CN110317898A - A kind of method for identifying cucumber variety authenticity and its combination of dedicated SSR primer - Google Patents

Abstract

The invention belongs to molecular labeling and its detection fields, and in particular to a kind of method for identifying cucumber variety authenticity and its combination of dedicated SSR primer.The site SSR for identifying cucumber variety authenticity, selected from following first site SSR to wantonly 1 to 19 kinds in the 19th site SSR；Above-mentioned SSR primer combination is selected from: the first SSR primer pair to the 19th SSR primer pair, it is respectively used to above-mentioned first site SSR to the 19th site SSR of PCR amplification, is respectively preferably the nucleotide sequence for being 85%-100% with the homology of the nucleotide sequence of the SEQ ID NO:1-38 in sequence table.The present invention can be used for carrying out early stage identification in seed or Seedling Stage to cucumber variety, guarantees the authenticity of kind, protects the equity of the producer and breeder conscientiously, and provides technical support for Cucumber Germplasm and New variety protection.

Description

A kind of method for identifying cucumber variety authenticity and its combination of dedicated SSR primer

Technical field

The invention belongs to molecular labeling and its detection fields, and in particular to it is a kind of identify cucumber variety authenticity method and Its dedicated SSR primer combination.

Background technique

Cucumber is the important vegetable crop in China, cultivated area up to 139.6 ten thousand hectares, Zhan Quanguo vegetable growing area 6.2%.With the fast development of cucumber industry since reform and opening-up, increased year by year by the kind of country and each province's authorization.According to According to " non-principal variety of crops registration method ", in April, 2017, the Ministry of Agriculture disclosed first non-principal variety of crops registration Catalogue, cucumber are among them.By the end of in April, 2019, cucumber registration kind quantity is only having reached 826 parts in two years.It is this It is to carry out registration variety managements and market surpervision that " blowout ", which breaks out phenomenon, before the mode of especially dependence field test proposes The challenge not having.Since breed cucumber enterprise is smaller and disperses, variety certification is mutually uncoordinated between each province and city, the product of cucumber Kind management fails effectively to follow up, and there are much fake and forged kinds, wherein many is derivative, the approximate kind of derivation.In addition breeding Production base managerial confusion, robber educates, kind of illegally buying up phenomenon is rampant, and xenogenesis phenomenon of the same name is serious, and seed quality accident happens occasionally. According to the requirement of " non-principal variety of crops registration guide ", breediness illustrates have with involved in kind DUS test report Character is closed if any clear associated gene, can directly submit DNA testing result.Therefore, China is badly in need of establishing and current cucumber product The DNA Fast Detection Technique that kind regulatory requirement matches.

In recent years, it is true that the advantages that SSR molecular marker is more with its quantity, variation is abundant, inheritance stability are widely used in kind Reality identification aspect.Currently, the DNA molecular detection of China's cucumber variety identification is mainly according to SSR molecular marker.But at that time SSR primer research and development without reference to Cucumber germplasm variation group big data support, to the sequence inside amplified fragments and SSR Motif The true variation situation of column is not grasped, and there are untrue variation situations；Kind negligible amounts used in SSR primer screening, and not The sale kind of China's Vehicles Collected from Market can be represented.Therefore current standard be easy to cause amplification unsuccessful, and authenticity, stability are bad The problems such as, it in turn results in Molecular Identification and determines result false positive, false negative.

Therefore, in the work using SSR molecular marker identification cucumber variety, one kind is needed to resurvey based on extensive cucumber Sequence and analysis cucumber variation group information, the stability and high efficiency site SSR and measuring method.

Summary of the invention

The present invention provides a kind of method for identifying cucumber variety authenticity and its combinations of dedicated SSR primer, it can be deduced that Stable, efficient qualification result: whether cucumber variety to be measured belongs to a certain kind in standard cucumber variety, and which is specifically Kind.

The present invention is realized by following technical solution:

Identify that the site SSR of cucumber variety authenticity, the site SSR are selected from following first site SSR to the 19th SSR Wantonly 1 to 19 kinds: the one site SSR in site is located at cucumber and refers to genome the 1st chromosome 7726518-7726537, Or its ortholog group segment；2nd site SSR is located at cucumber and refers to the 3rd chromosome 33305118- of genome 33305135 or its ortholog group segment；3rd site SSR is located at cucumber with reference to the 2nd chromosome of genome the 9328685-9328699 or its ortholog group segment；4th site SSR is located at cucumber and contaminates with reference to genome the 2nd Colour solid 20935940-20935954 or its ortholog group segment；5th site SSR is located at cucumber and refers to gene The 2nd chromosome 21903462-21903479 of group or its ortholog group segment；6th site SSR is located at cucumber With reference to the 3rd chromosome 18904506-18904526 of genome or its ortholog group segment；7th site SSR, The 5th chromosome 4094260-4094280 of genome or its ortholog group segment are referred to positioned at cucumber；8th SSR Site is located at cucumber and refers to the 6th chromosome 22367627-22367647 of genome or its ortholog group segment； 9th site SSR is located at cucumber and refers to the 5th chromosome 20995337-20995401 of genome or its ortholog Group segment；Tenth site SSR is located at cucumber and refers to the 1st chromosome 15183513-15183564 of genome or its inter-species Homologous gene group segment；11st site SSR is located at cucumber and refers to the 4th chromosome 21106675-21106714 of genome Position or its ortholog group segment；12nd site SSR is located at cucumber and refers to the 7th chromosome 251749- of genome 251781 or its ortholog group segment；13rd site SSR is located at cucumber with reference to the 6th chromosome of genome the 3719362-3719393 or its ortholog group segment；14th site SSR is located at cucumber and refers to genome the 3rd Chromosome 20507547-20507576 or its ortholog group segment；15th site SSR is located at cucumber and refers to The 4th chromosome 3220830-3220854 of genome or its ortholog group segment；16th site SSR, is located at Cucumber refers to the 1st chromosome 12303542-12303557 of genome or its ortholog group segment；17th SSR Site is located at cucumber and refers to the 3rd chromosome 32842873-32842888 of genome or its ortholog group segment； 18th site SSR is located at cucumber and refers to the 6th chromosome 22621037-22621060 of genome or the homologous base of its inter-species Because of a group segment；19th site SSR is located at cucumber and refers to the 4th chromosome 3220830-3220854 of genome or its kind Between homologous gene group segment.

Identify the SSR primer sets of cucumber variety authenticity, the SSR primer sets are for amplification respectively such as claim 1 institute The site SSR stated, the SSR primer sets include: the first SSR primer pair, for expanding the first site SSR；2nd SSR draws Object pair, for expanding the 2nd site SSR；3rd SSR primer pair, for expanding the 3rd site SSR；4th SSR draws Object pair, for expanding the 4th site SSR；5th SSR primer pair, for expanding the 5th site SSR；6th SSR draws Object pair, for expanding the 6th site SSR；7th SSR primer pair, for expanding the 7th site SSR；8th SSR draws Object pair, for expanding the 8th site SSR；9th SSR primer pair, for expanding the 9th site SSR；Tenth SSR draws Object pair, for expanding the tenth site SSR；11st SSR primer pair, for expanding the 11st site SSR；12nd SSR primer pair, for expanding the 12nd site SSR；13rd SSR primer pair, for expanding the described 13rd SSR Point；14th SSR primer pair, for expanding the 14th site SSR；15th SSR primer pair, for expanding the described tenth Five sites SSR；16th SSR primer pair, for expanding the 16th site SSR；17th SSR primer pair, for expanding 17th site SSR；18th SSR primer pair, for expanding the 18th site SSR；19th SSR primer pair, For expanding the 19th site SSR.

