DNA molecule shown as sequence 31 in the sequence table;
D2) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in D1).
The primer M16-R can be any one of the following single-stranded DNA molecules D3) or D4):
D3) a single-stranded DNA molecule shown as a sequence 32 in a sequence table;
D4) a single-stranded DNA molecule having 85% or more identity to the single-stranded DNA fragment defined in D3).
The SSR primer group may specifically include the primer pair M1, the primer pair M2, the primer pair M3, the primer pair M4, the primer pair M5, the primer pair M6, the primer pair M7, the primer pair M8, the primer pair M9, the primer pair M10, the primer pair M11, the primer pair M12, the primer pair M13, the primer pair M14, the primer pair M15, and the primer pair M16.
The application of any SSR primer group also belongs to the protection scope of the invention. The application of any SSR primer group can be any one of x1) to x 6):
x1) preparing a kit for identifying cucumber varieties;
x2) preparing a kit for identifying the authenticity of the cucumber variety;
x3) preparing a kit for analyzing genetic relationship of cucumber varieties;
x4) identifying cucumber varieties;
x5) identifying the authenticity of the cucumber variety;
x6) analyzing the genetic relationship of cucumber varieties.
A kit containing any SSR primer group is also in the protection scope of the invention.
The preparation method of the kit also belongs to the protection scope of the invention. The preparation method of the kit comprises the step of separately packaging each primer in any SSR primer group.
The application of the kit also belongs to the protection scope of the invention. The application of the kit can be x4) or x5) or x 6):
x4) identifying cucumber varieties;
x5) identifying the authenticity of the cucumber variety;
x6) analyzing the genetic relationship of cucumber varieties.
The invention provides a method for identifying which variety of 165 cucumbers to be detected belongs to, which comprises the following steps:
(1) taking the genome DNA of cucumber to be detected as a template, and carrying out PCR amplification by adopting any SSR primer group to obtain a PCR amplification product; performing PCR amplification by using the genome DNA of each cucumber variety in the standard cucumber population as a template and adopting any SSR primer group to obtain a PCR amplification product; the standard cucumber population consists of 165 cucumber varieties;
(2) and performing cluster analysis on the PCR amplification product of the cucumber to be detected and the PCR amplification product of each standard cucumber variety, wherein the cucumber to be detected and the standard cucumber variety in the cluster analysis are the same, and the cucumber to be detected and the standard cucumber variety belong to the same variety.
The invention also discloses a method for detecting whether the cucumber variety to be detected belongs to 165 cucumber varieties, which comprises the following steps:
(1) taking the genome DNA of cucumber to be detected as a template, and carrying out PCR amplification by adopting any SSR primer group to obtain a PCR amplification product; performing PCR amplification by using the genome DNA of each cucumber variety in the standard cucumber population as a template and adopting any SSR primer group to obtain a PCR amplification product; the standard cucumber population consists of 165 cucumber varieties;
(2) comparing the PCR amplification product of the cucumber variety to be detected with the PCR amplification product of each standard cucumber variety, counting the number of difference sites, and then judging as follows:
if the number of the difference sites between the cucumber variety to be detected and a standard cucumber variety is more than 2, the cucumber variety to be detected and the standard cucumber variety belong to different cucumber varieties; the more the number of the differential sites is, the farther the genetic relationship is;
if the number of the different sites of the cucumber variety to be detected and a standard cucumber variety is less than 2, the cucumber variety to be detected and the standard cucumber variety are or are suspected to be the same cucumber variety.
