CN111270004A

CN111270004A - Method for identifying authenticity of pepper variety and special SSR primer combination thereof

Info

Publication number: CN111270004A
Application number: CN202010210002.9A
Authority: CN
Inventors: 温常龙; 杜和山; 杨静静; 张建; 罗江
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2020-06-12
Anticipated expiration: 2040-03-23
Also published as: CN111270004B

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 pepper variety and a special SSR primer combination thereof. Identifying SSR sites of pepper variety authenticity, and performing data mining according to pepper reference genome Capsicum. The SSR primer combination is selected from: the first SSR primer pair to the twenty-second SSR primer pair are respectively used for PCR amplification of the first SSR locus to the twenty-second SSR locus, and are preferably the same as SEQ ID NO: 1-44 is 85% -100% homologous to the nucleotide sequence. The method can be used for identifying the authenticity of the pepper variety in the whole life cycle from seeds, and provides technical support for pepper germplasm resources and new variety protection.

Description

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

Background

The capsicum is a plant of the genus capsicum of the family solanaceae of the class magnoliaceae, is suitable for being eaten raw, cooked and used as a seasoning, is the most widely planted and eaten crop all over the world, originates from mexico to colombia, and is introduced to be cultivated widely all over the world.

Because of the universality of pepper cultivation and the edible universality, the pepper is a plant which is extremely popular with human beings, and because of certain difference in the actual requirements of the pepper all over the world, the breeding direction of the pepper is extremely vigorous and endless, and meanwhile, because the economic benefits generated by the pepper are rich, the pepper varieties on the market are almost difficult to count. In 2017, the 'registration and protection system for new crop varieties' is implemented in China, and by the end of 2019, the number of the registered pepper varieties exceeds 4000, so that a very severe test is provided for the approval and management work of the crop varieties in China, and how to quickly and effectively identify the pepper varieties becomes a problem to be solved urgently.

The problem of identifying the authenticity of pepper varieties always exists, and molecular marker identification means has been introduced since the number of the pepper varieties is huge. However, the prior SSR molecular identification technology is developed based on a small number of varieties and has no support of genome data, so that the identification capability and representativeness of the SSR molecular identification technology are greatly insufficient.

Disclosure of Invention

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

The invention is realized by the following technical scheme:

an SSR locus for identifying the authenticity of a pepper variety, wherein the SSR locus is selected from any 1 to 22 of a first SSR locus to a twenty-second SSR locus as follows: a first SSR site located at chromosome 3 of the pepper reference genome at position 37196662-37196880, or an interspecies homologous genomic fragment thereof; a second SSR site located at chromosome 4 of the pepper reference genome at position 9461660-9461928 or an interspecies homologous genomic fragment thereof; a third SSR locus located at chromosome 4 of the pepper reference genome at positions 13136291-13136448 or an interspecies homologous genomic fragment thereof; a fourth SSR locus located at chromosome 5 of the pepper reference genome at position 32758554-32758739 or an interspecies homologous genomic fragment thereof; a fifth SSR site located at chromosome 5 of the pepper reference genome at position 208120671-208120969, or an interspecies homologous genomic fragment thereof; a sixth SSR site located on chromosome 6 of the Capsicum annuum reference genome at positions 194333083-194333311, or an interspecies homologous genomic fragment thereof; a seventh SSR locus located at chromosome 7 of the pepper reference genome at position 204507520-204507743 or an interspecies homologous genomic fragment thereof; an eighth SSR site located at chromosome 9 of the pepper reference genome at position 173948946-173949110 or an interspecies homologous genomic fragment thereof; a ninth SSR site located at chromosome 10, position 118282068 and 118282357 of the pepper reference genome, or an interspecies homologous genomic fragment thereof; a tenth SSR locus located at the 163975350-163975616 th chromosome 11 of the capsicum reference genome or an interspecies homologous genomic fragment thereof; an eleventh SSR site located on chromosome 12 of the pepper reference genome at position 63723425-63723524, or an interspecies homologous genomic fragment thereof; a twelfth SSR locus located at chromosome 8, position 130825854 and 130826103 of the pepper reference genome, or an interspecies homologous genomic fragment thereof; a thirteenth SSR site located on chromosome 10 of the pepper reference genome at position 197117583 and 197117732, or an interspecies homologous genomic fragment thereof; a fourteenth SSR site located at chromosome 2 of the capsicum reference genome, position 110132463 and 110132662, or an interspecies homologous genomic fragment thereof; a fifteenth SSR site located at chromosome 146789152-146789252 not located in the pepper reference genome, or an interspecies homologous genomic fragment thereof; a sixteenth SSR locus which is positioned at the 4 th chromosome 213298719 and 213298732 of the pepper reference genome or an interspecies homologous genome fragment thereof; a seventeenth SSR site located at chromosome 10 of the pepper reference genome at position 33073195-33073206, or an interspecies homologous genomic fragment thereof; an eighteenth SSR locus which is located at the chromosome 9 of the pepper reference genome at positions 107586969 and 107586985, or an interspecies homologous genomic fragment thereof; a nineteenth SSR locus located at chromosome 9 of the pepper reference genome at positions 107585342 and 107585353 or an interspecies homologous genomic fragment thereof; a twentieth SSR site located at chromosome 4 of the pepper reference genome at positions 36880157 and 36880168, or an interspecies homologous genomic fragment thereof; the twenty-first SSR locus is positioned at the 7 th chromosome 215759919 and 215759930 of the capsicum reference genome or an interspecies homologous genome fragment thereof; a twenty-second SSR locus located at the 5 th chromosome 214376146-214376155 of the pepper reference genome or an interspecies homologous genome fragment thereof; the pepper reference genome is capsicum.