In a preferred embodiment, the first SSR primer pair, with SEQ ID NO:1 and the SEQ ID in sequence table The homology of the nucleotide sequence of NO:2 is more than or equal to 85%, preferably 100%；The 2nd SSR primer pair, in sequence table SEQ ID NO:3 and SEQ ID NO:4 nucleotide sequence homology be more than or equal to 85%, preferably 100%；Described Three SSR primer pairs are more than or equal to the homology of the nucleotide sequence of the SEQ ID NO:5 and SEQ ID NO:6 in sequence table 85%, preferably 100%；The 4th SSR primer pair, the nucleosides with the SEQ ID NO:7 and SEQ ID NO:8 in sequence table The homology of acid sequence is more than or equal to 85%, preferably 100%；The 5th SSR primer pair, with the SEQ ID in sequence table The homology of the nucleotide sequence of NO:9 and SEQ ID NO:10 is more than or equal to 85%, preferably 100%；6th SSR draws The homology of the nucleotide sequence of SEQ ID NO:11 and SEQ ID NO:12 in object pair, with sequence table is more than or equal to 85%, Preferably 100%；The 7th SSR primer pair, the nucleotide with the SEQ ID NO:13 and SEQ ID NO:14 in sequence table The homology of sequence is more than or equal to 85%, preferably 100%；The 8th SSR primer pair, and the SEQ ID NO in sequence table: The homology of the nucleotide sequence of 15 and SEQ ID NO:16 is more than or equal to 85%, preferably 100%；The 9th SSR primer Right, the homology with the nucleotide sequence of the SEQ ID NO:17 and SEQ ID NO:18 in sequence table is excellent more than or equal to 85% It is selected as 100%；The tenth SSR primer pair, the nucleotides sequence with the SEQ ID NO:19 and SEQ ID NO:20 in sequence table The homology of column is more than or equal to 85%, preferably 100%；The 11st SSR primer pair, and the SEQ ID NO in sequence table: The homology of the nucleotide sequence of 21 and SEQ ID NO:22 is more than or equal to 85%, preferably 100%；12nd SSR draws The homology of the nucleotide sequence of SEQ ID NO:23 and SEQ ID NO:24 in object pair, with sequence table is more than or equal to 85%, Preferably 100%；The 13rd SSR primer pair, the nucleosides with the SEQ ID NO:25 and SEQ ID NO:26 in sequence table The homology of acid sequence is more than or equal to 85%, preferably 100%；The 14th SSR primer pair, with the SEQ ID in sequence table The homology of the nucleotide sequence of NO:27 and SEQ ID NO:28 is more than or equal to 85%, preferably 100%；15th SSR The homology of the nucleotide sequence of SEQ ID NO:29 and SEQ ID NO:30 in primer pair, with sequence table is more than or equal to 85%, preferably 100%；The 16th SSR primer pair, with the SEQ ID NO:31 and SEQ ID NO:32's in sequence table The homology of nucleotide sequence is more than or equal to 85%, preferably 100%；The 17th SSR primer pair, in sequence table The homology of the nucleotide sequence of SEQ ID NO:33 and SEQ ID NO:34 is more than or equal to 85%, preferably 100%；Described 18 SSR primer pairs are greater than with the homology of the nucleotide sequence of the SEQ ID NO:35 and SEQ ID NO:36 in sequence table Equal to 85%, preferably 100%；The 19th SSR primer pair, and the SEQ ID NO:37 and SEQ ID NO in sequence table: The homology of 38 nucleotide sequence is more than or equal to 85%, preferably 100%；Preferably, one of each pair of primer pair draws Object is connected with fluorescent molecule, it is further preferred that the fluorescent molecule is selected from ROX, TAMRA, FAM, HEX.

Identify that the SSR kit of cucumber variety authenticity, the SSR kit are formulated as PCR reaction system；The PCR Reaction system includes:

The SSR primer sets, the upstream primer and downstream primer of every a pair in the SSR primer sets are in the system The ratio between concentration is 1:1；The upstream primer and the downstream primer final concentration in system are both preferably 0.25 μm of ol/L；Preferably, The system further include:

DNTPs: final concentration of every kind of 0.15mmol/L in system, magnesium chloride: final concentration of 2.5mmol/L, DNA in system Polymerase: final concentration of 0.05U/ μ L, PCR buffer in system: by 10-50mmol/L final concentration of in system potassium chloride with The Tris-HCL (pH7.5-9.0) of final concentration of 1-10mmol/L is formulated in system.

A kind of detection method for identifying cucumber variety authenticity, comprising the following steps:

Step 1: the genotype in the site SSR of cucumber to be measured is detected；Step 2: the kind of the cucumber to be measured is sentenced Determine step:

If certain in genotype and cucumber standard variety of the cucumber to be measured based on 19 sites SSR is specified The difference number of sites of the genotype based on 19 sites SSR of kind is 0, then cucumber to be measured and the cucumber standard variety The kind belong to identical kind；If genotype and cucumber standard of the cucumber to be measured based on 19 sites SSR The difference number of sites of the genotype based on 19 sites SSR of certain specified kind in kind is 1, then cucumber to be measured and institute The specified kind for stating cucumber standard variety belongs to approximate kind；If shown cucumber to be measured is based on 19 sites SSR Genotype and cucumber standard variety in each kind the genotype based on 19 sites SSR difference number of sites It is all larger than and is equal to 2, then cucumber to be measured and the kind of each cucumber standard variety are all different.

In a preferred embodiment, the SSR loci gene type step of detection cucumber to be measured include it is following step by step: Step by step one: respectively using the genomic DNA of the cucumber to be measured and the genomic DNA of cucumber standard variety as template, adopting respectively Primer sets in the combination of described in Claims 2 or 3 SSR primer carry out PCR amplification, obtain pcr amplification product；Step by step Two: the pcr amplification product being detected, the cucumber to be measured is obtained and the cucumber standard variety is based on described in 19 The genotype in the site SSR.

In a preferred embodiment, it is described step by step two detection method include: fluorescence signal detection: described in detection The fluorescence signal of pcr amplification product obtains the cucumber to be measured and the standard cucumber variety based on 19 sites SSR Genotype；Or: the detection of amplified production segment: detecting the clip size of the pcr amplification product, obtains cucumber to be measured and standard is yellow Genotype of the melon kind based on 19 sites SSR.

In a preferred embodiment, described to determine the result is that being obtained according to clustering in the step 2.