In any of the above methods, the 165 CUCUMBER varieties may be specifically north star of japan, zhongnong No. 19, zhuang melon, zhongnong No. 15, zhongnong No. 9, CUCUMBER1404, shenlv 64, jin you 12, jin chun No. 4, jin you No. 31, jin you No. 2, nin you 3, chun jade, shanza No. 6, bao za No. 2, ning yun No. 3, jin you No. 36, jin you 38, zhongnong No. 26, MC2065, jin you 20-11, sequarry, bo mei No. 11, emerald green, chun 1, lu san No. 3, C05-016, Dongnong 806, seolai old and young, jin you No. 48, shenho, nan estra No. 1, P01, nan estra CC No. 2, P02, qiu 3, ningfeng 09, jia quan full-female 09, HH-8-1-2, zuo-627, zuo-yun-3, jing H-1, jin you 401, jinyujin you # 401, jinyun 1, zuo # 401, zuo-1, zuo-yujin-1, zuo-1, zuo-yuzuo-1, zuo-yuzuo, zuo-yuzuo-1, zuo-yuzuo, zuo-1, zuo-6, zuo-kunji, zuo-zuo, zuo-6, zuo-zuo, zuo-6, zuo-6, zuo, jinyou No. 108, Huanai H1104, Jinyou No. 303, Qingmei, Qingshuang, Jiejuo, Bomei 5032, Yufengyuan No. 6, Bomei 517, Bomei No. 28, Delaute D19, Bomei No. 10, shouli HG1, shouli HG2, DS2121, Jiza 9, Cui Li, Jinyou 35, Biyu No. 2, Chuanqi No. 3, Liangliang Liang, Dongnong 804, Vinci dense spine, Jingyan mini No. 2, Jinyou 308, Bomei No. 8, Bomei 6913, 13AC230, Delaute 79, Delaute Y2, 13AC049, Delaut F16, Zhongnong No. 12, Condeza (Condez F1), Lvchun No. two, Lufengchun No. 5, Shuojin No. 316, Guaijing you No. 358, Miyao Shuangnong No. 37, Zhongnong Shenqiliang Shenyu No. 7, Cucumis Heiden Yu Ying Lu, Yu Jia Yu No. 7766, Cucumin Hao Yu, Cucumis Hakko No. 7, Cucumis Hakko Yu Ying Yu, Cucumis Hakko No. 6, Cucumis Hakko Yu, Cucumis No. 6, Cucumis Hakko No. 6, Cucumis Bayu Yu Ying Yu No. 6, Cucumis Hakko, Cucumis Hakkaiba Shi Yu No. 6, Cucumis Hakko No. 6, Cucumis Bayu No. 6, Cucumis Daihu, Cucumis No. 6, Cucumis Bayu, Jingbaiyu super white leaf No. 3, De Ruite No. 10, De Ruite cucumber L14-2, De Ruite cucumber L14-5, De Ruite cucumber GZ1603, Zhongnong No. 27, Zhongnong No. 8, Zhongnong No. 18, Xin Zhen No. four, Yongling 3-6, Mici King cucumber, Yameite (Thailand), AMATA765, Hu Gua, Lboqi No. seven, Lboqi No. eleven, Manguan, Tangchun 100, Chunmei, ao Guang, Jingfeng 298, Jing Min No. 3, Lxiu, Jing Cao, fruit type 101, Jing Min Hanbao No. 5, Jing Minqiumei, Jing Min 106, Jing Min No. 9, Jing Minchun Mei, Beijing 204, Beijing 403, Beijing Xia Minamei, Beijing Min No. 5, Ljinling No. 4, Jing Ming Lu 2, Jing Min 4, Jing Min Jia, Jing Min Ji, Jing Yun Mao, Jing Lu No. 1-13, Bai Mao, Bai Mao No. 1-13, Bei Mao, Bei Hao Mao, Bei shan Fei, Bei Mao Lang 1-13-Shi Fei, Bei Hao, Bei Mao, Bei Bai Mao, Bei Mao, Shi Mao, Bei shan No. 4, Bei Mao, Shi shan Dou Shi Hao, Shi Mao, Shi Hua Mao, Shi mu (Bei Mao, Shi mu (Bei mu No. 4, Yu Mao, Shi mu (Bei Mao, and so 1-6, and so on Shi mu No. 4, 15-1, 17, 23, 27, 38, 39, 40, a21, a24, a28, and a 30.
In any of the above-mentioned methods, the concentration of each primer in the reaction system for performing PCR amplification using any of the SSR primer sets described above may be specifically 0.2 μ M. The reaction procedure of performing PCR amplification by using any one of the SSR primer groups specifically comprises the following steps: 3min at 95 ℃; at 95 ℃ for 30s, at 60 ℃ for 4min, for 17 cycles; 4min at 72 ℃.