An SSR primer group for identifying the authenticity of pepper varieties, wherein the SSR primer group is used for amplifying the SSR loci respectively, 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; an eighteenth SSR primer pair for amplifying the eighteenth SSR locus; a nineteenth SSR primer pair for amplifying said nineteenth SSR site; a twenty-second SSR primer pair for amplifying said twenty-second SSR site.

In some embodiments, the first SSR primer pair is identical to SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the second SSR primer pair is similar to the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the third SSR primer pair is similar to the primer pair shown in SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fifth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the sixth SSR primer pair is similar to the sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the seventh SSR primer pair is similar to SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the eighth SSR primer pair is similar to SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the ninth SSR primer pair is similar to SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the tenth SSR primer pair is similar to SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SSR primer pair is similar to SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the twelfth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the thirteenth SSR primer pair is similar to SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the fifteenth SSR primer pair is similar to SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the sixteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SSR primer pair is similar to SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighteenth SSR primer pair is similar to SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the nineteenth SSR primer pair is similar to SEQ ID NO: 37 and SEQ ID NO: 38 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twentieth SSR primer pair is similar to SEQ ID NO: 39 and SEQ ID NO: 40 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-first SSR primer pair is similar to SEQ ID NO: 41 and SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the twenty-second SSR primer pair is similar to SEQ ID NO: 43 and SEQ ID NO: 44 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, one primer of each pair of said primers is linked to a fluorescent molecule, more preferably said fluorescent molecule is selected from the group consisting of ROX, TAMRA, FAM, HEX.

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

A detection method for identifying the authenticity of a pepper variety comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of the pepper to be detected; step two: and (3) judging the variety of the pepper to be detected: if the number of the difference loci of the 22 SSR loci-based genotypes of the to-be-detected pepper and a certain specified variety of the standard pepper variety based on the 22 SSR loci is 0-2, judging the to-be-detected pepper and the specified variety of the standard pepper variety to be a similar variety; if the number of different loci of the to-be-detected pepper based on the genotypes of the 22 SSR loci and the 22 SSR loci based on the genotypes of a certain specified variety in standard pepper varieties is more than 2, judging the specified variety of the to-be-detected pepper and the standard pepper variety to be a different variety; preferably, the result of the determination is obtained from a cluster analysis.

In some embodiments, the step of detecting the SSR locus genotype of the pepper to be detected comprises the following sub-steps: the method comprises the following steps: respectively taking the genome DNA of the pepper to be detected and the genome DNA of the pepper 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 detecting the PCR amplification product to obtain the genotypes of the to-be-detected pepper and the pepper standard variety based on the 22 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 pepper and the standard pepper variety based on the 22 SSR sites; or detecting the amplified product fragment: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the to-be-detected pepper and the standard pepper variety based on the 22 SSR sites.

In some embodiments, the standard pepper variety is selected from the following 108 pepper varieties: firstly, hong Wu, hong Jiang No. 1, Xiang Jiang No. 6, Zun spicy No. 7, Wang Tian hong No. three, seed pepper No. four, Xiang spicy No. four, B173, peak, green light early-growing, Xiang spicy No. 18, Xiang spicy Qiu Xiang, hong bird No. 1, Chang Zheng 58, SV8233HD, SV0108HA, crisp snail No. two, crisp snail No. one, Qu pepper No. 1, Yu hetero No. two, Sai Jia Mei, Su pepper No. 5 doctor king, Lanyuan Xin Yuan, Hunan Ying Zao No. 1, Yu Feng Techong meat Zanthoxylum (404), Su Capsici No. 5, Yang pepper No. 2, Fuqiang 0055, seed pepper No. eight, Su Capsici No. 15, Cheng Ma 219, Su Ma No. 17, Xinfeng ya 7318, Dong Huang yellow No. 1, Xiuyu No. 6, Ming Yang pepper No. 7, Gao Zao No. 1, HN Zao 14465, H Zao, Xin Zao No. 2, Xin Zao No. 107, Xin Zao hong Zao, kailai, Shen pepper No. three, 37-94, Luzun, Hengjiao No. 2, New science No. 8, PP1201, D pepper No. 7, Laobao 89, Kita, Van 1, Lafengshuliang, Yuanhualiang No. 4, resource No. 3, Huamei 1504, Yue pepper No. 8, Lamei No. 2, New science No. 18, Yu pepper No. 3, Yun pepper No. 2, resource No. 4, upturne 148, resource No. 5, H20, Zhongjiao No. 6, Xiang Jian No. 11, Zhongjiao No. 7, resource No. 6, resource No. 7, resource No. 8, resource No. 9, hong Da shuai, resource No. 10, Shenjian, Fu pepper No. six, Xinhuo No. one, Fengchi, Huangliren, Chanhuan, resource No. 11, resource No. 12, Honglong 16, Changsong, Chang pepper No. one, Wantian red pepper No. 98A, Luo pepper resource No. 18, resource No. 13, No. 15, resource No. 17, resource No.