In a preferred embodiment, the standard cucumber variety is selected from following 139 cucumber varieties: boat loses No. 1, Japan Northern star, middle peasant No. 19, strong melon, middle peasant No. 15, Zhongnong No.9, Shen Lv 72, CUCUMBER1404, Shen Lv 64, I1an, Luyitianshi, Spring king 9801, Jinyou No.12, saliva spring No. 4, Jinyou No.31, saliva is No. 2 excellent, Ning Jia 3, Vlaspik, spring jade, jasper, Shanghai are No. 6 miscellaneous, Precious miscellaneous No. 2, Ning Yun 3, Jinyou No.36, Jinyou No.35, saliva is No. 38 excellent, middle peasant No. 26, MC2065, lucky miscellaneous No. nine, the excellent 20- of saliva 11, select beautiful four, winter, rich U.S. 11, emerald green imperial, emerald green, 'Chunhua No.1', Shandong cucumber No. three, C05-016, eastern agriculture 806, Sui Ling always next Less, saliva is No. 48 excellent, saliva is No. 1 excellent, what drought, south are No. 1 female, P01, south are No. 2 female, P02, CC3, Ning Feng 09, excellent plus complete female 09, HH1-8- 1-2, U.S. be female 09,627, south is No. 3 female, H32, teenage girl, tangshan autumn melon, golden child, capital grind mini No. 1, capital grind 107, green sweet tea, it is green it is emerald green, Jinyou No.35, saliva are No. 401 excellent, saliva is No. 1 excellent, saliva is No. 108 excellent, the resistance to H1104 of China, the excellent No.1 of saliva, saliva is No. 303 excellent, blueness is beautiful, eastern agriculture 804, green refreshing, successively it is more, saliva is No. 3 green, rich U.S. 5032, garden Feng Yuan 6, rich U.S. 517, Jinyou No.10, rich U.S. No. 28, saliva excellent 35 Number, it is De Ruite D19, rich U.S. 10, longevity dish HG1, the saliva spring No. 3, longevity dish HG2, DS2121, rich U.S. 74, lucky miscellaneous 16, lucky miscellaneous 9, emerald green jasmine, P-2-5-1-1, saliva is excellent 35, spring and autumn king No. 2, jasper 2, spring and autumn king No. 3, river are No. 3 emerald green, bright beautiful, eastern agriculture 804, saliva are excellent No. 1, middle peasant No. 50, middle peasant No. 26, Wan Nong tri-, Chang Chun Mi Ci, capital grind No. 2 mini emerald, Jingyanmini No.2, saliva it is excellent 308, Moral Rett 4, Jinyou No.36, rich U.S. 8, rich U.S. 6913,13AC230, De Ruite 79, De Ruite Y2,13AC049, De Ruite F16, Zhongnong No. 12, Kant (Condesa) RZ F1, Lvchun County two, the four seasons are rich, Lvchun County's No.1, rich No. 5 large, saliva is No. 335 excellent, saliva Excellent No. 316, saliva is No. 358 excellent, Dong Fangxiu, saliva spring No. three, the green 21-10 of saliva, middle peasant No. 37, middle peasant No. 50, saliva are No. 315 excellent, universe moral 2 Number, universe moral 1217, universe moral 117, universe moral 777, rich No. eight large, Tian Jiao seven, Tian Jiao eight, emerald green dragon, the four seasons it is rich.

The site SSR described above or SSR primer combination or the SSR kit or the detection method, with The application of lower x1 or x2:

X1: identify whether the kind of cucumber to be measured belongs to a certain kind in standard cucumber variety；

X2: identify that the kind of cucumber to be measured is specially any in standard cucumber variety.

The present invention has the advantages that compared with prior art

1, SSR primer combination provided by the invention can be used for carrying out early stage identification in seed or Seedling Stage to cucumber variety, Guarantee the authenticity of kind, protects the equity of the producer and breeder conscientiously, and mention for Cucumber Germplasm and New variety protection For technical support.

2, method provided by the invention can be identified and be obtained: whether cucumber variety to be measured both to be measured belongs to standard cucumber variety In a certain kind, and it is specifically any.So method can both identify unknown cucumber variety, it can also be to known The authenticity of kind is identified.

3, there are method provided by the invention high-throughput, accurate, inexpensive, easy to operate, saving human and material resources etc. to have Point has very wide application prospect.

Detailed description of the invention

Fig. 1 is 139 dendrograms for trying cucumber variety established in 19 primer sets in embodiment 1.

Fig. 2 is that embodiment 2 is SSR marker number (i.e. the site SSR number) and distinguishes 139 relationships for trying cucumber variety Figure.

Fig. 3-Figure 21 is for 219 primer sets of embodiment in part for the SSR parting effect in examination cucumber variety.

Specific embodiment

It is defined as follows:

Cucumber variety authenticity: refer to the true correspondence an of cucumber variety Yu its genetic background.

Ortholog group segment: referring to except V2 is in the version number of the reference genome sequence of cucumber 9930, other The genomic fragment homologous for V2 with reference to the version number of genome sequence with cucumber 9930 in the cucumber of kind.For example, just specific Genomic fragment, for V2, there are identical with the version number of the reference genome sequence of cucumber 9930 in the kind in table 3 of the present invention Gene.

In a first aspect, the present invention provides the site SSR of identification cucumber variety authenticity, it is located at Cucumber germplasm, number Amount be 19, can be selected it is therein one or more, specifying information is as shown in table 1.

The above-mentioned site SSR is selected from following first site SSR to wantonly 1 to 19 kinds in the 19th site SSR:

It is same with reference to the 1st chromosome 7726518-7726537 of genome or its inter-species to be located at cucumber for first site SSR Source genomic fragment；

2nd site SSR is located at cucumber and refers to the 3rd chromosome 33305118-33305135 of genome or its inter-species Homologous gene group segment；

It is same with reference to the 2nd chromosome 9328685-9328699 of genome or its inter-species to be located at cucumber for 3rd site SSR Source genomic fragment；

4th site SSR is located at cucumber and refers to the 2nd chromosome 20935940-20935954 of genome or its inter-species Homologous gene group segment；

5th site SSR is located at cucumber and refers to the 2nd chromosome 21903462-21903479 of genome or its inter-species Homologous gene group segment；

6th site SSR is located at cucumber and refers to the 3rd chromosome 18904506-18904526 of genome or its inter-species Homologous gene group segment；

It is same with reference to the 5th chromosome 4094260-4094280 of genome or its inter-species to be located at cucumber for 7th site SSR Source genomic fragment；

8th site SSR is located at cucumber and refers to the 6th chromosome 22367627-22367647 of genome or its inter-species Homologous gene group segment；

9th site SSR is located at cucumber and refers to the 5th chromosome 20995337-20995401 of genome or its inter-species Homologous gene group segment；

Tenth site SSR is located at cucumber and refers to the 1st chromosome 15183513-15183564 of genome or its inter-species Homologous gene group segment；

11st site SSR is located at cucumber and refers to the 4th chromosome 21106675-21106714 of genome or its kind Between homologous gene group segment；

It is same with reference to the 7th chromosome 251749-251781 of genome or its inter-species to be located at cucumber for 12nd site SSR Source genomic fragment；

13rd site SSR is located at cucumber and refers to the 6th chromosome 3719362-3719393 of genome or its inter-species Homologous gene group segment；

14th site SSR is located at cucumber and refers to the 3rd chromosome 20507547-20507576 of genome or its kind Between homologous gene group segment；

15th site SSR is located at cucumber and refers to the 4th chromosome 3220830-3220854 of genome or its inter-species Homologous gene group segment；

16th site SSR is located at cucumber and refers to the 1st chromosome 12303542-12303557 of genome or its kind Between homologous gene group segment；

17th site SSR is located at cucumber and refers to the 3rd chromosome 32842873-32842888 of genome or its kind Between homologous gene group segment；

18th site SSR is located at cucumber and refers to the 6th chromosome 22621037-22621060 of genome or its kind Between homologous gene group segment；

19th site SSR is located at cucumber and refers to the 4th chromosome 3220830-3220854 of genome or its inter-species Homologous gene group segment.

Second aspect, the present invention provide the SSR primer sets of identification cucumber variety authenticity, can pass through pcr amplification reaction Obtain the pcr amplification product based on the above-mentioned site SSR.

Above-mentioned SSR primer combination is selected from: the first SSR primer pair to the 19th SSR primer pair is respectively used in PCR amplification State the first site SSR to the 19th site SSR.