The SSR primer group provided by the invention has the advantages of high polymorphism, good repeatability, stable and reliable marking, convenience in statistics and the like, and can reflect the genetic relationship of the cucumber variety to be tested to the greatest extent. In addition, the invention establishes the DNA fingerprint database for identifying the authenticity of the cucumber variety based on high-throughput sequencing for the first time, can be used for carrying out early identification on the cucumber variety in the seed or seedling stage, ensures the authenticity of the variety, practically protects the rights and interests of producers and breeders, and provides technical support for the protection of cucumber germplasm resources and new varieties. The method provided by the invention can be used for identifying unknown cucumber varieties and also can be used for identifying the authenticity of known varieties. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.
Drawings
FIG. 1 is a cluster map of 165 cucumber varieties established on 16 SSR primer pairs.
FIG. 2 is a graph showing the relationship between SSR primer pairs and 165 cucumber varieties.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 acquisition of SSR primer set for identifying the authenticity of cucumber varieties
The invention develops an SSR primer group which is suitable for high-throughput sequencing and used for identifying the authenticity of cucumber varieties based on the re-sequencing data of 49 parts of representative cucumber resources. The 49 cucumbers are rich in resource types, include North China (7), Indian (18), Japanese (3), south China (2), European fruit (5), American processed (4), Xishuangbanna (5) and intermediate (5), basically comprise the main ecological types and agronomic traits of the cucumbers, reflect germplasm representativeness as much as possible, and have higher genetic diversity.
Specifically, the SSR screening criteria are as follows: SNP and InDel are not existed in the motif region; MAF >0.3 and evenly distributed over the genome; the flanking sequence (200bp) connected with the motif region has no variation of a poly region, other SSRs, SNPs, indels and the like. Sequence information of the SSR primers is obtained by analyzing sequencing data of 49 cucumber resources. Finally, the inventors of the present invention developed an SSR primer set consisting of 16 SSR primer pairs uniformly distributed throughout the cucumber genome, with a higher polymorphism information content (PIC value). The nucleotide sequences of these 16 SSR primer pairs are shown in Table 1.
TABLE 1.16 names and nucleotide sequences of SSR primer pairs
Example 2, validation of SSR primer sets developed in example 1
The basic information of 165 cucumber varieties tested in this example is shown in table 2. The 165 cucumber varieties to be tested are all common fine varieties in production or partially foreign introduced varieties.
TABLE 2.165 cucumber species information
1. Acquisition of genomic DNA of cucumber varieties to be tested
Genomic DNAs of 165 leaves (cotyledons of 5 mixed seeds) of the cucumber species to be tested were extracted by the CTAB method, respectively, to obtain the genomic DNAs of the cucumber species to be tested. The quality and concentration of the genome DNA of the cucumber variety to be tested both need to meet the PCR requirement, and the standard of reaching the standard is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; detecting that the ratio of A260/A280 is about 1.8 and the ratio of A260/A230 is more than 2.0 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of genomic DNA of the cucumber variety tested was >50 ng/. mu.L.
2. Preparation of Panelmix primer mixture
Each primer shown in table 1 was synthesized manually, and then each primer was mixed to obtain a PanelMix primer mixture.
3. Preparation of sequencing libraries
Sequencing libraries were prepared for each cucumber variety tested. The specific steps for preparing the sequencing library for each cucumber variety tested were as follows:
(1) quantification of
By using
2.0dsDNA accurate quantification of genomic DNA of cucumber varieties tested.
(2) First round PCR amplification
And (3) carrying out first round PCR amplification by using the genome DNA of the cucumber variety to be tested as a template and adopting the Panelmix primer mixed solution prepared in the step (2) to obtain a first round PCR amplification product.
The reaction system for the first round of PCR amplification was 30. mu.L, consisting of 8. mu.L of the Panel Mix primer Mix, 50ng of the genomic DNA of the cucumber species tested, 10. mu.L of 3M enzyme and water. In this reaction system, the concentration of each primer was 0.2. mu.M.
Reaction procedure: 3min at 95 ℃; at 95 ℃ for 30s, at 60 ℃ for 4min, for 17 cycles; 4min at 72 ℃.
(3) Purification of
a) Adding a first round of PCR amplification product and magnetic beads (volume ratio is 2:1) into a centrifuge tube, gently and uniformly blowing by using a pipettor, and standing at room temperature for 2 min; then the centrifuge tube was placed on a magnetic stand and allowed to stand at room temperature, and the supernatant was collected.
b) Taking another new centrifuge tube, adding the supernatant and the magnetic beads (volume ratio is 10:7) collected in the step a), gently and uniformly blowing by using a pipette, and standing for 2min at room temperature; then placing the centrifuge tube on a magnetic frame for standing at room temperature, and removing the supernatant; adding 30 mu L of magnetic bead suspension into the centrifuge tube, resuspending, and standing at room temperature for 2 min; and finally, placing the centrifuge tube on a magnetic frame, standing at room temperature, and removing the supernatant.
c) After the step b) is finished, taking the centrifugal tube, adding 100 mu L of 80% (v/v) ethanol water solution, placing the centrifugal tube on a magnetic frame, and repeatedly adsorbing magnetic beads on two different surfaces by using the magnetic frame to fully wash the magnetic beads; then adsorbing for 2min by a magnetic frame, removing the supernatant, and standing at room temperature until the ethanol is completely volatilized.
(4) Second round of PCR amplification
a) And preparing a reaction system for the second round of PCR amplification.
The reaction system for the second round of PCR amplification was 30. mu.L, and consisted of magnetic beads after completion of step (3), 10. mu.L of 3M enzyme, 1. mu.L of primer F aqueous solution (10. mu.M concentration), 1. mu.L of primer R aqueous solution (10. mu.M concentration), and 18. mu.L of water.
Primer F：
5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG-3’；
Primer R：
5’-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCCTTGGCACCCGAGA-3' (the barcode sequence is underlined).
b) And c) after the step a) is finished, carrying out PCR amplification on the reaction system of the second round of PCR amplification to obtain a second round of PCR amplification product.
Reaction procedure: 3min at 95 ℃; 15s at 95 ℃, 15s at 58 ℃, 30s at 72 ℃ and 7 cycles; 4min at 72 ℃.
The second round of PCR amplification products added a linker and barcode compared to the first round PCR amplification products. Each sample corresponds to a specific barcode for the purpose of subsequent high throughput sequencing.
(5) Purification of
a) Adding a second round of PCR amplification product and magnetic beads (volume ratio is 1:1) into a centrifuge tube, gently and uniformly blowing by using a pipettor, and standing at room temperature for 2 min; then the centrifuge tube was placed on a magnetic stand and allowed to stand at room temperature, and the supernatant was discarded.
b) After the step a) is finished, taking the centrifugal tube, adding 30 mu L of magnetic bead suspension, carrying out resuspension, and standing for 2min at room temperature; and finally, placing the centrifuge tube on a magnetic frame, standing at room temperature, and removing the supernatant.
c) After the step b) is finished, taking the centrifugal tube, adding 100 mu L of 80% (v/v) ethanol water solution, placing the centrifugal tube on a magnetic frame, and repeatedly adsorbing magnetic beads on two different surfaces by using the magnetic frame to fully wash the magnetic beads; then adsorbing for 2min by a magnetic frame, removing the supernatant, and standing at room temperature until the ethanol is completely volatilized.
d) After step c) was completed, the tube was taken, resuspended in 23. mu.L of 10mM Tris-HCl buffer, pH8.0-8.5, and then allowed to stand at room temperature for 2 min.
e) And d), after the step d) is finished, taking the centrifuge tube, placing the centrifuge tube in a magnetic separator for 5min, and transferring the supernatant to the centrifuge tube to obtain a sequencing library of the tested cucumber variety.
4. Sequencing on machine
And respectively taking the sequencing library of each cucumber variety to be tested, and performing machine sequencing.
5. Cluster analysis and SSR fingerprint
And (3) obtaining the motif sequence variation information of the 165 cucumber varieties to be tested amplified in the 16 SSR primer pairs according to the sequencing result obtained in the step (4) (see table 3), and then carrying out cluster analysis on the 165 cucumber varieties to be tested by utilizing PowerMarker and MEGA7 software.
TABLE 3.16 basic information on SSR primers
The cluster map of 165 cucumber varieties tested, established on 16 SSR primer pairs, is shown in FIG. 1. The results show that the 16 SSR primer pairs can completely distinguish 165 cucumber varieties to be tested in the table 2. Therefore, the SSR primer group developed in the example 1 can be applied to the construction of a cucumber variety DNA fingerprint database and variety authenticity identification.