A detection method for identifying whether pepper varieties are the same is characterized in that: the peppers to be detected are two kinds of peppers of unknown variety; the detection method comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of the pepper to be detected; step two: and judging whether the varieties of the peppers to be detected are the same: if the number of the different loci of the pepper to be detected based on the genotypes of the 22 SSR loci is 0-2, judging the pepper to be detected to be a similar variety; and if the difference locus of the pepper to be detected based on the genotypes of the 22 SSR loci is more than 2, judging the pepper to be detected to be different varieties.

The SSR locus, or the SSR primer combination, or the SSR kit, or the detection method is applied to the following X1 or X2 or X3: x1: identifying whether the variety of the pepper to be detected belongs to one of standard pepper varieties; x2: identifying the variety of the pepper to be detected to be a standard pepper variety; x3: and identifying whether the pepper samples to be detected are the same varieties.

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

1. the invention provides an SSR molecular marker identification method which can quickly, accurately and stably detect variety authenticity. According to the invention, whole genome re-sequencing is carried out on a large number of pepper varieties, data mining is carried out by combining with a pepper reference genome, a large number of representative SSR sites are obtained, the markers are screened through various screening conditions, and finally a set of more reasonable SSR site combinations are obtained, so that the SSR site combinations have stronger discrimination capability and better representativeness to the whole genome compared with the original standard. The SSR primer combination can be used for carrying out early identification on pepper varieties in seed or seedling stages, and also can carry out authenticity identification of a full life cycle from seeds, so that the authenticity of the varieties is ensured, the problems of homonymy and heteronymy in seed and seedling markets in China can be solved, the rights and interests of producers and breeders are practically protected, and a powerful technical support is provided for pepper germplasm resources and new variety protection.

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

3. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

4.①, plant large-scale sequencing has just been rising for a few years, most of the previous SSR related technologies have no reference genome as data base, so that the SSR related technologies can only be selected blindly and randomly, the selected markers are completely luck, and the potential discrimination ability has not been mentioned at all. ② the invention invests a great deal of labor, carries out re-sequencing on 40 varieties, uses the data as data base, is used together with the reference genome to carry out big data analysis, carries out massive data mining and calculation, and uses the markers used by other people as verification materials compared with the prior art or selects the markers from some free databases to have essential differences. ③ the invention adopts varieties provided by breeding experts as verification materials, so that the discrimination ability of the SSR markers is fully guaranteed. ④ due to the data mining method used by the invention, the selected SSR markers can represent whole genome information very effectively, and simultaneously the adopted pepper variety materials cover all types of commercial pepper varieties, and the SSR markers selected by the invention have very strong potential discrimination ability.

5.① is quick, when identifying variety, it can use DNA extracted from seedling or even seed to detect, it is different from DUS identification that mature plant can be identified, the identification time is shortened from several months to several hours, ② is relative to phenotype identification method, the identification method based on DNA detection is not affected by external environment, it will not change due to environmental condition, its result is stable and reliable, ③ is identified by the method of the invention, it is identified in each phenological period of hot pepper, its result is stable and consistent, 22 SSR sites ④ are detected by Target-seq sequencing, 3730 fluorescence capillary detection and polyacrylamide gel electrophoresis, the height of multi-platform detection result is consistent, it is fully proved that the reliability of each SSR site of the invention, ⑤ is not needed to be accumulated by long time experience compared with DUS test.

Drawings

FIG. 1 is a cluster plot of 108 pepper varieties tested on 22 primer sets as set forth in example 1.

FIGS. 2 to 23 are graphs showing the SSR typing effects of the 22 primer sets in example 2 on a part of the tested pepper varieties.

Wherein, fig. 2: the primer is CaSSR028, the adopted variety is white noble, and the method is shown in figure 3: the primer is CaSSR032, the variety adopted is Pepper eight, and the method is shown in figure 4: primer CaSSR033, adopted breed is Hongniao No. 1, and figure 5: primer CaSSR040, the variety adopted is Xinxiu, FIG. 6: primer CaSSR042, variety adopted is spike, FIG. 7: primer CaSSR047, species great wall, figure 8: primer CaSSR053, adopted breed is Fuqiang 0055, figure 9: primer CaSSR063, adopted variety Ronieao, figure 10: the primer is CaSSR071, the adopted variety is Jinmom 219, and the sequence is shown in FIG. 11: primer CaSSR074, adopted variety resource No. 8, FIG. 12: primer CaSSR081, the breed of adoption is the star of the blue garden, figure 13: primer CaSSR088, adopted breed 148, FIG. 14: the primer is CaSSR098, the adopted variety is crisp snail No. two, and the method is shown in figure 15: the primer is CaSSR104, the adopted variety is Wangtianhong I, and the sequence is shown in figure 16: the primer is CaZQSSR10, the variety adopted is Zhu-Zhen No. 1, and the primer is shown in FIG. 17: the primer is CaSSr-3-001, the variety adopted is firstly red five, and the sequence is shown in figure 18: the primer is CaSSr-3-007, the adopted variety is green light early growth, and the sequence is shown in figure 19: the primer is CaSSr-3-010, the adopted variety is Bian jiao I, and the sequence is shown in figure 20: primer CaSSr-3-023, the variety used is yellow Susan, FIG. 21: primer CaSSr-3-033, adopted variety red bell pepper, FIG. 22: the primer is CaSSr-3-052, the adopted variety is Luzun, and FIG. 23: the primer is CaSSr-3-056, and the adopted variety is No. 7 Ming Pepper.