Above-mentioned first SSR primer pair, with the nucleotide sequence of the SEQ ID NO:1 and SEQ ID NO:2 in sequence table Homology is more than or equal to 85%, preferably 100%；

Above-mentioned 2nd SSR primer pair, with the nucleotide sequence of the SEQ ID NO:3 and SEQ ID NO:4 in sequence table Homology is more than or equal to 85%, preferably 100%；

Above-mentioned 3rd SSR primer pair, with the nucleotide sequence of the SEQ ID NO:5 and SEQ ID NO:6 in sequence table Homology is more than or equal to 85%, preferably 100%；

Above-mentioned 4th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:7 and SEQ ID NO:8 in sequence table Homology is more than or equal to 85%, preferably 100%；

Above-mentioned 5th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:9 and SEQ ID NO:10 in sequence table Homology is more than or equal to 85%, preferably 100%；

Above-mentioned 6th SSR primer pair, the nucleotide sequence with the SEQ ID NO:11 and SEQ ID NO:12 in sequence table Homology be more than or equal to 85%, preferably 100%；

Above-mentioned 7th SSR primer pair, the nucleotide sequence with the SEQ ID NO:13 and SEQ ID NO:14 in sequence table Homology be more than or equal to 85%, preferably 100%；

Above-mentioned 8th SSR primer pair, the nucleotide sequence with the SEQ ID NO:15 and SEQ ID NO:16 in sequence table Homology be more than or equal to 85%, preferably 100%；

Above-mentioned 9th SSR primer pair, the nucleotide sequence with the SEQ ID NO:17 and SEQ ID NO:18 in sequence table Homology be more than or equal to 85%, preferably 100%；

Above-mentioned tenth SSR primer pair, the nucleotide sequence with the SEQ ID NO:19 and SEQ ID NO:20 in sequence table Homology be more than or equal to 85%, preferably 100%；

Above-mentioned 11st SSR primer pair, the nucleotides sequence with the SEQ ID NO:21 and SEQ ID NO:22 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 12nd SSR primer pair, the nucleotides sequence with the SEQ ID NO:23 and SEQ ID NO:24 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 13rd SSR primer pair, the nucleotides sequence with the SEQ ID NO:25 and SEQ ID NO:26 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 14th SSR primer pair, the nucleotides sequence with the SEQ ID NO:27 and SEQ ID NO:28 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 15th SSR primer pair, the nucleotides sequence with the SEQ ID NO:29 and SEQ ID NO:30 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 16th SSR primer pair, the nucleotides sequence with the SEQ ID NO:31 and SEQ ID NO:32 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 17th SSR primer pair, the nucleotides sequence with the SEQ ID NO:33 and SEQ ID NO:34 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 18th SSR primer pair, the nucleotides sequence with the SEQ ID NO:35 and SEQ ID NO:36 in sequence table The homology of column is more than or equal to 85%, preferably 100%；

Above-mentioned 19th SSR primer pair, the nucleotides sequence with the SEQ ID NO:37 and SEQ ID NO:38 in sequence table The homology of column is more than or equal to 85%, preferably 100%.

In a preferred embodiment, above-mentioned SSR primer combination is one or more groups of in primer sets 01-19；It is above-mentioned The DNA sequence dna information of primer sets 01-19 is as shown in sequence table SEQ ID:1-38, referring to table 2.

In above-mentioned primer sets, 5 ' ends of upstream primer can be with fluorescence labels sequence so as to fluorescent PCR detection, such as FAM The fluorescence signal of fluorescence labels sequence is blue, and the fluorescence signal of HEX fluorescence labels sequence is red.

The third aspect, the present invention provide the SSR kit of identification cucumber variety authenticity, and SSR preparation of reagents is PCR reaction System, the system preferably include:

The ratio between final concentration of above-mentioned SSR primer sets, upstream primer and downstream primer is 1:1.

Fourth aspect, the present invention provide a kind of detection method for identifying cucumber variety authenticity, comprising the following steps:

Step 1: the SSR loci gene type of cucumber to be measured is detected.

Step by step one: respectively using the genomic DNA of above-mentioned cucumber to be measured and the genomic DNA of cucumber standard variety as mould Plate is respectively adopted the carry out pcr amplification reaction of the primer sets in above-mentioned SSR primer combination, obtains pcr amplification product；

Step by step two: above-mentioned pcr amplification product being detected, cucumber and cucumber standard variety to be measured is obtained and is based on 19 The genotype in the above-mentioned site SSR.

The detection can detect for fluorescence signal: detecting the fluorescence signal of above-mentioned pcr amplification product, obtain cucumber to be measured Genotype with standard cucumber variety based on above-mentioned 19 sites SSR；

Above-mentioned detection may be the detection of amplified production segment: utilize the above-mentioned pcr amplification product of capillary electrophoresis detection Clip size obtains the genotype of cucumber to be measured and standard cucumber variety based on above-mentioned 19 sites SSR.

Step 2: the kind determination step of cucumber to be measured:

It is drawn a conclusion by clustering cucumber to be measured and standard cucumber variety based on the genotype in above-mentioned 19 sites SSR Following result:

If certain kind in genotype and cucumber standard variety of the above-mentioned cucumber to be measured based on above-mentioned 19 sites SSR The difference number of sites of genotype based on above-mentioned 19 sites SSR is 0, i.e., completely the same, then cucumber to be measured and above-mentioned cucumber mark Certain kind in quasi- kind belongs to the same kind；

If certain kind in genotype and cucumber standard variety of the above-mentioned cucumber to be measured based on above-mentioned 19 sites SSR The difference number of sites of genotype based on above-mentioned 19 sites SSR is 1, then certain in cucumber to be measured and above-mentioned cucumber standard variety Kind belongs to approximate kind；

If each kind in genotype and cucumber standard variety of the cucumber to be measured based on 19 sites SSR The difference number of sites of genotype based on above-mentioned 19 sites SSR is all larger than equal to 2, then cucumber to be measured and each cucumber standard items The kind of kind is all different.

The program of above-mentioned pcr amplification reaction is preferred are as follows:

94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 45s, 0.8 DEG C of every cycle down, totally 12 A circulation；94 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 45s, totally 25 recycle；72 DEG C extend 10min eventually.Amplification produces It is saved before object electrophoresis in -20 DEG C or on ice.