6. Evaluation of efficiency
The variety authenticity identification can reduce the workload by adopting a sequential analysis mode. The inventor of the invention compares the relation between the SSR primer pair number and the identification rate for distinguishing 165 cucumber varieties to be tested. The experimental results show (figure 2) that the recognition rate of 16 SSR primer pairs in 165 cucumber varieties to be tested reaches 100%.
Example 3 method for detecting whether cucumber variety to be tested belongs to one of 165 cucumber varieties to be tested
1. Obtaining of genome DNA of cucumber variety to be tested
The leaves of the cucumber variety to be tested are 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 leaves of the cucumber variety to be tested are replaced by the leaves of the cucumber variety to be tested, and other steps are not changed, so that the genome DNA of the cucumber variety to be tested is obtained.
2. Preparation of Panelmix primer mixture
Same as step 2 in example 2.
3. Preparation of sequencing libraries
According to the method of step 3 in the embodiment 2, the 'genome DNA of the cucumber variety to be tested' is replaced by the 'genome DNA of the cucumber variety to be tested', and other steps are not changed, so that the sequencing library of the cucumber variety to be tested is obtained.
4. Sequencing on machine
And (4) taking a sequencing library of the cucumber variety to be detected, and sequencing.
The sequencing results of 16 SSR amplification products of cucumber varieties to be tested are respectively compared with 16 SSR loci of 165 cucumber varieties to be tested (shown in Table 2), the number of difference loci of the two cucumber varieties is counted, and then the following judgment is carried out:
if the number of the difference sites between the cucumber variety to be detected and a standard cucumber variety is more than 2, the cucumber variety to be detected and the standard cucumber variety belong to different cucumber varieties; the more the number of the differential sites is, the farther the genetic relationship is;
if the number of the different sites of the cucumber variety to be detected and a standard cucumber variety is less than 2, the cucumber variety to be detected and the standard cucumber variety are or are suspected to be the same cucumber variety.
The result shows that the number of the difference sites of the cucumber variety to be detected and 165 cucumber varieties to be tested on the 16 SSR sites is more than 4, so that the cucumber variety to be detected does not belong to any one of the 165 cucumber varieties to be tested, namely the cucumber variety to be detected is different from any one of the 165 cucumber varieties to be tested.
<110> agriculture and forestry academy of sciences of Beijing City
<120> method for identifying cucumber variety authenticity and special SSR primer group thereof
<160> 32
<170> PatentIn version 3.5
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actcgctttg cattgtgtcg 20
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Claims (6)
- An SSR primer group, which comprises a primer pair M1, a primer pair M2, a primer pair M3, a primer pair M4, a primer pair M5, a primer pair M6, a primer pair M7, a primer pair M8, a primer pair M9, a primer pair M10, a primer pair M11, a primer pair M12, a primer pair M13, a primer pair M14, a primer pair M15 and a primer pair M16;the primer pair M1 consists of a primer M1-F and a primer M1-R; the primer pair M2 consists of a primer M2-F and a primer M2-R; the primer pair M3 consists of a primer M3-F and a primer M3-R; the primer pair M4 consists of a primer M4-F and a primer M4-R; the primer pair M5 consists of a primer M5-F and a primer M5-R; the primer pair M6 consists of a primer M6-F and a primer M6-R; the primer pair M7 consists of a primer M7-F and a primer M7-R; the primer pair M8 consists of a primer M8-F and a primer M8-R; the primer pair M9 consists of a primer M9-F and a primer M9-R; the primer pair M10 consists of a primer M10-F and a primer M10-R; the primer pair M11 consists of a primer M11-F and a primer M11-R; the primer pair M12 consists of a primer M12-F and a primer M12-R; the primer pair M13 consists of a primer M13-F and a primer M13-R; the primer pair M14 consists of a primer M14-F and a primer M14-R; the primer pair M15 consists of a primer M15-F and a primer M15-R; the primer pair M16 consists of a primer M16-F and a primer M16-R;the primer M1-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;the primer M1-R is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;the primer M2-F is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;the primer M2-R is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;the primer M3-F is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;the primer M3-R is a single-stranded DNA molecule shown in a sequence 6 in a sequence table;the primer M4-F is a single-stranded DNA molecule shown in a sequence 7 in a sequence table;the primer M4-R is a single-stranded DNA molecule shown in a sequence 8 of a sequence table;the primer M5-F is a single-stranded DNA molecule shown in a sequence 9 in a sequence table;the primer M5-R is a single-stranded DNA molecule shown in a sequence 10 of a sequence table;the primer M6-F is a single-stranded DNA molecule shown in a sequence 11 in a sequence table;the primer M6-R is a single-stranded DNA molecule shown in a sequence 12 in a sequence table;the primer M7-F is a single-stranded DNA molecule shown in a sequence 13 in a sequence table;the primer M7-R is a single-stranded DNA molecule shown in a sequence 14 in a sequence table;the primer M8-F is a single-stranded DNA molecule shown in a sequence 15 in a sequence table;the primer M8-R is a single-stranded DNA molecule shown in a sequence 16 in a sequence table;the primer M9-F is a single-stranded DNA molecule shown in a sequence 17 in a sequence table;the primer M9-R is a single-stranded DNA molecule shown in a sequence 18 in a sequence table;the primer M10-F is a single-stranded DNA molecule shown in a sequence 19 in a sequence table;the primer M10-R is a single-stranded DNA molecule shown in a sequence 20 in a sequence table;the primer M11-F is a single-stranded DNA molecule shown in a sequence 21 in a sequence table;the primer M11-R is a single-stranded DNA molecule shown in a sequence 22 of a sequence table;the primer M12-F is a single-stranded DNA molecule shown in a sequence 23 in a sequence table;the primer M12-R is a single-stranded DNA molecule shown in a sequence 24 of a sequence table;the primer M13-F is a single-stranded DNA molecule shown in a sequence 25 in a sequence table;the primer M13-R is a single-stranded DNA molecule shown in a sequence 26 in a sequence table;the primer M14-F is a single-stranded DNA molecule shown in a sequence 27 in a sequence table;the primer M14-R is a single-stranded DNA molecule shown in a sequence 28 of a sequence table;the primer M15-F is a single-stranded DNA molecule shown in a sequence 29 in a sequence table;the primer M15-R is a single-stranded DNA molecule shown in a sequence 30 in a sequence table;the primer M16-F is a single-stranded DNA molecule shown in a sequence 31 in a sequence table;the primer M16-R is a single-stranded DNA molecule shown in a sequence 32 of a sequence table.
- 2. The SSR primer set of claim 1, characterized in that:the SSR primer group consists of the primer pair M1, the primer pair M2, the primer pair M3, the primer pair M4, the primer pair M5, the primer pair M6, the primer pair M7, the primer pair M8, the primer pair M9, the primer pair M10, the primer pair M11, the primer pair M12, the primer pair M13, the primer pair M14, the primer pair M15 and the primer pair M16.
- 3. The use of the SSR primer set of claim 1 or 2, being any one of x1) to x 6):x1) preparing a kit for identifying cucumber varieties;x2) preparing a kit for identifying the authenticity of the cucumber variety;x3) preparing a kit for analyzing genetic relationship of cucumber varieties;x4) identifying cucumber varieties;x5) identifying the authenticity of the cucumber variety;x6) analyzing the genetic relationship of cucumber varieties.
- 4. A kit containing a SSR primer set according to claim 1 or 2.
- 5. A method of making a kit according to claim 4 comprising the step of packaging each primer of the SSR primer sets according to claim 1 or 2 separately.
- 6. The use of the kit of claim 4, being x4) or x5) or x 6):x4) identifying cucumber varieties;x5) identifying the authenticity of the cucumber variety;x6) analyzing the genetic relationship of cucumber varieties.
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| CN109536615B (en) * | 2018-10-23 | 2020-09-18 | 中国水产科学研究院南海水产研究所 | Development method and application of microsatellite marker primer |
| CN110317898A (en) * | 2019-06-28 | 2019-10-11 | 北京市农林科学院 | A kind of method for identifying cucumber variety authenticity and its combination of dedicated SSR primer |
| CN111411165B (en) * | 2020-04-20 | 2021-04-27 | 北京市农林科学院 | SNP (Single nucleotide polymorphism) site primer combination for identifying cucumber germplasm authenticity and application |
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