FIG. 24 is a difference marker chart of the number of SSR markers (i.e., the number of SSR loci) and the differentiation of 108 tested pepper varieties in example 2.

Detailed Description

The definition is as follows:

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

Interspecies homologous genomic fragments: refers to a genomic fragment that is homologous to a reference genomic sequence of Capsicum annuum L in other varieties of Capsicum annuum L, in addition to the reference genomic sequence of Capsicum annuum L. For example, for a particular genomic fragment, there are identical genomic fragments to the capsicum. annuum.l reference genomic sequence in 108 standard varieties of the invention.

In a first aspect, the invention provides SSR loci for identifying the authenticity of pepper varieties, wherein the SSR loci are respectively located in pepper genomes, the number of the SSR loci is 22, 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 22 of the following first SSR site to twenty-second SSR site: a first SSR site located at chromosome 3 of the pepper reference genome at position 37196662-37196880, or an interspecies homologous genomic fragment thereof;

a second SSR site located at chromosome 4 of the pepper reference genome at position 9461660-9461928 or an interspecies homologous genomic fragment thereof;

a third SSR locus located at chromosome 4 of the pepper reference genome at positions 13136291-13136448 or an interspecies homologous genomic fragment thereof;

a fourth SSR locus located at chromosome 5 of the pepper reference genome at position 32758554-32758739 or an interspecies homologous genomic fragment thereof;

a fifth SSR site located at chromosome 5 of the pepper reference genome at position 208120671-208120969, or an interspecies homologous genomic fragment thereof;

a sixth SSR site located on chromosome 6 of the Capsicum annuum reference genome at positions 194333083-194333311, or an interspecies homologous genomic fragment thereof;

a seventh SSR locus located at chromosome 7 of the pepper reference genome at position 204507520-204507743 or an interspecies homologous genomic fragment thereof;

an eighth SSR site located at chromosome 9 of the pepper reference genome at position 173948946-173949110 or an interspecies homologous genomic fragment thereof;

a ninth SSR site located at chromosome 10, position 118282068 and 118282357 of the pepper reference genome, or an interspecies homologous genomic fragment thereof;

a tenth SSR locus located at the 163975350-163975616 th chromosome 11 of the capsicum reference genome or an interspecies homologous genomic fragment thereof;

an eleventh SSR site located on chromosome 12 of the pepper reference genome at position 63723425-63723524, or an interspecies homologous genomic fragment thereof;

a twelfth SSR locus located at chromosome 8, position 130825854 and 130826103 of the pepper reference genome, or an interspecies homologous genomic fragment thereof;

a thirteenth SSR site located on chromosome 10 of the pepper reference genome at position 197117583 and 197117732, or an interspecies homologous genomic fragment thereof;

a fourteenth SSR site located at chromosome 2 of the capsicum reference genome, position 110132463 and 110132662, or an interspecies homologous genomic fragment thereof;

a fifteenth SSR site located at chromosome 146789152-146789252 not located in the pepper reference genome, or an interspecies homologous genomic fragment thereof;

a sixteenth SSR locus which is positioned at the 4 th chromosome 213298719 and 213298732 of the pepper reference genome or an interspecies homologous genome fragment thereof;

a seventeenth SSR site located at chromosome 10 of the pepper reference genome at position 33073195-33073206, or an interspecies homologous genomic fragment thereof;

an eighteenth SSR locus which is located at the chromosome 9 of the pepper reference genome at positions 107586969 and 107586985, or an interspecies homologous genomic fragment thereof;

a nineteenth SSR locus located at chromosome 9 of the pepper reference genome at positions 107585342 and 107585353 or an interspecies homologous genomic fragment thereof;

a twentieth SSR site located at chromosome 4 of the pepper reference genome at positions 36880157 and 36880168, or an interspecies homologous genomic fragment thereof;

the twenty-first SSR locus is positioned at the 7 th chromosome 215759919 and 215759930 of the capsicum reference genome or an interspecies homologous genome fragment thereof;

the twenty-second SSR locus is located at the 5 th chromosome 214376146-214376155 of the pepper reference genome or an interspecies homologous genome fragment thereof.

The pepper reference genome is capsicum.