Above-mentioned standard cucumber variety includes following 139 cucumber varieties: boat lose No. 1, the northern star of Japan, middle peasant No. 19, strong melon, Middle peasant No. 15, Zhongnong No.9, Shen Lv 72, CUCUMBER1404, Shen Lv 64, I1an, Luyitianshi, spring king 9801, Jinyou No.12, Saliva spring No. 4, Jinyou No.31, saliva is No. 2 excellent, Ning Jia 3, Vlaspik, spring jade, jasper, Shanghai are No. 6 miscellaneous, precious miscellaneous No. 2, Ning Yun 3, saliva Excellent No. 36, Jinyou No.35, saliva is No. 38 excellent, middle peasant No. 26, MC2065, miscellaneous No. nine lucky, the excellent 20-11 of saliva, selects that four, winter is beautiful, rich U.S. 11 Number, emerald green imperial, emerald green, 'Chunhua No.1', Shandong cucumber No. three, C05-016, eastern agriculture 806, peaceful mound an old person having a young heart' a person old in age but young in spirit, saliva is No. 48 excellent, saliva is No. 1 excellent, What drought, south is No. 1 female, P01, south are No. 2 female, P02, CC3, Ning Feng 09, excellent plus complete female 09, HH1-8-1-2, U.S. are female 09,627, south female 3 Number, H32, teenage girl, tangshan autumn melon, golden child, capital grind mini No. 1, capital grind that 107, green sweet tea, green kingfisher, Jinyou No.35, saliva is No. 401 excellent, saliva Excellent No. 1, saliva is No. 108 excellent, the resistance to H1104 of China, the excellent No.1 of saliva, saliva are No. 303 excellent, green beauty, eastern agriculture 804, green refreshing, more, saliva green 3 successively Number, rich U.S. 5032, garden Feng Yuan 6, rich U.S. 517, Jinyou No.10, rich U.S. 28, Jinyou No.35, De Ruite D19, rich U.S. 10, Longevity dish HG1, saliva spring No. 3, longevity dish HG2, DS2121, rich U.S. 74, lucky miscellaneous 16, lucky miscellaneous 9, emerald green jasmine, P-2-5-1-1, saliva are excellent 35, spring and autumn king No. 2, jasper 2, spring and autumn king No. 3, river be No. 3 emerald green, bright beautiful, eastern agriculture 804, saliva are No. 1 excellent, middle peasant No. 50, middle peasant 26 Number, Wan Nong tri-, Chang Chun Mi Ci, capital grind No. 2 mini emerald, Jingyanmini No.2, saliva excellent 308, De Ruite 4, Jinyou No.36, Rich U.S. 8, rich U.S. 6913,13AC230, De Ruite 79, De Ruite Y2,13AC049, De Ruite F16, Zhongnong No. 12, Kant (Condesa) RZ F1, Lvchun County two, the four seasons are rich, Lvchun County's No.1, rich No. 5 large, saliva is No. 335 excellent, saliva is No. 316 excellent, saliva excellent 358 Number, Dong Fangxiu, saliva spring No. three, the green 21-10 of saliva, middle peasant No. 37, middle peasant No. 50, saliva is No. 315 excellent, universe moral 2, universe moral 1217, dry Moral 117, universe moral 777, rich No. eight large, Tian Jiao seven, Tian Jiao eight, emerald green dragon, the four seasons are rich.

5th aspect, the present invention provide the above-mentioned site SSR, the combination of SSR primer, and SSR kit states detection method, with The application of lower x1 or x2:

X1: identify whether the kind of cucumber to be measured belongs to a certain kind in standard cucumber variety；

X2: identify that the kind of cucumber to be measured is specially any in standard cucumber variety.

X1 and X2 belongs to the application of identification cucumber variety authenticity.

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.

Embodiment 1

The acquisition combined for identifying the SSR primer of cucumber variety authenticity

One, the discovery in 19 sites SSR

The present invention is based on the heavy sequencing datas (NCBI sequence number SRA056480) that 115 portions of cucumber represent resource, find for the first time And obtain 19 sites SSR.This 115 parts of cucumber resource types are abundant, cover North-China Type, indica-type, Japanese type, South China type, Europe Continent fruit type, America processing type, Xishuangbanna type and osculant include the main ecology of existing cucumber in the market substantially Type and economical character embody germplasm representativeness as much as possible, and genetic diversity with higher is (referring to A genomic variation map provides insights into the genetic basis of cucumber Domestication and diversity, 2013, PMID:24141363).

Specifically, the screening criteria in the site SSR is as follows: selection position is uniform within the scope of full-length genome, polymorphism is good, miscellaneous Right small, MAF > 0.3, PCA Clustering Effect is good, discrimination is high and both wings 50bp sequence preservative (no InDel, no SSR, without other SSR the site SSR).The essential information in 19 sites SSR the 1st to 4 column during see Table 1 for details.Wherein SSR site is on chromosome Position and motif information are to compare determination with reference to genome sequence based on cucumber 9930, and the cucumber 9930 refers to genome The version number of sequence be V2 (download address are as follows: http://cucurbitgenomics.org/organism/2, and Https: //www.ncbi.nlm.nih.gov/genome/? term=cucumber).

The essential information in 1. 19 sites SSR of table

Wherein, " main allelic variation " is to be counted according to 139 in table 3 for examination cucumber variety.Such as: certain sample Only there is 1 allelic variation in some site, size 150bp then writes in the genotype of the main allelic variation in the site 150/150；There are two allelic variations in some site for certain sample, and size is respectively 141bp, 150bp, then in the main of the site The genotype writing 141/150 of allelic variation.

Two, the acquisition combined for identifying the SSR primer of cucumber variety authenticity

Based on 19 sites SSR of step 1 discovery, present inventors have developed polymorphism informations with higher (i.e. PIC value, PIC value refers to a label to amount, for the value in crowd surveillance polymorphism；PIC value depend on detection etc. The number and allele their frequency distribution of position gene；PIC value be equal to 1 subtract all gene frequencies square it is total With.Be shown in Table in 1 the 5th column) the SSR primer combination for identifying cucumber variety authenticity.Based on the aforementioned site SSR in cucumber The version number of 9930 reference genome sequences is the upstream and downstream primers in V2, and SSR primer is combined by 19 primer sets Composition, the title of each primer sets are shown in Table the 2nd column in 2.Each primer sets are made of 2 primer sequences, for expanding a SSR Site.The nucleotide sequence of each primer is as shown in the 4th column in table 2 in 19 primer sets.

Table 2

Embodiment 2

The present embodiment is the validity check for the SSR primer combination that embodiment 1 is developed.

139 in the present embodiment are shown in Table 3 for trying the essential information of cucumber variety.139 are normal for examination cucumber variety The external introduced variety of excellent variety or part seen.

3:139, the table essential informations for trying cucumber variety

1, for the acquisition of the genomic DNA of examination cucumber variety

139 blade (the mixed true leaf for taking 30 seeds, i.e., each product for trying cucumber variety are extracted using CTAB method respectively The true leaf for kind taking 30 seeds longer, refer to: same kind takes the true leaf of 30 different plants to mix) genomic DNA, Obtain the genomic DNA for trying cucumber variety.

The operation of above-mentioned CTAB method specifically:

The blade for winning above-mentioned 139 kinds of Seedling Stage respectively is placed in freeze drier (CoolSafe 55-4) and takes off Water；Then blade is smashed with high-throughput beveller (Geno/Grind6875), then takes 20 μ g blade dry powder, 800 μ are added thereto L CTAB extracting solution (2%CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0,20mM EDTA pH8.0,1%PVP- 40,0.2% beta -mercaptoethanol), it mixes, in 65 DEG C of water-bath 30min, isometric chloroform/isoamyl alcohol (24:1) is added, It is centrifuged 10 minutes under the conditions of 10000rpm/min, Aspirate supernatant is transferred in new centrifuge tube, and 0.8 times of volume pre-cooling is added Isopropanol is gently mixed by inversion, and is centrifuged 10min in 4 DEG C, 12,000r/min after -20 DEG C of placement 30min.Supernatant is abandoned, is added 70% ethanol solution washs 2 times, and dry plus 100 μ L ddH2O dissolving DNAs under natural conditions are obtained for trying cucumber variety genome DNA, after detectable concentration 4 DEG C it is spare.

PCR requirement, Passing Criteria are as follows: ultraviolet point must be met for the quality and concentration for trying the genomic DNA of cucumber variety Light photometer Nanodrop2000 (Thermo) detects A260/A280 ratio and is greater than 1.8 in 1.8 or so, A260/A230 ratio； For try cucumber variety genomic DNA concentration in 10-30ng/ μ L.

2, the genomic DNA using 139 for trying cucumber variety is respectively adopted 19 primer sets and carries out PCR as template respectively Amplification, obtains pcr amplification product.In each PCR reaction system, drawing containing " R " in the primer and title in title containing " F " Object concentration ratio is 1:1.