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

The SSR primer combination is selected from: and the first SSR primer pair to the twenty-second SSR primer pair are respectively used for PCR amplification of the first SSR locus to the twenty-second SSR locus. The first SSR primer pair is similar to SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second SSR primer pair is similar to SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the third SSR primer pair is similar to SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourth SSR primer pair is similar to SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fifth SSR primer pair is similar to SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the sixth SSR primer pair is similar to SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the seventh SSR primer pair is similar to SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighth SSR primer pair is similar to SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the ninth SSR primer pair is similar to SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the tenth SSR primer pair is similar to SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SSR primer pair, together with SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twelfth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the thirteenth SSR primer pair, as described above, is identical to SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourteenth SSR primer pair is a primer pair that binds to SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the fifteenth SSR primer pair, together with SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the sixteenth SSR primer pair is similar to SEQ id no: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SSR primer pair, in combination with SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighteenth SSR primer pair is similar to SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the nineteenth SSR primer pair is similar to SEQ ID NO: 37 and SEQ ID NO: 38 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twentieth SSR primer pair is similar to SEQ ID NO: 39 and SEQ ID NO: 40 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-first SSR primer pair is similar to SEQ ID NO: 41 and SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the twenty-second SSR primer pair is similar to SEQ ID NO: 43 and SEQ ID NO: 44 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%.

In a preferred embodiment, the SSR primer combination is selected from one or more of primer sets 01-22; the DNA sequence information of the primer groups 01-22 is shown in a sequence table SEQ ID: 1-44, see table 2.

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

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

A reaction system, preferably comprising:

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

In a fourth aspect, the invention provides a detection method for identifying the authenticity of a pepper variety, comprising the following steps:

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

The method comprises the following steps: respectively taking the genomic DNA of the pepper to be detected and the genomic DNA of the pepper 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 detecting the PCR amplification product to obtain the genotypes of the hot pepper to be detected and the hot pepper standard variety based on 22 SSR loci.

The detection may be a fluorescence signal detection: detecting the fluorescent signal of the PCR amplification product to obtain the genotypes of the hot pepper to be detected and the standard hot pepper variety based on the 22 SSR loci;

the detection can also be the detection of amplified product fragments: and (3) detecting the fragment size of the PCR amplification product by utilizing capillary electrophoresis to obtain the genotypes of the to-be-detected pepper and the standard pepper variety based on the 22 SSR sites.

Step two: judging the variety of the pepper to be detected:

by clustering and analyzing the varieties of the hot pepper to be detected and the standard hot pepper, judging the standard references GB/T3543.1, GB/T3543.5 and NY/T2594, and obtaining the following results based on the genotypes of the 22 SSR loci:

if the number of the difference loci of the 22 SSR loci-based genotypes of the to-be-detected pepper and the 22 SSR loci-based genotypes of a certain specified variety in the standard pepper variety is 0-2, the to-be-detected pepper and the variety of the standard pepper variety belong to similar varieties;

and if the number of different loci of the genotype of the hot pepper to be detected based on the 22 SSR loci and the genotype of a certain specified variety in the hot pepper standard variety based on the 22 SSR loci is more than 2, judging the specified variety of the hot pepper to be detected and the hot pepper standard variety to be different varieties.

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 pepper varieties comprise the following 108 pepper varieties:

firstly, hong Wu, hong Jiang No. 1, Xiang Jiang No. 6, Zun spicy No. 7, Wang Tian hong No. three, seed pepper No. four, Xiang spicy No. four, B173, peak, green light early-growing, Xiang spicy No. 18, Xiang spicy Qiu Xiang, hong bird No. 1, Chang Zheng 58, SV8233HD, SV0108HA, crisp snail No. two, crisp snail No. one, Qu pepper No. 1, Yu hetero No. two, Sai Jia Mei, Su pepper No. 5 doctor king, Lanyuan Xin Yuan, Hunan Ying Zao No. 1, Yu Feng Techong meat Zanthoxylum (404), Su Capsici No. 5, Yang pepper No. 2, Fuqiang 0055, seed pepper No. eight, Su Capsici No. 15, Cheng Ma 219, Su Ma No. 17, Xinfeng ya 7318, Dong Huang yellow No. 1, Xiuyu No. 6, Ming Yang pepper No. 7, Gao Zao No. 1, HN Zao 14465, H Zao, Xin Zao No. 2, Xin Zao No. 107, Xin Zao hong Zao, kailai, Shen pepper No. three, 37-94, Luzun, Hengjiao No. 2, New science No. 8, PP1201, D pepper No. 7, Laobao 89, Kita, Van 1, Lafengshuliang, Yuanhualiang No. 4, resource No. 3, Huamei 1504, Yue pepper No. 8, Lamei No. 2, New science No. 18, Yu pepper No. 3, Yun pepper No. 2, resource No. 4, upturne 148, resource No. 5, H20, Zhongjiao No. 6, Xiang Jian No. 11, Zhongjiao No. 7, resource No. 6, resource No. 7, resource No. 8, resource No. 9, hong Da shuai, resource No. 10, Shenjian, Fu pepper No. six, Xinhuo No. one, Fengchi, Huangliren, Chanhuan, resource No. 11, resource No. 12, Honglong 16, Changsong, Chang pepper No. one, Wantian red pepper No. 98A, Luo pepper resource No. 18, resource No. 13, No. 15, resource No. 17, resource No.

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

Wherein, the peppers to be detected are two kinds of peppers of unknown variety;

the detection method comprises the following steps:

the method comprises the following steps: detecting the genotypes of the 22 SSR loci of the pepper to be detected; the detection method is as described in the fourth aspect.