Reaction system includes:

Upstream primer (containing " F " in title) and downstream primer (containing " R " in title) the ratio between concentration in system are 1: 1。

Response procedures are as follows: initial denaturation: 94 DEG C of 5min；Amplification: 94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 45s, 0.8 DEG C of every cycle down, totally 12 recycle；94 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 45s, totally 25 recycle；Prolong eventually It stretches: 72 DEG C of 10min.It is saved before the amplified production electrophoresis of formation in 4 DEG C.

3, fluorescent capillary electrophoresis tube

It, can be more according to different instrument selections according to the difference of SSR molecular marker amplified fragments size after completing step 2 A primer combination carries out electrophoresis.According to predetermined combination primer, the different fluorescence of isometric same combination primer are taken respectively The amplified production of label, TAMRA dilute 50 times, and other fluorescence-causing substances mix well after diluting 100 times.2 μ are drawn from mixed liquor L is added in the dedicated loading plate hole of DNA analysis instrument.Each hole is separately added into 0.1 μ L molecular weight internal standard and 8.9 μ L deionization first again Amide, 95 DEG C of denaturation 5min in PCR instrument take out and are set in -20 DEG C of refrigerators immediately or on ice, cool down 5min.After brief centrifugation 10s It is placed on DNA analysis instrument.Open DNA analysis instrument, inspection apparatus working condition and reagent state.The upper template of sample will be housed It is placed on specimen holder pedestal, the buffer plate equipped with electrode buffer is placed on buffer grillage pedestal, open data Software is collected, is operated according to the service manual of DNA analysis instrument.Automatic running parameter is saved electrophoresis original by DNA analysis instrument Beginning data.Detect excitation wavelength and color used in fluorescent primer referring to instrument default value (the maximum excitation wavelength 494nm of FAM, The maximum excitation wavelength 587nm of maximum excitation the wavelength 560nm, ROX of maximum excitation the wavelength 535nm, TAMRA of HEX), it needs Spectrum correction periodically is carried out to capillary electrophoresis.

Partial results are shown in Fig. 3.The result shows that each primer sets available good parting in for examination cucumber variety is imitated Fruit.

4, clustering

According to 139 for trying genotype of the cucumber variety based on 19 sites SSR, using MEGA7 software to 139 for examination Cucumber variety carries out clustering.

It is as shown in Figure 1 to establish dendrogram of 139 in 19 primer sets for trying cucumber variety.The result shows that 19 Primer sets can distinguish 139 in table 3 for trying cucumber variety completely.It can be seen that the SSR primer combination that embodiment 1 is developed It can be applied to cucumber variety DNA fingerprint database sharing and Varieties identification.

5, efficiency rating

Varieties identification can reduce workload using sequential analysis mode.The present inventor compares SSR It marks number (i.e. primer sets number) and distinguishes 139 relationships for trying cucumber variety differentiation rate.

139 kinds as shown in Figure 2 between any two relatively and the difference reference numerals that count, wherein comparing between any two As a result number is C¹³⁹ ₂=139 × 128 ÷ 2=9591；In this 9591 results, difference site number is 12 to account for about The 17% of sum, account for about sum 14% that difference site number is 13, account for about sum 9% that difference site number is 14, Account for about sum 4% that difference site number is 15, account for about sum 1% that difference site number is 16, difference site number For 17-19 account for about sum 0%, therefore the result in 12 or more difference sites account for about sum 45%, this result explanation use this A little labels are good in the polymorphism of 139 kinds；19 primer sets (i.e. 19 SSR marker numbers) are at 139 for trying cucumber variety In differentiation rate reach 100%.

Embodiment 3

Detect whether cucumber variety to be measured belongs to 139 methods for the kind in examination cucumber variety

1, the acquisition of the genomic DNA of cucumber variety to be measured

The blade of cucumber variety to be measured is derived from Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences proving ground.

According to the method for the step 1 in embodiment 2, " cucumber variety to be measured will be replaced with " for trying the blade of cucumber variety " Blade ", other steps are constant, obtain the genomic DNA of cucumber variety to be measured.

2, the configuration of SSR primer and PCR reaction system

According to the method for the step 2 in embodiment 2, " cucumber to be measured will be replaced with " for trying the genomic DNA of cucumber variety " The genomic DNA of kind ", other steps are constant, obtain the PCR product of cucumber variety to be measured.

3, fluorescent capillary electrophoresis tube detects

Take the PCR product of cucumber variety to be measured.

By the clip size of 19 SSR amplified productions of cucumber variety to be measured respectively with 139 for trying cucumber variety (table 3 It is shown) 19 sites SSR compare, count the difference number of sites of cucumber variety to be measured and 19 standard cucumber varieties, so After make the following judgment:

If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is 2 or more, cucumber product to be measured Kind and the standard cucumber variety belong to different cucumber varieties；Difference number of sites is more, and genetic relationship is remoter.

If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is 1 or 0, cucumber variety to be measured and The standard cucumber variety be or it is doubtful be same cucumber variety.

The result shows that cucumber variety to be measured is equal with 139 difference number of sites for trying cucumber variety on 19 sites SSR It is 4 or more, therefore, cucumber variety to be measured is not belonging to 139 for any one of examination cucumber variety kind, i.e., cucumber to be measured Kind is different from 139 for any one of examination cucumber variety kind.

Embodiment 4

The present embodiment is to compare clip size using Capillary Electrophoresis to determine cucumber variety, rather than use fluorescence signal Determine.

Present case is to be grasped if using other platforms according to equipment with 3730 fluorescent capillary pipe detection platform of ABI for reference It is required and adjusts accordingly.

According to the difference of SSR molecular marker amplified fragments size, can be combined according to the multiple primers of different instrument selections into Row electrophoresis.

S1: according to predetermined combination primer, the expansion of the different fluorescent markers of isometric same combination primer is taken respectively Increase production object, TAMRA dilutes 50 times, and other fluorescence-causing substances mix well after diluting 100 times.1 μ L is drawn from mixed liquor, is added to In the dedicated loading plate hole of DNA analysis instrument.Each hole is separately added into 0.1 μ L molecular weight internal standard and 8.9 μ L deionized formamides again, 95 DEG C of denaturation 1min in PCR instrument, taking-up are set on ice immediately, cooling 5min.It is placed to after brief centrifugation 10s on DNA analysis instrument.

S2: 3730 DNA analysis instrument of ABI, inspection apparatus working condition and reagent state are opened.The loading of sample will be housed Plate is placed on specimen holder pedestal, and the buffer plate equipped with electrode buffer is placed on buffer grillage pedestal, opens number According to software is collected, operated according to the service manual of DNA analysis instrument.Automatic running parameter is saved electrophoresis by DNA analysis instrument Initial data.Excitation wavelength used in fluorescent primer and color are detected referring to instrument default value.

S3: export electrophoresis raw data file carries out data examination according to the following steps using Data Analysis Software: in number According to pre-setting SSR Primer and fluorescence classification, molecular weight internal standard, the amplified fragments of corresponding primer are big in analysis software It is small；Electrophoresis raw data file is imported into analysis software, panel, molecular weight internal standard, Bin, quality-controlling parameters etc. is selected to carry out Analysis；Analysis software can to detection quality be assigned to it is color-coded score, green indicate reliable in quality without intervene, red table Show that quality is unqualified or do not fall within the scope of regulation clip size, yellow expression, which has a question, needs to check original image progress really Recognize.

S4: pass through the standard sample tested simultaneously, reference sample (foundation primer select a small amount of control), school respectively Amplified fragments size is read again after data deviation between quasi- difference electrophoresis plate.If the specific peak after examination falls into defined segment In magnitude range, then amplified fragments size is directly read；If its peak is mostly not within the specified scope, can be by its integral translation as far as possible Data are read after falling peak in setting range.

S5: by the clip size of 19 SSR amplified productions of cucumber variety to be measured respectively with 139 for trying cucumber variety 19 sites SSR of (shown in table 3) compare, and count the difference site of cucumber variety to be measured and 19 standard cucumber varieties Number, then makes the following judgment:

If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is 2 or more, cucumber product to be measured Kind and the standard cucumber variety belong to different cucumber varieties；Difference number of sites is more, and genetic relationship is remoter.

If the difference number of sites of cucumber variety to be measured and certain standard cucumber variety is 1 or 0, cucumber variety to be measured and The standard cucumber variety be or it is doubtful be same cucumber variety.

It should be noted last that the above specific embodiment is only used to illustrate the technical scheme of the present invention and not to limit it, Although being described the invention in detail referring to preferred embodiment, those skilled in the art should understand that, it can be right Technical solution of the present invention is modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention, It is intended to be within the scope of the claims of the invention.

Sequence table

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Claims (10)

1. identifying the site SSR of cucumber variety authenticity, the site SSR is selected from following first site SSR to the 19th SSR Wantonly 1 to 19 kinds in point:

First site SSR is located at cucumber and refers to the 1st chromosome 7726518-7726537 of genome or the homologous base of its inter-species Because of a group segment；

It is homologous with reference to the 3rd chromosome 33305118-33305135 of genome or its inter-species to be located at cucumber for 2nd site SSR Genomic fragment；

3rd site SSR is located at cucumber and refers to the 2nd chromosome 9328685-9328699 of genome or the homologous base of its inter-species Because of a group segment；

It is homologous with reference to the 2nd chromosome 20935940-20935954 of genome or its inter-species to be located at cucumber for 4th site SSR Genomic fragment；

It is homologous with reference to the 2nd chromosome 21903462-21903479 of genome or its inter-species to be located at cucumber for 5th site SSR Genomic fragment；

It is homologous with reference to the 3rd chromosome 18904506-18904526 of genome or its inter-species to be located at cucumber for 6th site SSR Genomic fragment；

7th site SSR is located at cucumber and refers to the 5th chromosome 4094260-4094280 of genome or the homologous base of its inter-species Because of a group segment；

It is homologous with reference to the 6th chromosome 22367627-22367647 of genome or its inter-species to be located at cucumber for 8th site SSR Genomic fragment；

It is homologous with reference to the 5th chromosome 20995337-20995401 of genome or its inter-species to be located at cucumber for 9th site SSR Genomic fragment；

It is homologous with reference to the 1st chromosome 15183513-15183564 of genome or its inter-species to be located at cucumber for tenth site SSR Genomic fragment；

It is same with reference to the 4th chromosome 21106675-21106714 of genome or its inter-species to be located at cucumber for 11st site SSR Source genomic fragment；

12nd site SSR is located at cucumber and refers to the 7th chromosome 251749-251781 of genome or the homologous base of its inter-species Because of a group segment；

It is homologous with reference to the 6th chromosome 3719362-3719393 of genome or its inter-species to be located at cucumber for 13rd site SSR Genomic fragment；

It is same with reference to the 3rd chromosome 20507547-20507576 of genome or its inter-species to be located at cucumber for 14th site SSR Source genomic fragment；

It is homologous with reference to the 4th chromosome 3220830-3220854 of genome or its inter-species to be located at cucumber for 15th site SSR Genomic fragment；

It is same with reference to the 1st chromosome 12303542-12303557 of genome or its inter-species to be located at cucumber for 16th site SSR Source genomic fragment；

It is same with reference to the 3rd chromosome 32842873-32842888 of genome or its inter-species to be located at cucumber for 17th site SSR Source genomic fragment；

It is same with reference to the 6th chromosome 22621037-22621060 of genome or its inter-species to be located at cucumber for 18th site SSR Source genomic fragment；

It is homologous with reference to the 4th chromosome 3220830-3220854 of genome or its inter-species to be located at cucumber for 19th site SSR Genomic fragment.

2. identifying the SSR primer sets of cucumber variety authenticity, the SSR primer sets are as described in claim 1 for amplification respectively The site SSR, the SSR primer sets include:

First SSR primer pair, for expanding the first site SSR；

2nd SSR primer pair, for expanding the 2nd site SSR；

3rd SSR primer pair, for expanding the 3rd site SSR；

4th SSR primer pair, for expanding the 4th site SSR；

5th SSR primer pair, for expanding the 5th site SSR；

6th SSR primer pair, for expanding the 6th site SSR；

7th SSR primer pair, for expanding the 7th site SSR；

8th SSR primer pair, for expanding the 8th site SSR；

9th SSR primer pair, for expanding the 9th site SSR；

Tenth SSR primer pair, for expanding the tenth site SSR；

11st SSR primer pair, for expanding the 11st site SSR；

12nd SSR primer pair, for expanding the 12nd site SSR；

13rd SSR primer pair, for expanding the 13rd site SSR；

14th SSR primer pair, for expanding the 14th site SSR；

15th SSR primer pair, for expanding the 15th site SSR；

16th SSR primer pair, for expanding the 16th site SSR；

17th SSR primer pair, for expanding the 17th site SSR；

18th SSR primer pair, for expanding the 18th site SSR；

19th SSR primer pair, for expanding the 19th site SSR.

3. SSR primer sets according to claim 2, it is characterised in that:

The first SSR primer pair, it is homologous with the nucleotide sequence of the SEQ ID NO:1 and SEQ ID NO:2 in sequence table Property be more than or equal to 85%, preferably 100%；The 2nd SSR primer pair, with SEQ ID NO:3 and the SEQ ID in sequence table The homology of the nucleotide sequence of NO:4 is more than or equal to 85%, preferably 100%；

The 3rd SSR primer pair, it is homologous with the nucleotide sequence of the SEQ ID NO:5 and SEQ ID NO:6 in sequence table Property be more than or equal to 85%, preferably 100%；

The 4th SSR primer pair, it is homologous with the nucleotide sequence of the SEQ ID NO:7 and SEQ ID NO:8 in sequence table Property be more than or equal to 85%, preferably 100%；

The 5th SSR primer pair, it is homologous with the nucleotide sequence of the SEQ ID NO:9 and SEQ ID NO:10 in sequence table Property be more than or equal to 85%, preferably 100%；

The 6th SSR primer pair, it is same with the nucleotide sequence of the SEQ ID NO:11 and SEQ ID NO:12 in sequence table Source property is more than or equal to 85%, preferably 100%；

The 7th SSR primer pair, it is same with the nucleotide sequence of the SEQ ID NO:13 and SEQ ID NO:14 in sequence table Source property is more than or equal to 85%, preferably 100%；

The 8th SSR primer pair, it is same with the nucleotide sequence of the SEQ ID NO:15 and SEQ ID NO:16 in sequence table Source property is more than or equal to 85%, preferably 100%；

The 9th SSR primer pair, it is same with the nucleotide sequence of the SEQ ID NO:17 and SEQ ID NO:18 in sequence table Source property is more than or equal to 85%, preferably 100%；

The tenth SSR primer pair, it is same with the nucleotide sequence of the SEQ ID NO:19 and SEQ ID NO:20 in sequence table Source property is more than or equal to 85%, preferably 100%；

The 11st SSR primer pair, with the nucleotide sequence of the SEQ ID NO:21 and SEQ ID NO:22 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 12nd SSR primer pair, with the nucleotide sequence of the SEQ ID NO:23 and SEQ ID NO:24 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 13rd SSR primer pair, with the nucleotide sequence of the SEQ ID NO:25 and SEQ ID NO:26 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 14th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:27 and SEQ ID NO:28 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 15th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:29 and SEQ ID NO:30 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 16th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:31 and SEQ ID NO:32 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 17th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:33 and SEQ ID NO:34 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 18th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:35 and SEQ ID NO:36 in sequence table Homology is more than or equal to 85%, preferably 100%；

The 19th SSR primer pair, with the nucleotide sequence of the SEQ ID NO:37 and SEQ ID NO:38 in sequence table Homology is more than or equal to 85%, preferably 100%；

Preferably, a primer of each pair of primer pair is connected with fluorescent molecule, it is further preferred that the fluorescent molecule be selected from ROX, TAMRA、FAM、HEX。

4. identifying the SSR kit of cucumber variety authenticity, it is characterised in that: the SSR kit is formulated as PCR reactant System；The PCR reaction system includes:

SSR primer sets described in Claims 2 or 3, the upstream primer and downstream primer of every a pair in the SSR primer sets are in institute Stating the ratio between concentration in system is 1:1；The upstream primer and the downstream primer final concentration in system are both preferably 0.25 μm of ol/L；

Preferably, the system further include:

DNTPs: final concentration of every kind of 0.15mmol/L in system,

Magnesium chloride: final concentration of 2.5mmol/L in system,

Archaeal dna polymerase: final concentration of 0.05U/ μ L in system,

PCR buffer: by final concentration of 1-10mmol/L in the potassium chloride and system of 10-50mmol/L final concentration of in system Tris-HCL (pH7.5-9.0) is formulated.

5. a kind of detection method for identifying cucumber variety authenticity, it is characterised in that: described detection method includes the following steps:

Step 1: the genotype in the site SSR as described in claim 1 of cucumber to be measured is detected；

Step 2: the kind determination step of the cucumber to be measured:

If certain specified kind in genotype and cucumber standard variety of the cucumber to be measured based on 19 sites SSR The difference number of sites of the genotype based on 19 sites SSR be 0, then cucumber to be measured and the cucumber standard variety should Kind belongs to identical kind；

If certain specified kind in genotype and cucumber standard variety of the cucumber to be measured based on 19 sites SSR The difference number of sites of the genotype based on 19 sites SSR be 1, then cucumber to be measured and the cucumber standard variety should Specified kind belongs to approximate kind；

If each kind in genotype and cucumber standard variety of the shown cucumber to be measured based on 19 sites SSR The difference number of sites of genotype based on 19 sites SSR is all larger than equal to 2, then cucumber to be measured and each cucumber mark The kind of quasi- kind is all different.

6. detection method according to claim 5: it is characterized by:

The SSR loci gene type step of detection cucumber to be measured include it is following step by step:

Step by step one: respectively using the genomic DNA of the cucumber to be measured and the genomic DNA of cucumber standard variety as template, point Not Cai Yong primer sets in the combination of SSR primer described in Claims 2 or 3 carry out PCR amplification, obtain pcr amplification product；

Step by step two: the pcr amplification product being detected, the cucumber to be measured is obtained and the cucumber standard variety is based on The genotype in 19 sites SSR.

7. detection method according to claim 6: it is characterized by:

It is described step by step two detection method include:

Fluorescence signal detection: detecting the fluorescence signal of the pcr amplification product, obtains the cucumber to be measured and the standard cucumber Genotype of the kind based on 19 sites SSR；Or:

The detection of amplified production segment: the clip size of the pcr amplification product is detected, cucumber to be measured and standard cucumber variety are obtained Genotype based on 19 sites SSR.

8. detection method according to claim 5: it is characterized by: in the step 2, it is described to determine the result is that according to poly- What alanysis obtained.

9. according to any detection method of claim 5-8, it is characterised in that: the standard cucumber variety is selected from following 139 A cucumber variety: boat lose No. 1, the northern star of Japan, middle peasant No. 19, strong melon, middle peasant No. 15, Zhongnong No.9, Shen Lv 72, CUCUMBER1404, Shen Lv 64, I1an, Luyitianshi, spring king 9801, Jinyou No.12, saliva spring No. 4, Jinyou No.31, saliva is No. 2 excellent, Peaceful good No. 3, Vlaspik, spring jade, jasper, Shanghai is No. 6 miscellaneous, treasured is No. 2 miscellaneous, Ning Yun 3, Jinyou No.36, Jinyou No.35, saliva are No. 38 excellent, Middle peasant No. 26, miscellaneous No. nine lucky, the excellent 20-11 of saliva, selects beautiful four, winter, rich U.S. 11, emerald green imperial, emerald green, 'Chunhua No.1', Shandong Huang at MC2065 Melon three, C05-016, eastern agriculture 806, the mound an old person having a young heart' a person old in age but young in spirit that pacifies, saliva is No. 48 excellent, saliva is No. 1 excellent, what drought, south are No. 1 female, P01, south are No. 2 female, P02, CC3, Ning Feng 09, excellent plus complete female 09, HH1-8-1-2, U.S. be female 09,627, south female No. 3, H32, teenage girl, tangshan autumn melon, gold Virgin, capital grinds mini No. 1, capital grinds 107, green sweet tea, green kingfisher, Jinyou No.35, saliva is No. 401 excellent, saliva is No. 1 excellent, saliva is No. 108 excellent, China is resistance to The excellent No.1 of H1104, saliva, saliva is No. 303 excellent, green beauty, eastern agriculture 804, it is green it is refreshing, successively it is more, saliva is No. 3 green, rich U.S. 5032, garden Feng Yuan 6, Rich U.S. 517, Jinyou No.10, rich U.S. 28, Jinyou No.35, De Ruite D19, rich U.S. No. 10, longevity dish HG1, saliva spring No. 3, longevity dish No. HG2, DS2121, rich U.S. 74, lucky miscellaneous 16, lucky miscellaneous 9, emerald green jasmine, P-2-5-1-1, saliva is excellent 35, spring and autumn king No. 2, jasper 2, spring Autumn king No. 3, river be No. 3 emerald green, bright beautiful, eastern agriculture 804, saliva are No. 1 excellent, middle peasant No. 50, middle peasant No. 26, Wan Nong tri-, Chang Chun Mi Ci, capital are ground Emerald is No. 2 mini, Jingyanmini No.2, saliva are excellent 308, De Ruite 4, Jinyou No.36, rich U.S. 8, rich U.S. 6913,13AC230, Moral Rett 79, De Ruite Y2,13AC049, De Ruite F16, Zhongnong No. 12, Kant (Condesa) RZ F1, Lvchun County two, the four seasons Rich, Lvchun County's No.1, rich No. 5 large, saliva is No. 335 excellent, saliva is No. 316 excellent, saliva is No. 358 excellent, Dong Fangxiu, the saliva spring No. three, the green 21-10 of saliva, Middle peasant No. 37, middle peasant No. 50, saliva is No. 315 excellent, universe moral 2, universe moral 1217, universe moral 117, universe moral 777, rich No. eight large, Tian Jiao seven Number, Tian Jiao eight, emerald green dragon, the four seasons it is rich.

10. SSR described in the combination of SSR primer described in the site SSR or Claims 2 or 3 described in claim 1 or claim 4 Kit or any detection method of claim 5-9, in the application of following x1 or x2:

X1: identify whether the kind of cucumber to be measured belongs to a certain kind in standard cucumber variety；

X2: identify that the kind of cucumber to be measured is specially any in standard cucumber variety.