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

if the number of the difference loci of the two unknown pepper varieties based on the genotypes of the 22 SSR loci is 0-2, judging the two unknown pepper varieties as similar varieties;

and if the difference loci of the two unknown pepper varieties based on the genotypes of the 22 SSR loci are more than 2, judging the two unknown pepper varieties to be different varieties.

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

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

x2: identifying the variety of the pepper to be detected to be a standard pepper variety;

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

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

Discovery of one, 22 SSR sites: these sites were obtained by data mining of the reference genome capsicum annuum l of the closely related species of capsicum and 40 germplasm resources genome-wide re-sequencing data.

1. The invention is different from other molecular marker identification methods, and relates to a method for randomly and blindly selecting SSR markers. The method is characterized in that different detection means such as Target-Seq simplified sequencing technology, ABI3730 fluorescence capillary electrophoresis and the like are used for verification, and finally 22 SSR sites with the strongest identification ability are selected as the SSR sites used in the method by combining 40 parts of actual materials.

The invention discovers and obtains 22 SSR loci for the first time based on the re-sequencing data of 40 parts of pepper representative resources. The 40 parts of pepper has rich resource types, covers the main ecological types and the agronomic traits of the existing peppers in the market, embodies germplasm representativeness as much as possible and has higher genetic diversity.

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

firstly, comparing 40 parts of pepper material with a reference genome sequence of Capsicum annuum L to find 14282 SSR loci, and according to the screening conditions: the data deletion is less than 10%, the heterozygosity is lower than 15%, the PIC is greater than 0.25, indels are not provided with SNP within 150bp of two ends, 2207 SSR sites are remained, finally, the SSR sites with the most representativeness are selected according to a data dimension reduction method, and 106 SSR sites are remained.

After the 106 SSR primers are used for amplifying 108 varieties (see example 2), SSR sequence information of the varieties is obtained by a TargetSeq technology, data analysis is carried out, and the remaining 99 SSR loci with deletion proportion larger than 10% of SSR locus data are removed and used as alternative loci. And then selecting 22 SSR sites by using a minimarker algorithm, wherein the algorithm preferentially considers the discrimination capability of the algorithm on 108 pepper varieties and comprehensively considers the conditions of SSR site polymorphism and the like.

In order to determine the reliability of the discrimination ability of the 22 SSR loci, the inventors redesign primers suitable for fluorescent capillary electrophoresis detection, perform PCR amplification on 108 pepper varieties by using the 22 pairs of primers, and perform fragment length detection by using an AB 3730 fluorescent capillary electrophoresis system, and the results prove that the selected 22 SSR loci have very high discrimination ability and reliability.

3. Specifically, the screening criteria for SSR sites are as follows: SSR sites which have uniform positions, good polymorphism, small heterozygosity, MAF >0.3, good PCA clustering effect and high discrimination and have 150bp sequence conservation (no InDel, no SNP and no other SSR) on both wings are selected in the whole genome range. The basic information of the 22 SSR sites is detailed in Table 1. Wherein the position of SSR loci on chromosome and motif information are determined based on alignment of pepper reference genome sequences, wherein the pepper reference genome sequences are shown in the following websites: website 1: ftp:// ftp. solgenomics. net/genes/Capsicum _ annuum/C.annuum _ zunla/associations/.

The PIC values and the major allelic variant amplification lengths in Table 1 were obtained for 108 varieties according to example 2.

TABLE 1.22 basic information of SSR loci

Second, obtaining SSR primer combination for identifying authenticity of pepper varieties

Based on the 22 SSR sites found in step one, the inventors of the present invention developed an SSR primer combination for identifying the authenticity of pepper varieties that has a high amount of polymorphism information (i.e., PIC value, which refers to the value of one marker for detecting polymorphisms in a population; PIC value depends on the number of detected alleles and their frequency distribution of alleles; PIC value is equal to 1 minus the sum of the squares of all allele frequencies). Primers are designed based on upstream and downstream sequences of the SSR locus in a pepper capsicum. 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 22 primer sets are shown in Table 2.

TABLE 2.22 information on primer sets

Example 2

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

The 108 tested pepper varieties in this example are all common fine varieties or partially foreign introduced varieties, and the specific varieties are as follows:

1. acquisition of genomic DNA of test Capsicum species

The genome DNA of 108 leaves of the pepper variety to be tested (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 pepper variety to be tested is obtained.

The CTAB method is specifically operated as follows:

respectively picking 108 leaves of the above variety in seedling stage, dewatering in a freeze dryer (CoolSafe 55-4), breaking the leaves with a high-throughput grinder (Geno/Grind68108), collecting 200mg dry powder of the leaves, adding 800 μ L CTAB extract (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1% PVP-40, 0.2% β -mercaptoethanol), mixing, placing in a water bath at 65 deg.C for 30min, adding equal volume of chloroform/isoamyl alcohol (v: v 20: 1), centrifuging at 10000rpm/min for 10min, transferring the supernatant to a new centrifuge tube, adding 0.8 volume of precooled isopropanol, mixing, standing at-20 deg.C for 30min, centrifuging at 4 deg.C for 12,000r/min for 10min, adding 70% ethanol solution, washing for 2 times, drying under natural ploidy condition, and adding 100 μ L ddH₂And dissolving DNA by O to obtain the genome DNA of the pepper variety to be tested, and detecting the concentration for later use at 4 ℃.

The quality and concentration of the genome DNA of the pepper variety to be tested meet the PCR requirement, and the standard of the standard is as follows: 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 genomic DNA of the pepper variety to be tested was 30-50 ng/. mu.L.

2. Respectively taking the genome DNA of 108 tested pepper varieties as templates, and respectively adopting 22 primer groups to carry out PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name and the primer containing "R" in the name was 1: 1.

The reaction system comprises:

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

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

3. Fluorescence capillary electrophoresis

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

Partial results are shown in FIGS. 2-23. The results show that each primer group can obtain good typing effect in the tested pepper varieties.

4. Cluster analysis

According to the genotypes of the 108 tested pepper varieties based on the 22 SSR loci, clustering analysis is carried out on the 108 tested pepper varieties by utilizing MEGA7 software.

The cluster map of 108 tested pepper varieties built on 22 primer sets is shown in FIG. 1. The result shows that 22 primer groups can completely distinguish 108 pepper varieties to be tested. Therefore, the SSR primer combination developed in the example 1 can be applied to the construction of a pepper variety DNA fingerprint database and variety authenticity identification.

5. Evaluation of efficiency

The variety authenticity identification can reduce the workload by adopting a sequential analysis mode. The inventors of the present invention compared the relationship between the number of SSR markers (i.e., the number of primer sets) and the discrimination rate for discriminating 108 pepper varieties to be tested.

The number of the difference markers compared and counted between 108 varieties as shown in FIG. 24, wherein the number of the results compared between two varieties is C2₁ ₀₈108 × 107 ÷ 2 ÷ 5778; in these results, the number of difference sites was 0.11% of the total number of 2, the number of difference sites was 0.24% of the total number of 3, the number of difference sites was 0.41% of the total number of 4, the number of difference sites was 1.34% of the total number of 5, the number of difference sites was 4.16% of the total number of 6, the number of difference sites was 6.79% of the total number of 7, the number of difference sites was 10.42% of the total number of 8, the number of difference sites was 13.54% of 9, the number of difference sites was 14.38% of 10, the number of difference sites was 16.22% of 11, the number of difference sites was 13.86% of 12, the number of difference sites was 8.32% of 13, the number of difference sites was 4.00% of 14, the number of difference sites was 2.18% of 15, the number of difference sites was 1.39% of 16, and the number of difference sites was 1.30% of 17, the number of differential sites was 0.96% of the total number of 18, 0.22% of the total number of 19, 0.16% of the total number of 20, and 0% of the total number of 21 and 22. This result indicates that the polymorphisms were good in 108 varieties with these markers; the discrimination rate of 22 primer groups (namely 22 SSR markers) in 108 pepper varieties to be tested reaches 100 percent.

Example 3

The present embodiment is a method for detecting whether a to-be-detected pepper variety belongs to 108 pepper varieties to be detected, where the pepper variety to be detected is unknown, and whether the pepper variety to be detected is one of the 108 pepper varieties needs to be obtained through the detection method of the present embodiment.

1. Obtaining of genomic DNA of pepper variety to be tested

The leaves of the pepper 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 leaf of the pepper variety to be tested is replaced by the leaf of the pepper variety to be tested, and the genome DNA of the pepper variety to be tested is obtained without changing other steps.

2. SSR primer and configuration of PCR reaction system

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

3. Fluorescence capillary electrophoresis detection

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

Comparing the fragment sizes of the 22 SSR amplification products of the pepper variety to be detected with the 22 SSR loci of the 108 pepper varieties to be detected in the embodiment 2, counting the number of difference loci of the pepper variety to be detected and the 22 standard pepper varieties, and then judging as follows:

if the number of the ectopic difference points between the pepper variety to be detected and a standard pepper variety is more than 2, judging the pepper variety to be detected and the standard pepper variety to be different pepper varieties; the greater the number of differential sites, the more distant the genetic relationship.

And if the number of the difference potential points of the pepper variety to be detected and a standard pepper variety is 0-2, judging the pepper variety to be detected and the standard pepper variety to be similar pepper varieties.

The result shows that the number of the different sites of the pepper variety to be tested and 108 pepper varieties to be tested on 22 SSR sites is more than 2, so that the pepper variety to be tested does not belong to any one of the 108 pepper varieties to be tested, namely the pepper variety to be tested is not one of the 108 pepper varieties to be tested.

Example 4

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

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

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

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

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

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

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

S5: comparing the fragment sizes of the 22 SSR amplification products of the pepper variety to be detected with the 22 SSR loci of 108 pepper varieties to be detected, counting the number of difference loci of the pepper variety to be detected and 22 standard pepper varieties, and then judging as follows:

And if the number of the difference potential points of the pepper variety to be detected and a standard pepper variety is 0-2, judging the pepper variety to be detected and the standard pepper variety to be similar to the pepper variety.

Example 5

In this embodiment, it is detected whether two unknown pepper varieties are the same, and the pepper varieties to be detected in this embodiment are the two unknown pepper varieties.

1. Obtaining of genomic DNA of pepper variety to be tested

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

2. SSR primer and configuration of PCR reaction system

3. Fluorescence capillary electrophoresis detection and data recording

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

4. In step 3, 22 SSR site data of two unknown pepper varieties (pepper varieties to be detected) are respectively obtained, the data of the two unknown pepper varieties on each SSR site in the 22 same SSR sites are respectively compared, and the number of the different sites is recorded.

If the number of the ectopic difference points of the two unknown pepper varieties at the 22 SSR sites is more than 2, judging the two unknown pepper varieties as different pepper varieties; the more the number of the differential sites is, the farther the genetic relationship is;

and if the number of the ectopic points of the two unknown pepper varieties at the 22 SSR sites is 0-2, judging the two unknown pepper varieties as similar pepper varieties.

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

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<211>25

<212>DNA

<213>Artificial Sequence

<400>48

ggattcacag gatgttgaag tgttt 25

Claims

1. An SSR locus for identifying the authenticity of a pepper variety, wherein the SSR locus is selected from any 1 to 22 of a first SSR locus to a twenty-second SSR locus as follows:

a first SSR site located at chromosome 3 of the pepper reference genome at position 37196662-37196880, or an interspecies homologous genomic fragment thereof;

the fifteenth SSR site located on the unmarked chromosome of the pepper reference genome

146789152-146789252 site, or an interspecies homologous genomic fragment thereof;

a twenty-second SSR locus located at the 5 th chromosome 214376146-214376155 of the pepper reference genome or an interspecies homologous genome fragment thereof;

the pepper reference genome is capsicum.

2. An SSR primer set for identifying the authenticity of pepper varieties, which is used for respectively amplifying the SSR loci according to claim 1, and which 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;

an eighteenth SSR primer pair for amplifying the eighteenth SSR locus;

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

a twenty-second SSR primer pair for amplifying said twenty-second SSR site.

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

the first SSR primer pair is similar to SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the second SSR primer pair is similar to the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the third SSR primer pair is similar to the primer pair shown in SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the fourth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the fifth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the sixth SSR primer pair is similar to the sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the seventh SSR primer pair is similar to SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the eighth SSR primer pair is similar to SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the ninth SSR primer pair is similar to SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the tenth SSR primer pair is similar to SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the eleventh SSR primer pair is similar to SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the twelfth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the thirteenth SSR primer pair is similar to SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the fourteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

the fifteenth SSR primer pair is similar to SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

and the sixteenth SSR primer pair is similar to the primer pair shown in SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

the seventeenth SSR primer pair is similar to SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the eighteenth SSR primer pair is similar to SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the nineteenth SSR primer pair is similar to SEQ ID NO: 37 and SEQ ID NO: 38 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the twentieth SSR primer pair is similar to SEQ ID NO: 39 and SEQ ID NO: 40 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the twenty-first SSR primer pair is similar to SEQ ID NO: 41 and SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the twenty-second SSR primer pair is similar to SEQ ID NO: 43 and SEQ ID NO: 44 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. SSR kit of appraisal hot pepper variety authenticity, its characterized in that: the SSR kit is prepared into a PCR reaction system; the PCR reaction system comprises:

the SSR primer set according to claim 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; 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 pepper 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 site of claim 1 in a pepper to be detected;

step two: and (3) judging the variety of the pepper to be detected:

if the number of the difference loci of the 22 SSR loci-based genotypes of the to-be-detected pepper and a certain specified variety of the standard pepper variety based on the 22 SSR loci is 0-2, judging the to-be-detected pepper and the specified variety of the standard pepper variety to be a similar variety;

if the number of different loci of the to-be-detected pepper based on the genotypes of the 22 SSR loci and the 22 SSR loci based on the genotypes of a certain specified variety in standard pepper varieties is more than 2, judging the specified variety of the to-be-detected pepper and the standard pepper variety to be a different variety;

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

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

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

the method comprises the following steps: respectively carrying out PCR amplification by using the genomic DNA of the pepper to be detected and the genomic DNA of a pepper 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 detecting the PCR amplification product to obtain the genotypes of the to-be-detected pepper and the pepper standard variety based on the 22 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 pepper and the standard pepper variety based on the 22 SSR sites; or:

detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the to-be-detected pepper and the standard pepper variety based on the 22 SSR sites.

8. The detection method according to any one of claims 5 to 7, wherein: the standard pepper variety is selected from the following 108 pepper varieties:

9. A detection method for identifying whether pepper varieties are the same is characterized in that:

the peppers to be detected are two kinds of peppers of unknown variety;

the detection method comprises the following steps:

the method comprises the following steps: detecting the genotype of the SSR site of claim 1 in the pepper to be detected;

step two: and judging whether the varieties of the peppers to be detected are the same:

judging the pepper to be detected as a similar variety if the number of the difference loci of the pepper to be detected based on the genotypes of the 22 SSR loci as claimed in claim 1 is 0-2;

and if the difference site of the pepper to be detected based on the genotypes of the 22 SSR sites in the claim 1 is more than 2, judging the pepper to be detected to be different varieties.

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 or X3: