CN109517923A - A kind of method for identifying cucumber variety authenticity and its combination of dedicated SNP primer - Google Patents

A kind of method for identifying cucumber variety authenticity and its combination of dedicated SNP primer Download PDF

Info

Publication number
CN109517923A
CN109517923A CN201811634016.2A CN201811634016A CN109517923A CN 109517923 A CN109517923 A CN 109517923A CN 201811634016 A CN201811634016 A CN 201811634016A CN 109517923 A CN109517923 A CN 109517923A
Authority
CN
China
Prior art keywords
sequence
primer
forward primer
cucumber
sets
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811634016.2A
Other languages
Chinese (zh)
Other versions
CN109517923B (en
Inventor
温常龙
张建
杨静静
罗江
张峰
毛爱军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Academy of Agriculture and Forestry Sciences
Original Assignee
Beijing Academy of Agriculture and Forestry Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Academy of Agriculture and Forestry Sciences filed Critical Beijing Academy of Agriculture and Forestry Sciences
Priority to CN201811634016.2A priority Critical patent/CN109517923B/en
Publication of CN109517923A publication Critical patent/CN109517923A/en
Application granted granted Critical
Publication of CN109517923B publication Critical patent/CN109517923B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of method for identifying cucumber variety authenticity and its combinations of dedicated SNP primer.SNP primer combination provided by the present invention is made of 32 primer sets.Each primer sets are made of 3 primer sequences, for expanding a SNP site.The nucleotide sequence of each primer is successively as shown in sequence 1 to sequence 96 in sequence table in 32 primer sets.SNP primer combination provided by the 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.Method provided by the invention can both identify unknown cucumber variety, can also identify the authenticity of known kind.Method provided by the invention has high-throughput, accurate, inexpensive, easy to operate, saving human and material resources etc. a little, has very wide application prospect.

Description

A kind of method for identifying cucumber variety authenticity and its combination of dedicated SNP primer
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method of cucumber variety authenticity and its dedicated identified The combination of SNP primer.
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%.Increased year by year by the kind of country and each province's authorization.Foundation " non-principal variety of crops registration method ", 2017 April, the Ministry of Agriculture disclosed first non-principal variety of crops registration catalogue, and cucumber is among them.Currently, the whole nation passes through mirror Kind that is fixed, assert, register is a up to more than 1000, but cucumber variety quantity in the market is digital considerably beyond this.Due to Huang Melon breeding enterprise is smaller and disperses, and variety certification is mutually uncoordinated between each province and city, the variety managements of cucumber fail effectively with Into, there are many fake and forged kinds, wherein it is many for it is derivative, derive from approximate kind.In addition the management of breeding production base is mixed Random, 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 " non-principal agriculture Crop varieties register guide " requirement, breediness illustrates with related character involved in kind DUS test report if any bright True associated gene can directly submit DNA testing result.Therefore, it establishes a set of DNA fingerprint that is based on and effectively carries out cucumber variety Resource authenticity and the standard of Purity are particularly important.
In recent years, the advantages that SNP is as third generation molecular labeling, more with its quantity, distribution is wide, inheritance stability is by extensive Pay attention to.With the development of high throughput sequencing technologies and the continuous reduction of sequencing cost, extensive cucumber resurveys sequence and is possibly realized. Based on analysis cucumber variation group information, the SNP site of more stability and high efficiency can be excavated.It is competitive special using allele PCR method develops its specific primer, and the final sample that obtains is in the genotype of SNP site.
Currently, China's cucumber variety identification of dna Molecular Detection is mainly according to SSR molecular marker method, but SSR primer is ground Hair is without reference to Cucumber germplasm variation group information, and there are untrue variation situations；Kind quantity used in SSR primer screening It is less, and the sale kind of China's Vehicles Collected from Market cannot be represented；In addition it is limited to SSR detection mode easily and causes and is untrue, false Positive, false negative result；Do not adapt to automation, high-throughput, requirement on a large scale.Compared with SSR marker, SNP has multiple The advantage of aspect: the steady and audible easy detection of variation, authenticity accuracy are high；Each crop have millions of SNP for Selection；It is suitable for high-throughput, low cost, automation quickly detection, human error can be reduced；SNP parting is not necessarily to check variety, Result is presented with accurate base.
Summary of the invention
The purpose of the present invention is identify cucumber variety.
The present invention protects SNP site to combine first, it may include 32 SNP sites of Cucumber germplasm；Described 32 SNP Point is as follows: the site CuSNP01 is the 1976800th nucleotide on No. 1 chromosome；The site CuSNP02 is on No. 1 chromosome The 8433330th nucleotide；The site CuSNP03 is the 13502481st nucleotide on No. 1 chromosome；The site CuSNP04 For the 17508118th nucleotide on No. 1 chromosome；The site CuSNP05 is the 25296763rd nucleosides on No. 1 chromosome Acid；The site CuSNP06 is the 1200640th nucleotide on No. 2 chromosomes；The site CuSNP07 is the on No. 2 chromosomes 2563528 nucleotide；The site CuSNP08 is the 5891488th nucleotide on No. 2 chromosomes；The site CuSNP09 is No. 2 The 6045153rd nucleotide on chromosome；The site CuSNP10 is the 11947823rd nucleotide on No. 2 chromosomes； The site CuSNP11 is the 40726th nucleotide on No. 3 chromosomes；The site CuSNP12 is the on No. 3 chromosomes 1001975 nucleotide；The site CuSNP13 is the 10713052nd nucleotide on No. 3 chromosomes；The site CuSNP14 is 3 The 14161884th nucleotide on number chromosome；The site CuSNP15 is the 18678235th nucleotide on No. 3 chromosomes； The site CuSNP16 is the 27913661st nucleotide on No. 3 chromosomes；The site CuSNP17 is the on No. 4 chromosomes 16975775 nucleotide；The site CuSNP18 is the 21068185th nucleotide on No. 4 chromosomes；The site CuSNP19 is 5 The 2133319th nucleotide on number chromosome；The site CuSNP20 is the 4054461st nucleotide on No. 5 chromosomes； The site CuSNP21 is the 8103814th nucleotide on No. 5 chromosomes；The site CuSNP22 is the on No. 5 chromosomes 9582207 nucleotide；The site CuSNP23 is the 17245080th nucleotide on No. 5 chromosomes；The site CuSNP24 is 6 The 195377th nucleotide on number chromosome；The site CuSNP25 is the 2397556th nucleotide on No. 6 chromosomes； The site CuSNP26 is the 18079846th nucleotide on No. 6 chromosomes；The site CuSNP27 is the on No. 6 chromosomes 20929474 nucleotide；The site CuSNP28 is the 24439780th nucleotide on No. 6 chromosomes；The site CuSNP29 is 7 The 2085399th nucleotide on number chromosome；The site CuSNP30 is the 4866176th nucleotide on No. 7 chromosomes； The site CuSNP31 is the 6739101st nucleotide on No. 7 chromosomes；The site CuSNP32 is the on No. 7 chromosomes 15415870 nucleotide.
The SNP site combination can be specifically made of above-mentioned 32 SNP sites.
The present invention also protects SNP primer to combine, it may include for expanding the primer sets 1 of the CuSNP01, for expanding State the primer sets 2 of CuSNP02, the primer sets 3 for expanding the CuSNP03, the primer sets for expanding the CuSNP04 4, for expanding the primer sets 5 of the CuSNP05, the primer sets 6 for expanding the CuSNP06, described for expanding The primer sets 7 of CuSNP07, the primer sets 8 for expanding the CuSNP08, the primer sets 9 for expanding the CuSNP09, For expanding the primer sets 10 of the CuSNP010, the primer sets 11 for expanding the CuSNP11, described for expanding The primer sets 12 of CuSNP12, the primer sets 13 for expanding the CuSNP13, the primer sets for expanding the CuSNP14 14, for expanding the primer sets 15 of the CuSNP15, the primer sets 16 for expanding the CuSNP16, described for expanding The primer sets 17 of CuSNP17, the primer sets 18 for expanding the CuSNP18, the primer sets for expanding the CuSNP19 19, for expanding the primer sets 20 of the CuSNP20, the primer sets 21 for expanding the CuSNP21, described for expanding The primer sets 22 of CuSNP22, the primer sets 23 for expanding the CuSNP23, the primer sets for expanding the CuSNP24 24, for expanding the primer sets 25 of the CuSNP25, the primer sets 26 for expanding the CuSNP26, described for expanding The primer sets 27 of CuSNP27, the primer sets 28 for expanding the CuSNP28, the primer sets for expanding the CuSNP29 29, described for expanding the primer sets 30 of the CuSNP30, the primer sets 31 for expanding the CuSNP31 and for expanding The primer sets 32 of CuSNP32.
In SNP primer combination, the primer sets 1 forward primer 01F1 as shown in sequence 1, shown in sequence 2 The composition of reverse primer 01R shown in forward primer 01F2 and sequence 3.The primer sets 2 can the forward primer as shown in sequence 4 The composition of reverse primer 02R shown in forward primer 02F2 shown in 02F1, sequence 5 and sequence 6.The primer sets 3 can be by sequence The composition of reverse primer 03R shown in forward primer 03F2 shown in forward primer 03F1, sequence 8 shown in 7 and sequence 9.It is described It is anti-shown in the forward primer 04F1 as shown in sequence 10 of primer sets 4, forward primer 04F2 and sequence 12 shown in sequence 11 It is formed to primer 04R.The primer sets 5 forward primer 05F1 as shown in sequence 13, forward primer shown in sequence 14 The composition of reverse primer 05R shown in 05F2 and sequence 15.The primer sets 6 forward primer 06F1, sequence as shown in sequence 16 The composition of reverse primer 06R shown in forward primer 06F2 and sequence 18 shown in column 17.The primer sets 7 can be by 19 institute of sequence Reverse primer 07R composition shown in forward primer 07F2 shown in the forward primer 07F1 that shows, sequence 20 and sequence 21.It is described It is anti-shown in the forward primer 08F1 as shown in sequence 22 of primer sets 8, forward primer 08F2 and sequence 24 shown in sequence 23 It is formed to primer 08R.The primer sets 9 forward primer 09F1 as shown in sequence 25, forward primer shown in sequence 26 The composition of reverse primer 09R shown in 09F2 and sequence 27.The primer sets 10 forward primer 10F1 as shown in sequence 28, The composition of reverse primer 10R shown in forward primer 10F2 and sequence 30 shown in sequence 29.The primer sets 11 can be by sequence 31 Shown in reverse primer 11R composition shown in forward primer 11F1, forward primer 11F2 and sequence 33 shown in sequence 32.Institute It states shown in the forward primer 12F1 as shown in sequence 34 of primer sets 12, forward primer 12F2 and sequence 36 shown in sequence 35 Reverse primer 12R composition.The primer sets 13 forward primer 13F1 as shown in sequence 37, forward direction shown in sequence 38 The composition of reverse primer 13R shown in primer 13F2 and sequence 39.The primer sets 14 can the forward primer as shown in sequence 40 The composition of reverse primer 14R shown in forward primer 14F2 shown in 14F1, sequence 41 and sequence 42.The primer sets 15 can be by Reverse primer 15R group shown in forward primer 15F2 shown in forward primer 15F1, sequence 44 shown in sequence 43 and sequence 45 At.The primer sets 16 forward primer 16F1 as shown in sequence 46, forward primer 16F2 and sequence 48 shown in sequence 47 Shown in reverse primer 16R composition.The primer sets 17 forward primer 17F1 as shown in sequence 49, shown in sequence 50 The composition of reverse primer 17R shown in forward primer 17F2 and sequence 51.The primer sets 18 can the forward direction as shown in sequence 52 draw The composition of reverse primer 18R shown in forward primer 18F2 shown in object 18F1, sequence 53 and sequence 54.The primer sets 19 can Reverse primer 19R shown in forward primer 19F2 shown in forward primer 19F1, sequence 56 as shown in sequence 55 and sequence 57 Composition.The primer sets 20 forward primer 20F1 as shown in sequence 58, forward primer 20F2 and sequence shown in sequence 59 The composition of reverse primer 20R shown in 60.The primer sets 21 forward primer 21F1 as shown in sequence 61, shown in sequence 62 Forward primer 21F2 and sequence 63 shown in reverse primer 21R composition.The primer sets 22 can the forward direction as shown in sequence 64 The composition of reverse primer 22R shown in forward primer 22F2 shown in primer 2 2F1, sequence 65 and sequence 66.The primer sets 23 The forward primer 23F1 as shown in sequence 67, reverse primer shown in forward primer 23F2 and sequence 69 shown in sequence 68 23R composition.The primer sets 24 forward primer 24F1 as shown in sequence 70, forward primer 24F2 shown in sequence 71 and The composition of reverse primer 24R shown in sequence 72.The primer sets 25 forward primer 25F1 as shown in sequence 73, sequence 74 Shown in reverse primer 25R composition shown in forward primer 25F2 and sequence 75.The primer sets 26 can be as shown in sequence 76 The composition of reverse primer 26R shown in forward primer 26F2 shown in forward primer 26F1, sequence 77 and sequence 78.The primer It is reversed shown in the 27 forward primer 27F1 as shown in sequence 79 of group, forward primer 27F2 and sequence 81 shown in sequence 80 Primer 2 7R composition.The primer sets 28 forward primer 28F1 as shown in sequence 82, forward primer shown in sequence 83 The composition of reverse primer 28R shown in 28F2 and sequence 84.The primer sets 29 forward primer 29F1 as shown in sequence 85, The composition of reverse primer 29R shown in forward primer 29F2 and sequence 87 shown in sequence 86.The primer sets 30 can be by sequence 88 Shown in reverse primer 30R composition shown in forward primer 30F1, forward primer 30F2 and sequence 90 shown in sequence 89.Institute It states shown in the forward primer 31F1 as shown in sequence 91 of primer sets 31, forward primer 31F2 and sequence 93 shown in sequence 92 Reverse primer 31R composition.The primer sets 32 forward primer 32F1 as shown in sequence 94, forward direction shown in sequence 95 The composition of reverse primer 32R shown in primer 32F2 and sequence 96.
In SNP primer combination, the primer sets 1 can by sequence 1 from 5 ' ends forward direction shown in the 22nd to 46 Primer 01F1, sequence 2 the reverse primer 01R shown in forward primer 01F2 and sequence 3 shown in the 22nd to 46 from 5 ' ends Composition.The primer sets 2 can forward primer 02F1, sequence 5 shown in the 22nd to 46 be last from 5 ' from 5 ' ends by sequence 4 The composition of reverse primer 02R shown in forward primer 02F2 and sequence 6 shown in having held the 22nd to 43.The primer sets 3 can be by Sequence 7 from 5 ' ends forward primer 03F1, sequence 8 shown in the 22nd to 48 from 5 ' ends shown in the 22nd to 50 The composition of reverse primer 03R shown in forward primer 03F2 and sequence 9.The primer sets 4 can be by sequence 10 the 22nd from 5 ' ends To forward primer 04F1, sequence 11 forward primer 04F2 and sequence shown in the 22nd to 51 from 5 ' ends shown in 52 The composition of reverse primer 04R shown in 12.The primer sets 5 can by sequence 13 from 5 ' ends forward direction shown in the 22nd to 47 Primer 05F1, sequence 14 reverse primer shown in forward primer 05F2 and sequence 15 shown in the 22nd to 47 from 5 ' ends 05R composition.The primer sets 6 can by sequence 16 from 5 ' ends forward primer 06F1, sequence 17 shown in the 22nd to 51 from 5 ' ends play the 22nd to 51 shown in reverse primer 06R composition shown in forward primer 06F2 and sequence 18.The primer sets 7 can by sequence 19 from 5 ' ends forward primer 07F1, sequence 20 shown in the 22nd to 46 the 22nd to 48 from 5 ' ends Shown in reverse primer 07R composition shown in forward primer 07F2 and sequence 21.The primer sets 8 can be last from 5 ' by sequence 22 The forward primer shown in the 22nd to 48 from 5 ' ends of forward primer 08F1, sequence 23 shown in having held the 22nd to 48 The composition of reverse primer 08R shown in 08F2 and sequence 24.The primer sets 9 can be by sequence 25 the 22nd to 48 from 5 ' ends Shown in forward primer 09F1, sequence 26 from 5 ' ends shown in forward primer 09F2 and sequence 27 shown in the 22nd to 47 Reverse primer 09R composition.The primer sets 10 can by sequence 28 from 5 ' ends forward primer shown in the 22nd to 51 10F1, sequence 29 the reverse primer 10R group shown in forward primer 10F2 and sequence 30 shown in the 22nd to 51 from 5 ' ends At.The primer sets 11 can forward primer 11F1, sequence 32 shown in the 22nd to 46 be last from 5 ' from 5 ' ends by sequence 31 The composition of reverse primer 11R shown in forward primer 11F2 and sequence 33 shown in having held the 22nd to 45.The primer sets 12 can By sequence 34 from 5 ' ends the 22nd to 47 institute from 5 ' ends of forward primer 12F1, sequence 35 shown in the 22nd to 46 The composition of reverse primer 12R shown in the forward primer 12F2 and sequence 36 shown.The primer sets 13 can be by sequence 37 from 5 ' ends Forward primer 13F1, sequence 38 the forward primer 13F2 shown in the 22nd to 44 from 5 ' ends shown in playing the 22nd to 43 It is formed with reverse primer 13R shown in sequence 39.The primer sets 14 can be by sequence 40 from 5 ' ends shown in the 22nd to 49 Forward primer 14F1, sequence 41 it is anti-shown in forward primer 14F2 and sequence 42 shown in the 22nd to 49 from 5 ' ends It is formed to primer 14R.The primer sets 15 can by sequence 43 from 5 ' ends forward primer 15F1 shown in the 22nd to 48, The reverse primer 15R shown in forward primer 15F2 and sequence 45 shown in the 22nd to 49 from 5 ' ends of sequence 44 is formed.Institute State primer sets 16 can by sequence 46 from 5 ' ends forward primer 16F1, sequence 47 shown in the 22nd to 46 from 5 ' ends The composition of reverse primer 16R shown in forward primer 16F2 and sequence 48 shown in 22nd to 48.The primer sets 17 can be by sequence Column 49 from 5 ' ends forward primer 17F1, sequence 50 shown in the 22nd to 51 from 5 ' ends shown in the 22nd to 52 The composition of reverse primer 17R shown in forward primer 17F2 and sequence 51.The primer sets 18 can be by sequence 52 from 5 ' ends Forward primer 18F1, sequence 53 shown in 22 to 46 forward primer 18F2 and sequence shown in the 22nd to 44 from 5 ' ends The composition of reverse primer 18R shown in column 54.The primer sets 19 can by sequence 55 from 5 ' ends shown in the 22nd to 48 just Reversely draw shown in forward primer 19F2 and sequence 57 shown in the 22nd to 49 from 5 ' ends to primer 19F1, sequence 56 Object 19R composition.The primer sets 20 can by sequence 58 from 5 ' ends forward primer 20F1, sequence shown in the 22nd to 48 59 from 5 ' ends reverse primer 20R shown in forward primer 20F2 and sequence 60 shown in the 22nd to 47 form.It is described to draw Object group 21 can by sequence 61 from 5 ' ends forward primer 21F1, sequence 62 shown in the 22nd to 43 the 22nd from 5 ' ends To reverse primer 21R composition shown in forward primer 21F2 shown in 42 and sequence 63.The primer sets 22 can be by sequence 64 The forward direction shown in the 22nd to 46 from 5 ' ends of forward primer 22F1, sequence 65 shown in the 22nd to 47 from 5 ' ends The composition of reverse primer 22R shown in primer 2 2F2 and sequence 66.The primer sets 23 can by sequence 67 from 5 ' ends the 22nd to The forward primer 23F2 and sequence 69 shown in the 22nd to 46 from 5 ' ends of forward primer 23F1, sequence 68 shown in 46 Shown in reverse primer 23R composition.The primer sets 24 can forward direction shown in the 22nd to 43 be drawn from 5 ' ends by sequence 70 Object 24F1, sequence 71 the reverse primer 24R shown in forward primer 24F2 and sequence 72 shown in the 22nd to 44 from 5 ' ends Composition.The primer sets 25 can by sequence 73 from 5 ' ends forward primer 25F1, sequence 74 from 5 ' shown in the 22nd to 51 End play the 22nd to 51 shown in reverse primer 25R composition shown in forward primer 25F2 and sequence 75.The primer sets 26 Can by sequence 76 from 5 ' ends forward primer 26F1, sequence 77 shown in the 22nd to 43 the 22nd to 42 from 5 ' ends Shown in reverse primer 26R composition shown in forward primer 26F2 and sequence 78.The primer sets 27 can be last from 5 ' by sequence 79 The forward primer shown in the 22nd to 48 from 5 ' ends of forward primer 27F1, sequence 80 shown in having held the 22nd to 49 The composition of reverse primer 27R shown in 27F2 and sequence 81.The primer sets 28 can be by sequence 82 the 22nd to 54 from 5 ' ends Shown in forward primer 28F1, sequence 83 from 5 ' ends shown in forward primer 28F2 and sequence 84 shown in the 22nd to 54 Reverse primer 28R composition.The primer sets 29 can by sequence 85 from 5 ' ends forward primer shown in the 22nd to 46 29F1, sequence 86 the reverse primer 29R group shown in forward primer 29F2 and sequence 87 shown in the 22nd to 45 from 5 ' ends At.The primer sets 30 can forward primer 30F1, sequence 89 shown in the 22nd to 52 be last from 5 ' from 5 ' ends by sequence 88 The composition of reverse primer 30R shown in forward primer 30F2 and sequence 90 shown in having held the 22nd to 52.The primer sets 31 can By sequence 91 from 5 ' ends the 22nd to 53 institute from 5 ' ends of forward primer 31F1, sequence 92 shown in the 22nd to 54 The composition of reverse primer 31R shown in the forward primer 31F2 and sequence 93 shown.The primer sets 32 can be by sequence 94 from 5 ' ends Forward primer 32F1, sequence 95 the forward primer 32F2 shown in the 22nd to 56 from 5 ' ends shown in playing the 22nd to 56 It is formed with reverse primer 32R shown in sequence 96.
In any of the above-described primer sets, the primer and title of " F2 " are contained in the primer, title in title containing " F1 " In the primer containing " R " molar ratio concretely 2:2:5.
Any of the above-described SNP primer combination specifically can be by the primer sets 1, the primer sets 2, the primer sets 3, institute State the primer sets 4, primer sets 5, the primer sets 6, the primer sets 7, the primer sets 8, the primer sets 9, described Primer sets 10, the primer sets 11, the primer sets 12, the primer sets 13, the primer sets 14, the primer sets 15, institute State primer sets 16, the primer sets 17, the primer sets 18, the primer sets 19, the primer sets 20, the primer sets 21, The primer sets 22, the primer sets 23, the primer sets 24, the primer sets 25, the primer sets 26, the primer sets 27, the primer sets 28, the primer sets 29, the primer sets 30, the primer sets 31 and the primer sets 32 form.
Above, the nucleotides sequence shown in the 1st to 21 from 5 ' ends of sequence 1 is classified as fluorescence labels sequence in sequence table (i.e. FAM fluorescence labels sequence), fluorescence signal are specially blue.Sequence 2 is from 5 ' ends shown in the 1st to 21 in sequence table Nucleotide sequence be also fluorescence labels sequence (i.e. HEX fluorescence labels sequence), fluorescence signal is specially red.
Kit containing any of the above-described SNP primer combination also belongs to protection scope of the present invention.
The preparation method of the kit also belongs to protection scope of the present invention.The preparation method of the kit includes will The step of each primer in any of the above-described primer sets is individually packed.
The application of the kit also belongs to protection scope of the present invention.The application of the kit can be x3) or x4): X3 cucumber variety) is identified；X4) identify cucumber variety true or false.
The application of any of the above-described SNP site combination or any of the above-described SNP primer combination, can be x1) into x4) It is any: x1) prepare the kit for identifying cucumber variety；X2 the reagent for identifying cucumber variety true or false) is prepared Box；X3 cucumber variety) is identified；X4) identify cucumber variety true or false.
The present invention also protects a kind of method identified cucumber to be measured and belong to which kind in 241 cucumber varieties, it may include Following steps: the genotype of cucumber to be measured and 241 cucumber varieties based on 32 SNP sites is detected respectively, is then carried out Following judgement: if certain kind is based in genotype and 241 cucumber varieties of the cucumber to be measured based on 32 SNP sites The genotype of 32 SNP sites is completely the same, then cucumber to be measured and the cucumber variety belong to the same kind；If to It surveys each kind in genotype and 241 cucumber varieties of the cucumber based on 32 SNP sites and is based on described 32 SNP The genotype of point is inconsistent, then cucumber to be measured and the kind of 241 cucumber varieties are all different.
It is described " to detect the gene of cucumber to be measured and 241 cucumber varieties based on 32 SNP sites in the above method The step of type ", can be as follows:
(1) it is respectively adopted respectively using the genomic DNA of the genomic DNA of cucumber to be measured and 241 cucumber varieties as template The carry out PCR amplification of primer sets, obtains pcr amplification product in any of the above-described SNP primer combination；
(2) after completing step (1), using the fluorescence signal of instrument detection pcr amplification product, according to the color of fluorescence signal Obtain the genotype of cucumber to be measured and 241 cucumber varieties based on 32 SNP sites.
In the above method, described " detection detects cucumber to be measured and 241 cucumber varieties based on 32 SNP sites The step of genotype ", can be as follows:
(1) it is respectively adopted respectively using the genomic DNA of the genomic DNA of cucumber to be measured and 241 cucumber varieties as template The carry out PCR amplification of primer sets, obtains pcr amplification product in any of the above-described SNP primer combination；
(2) pcr amplification product for taking step (1) to obtain, sequencing；
(3) sequencing result obtained according to step (2), obtains cucumber to be measured and 241 cucumber varieties are based on described 32 The genotype of SNP site.
The present invention also protects a kind of method identified cucumber to be measured and belong to which kind in 241 cucumber varieties, it may include Following steps:
(1) using the genomic DNA of cucumber to be measured as template, primer in any of the above-described SNP primer combination is respectively adopted The carry out PCR amplification of group, obtains pcr amplification product；With the genomic DNA of each cucumber variety in standard cucumber variety group For template, the carry out PCR amplification of primer sets in any of the above-described SNP primer combination is respectively adopted, obtains pcr amplification product； The standard cucumber variety group is made of 241 cucumber varieties；
(2) each pcr amplification product for obtaining cucumber to be measured pcr amplification product corresponding with each standard cucumber variety Clustering is carried out, cucumber to be measured and which standard cucumber variety are same class, the cucumber to be measured and the standard in clustering Cucumber variety belongs to the same kind.
In any of the above-described method, 241 cucumber varieties can be emerald green, lucky miscellaneous No. 16, middle peasant No. 20, Shandong cucumber 9 Number, middle peasant No. 29, Diana, in grind fan 8F1, Bertha, in grind 23F1, golden embryo 99-3F1, in grind 17F1, golden embryo 99-2F1, gold Embryo 99F1, golden embryo 99-1F1, golden embryo 98F1, golden embryo 98-1F1, in grind 19F1, silver-colored embryo 88F1,128 half-bloods, middle peasant No. 6, Middle peasant No. 10, the good elegant, capital of northern agriculture grind 106, surmount cucumber F1, peaceful star all-round spring and autumn F1, Ya Meite, AMATA765, Kingsoft strong yellow Melon, capital grind mini No. 9, capital grinds mini No. 3, capital grinds mini No. 5, capital grinds mini No. 4, that beauty fruit grinds in smart No. 4 green, China is yellow Melon, capital grind pickling treasured, white highest-ranking imperial concubine's Fruit Cucumber, Beijing white jade three, spring beauty, fruit type 101, adopted king's Mini cucumber, Tangshan kingfisher Precious F1, fine work Bai Yesan, Tang Chun 100, cucumber, light difficult to understand, Man Guan, all-round spring and autumn F1, capital grind green exquisite No. 2, capital grind drought treasured 5, Excellent bright the best, capital grind Xia Mei, capital grind winning, capital is rich 298, Jinyou No.35, Qiu Mei is ground in capital, spring beauty is ground in capital, green rich ride on Bus No. 11, Green rich No. seven, Beijing 403, green show, Beijing 204, it is new grind No. four, saliva grinds No. four, close thorn king cucumber, plucks unbeaten open country king, dragon's fountain The green hat of king-dragon's fountain, XSBN4, XSBN6, XSBN8, XSBN11, XSBN16, XSBN1, XSBN2, XSBN3, XSBN5, XSBN7, XSBN9, XSBN10, XSBN12, XSBN13, XSBN14, XSBN15, XSBN17, XSBN18, XSBN19, saliva are No. 401 excellent, saliva is excellent No. 108, Vlaspik, Ning Yun No. 3 numbers, middle peasant No. 19, spring jade, spring and autumn king No. 3, P-2-5-1-1, spring and autumn king No. 2, strong melon, Shen Lv 72, CUCUMBER1404, Shen Lv 64, I1an, capital grind No. 2 mini emerald, 13AC230, jasper, green emerald green, capital grind mini No. 1, it is bright Beautiful, eastern agriculture 804, Ning Jia 3, excellent plus complete female 09, what drought, teenage girl, Jinyou No.12, saliva spring No. 4, Shanghai is No. 6 miscellaneous, it is miscellaneous No. 2 precious, select Four, saliva is No. 2 excellent, the winter is beautiful, Jinyou No.36, MC2065, middle peasant No. 15, the northern star of Japan, middle peasant No. 26, saliva are No. 38 excellent, saliva excellent 31 Number, rich U.S. 11, rich U.S. 74, the saliva spring No. three, saliva is No. 3 green, miscellaneous No. nine lucky, Tian Jiao eight, rich No. eight large, the four seasons are rich, emerald green Dragon, spring king 9801, Luyitianshi, boat lose No. 1, Shandong cucumber No. three, Jinyou No.10, rich U.S. 6913, dry moral 117, saliva spring No. 3, saliva Excellent No. 315, saliva is excellent 308, P02, Wan Nong No. tri- numbers, 9930, prince charming, local king cucumber, De Ruite 723, extra large promise No.1, army Section 18, dark green section at, European No.1, strong fruit nodule 80, capital A008, rich U.S. 8, capital AK42, the rich melon king of U.S., capital AK18, The good glossy continuous cropping king of Dare LD-1, De Ruite F16, De Ruite Y8, ancient cooking vessel, dirty oil be bright 999, the positive A207 of saliva, green mound 8, De Rui Spy 727, De Ruite 7876, De Ruite, Dare LD-3, glossy dark green prince, capital grind 118, converge win 15, the big Trivhosantnes Kirilouii Manim of cyanine, it is No. 3 mini, Saliva is prosperous 203, emerald green U.S. Fruit Cucumber, green emerald green precious, large nine, Tian Yi, odd sword 1, green excellent 188, spring happiness F1, strong non-irrigated nodule, Xing Yansi Number, Xing Yan bis-, dirty oil be bright 507, odd sword 2, Xinjin spring No. four, saliva are excellent 409, the sharp nova of grain rains U.S., 83-ms702, capital are ground Mini No. 2, Dai Duoxing, smart No. 2 green, CU003, tie up lucky tail Asia, be CU001, stoke 9006, CU002, smart No. 5 green, green No. 3 smart, the short cucumber 3966 of stingless fruit, 88-ms701, Ma Sha's cucumber pickling type, lucky miscellaneous mini, Bai Jingling 2, monarch excellent 66 Cucumber, De Ruite cucumber 136-7, Peking silicite ultrawhite leaf three, special Ji are No. four miscellaneous, green exquisite, emerald green hat bright star, Tian Jiao 7 are ground in capital South China type, auspicious light two, the green excellent cucumber of brothers, selected leaf three, grain rains beauty, grain rains dry land cucumber, same to 52, De Ruite cucumber L14-2, De Ruite cucumber GZ1601, De Ruite cucumber L14-5, De Ruite cucumber, South Korea cucumber 2-06-97-164, Sheng Feng 908, the close thorn cucumber of De Ruite 15-10, Jin Dongke moisten that 99 close thorn cucumber, middle peasant No. 106, middle peasant No. 27, saliva is No. 4 excellent, saliva excellent 11 Number, saliva is No. 2 miscellaneous, Zhongnong No.16, saliva are excellent 38, PI197088, H9, WI2757, true lemon, 1,364 2014.11 and Z298 2013.11。
In any of the above-described method, " progress of primer sets in any of the above-described SNP primer combination is respectively adopted The response procedures of PCR amplification " are concretely: 94 DEG C of initial denaturations, 15min；94 DEG C of denaturation 20s, 61 DEG C -55 DEG C (are selected touch Down program, every circulation reduce by 0.6 DEG C), 1min expands 10 circulations；94 DEG C of denaturation 20s, 55 DEG C of renaturation & extend 1min, after 26 circulations of continuous amplification.If fluorescence signal is weak after PCR amplification, data analysis is influenced, circulation (94 DEG C of denaturation can be added 20s, 55 DEG C of renaturation and extension 1min, 5 circulations), until result is satisfied.
SNP primer combination provided by the invention can be used for carrying out early stage identification in seed or Seedling Stage to cucumber variety, protect The authenticity of kind is demonstrate,proved, protects the equity of the producer and breeder conscientiously, and is provided for Cucumber Germplasm and New variety protection Technical support.Method provided by the invention can both identify unknown cucumber variety, can also be to the true of known kind Property is identified.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 241 dendrograms for trying cucumber variety established in 32 primer sets.
Fig. 2 is SNP marker number (i.e. SNP site number) and distinguishes 241 relational graphs for trying cucumber variety.
Fig. 3 is for 32 primer sets in part for the SNP parting effect in examination cucumber variety.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
The acquisition that embodiment 1, the SNP primer for identifying cucumber variety authenticity combine
One, the discovery of 32 SNP sites
The present invention is based on the heavy sequencing datas that 49 portions of cucumber represent resource, obtain 32 SNP sites.This 49 parts of cucumber resources Type is abundant, covers North-China Type (7 parts), indica-type (18 parts), Japanese type (3 parts), South China type (2 parts), European fruit type (5 Part), America processing type (4 parts), Xishuangbanna type (5 parts) and osculant (5 parts), substantially include the main ecology of cucumber Type and economical character embody germplasm representativeness, genetic diversity with higher as much as possible.
Specifically, the screening criteria of SNP site 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 SNP SNP site).
The essential information of 32 SNP sites the 1st to 4 column during see Table 1 for details.Wherein the position of SNP site on chromosome is Determination is compared with reference to genome sequence based on cucumber 9930, the cucumber 9930 is V2 with reference to the version number of genome sequence (download address are as follows: http://cucurbitgenomics.org/organism/2).
The essential information of 1.32 SNP sites of table
Two, the acquisition combined for identifying the SNP primer of cucumber variety authenticity
Based on 32 SNP sites of step 1 discovery, present inventors have developed polymorphism informations with higher The SNP primer combination for identifying cucumber variety authenticity of amount (i.e. PIC value is shown in Table the 5th column in 1).
The combination of SNP primer is made of 32 primer sets, and the title of each primer sets is shown in Table the 2nd column in 2.Each primer sets by 3 primer sequence compositions, for expanding a SNP site.In 32 primer sets in the nucleotide sequence such as table 2 of each primer Shown in 4th column.
Table 2
Note: single underscore is FAM fluorescence labels sequence, and double underline is HEX fluorescence labels sequence.
The validity check for the SNP primer combination that embodiment 2, embodiment are developed
241 in the present embodiment are shown in Table 3 for trying the essential information of cucumber variety.241 are normal for examination cucumber variety The external introduced variety of excellent variety or part seen.
3.241, the table essential informations for trying cucumber variety
1, for the acquisition of the genomic DNA of examination cucumber variety
Extract the genome of 241 blades (the mixed true leaf for taking 30 seeds) for trying cucumber variety respectively using CTAB method DNA obtains the genomic DNA for trying cucumber variety.
PCR requirement, Passing Criteria are as follows: agarose must be met for the quality and concentration for trying the genomic DNA of cucumber variety Electrophoresis showed DNA band is single, without obvious disperse；Ultraviolet specrophotometer Nanodrop2000 (Thermo) detects A260/ A280 ratio is greater than 1.8 in 1.8 or so, A260/A230 ratio；For try cucumber variety genomic DNA concentration in 10- 30ng/μL。
2, the genomic DNA using 241 for trying cucumber variety is respectively adopted 32 primer sets and carries out PCR as template respectively Amplification, obtains pcr amplification product.In each PCR reaction system, contain " F2 " in the primer, title in title containing " F1 " Primer and title in containing " R " primer concentration ratio be 2:2:5.
Response procedures are as follows: 94 DEG C of initial denaturations, 15min；94 DEG C of denaturation 20s, 61 DEG C -55 DEG C (touch down program is selected, Every circulation reduces by 0.6 DEG C), 1min expands 10 circulations；94 DEG C of denaturation 20s, 55 DEG C of renaturation & extension 1min continue amplification 26 A circulation.
3, after completing step 2, pass through FAM, HEX light beam of microplate reader when pcr amplification product temperature is down to 40 DEG C or less Fluorescent value is read in scanning, and (FAM fluorescence labels sequence emits in exciting light 485nm and observes readings under light 520nm wavelength, HEX is glimmering Optical label sequence emits in exciting light 528nm and observes readings under light 560nm wavelength), 241 are judged according to fluorescence signal color For trying genotype of the cucumber variety based on each SNP site.Specific judgment principle is as follows: if certain is for trying cucumber variety base Mr. Yu's SNP site is displayed in blue fluorescence signal, then this " is expanded and be somebody's turn to do based on the genotype of the SNP site for examination cucumber variety The complementary base of the 1st base in 3 ' ends of primer in SNP site and title containing " F1 " " is homozygous；If certain is yellow for examination Melon kind is based on certain SNP site and is displayed in red fluorescence signal, then this is for examination genotype of the cucumber variety based on the SNP site " complementary base for expanding the 1st base in 3 ' ends of the primer in the SNP site and title containing " F2 " " is homozygous；If Certain is based on certain SNP site for examination cucumber variety and shows green florescent signal, then this is for trying base of the cucumber variety based on the SNP site Because type is heterozygous, a base is " to expand the base of 3 ' ends the 1st of the primer in the SNP site and title containing " F1 " Complementary base ", another base " expands the base of 3 ' ends the 1st of the primer in the SNP site and title containing " F2 " Complementary base ".
It should be noted that if fluorescence signal is weak after PCR amplification, data analysis is influenced, circulation (94 DEG C of changes can be added Property 20s, 55 DEG C of renaturation and extend 1min, 5 circulation), until result be satisfied with until.
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
It is soft using MiniMarker and MEGA7 according to 241 for trying genotype of the cucumber variety based on 32 SNP sites Part carries out clustering for examination cucumber variety to 241.
It is as shown in Figure 1 to establish dendrogram of 241 in 32 primer sets for trying cucumber variety.The result shows that 32 Primer sets can distinguish 241 in table 3 for trying cucumber variety completely.It can be seen that the SNP 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 SNP It marks number (i.e. primer sets number) and distinguishes 241 relationships for trying cucumber variety differentiation rate.
The experimental results showed that (Fig. 2), 32 primer sets (i.e. 32 SNP marker numbers) are at 241 in examination cucumber variety Differentiation rate reach 100%.
<110>Beijing City Agriculture and Forestry Institute
<120>a kind of method for identifying cucumber variety authenticity and its combination of dedicated SNP primer
<160> 96
<170> PatentIn version 3.5
<210> 1
<211> 46
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
gaaggtgacc aagttcatgc tttatcaaac aactcgacgg cttcga 46
<210> 2
<211> 46
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
gaaggtcgga gtcaacggat tttatcaaac aactcgacgg cttcgt 46
<210> 3
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
gattgcactt tatgggaaat gtgggttttt 30
<210> 4
<211> 46
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 4
gaaggtgacc aagttcatgc tttaaatggt cttctggctg ccttca 46
<210> 5
<211> 43
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 5
gaaggtcgga gtcaacggat taatggtctt ctggctgcct tcg 43
<210> 6
<211> 27
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
gctacaagtt cgaatggagg tgcaaaa 27
<210> 7
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 7
gaaggtgacc aagttcatgc ttttcatttt gttccctttt ggcttcag 48
<210> 8
<211> 50
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 8
gaaggtcgga gtcaacggat tcatttcatt ttgttccctt ttggcttcaa 50
<210> 9
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 9
gaaaaatagt ctaccacaat tttgaaaccg aa 32
<210> 10
<211> 52
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 10
gaaggtgacc aagttcatgc tgaatgaaag tttaatcaag gatgcaacta tt 52
<210> 11
<211> 51
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 11
gaaggtcgga gtcaacggat taatgaaagt ttaatcaagg atgcaactat g 51
<210> 12
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 12
agattttgtg ccaatagagc aaagtcatat a 31
<210> 13
<211> 47
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 13
gaaggtgacc aagttcatgc tgttgatgac caatgaccat ttcgtag 47
<210> 14
<211> 47
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 14
gaaggtcgga gtcaacggat tgttgatgac caatgaccat ttcgtac 47
<210> 15
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 15
cgacattaac cccaatcttg gtaaataatt a 31
<210> 16
<211> 51
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 16
gaaggtgacc aagttcatgc tgttcaatgc ttggatcttt actttcatta a 51
<210> 17
<211> 51
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 17
gaaggtcgga gtcaacggat tgttcaatgc ttggatcttt actttcatta t 51
<210> 18
<211> 28
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 18
gtttcaagcc tattcgcatc atcttctt 28
<210> 19
<211> 46
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 19
gaaggtgacc aagttcatgc tctcaagcgt tttgctaaat gcatgc 46
<210> 20
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 20
gaaggtcgga gtcaacggat taactcaagc gttttgctaa atgcatgt 48
<210> 21
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 21
gttcaaaaaa ggaagactca gtcaagtcaa 30
<210> 22
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 22
gaaggtgacc aagttcatgc tacctgtgtt gtttgcatct agcataaa 48
<210> 23
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 23
gaaggtcgga gtcaacggat tacctgtgtt gtttgcatct agcataat 48
<210> 24
<211> 27
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 24
cgccatcttt tccgtaagcg atgttat 27
<210> 25
<211> 48
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 25
gaaggtgacc aagttcatgc tcttgtctgt tggacttcca caattttt 48
<210> 26
<211> 47
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 26
gaaggtcgga gtcaacggat tttgtctgtt ggacttccac aattttg 47
<210> 27
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 27
aaaccaacgg tcactctcag gcaaa 25
<210> 28
<211> 51
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 28
gaaggtgacc aagttcatgc taaagtgatt gttaatatcg attgcttgtt g 51
<210> 29
<211> 51
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 29
gaaggtcgga gtcaacggat taaagtgatt gttaatatcg attgcttgtt c 51
<210> 30
<211> 28
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 30
atccagaaaa gcatcccaca attacgta 28
<210> 31
<211> 46
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 31
gaaggtgacc aagttcatgc tgtctctgct tgcataagac tggtta 46
<210> 32
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 32
gaaggtcgga gtcaacggat ttctctgctt gcataagact ggttg 45
<210> 33
<211> 31
<212> DNA
<213>artificial sequence
<400> 33
tacaccaggt ttcagtaata acattaacca t 31
<210> 34
<211> 46
<212> DNA
<213>artificial sequence
<400> 34
gaaggtgacc aagttcatgc tactaggttt ggaactttgg agtgtc 46
<210> 35
<211> 47
<212> DNA
<213>artificial sequence
<400> 35
gaaggtcgga gtcaacggat tgactaggtt tggaactttg gagtgtt 47
<210> 36
<211> 34
<212> DNA
<213>artificial sequence
<400> 36
caacgtataa ttttagcatg ttagaactta gaaa 34
<210> 37
<211> 43
<212> DNA
<213>artificial sequence
<400> 37
gaaggtgacc aagttcatgc tgtcatcaag agtctcagct gac 43
<210> 38
<211> 44
<212> DNA
<213>artificial sequence
<400> 38
gaaggtcgga gtcaacggat tcgtcatcaa gagtctcagc tgaa 44
<210> 39
<211> 22
<212> DNA
<213>artificial sequence
<400> 39
tgtcgttgct ggccacccag aa 22
<210> 40
<211> 49
<212> DNA
<213>artificial sequence
<400> 40
gaaggtgacc aagttcatgc tgaatcgaaa acttctgtgt gcatcaaat 49
<210> 41
<211> 49
<212> DNA
<213>artificial sequence
<400> 41
gaaggtcgga gtcaacggat tgaatcgaaa acttctgtgt gcatcaaaa 49
<210> 42
<211> 31
<212> DNA
<213>artificial sequence
<400> 42
tgcaggtaac ttagttcaat tgtagagaat t 31
<210> 43
<211> 48
<212> DNA
<213>artificial sequence
<400> 43
gaaggtgacc aagttcatgc ttttgcctaa gtgaacacat acttagtg 48
<210> 44
<211> 49
<212> DNA
<213>artificial sequence
<400> 44
gaaggtcgga gtcaacggat tgtttgccta agtgaacaca tacttagtt 49
<210> 45
<211> 28
<212> DNA
<213>artificial sequence
<400> 45
gtgcacataa aaaggtatgg caagtcta 28
<210> 46
<211> 46
<212> DNA
<213>artificial sequence
<400> 46
gaaggtgacc aagttcatgc ttgctgtctg aagagaaaat gcatgc 46
<210> 47
<211> 48
<212> DNA
<213>artificial sequence
<400> 47
gaaggtcgga gtcaacggat ttttgctgtc tgaagagaaa atgcatgt 48
<210> 48
<211> 34
<212> DNA
<213>artificial sequence
<400> 48
ctatttcatt tgtatggaaa gacagatatt tcat 34
<210> 49
<211> 51
<212> DNA
<213>artificial sequence
<400> 49
gaaggtgacc aagttcatgc tataaatata aggctagact agtagcaaaa g 51
<210> 50
<211> 52
<212> DNA
<213>artificial sequence
<400> 50
gaaggtcgga gtcaacggat tgataaatat aaggctagac tagtagcaaa aa 52
<210> 51
<211> 31
<212> DNA
<213>artificial sequence
<400> 51
gaagaagtct atgttttccc tctatctaaa a 31
<210> 52
<211> 46
<212> DNA
<213>artificial sequence
<400> 52
gaaggtgacc aagttcatgc tttcggtcag tgctttaatc cccttt 46
<210> 53
<211> 44
<212> DNA
<213>artificial sequence
<400> 53
gaaggtcgga gtcaacggat tcggtcagtg ctttaatccc cttc 44
<210> 54
<211> 25
<212> DNA
<213>artificial sequence
<400> 54
cagcagattc tgcaatggca acgtt 25
<210> 55
<211> 48
<212> DNA
<213>artificial sequence
<400> 55
gaaggtgacc aagttcatgc tagactagca aagagtaata tggttgac 48
<210> 56
<211> 49
<212> DNA
<213>artificial sequence
<400> 56
gaaggtcgga gtcaacggat tgagactagc aaagagtaat atggttgat 49
<210> 57
<211> 31
<212> DNA
<213>artificial sequence
<400> 57
tacatccgtt atggaatttt gagttacact a 31
<210> 58
<211> 48
<212> DNA
<213>artificial sequence
<400> 58
gaaggtgacc aagttcatgc tcggcataat atgaatcaac ctggtaaa 48
<210> 59
<211> 47
<212> DNA
<213>artificial sequence
<400> 59
gaaggtcgga gtcaacggat tggcataata tgaatcaacc tggtaag 47
<210> 60
<211> 24
<212> DNA
<213>artificial sequence
<400> 60
gaccaccaag gcctatcact ccaa 24
<210> 61
<211> 43
<212> DNA
<213>artificial sequence
<400> 61
gaaggtgacc aagttcatgc tgggatgtaa tagtcggagc cct 43
<210> 62
<211> 42
<212> DNA
<213>artificial sequence
<400> 62
gaaggtcgga gtcaacggat tggatgtaat agtcggagcc cc 42
<210> 63
<211> 29
<212> DNA
<213>artificial sequence
<400> 63
gaatatacaa aagccaagcc caccaaaaa 29
<210> 64
<211> 47
<212> DNA
<213>artificial sequence
<400> 64
gaaggtgacc aagttcatgc tcaacgagtt gttaatgctt tggagct 47
<210> 65
<211> 46
<212> DNA
<213>artificial sequence
<400> 65
gaaggtcgga gtcaacggat taacgagttg ttaatgcttt ggagcc 46
<210> 66
<211> 25
<212> DNA
<213>artificial sequence
<400> 66
actgacactc caggagcaat ctcaa 25
<210> 67
<211> 46
<212> DNA
<213>artificial sequence
<400> 67
gaaggtgacc aagttcatgc tattcgagat tactccaagt tgtccc 46
<210> 68
<211> 46
<212> DNA
<213>artificial sequence
<400> 68
gaaggtcgga gtcaacggat tattcgagat tactccaagt tgtccg 46
<210> 69
<211> 31
<212> DNA
<213>artificial sequence
<400> 69
aagtcccaag ccataaaaaa agaaaggaaa t 31
<210> 70
<211> 43
<212> DNA
<213>artificial sequence
<400> 70
gaaggtgacc aagttcatgc tctgaggtcg ttatggtgac cac 43
<210> 71
<211> 44
<212> DNA
<213>artificial sequence
<400> 71
gaaggtcgga gtcaacggat tcctgaggtc gttatggtga ccaa 44
<210> 72
<211> 31
<212> DNA
<213>artificial sequence
<400> 72
aaataaagaa acaaaagaat gggcgtagca a 31
<210> 73
<211> 51
<212> DNA
<213>artificial sequence
<400> 73
gaaggtgacc aagttcatgc taacaaacaa atggagcttt aagtaaagta c 51
<210> 74
<211> 52
<212> DNA
<213>artificial sequence
<400> 74
gaaggtcgga gtcaacggat tcaacaaaca aatggagctt taagtaaagt aa 52
<210> 75
<211> 32
<212> DNA
<213>artificial sequence
<400> 75
gccttttgac tcgtatgttt tactttactt ta 32
<210> 76
<211> 43
<212> DNA
<213>artificial sequence
<400> 76
gaaggtgacc aagttcatgc ttgaacagct gagggccatt gca 43
<210> 77
<211> 42
<212> DNA
<213>artificial sequence
<400> 77
gaaggtcgga gtcaacggat tgaacagctg agggccattg cg 42
<210> 78
<211> 27
<212> DNA
<213>artificial sequence
<400> 78
cacaacgtct tcctcttggc tagttat 27
<210> 79
<211> 49
<212> DNA
<213>artificial sequence
<400> 79
gaaggtgacc aagttcatgc tcatatacac acaatacata gctgttgga 49
<210> 80
<211> 48
<212> DNA
<213>artificial sequence
<400> 80
gaaggtcgga gtcaacggat tatatacaca caatacatag ctgttggg 48
<210> 81
<211> 31
<212> DNA
<213>artificial sequence
<400> 81
ctctgccaga aacaaacaat aacttttgta a 31
<210> 82
<211> 54
<212> DNA
<213>artificial sequence
<400> 82
gaaggtgacc aagttcatgc tatgaggaag aaatgagcat atatttattt gttt 54
<210> 83
<211> 54
<212> DNA
<213>artificial sequence
<400> 83
gaaggtcgga gtcaacggat tatgaggaag aaatgagcat atatttattt gtta 54
<210> 84
<211> 30
<212> DNA
<213>artificial sequence
<400> 84
gattaggagg ggtagaagtg attaatgaat 30
<210> 85
<211> 46
<212> DNA
<213>artificial sequence
<400> 85
gaaggtgacc aagttcatgc tgatgtgtgc agatgacgat gataga 46
<210> 86
<211> 45
<212> DNA
<213>artificial sequence
<400> 86
gaaggtcgga gtcaacggat tatgtgtgca gatgacgatg atagg 45
<210> 87
<211> 34
<212> DNA
<213>artificial sequence
<400> 87
acttagtttg gaattattga taatttcctg caat 34
<210> 88
<211> 52
<212> DNA
<213>artificial sequence
<400> 88
gaaggtgacc aagttcatgc tgtttattgg gtttatttaa actaagacat cg 52
<210> 89
<211> 52
<212> DNA
<213>artificial sequence
<400> 89
gaaggtcgga gtcaacggat tgtttattgg gtttatttaa actaagacat cc 52
<210> 90
<211> 31
<212> DNA
<213>artificial sequence
<400> 90
agagaagaag ccaaatatga atggaattct t 31
<210> 91
<211> 54
<212> DNA
<213>artificial sequence
<400> 91
gaaggtgacc aagttcatgc ttctttttct cttttctttt ttctgacttc ttta 54
<210> 92
<211> 53
<212> DNA
<213>artificial sequence
<400> 92
gaaggtcgga gtcaacggat tctttttctc ttttcttttt tctgacttct ttg 53
<210> 93
<211> 30
<212> DNA
<213>artificial sequence
<400> 93
ctgaaacatg cgtacaacta aactaagcta 30
<210> 94
<211> 56
<212> DNA
<213>artificial sequence
<400> 94
gaaggtgacc aagttcatgc ttttgtttcc aatgattaga agtatattta tatatc 56
<210> 95
<211> 56
<212> DNA
<213>artificial sequence
<400> 95
gaaggtcgga gtcaacggat ttttgtttcc aatgattaga agtatattta tatatg 56
<210> 96
<211> 36
<212> DNA
<213>artificial sequence
<400> 96
ctttgttcca attttctaaa ttttgttttt ccatta 36

Claims (10)

1.SNP Sites Combination, 32 SNP sites including Cucumber germplasm；32 SNP sites are as follows: the site CuSNP01 For the 1976800th nucleotide on No. 1 chromosome；The site CuSNP02 is the 8433330th nucleotide on No. 1 chromosome； The site CuSNP03 is the 13502481st nucleotide on No. 1 chromosome；The site CuSNP04 is the on No. 1 chromosome 17508118 nucleotide；The site CuSNP05 is the 25296763rd nucleotide on No. 1 chromosome；The site CuSNP06 is 2 The 1200640th nucleotide on number chromosome；The site CuSNP07 is the 2563528th nucleotide on No. 2 chromosomes； The site CuSNP08 is the 5891488th nucleotide on No. 2 chromosomes；The site CuSNP09 is the on No. 2 chromosomes 6045153 nucleotide；The site CuSNP10 is the 11947823rd nucleotide on No. 2 chromosomes；The site CuSNP11 is No. 3 The 40726th nucleotide on chromosome；The site CuSNP12 is the 1001975th nucleotide on No. 3 chromosomes；CuSNP13 Site is the 10713052nd nucleotide on No. 3 chromosomes；The site CuSNP14 is the 14161884th on No. 3 chromosomes Nucleotide；The site CuSNP15 is the 18678235th nucleotide on No. 3 chromosomes；The site CuSNP16 is on No. 3 chromosomes The 27913661st nucleotide；The site CuSNP17 is the 16975775th nucleotide on No. 4 chromosomes；The site CuSNP18 For the 21068185th nucleotide on No. 4 chromosomes；The site CuSNP19 is the 2133319th nucleosides on No. 5 chromosomes Acid；The site CuSNP20 is the 4054461st nucleotide on No. 5 chromosomes；The site CuSNP21 is the on No. 5 chromosomes 8103814 nucleotide；The site CuSNP22 is the 9582207th nucleotide on No. 5 chromosomes；The site CuSNP23 is No. 5 The 17245080th nucleotide on chromosome；The site CuSNP24 is the 195377th nucleotide on No. 6 chromosomes； The site CuSNP25 is the 2397556th nucleotide on No. 6 chromosomes；The site CuSNP26 is the on No. 6 chromosomes 18079846 nucleotide；The site CuSNP27 is the 20929474th nucleotide on No. 6 chromosomes；The site CuSNP28 is 6 The 24439780th nucleotide on number chromosome；The site CuSNP29 is the 2085399th nucleotide on No. 7 chromosomes； The site CuSNP30 is the 4866176th nucleotide on No. 7 chromosomes；The site CuSNP31 is the on No. 7 chromosomes 6739101 nucleotide；The site CuSNP32 is the 15415870th nucleotide on No. 7 chromosomes.
2.SNP primer combination, including for expanding CuSNP01 described in claim 1 primer sets 1, for expanding claim 1 The primer sets 2 of the CuSNP02, the primer sets 3 for expanding CuSNP03 described in claim 1, for expanding claim 1 The primer sets 4 of the CuSNP04, the primer sets 5 for expanding CuSNP05 described in claim 1, for expanding claim 1 The primer sets 6 of the CuSNP06, the primer sets 7 for expanding CuSNP07 described in claim 1, for expanding claim 1 The primer sets 8 of the CuSNP08, the primer sets 9 for expanding CuSNP09 described in claim 1, for expanding claim 1 The primer sets 10 of the CuSNP010, the primer sets 11 for expanding CuSNP11 described in claim 1 are wanted for expanding right Ask the primer sets 12 of 1 CuSNP12, the primer sets 13 for expanding CuSNP13 described in claim 1, for expanding right It is required that the primer sets 14 of 1 CuSNP14, the primer sets 15 for expanding CuSNP15 described in claim 1, for expand power Benefit requires the primer sets 16 of 1 CuSNP16, the primer sets 17 for expanding CuSNP17 described in claim 1, for expanding The primer sets 18 of CuSNP18 described in claim 1, the primer sets 19 for expanding CuSNP19 described in claim 1, for expanding Increase the primer sets 20 of CuSNP20 described in claim 1, the primer sets 21 for expanding CuSNP21 described in claim 1, be used for It expands the primer sets 22 of CuSNP22 described in claim 1, the primer sets 23 for expanding CuSNP23 described in claim 1, use The primer sets 24 of CuSNP24 described in amplification claim 1, the primer sets 25 for expanding CuSNP25 described in claim 1, Primer sets 26 for expanding CuSNP26 described in claim 1, the primer sets for expanding CuSNP27 described in claim 1 27, the primer sets 28 for expanding CuSNP28 described in claim 1, the primer for expanding CuSNP29 described in claim 1 Organize the 29, primer sets 30 for expanding CuSNP30 described in claim 1, for expanding drawing for CuSNP31 described in claim 1 Object group 31 and primer sets 32 for expanding CuSNP32 described in claim 1.
3. SNP primer combination as claimed in claim 2, it is characterised in that:
Shown in forward primer 01F2 shown in the primer sets 1 forward primer 01F1, sequence 2 as shown in sequence 1 and sequence 3 Reverse primer 01R composition；Forward primer 02F2 shown in the primer sets 2 forward primer 02F1, sequence 5 as shown in sequence 4 It is formed with reverse primer 02R shown in sequence 6；Shown in the primer sets 3 forward primer 03F1, sequence 8 as shown in sequence 7 The composition of reverse primer 03R shown in forward primer 03F2 and sequence 9；The forward primer as shown in sequence 10 of primer sets 4 The composition of reverse primer 04R shown in forward primer 04F2 shown in 04F1, sequence 11 and sequence 12；The primer sets 5 are by sequence The composition of reverse primer 05R shown in forward primer 05F2 shown in forward primer 05F1, sequence 14 shown in 13 and sequence 15；Institute It states anti-shown in forward primer 06F2 shown in primer sets 6 forward primer 06F1, sequence 17 as shown in sequence 16 and sequence 18 It is formed to primer 06R；Forward primer 07F2 shown in the primer sets 7 forward primer 07F1, sequence 20 as shown in sequence 19 It is formed with reverse primer 07R shown in sequence 21；The primer sets 8 forward primer 08F1 as shown in sequence 22,23 institute of sequence The composition of reverse primer 08R shown in the forward primer 08F2 and sequence 24 shown；The forward direction as shown in sequence 25 of primer sets 9 is drawn The composition of reverse primer 09R shown in forward primer 09F2 shown in object 09F1, sequence 26 and sequence 27；The primer sets 10 are by sequence The composition of reverse primer 10R shown in forward primer 10F2 shown in forward primer 10F1, sequence 29 shown in column 28 and sequence 30； Shown in forward primer 11F2 shown in the primer sets 11 forward primer 11F1, sequence 32 as shown in sequence 31 and sequence 33 Reverse primer 11R composition；Forward primer shown in the primer sets 12 forward primer 12F1, sequence 35 as shown in sequence 34 The composition of reverse primer 12R shown in 12F2 and sequence 36；The primer sets 13 forward primer 13F1, sequence as shown in sequence 37 The composition of reverse primer 13R shown in forward primer 13F2 and sequence 39 shown in 38；The primer sets 14 are as shown in sequence 40 The composition of reverse primer 14R shown in forward primer 14F2 shown in forward primer 14F1, sequence 41 and sequence 42；The primer sets Reverse primer shown in forward primer 15F2 shown in 15 forward primer 15F1, sequence 44 as shown in sequence 43 and sequence 45 15R composition；Forward primer 16F2 and sequence shown in the primer sets 16 forward primer 16F1, sequence 47 as shown in sequence 46 The composition of reverse primer 16R shown in 48；Shown in the primer sets 17 forward primer 17F1, sequence 50 as shown in sequence 49 just It is formed to reverse primer 17R shown in primer 17F2 and sequence 51；The forward primer as shown in sequence 52 of primer sets 18 The composition of reverse primer 18R shown in forward primer 18F2 shown in 18F1, sequence 53 and sequence 54；The primer sets 19 are by sequence The composition of reverse primer 19R shown in forward primer 19F2 shown in forward primer 19F1, sequence 56 shown in 55 and sequence 57；Institute It states anti-shown in forward primer 20F2 shown in primer sets 20 forward primer 20F1, sequence 59 as shown in sequence 58 and sequence 60 It is formed to primer 2 0R；Forward primer 21F2 shown in the primer sets 21 forward primer 21F1, sequence 62 as shown in sequence 61 It is formed with reverse primer 21R shown in sequence 63；The primer sets 22 forward primer 22F1 as shown in sequence 64,65 institute of sequence The composition of reverse primer 22R shown in the forward primer 22F2 and sequence 66 shown；The forward direction as shown in sequence 67 of primer sets 23 The composition of reverse primer 23R shown in forward primer 23F2 shown in primer 2 3F1, sequence 68 and sequence 69；The primer sets 24 by Reverse primer 24R group shown in forward primer 24F2 shown in forward primer 24F1, sequence 71 shown in sequence 70 and sequence 72 At；75 institute of forward primer 25F2 shown in the primer sets 25 forward primer 25F1, sequence 74 as shown in sequence 73 and sequence The reverse primer 25R composition shown；Forward direction shown in the primer sets 26 forward primer 26F1, sequence 77 as shown in sequence 76 is drawn The composition of reverse primer 26R shown in object 26F2 and sequence 78；The primer sets 27 forward primer 27F1, sequence as shown in sequence 79 The composition of reverse primer 27R shown in forward primer 27F2 and sequence 81 shown in column 80；The primer sets 28 are as shown in sequence 82 Forward primer 28F1, forward primer 28F2 and sequence 84 shown in sequence 83 shown in reverse primer 28R composition；The primer Organize reverse primer shown in forward primer 29F2 shown in 29 forward primer 29F1, sequence 86 as shown in sequence 85 and sequence 87 29R composition；Forward primer 30F2 and sequence shown in the primer sets 30 forward primer 30F1, sequence 89 as shown in sequence 88 The composition of reverse primer 30R shown in 90；Shown in the primer sets 31 forward primer 31F1, sequence 92 as shown in sequence 91 just It is formed to reverse primer 31R shown in primer 31F2 and sequence 93；The forward primer as shown in sequence 94 of primer sets 32 The composition of reverse primer 32R shown in forward primer 32F2 shown in 32F1, sequence 95 and sequence 96.
4. SNP primer as claimed in claim 2 combination, it is characterised in that: the primer sets 1 are by sequence 1 the from 5 ' ends The forward primer 01F2 and sequence 3 shown in the 22nd to 46 from 5 ' ends of forward primer 01F1, sequence 2 shown in 22 to 46 Shown in reverse primer 01R composition；The primer sets 2 forward primer shown in the 22nd to 46 from 5 ' ends by sequence 4 02F1, sequence 5 the reverse primer 02R shown in forward primer 02F2 and sequence 6 shown in the 22nd to 43 from 5 ' ends are formed； The primer sets 3 by sequence 7 from 5 ' ends forward primer 03F1, sequence 8 shown in the 22nd to 48 the 22nd from 5 ' ends To reverse primer 03R composition shown in forward primer 03F2 shown in 50 and sequence 9；The primer sets 4 are by sequence 10 from 5 ' Forward primer 04F1, sequence 11 forward primer shown in the 22nd to 51 from 5 ' ends shown in end the 22nd to 52 The composition of reverse primer 04R shown in 04F2 and sequence 12；The primer sets 5 are by sequence 13 from 5 ' ends shown in the 22nd to 47 Forward primer 05F1, sequence 14 it is reversed shown in forward primer 05F2 and sequence 15 shown in the 22nd to 47 from 5 ' ends Primer 05R composition；The primer sets 6 forward primer 06F1, sequence 17 shown in the 22nd to 51 from 5 ' ends by sequence 16 Reverse primer 06R shown in forward primer 06F2 and sequence 18 shown in the 22nd to 51 is formed from 5 ' ends；The primer Group 7 by sequence 19 from 5 ' ends forward primer 07F1, sequence 20 shown in the 22nd to 46 the 22nd to 48 from 5 ' ends Shown in reverse primer 07R composition shown in forward primer 07F2 and sequence 21；The primer sets 8 are by sequence 22 from 5 ' ends Forward primer 08F1, sequence 23 shown in 22nd to 48 forward primer 08F2 and sequence shown in the 22nd to 48 from 5 ' ends The composition of reverse primer 08R shown in column 24；The primer sets 9 by sequence 25, draw by the forward direction shown in the 22nd to 48 from 5 ' ends Object 09F1, sequence 26 the reverse primer 09R shown in forward primer 09F2 and sequence 27 shown in the 22nd to 47 from 5 ' ends Composition；Forward primer 10F1, sequence 29 shown in the 22nd to 51 from 5 ' ends are last from 5 ' by sequence 28 for the primer sets 10 The composition of reverse primer 10R shown in forward primer 10F2 and sequence 30 shown in having held the 22nd to 51；The primer sets 11 by Sequence 31 from 5 ' ends forward primer 11F1, sequence 32 shown in the 22nd to 46 from 5 ' ends shown in the 22nd to 45 The composition of reverse primer 11R shown in forward primer 11F2 and sequence 33；The primer sets 12 are by sequence 34 the 22nd from 5 ' ends To forward primer 12F1, sequence 35 forward primer 12F2 and sequence 36 shown in the 22nd to 47 from 5 ' ends shown in 46 Shown in reverse primer 12R composition；The primer sets 13 forward primer shown in the 22nd to 43 from 5 ' ends by sequence 37 13F1, sequence 38 the reverse primer 13R group shown in forward primer 13F2 and sequence 39 shown in the 22nd to 44 from 5 ' ends At；The primer sets 14 by sequence 40 from 5 ' ends forward primer 14F1, sequence 41 shown in the 22nd to 49 from 5 ' ends The composition of reverse primer 14R shown in forward primer 14F2 and sequence 42 shown in playing the 22nd to 49；The primer sets 15 are by sequence Column 43 from 5 ' ends forward primer 15F1, sequence 44 shown in the 22nd to 48 from 5 ' ends shown in the 22nd to 49 just It is formed to reverse primer 15R shown in primer 15F2 and sequence 45；The primer sets 16 by sequence 46 from 5 ' ends the 22nd to 48 institute of forward primer 16F2 shown in the 22nd to 48 and sequence from 5 ' ends of forward primer 16F1, sequence 47 shown in 46 The reverse primer 16R composition shown；The primer sets 17 forward primer shown in the 22nd to 51 from 5 ' ends by sequence 49 17F1, sequence 50 the reverse primer 17R group shown in forward primer 17F2 and sequence 51 shown in the 22nd to 52 from 5 ' ends At；The primer sets 18 by sequence 52 from 5 ' ends forward primer 18F1, sequence 53 shown in the 22nd to 46 from 5 ' ends The composition of reverse primer 18R shown in forward primer 18F2 and sequence 54 shown in playing the 22nd to 44；The primer sets 19 are by sequence Column 55 from 5 ' ends forward primer 19F1, sequence 56 shown in the 22nd to 48 from 5 ' ends shown in the 22nd to 49 just It is formed to reverse primer 19R shown in primer 19F2 and sequence 57；The primer sets 20 by sequence 58 from 5 ' ends the 22nd to 60 institute of forward primer 20F2 shown in the 22nd to 47 and sequence from 5 ' ends of forward primer 20F1, sequence 59 shown in 48 The reverse primer 20R composition shown；The primer sets 21 forward primer shown in the 22nd to 43 from 5 ' ends by sequence 61 21F1, sequence 62 the reverse primer 21R group shown in forward primer 21F2 and sequence 63 shown in the 22nd to 42 from 5 ' ends At；The primer sets 22 by sequence 64 from 5 ' ends forward primer 22F1, sequence 65 shown in the 22nd to 47 from 5 ' ends The composition of reverse primer 22R shown in forward primer 22F2 and sequence 66 shown in playing the 22nd to 46；The primer sets 23 are by sequence Column 67 from 5 ' ends forward primer 23F1, sequence 68 shown in the 22nd to 46 from 5 ' ends shown in the 22nd to 46 just It is formed to reverse primer 23R shown in primer 2 3F2 and sequence 69；The primer sets 24 by sequence 70 from 5 ' ends the 22nd to 72 institute of forward primer 24F2 shown in the 22nd to 44 and sequence from 5 ' ends of forward primer 24F1, sequence 71 shown in 43 The reverse primer 24R composition shown；The primer sets 25 forward primer shown in the 22nd to 51 from 5 ' ends by sequence 73 25F1, sequence 74 the reverse primer 25R group shown in forward primer 25F2 and sequence 75 shown in the 22nd to 51 from 5 ' ends At；The primer sets 26 by sequence 76 from 5 ' ends forward primer 26F1, sequence 77 shown in the 22nd to 43 from 5 ' ends The composition of reverse primer 26R shown in forward primer 26F2 and sequence 78 shown in playing the 22nd to 42；The primer sets 27 are by sequence Column 79 from 5 ' ends forward primer 27F1, sequence 80 shown in the 22nd to 49 from 5 ' ends shown in the 22nd to 48 just It is formed to reverse primer 27R shown in primer 2 7F2 and sequence 81；The primer sets 28 by sequence 82 from 5 ' ends the 22nd to 84 institute of forward primer 28F2 shown in the 22nd to 54 and sequence from 5 ' ends of forward primer 28F1, sequence 83 shown in 54 The reverse primer 28R composition shown；The primer sets 29 forward primer shown in the 22nd to 46 from 5 ' ends by sequence 85 29F1, sequence 86 the reverse primer 29R group shown in forward primer 29F2 and sequence 87 shown in the 22nd to 45 from 5 ' ends At；The primer sets 30 by sequence 88 from 5 ' ends forward primer 30F1, sequence 89 shown in the 22nd to 52 from 5 ' ends The composition of reverse primer 30R shown in forward primer 30F2 and sequence 90 shown in playing the 22nd to 52；The primer sets 31 are by sequence Column 91 from 5 ' ends forward primer 31F1, sequence 92 shown in the 22nd to 54 from 5 ' ends shown in the 22nd to 53 just It is formed to reverse primer 31R shown in primer 31F2 and sequence 93；The primer sets 32 by sequence 94 from 5 ' ends the 22nd to 96 institute of forward primer 32F2 shown in the 22nd to 56 and sequence from 5 ' ends of forward primer 32F1, sequence 95 shown in 56 The reverse primer 32R composition shown.
5. the application of the combination of SNP site described in claim 1 or any SNP primer combination of claim 2 to 4, is x1) extremely Any one of x4):
X1 the kit for identifying cucumber variety) is prepared；
X2 the kit for identifying cucumber variety true or false) is prepared；
X3 cucumber variety) is identified；
X4) identify cucumber variety true or false.
6. a kind of method identified cucumber to be measured and belong to which kind in 241 cucumber varieties, includes the following steps: to detect respectively Cucumber to be measured and 241 genotype of the cucumber variety based on 32 SNP sites described in claim 1, then make the following judgment: If certain kind is based on 32 SNP in genotype and 241 cucumber varieties of the cucumber to be measured based on 32 SNP sites The genotype in site is completely the same, then cucumber to be measured and the cucumber variety belong to the same kind；If cucumber to be measured is based on institute State in the genotype and 241 cucumber varieties of 32 SNP sites genotype of each kind based on 32 SNP sites not Unanimously, then cucumber to be measured and the kind of 241 cucumber varieties are all different.
7. method as claimed in claim 6, it is characterised in that: described " to detect cucumber to be measured and 241 cucumber varieties are based on power Benefit requires the genotype of 1 32 SNP sites " the step of it is as follows:
(1) right is respectively adopted using the genomic DNA of the genomic DNA of cucumber to be measured and 241 cucumber varieties as template respectively It is required that in the 3 SNP primers combinations primer sets carry out PCR amplification, obtain pcr amplification product；
(2) it after completing step (1), using the fluorescence signal of instrument detection pcr amplification product, is obtained according to the color of fluorescence signal The genotype of cucumber to be measured and 241 cucumber varieties based on 32 SNP sites.
8. method as claimed in claim 6, it is characterised in that: described " detection detects cucumber to be measured and 241 cucumber variety bases The step of genotype of 32 SNP sites described in claim 1 ", is as follows:
(1) right is respectively adopted using the genomic DNA of the genomic DNA of cucumber to be measured and 241 cucumber varieties as template respectively It is required that in the 4 SNP primers combinations primer sets carry out PCR amplification, obtain pcr amplification product；
(2) pcr amplification product for taking step (1) to obtain, sequencing；
(3) sequencing result obtained according to step (2), obtains cucumber to be measured and 241 cucumber varieties are based on described 32 SNP The genotype of point.
9. a kind of method identified cucumber to be measured and belong to which kind in 241 cucumber varieties, includes the following steps:
(1) using the genomic DNA of cucumber to be measured as template, primer sets in the combination of SNP primer described in claim 4 are respectively adopted PCR amplification is carried out, pcr amplification product is obtained；Using the genomic DNA of each cucumber variety in standard cucumber variety group as mould Plate is respectively adopted the carry out PCR amplification of primer sets in the combination of SNP primer described in claim 4, obtains pcr amplification product；It is described Standard cucumber variety group is made of 241 cucumber varieties；
(2) each pcr amplification product for obtaining cucumber to be measured pcr amplification product corresponding with each standard cucumber variety carries out Clustering, cucumber to be measured and which standard cucumber variety are same class, the cucumber to be measured and the standard cucumber in clustering Kind belongs to the same kind.
10. the method as described in claim 6 to 9 is any, it is characterised in that: 241 cucumber varieties are emerald green, Ji miscellaneous 16 Number, middle peasant No. 20, Shandong cucumber No. 9, middle peasant No. 29, Diana, in grind fan 8F1, Bertha, in grind 23F1, golden embryo 99-3F1, in grind 17F1, golden embryo 99-2F1, golden embryo 99F1, golden embryo 99-1F1, golden embryo 98F1, golden embryo 98-1F1, in grind 19F1, silver-colored embryo 88F1,128 Half-blood, middle peasant No. 6, middle peasant No. 10, the good elegant, capital of northern agriculture grind 106, surmount cucumber F1, peaceful star all-round spring and autumn F1, Ya Meite, AMATA765, Kingsoft old cucumber, capital grind mini No. 9, capital grind mini No. 3, capital grind mini No. 5, capital grind mini No. 4, green smart 4 Number, beauty's Fruit Cucumber grinds in China, pickling precious, white highest-ranking imperial concubine's Fruit Cucumber, Beijing white jade three, spring beauty, fruit type 101, justice king are ground in capital Mini cucumber, the emerald green treasured F1 in Tangshan, fine work Bai Yesan, Tang Chun 100, cucumber, Austria's light, Man Guan, all can spring and autumn F1, capital grind it is green exquisite No. 2, capital grinds non-irrigated precious No. 5, Xia Mei is ground in excellent bright the best, capital, capital grinds that winning, capital is rich 298, Jinyou No.35, Qiu Mei is ground in capital, the spring is ground in capital Beautiful, green rich ride on Bus No. 11, rich No. seven green, Beijing 403, green show, Beijing 204, it is new grind No. four, saliva grinds No. four, close thorn king cucumber, plucks not Be exposed ground king, the green hat of dragon's fountain king-dragon's fountain, XSBN4, XSBN6, XSBN8, XSBN11, XSBN16, XSBN1, XSBN2, XSBN3, XSBN5, XSBN7, XSBN9, XSBN10, XSBN12, XSBN13, XSBN14, XSBN15, XSBN17, XSBN18, XSBN19, saliva Excellent No. 401, saliva is No. 108 excellent, Vlaspik, Ning Yun No. 3 numbers, middle peasant No. 19, spring jade, spring and autumn king No. 3, P-2-5-1-1, spring and autumn king 2 Number, strong melon, Shen Lv 72, CUCUMBER1404, Shen Lv 64, I1an, capital grind No. 2 mini emerald, 13AC230, jasper, green emerald green, capital Grind mini No. 1, bright beautiful, eastern agriculture 804, Ning Jia 3, excellent plus complete female 09, what drought, teenage girl, Jinyou No.12, saliva spring No. 4, Shanghai it is No. 6 miscellaneous, Precious miscellaneous No. 2, select that four, saliva is No. 2 excellent, the winter is beautiful, Jinyou No.36, MC2065, middle peasant No. 15, Japan northern star, middle peasant No. 26, saliva excellent 38 Number, Jinyou No.31, rich U.S. 11, rich U.S. 74, saliva spring No. three, saliva is No. 3 green, miscellaneous No. nine lucky, Tian Jiao eight, it is rich No. eight large, four Ji Feng, emerald green dragon, spring king 9801, Luyitianshi, boat lose No. 1, Shandong cucumber No. three, Jinyou No.10, rich U.S. 6913, universe moral 117, saliva spring No. 3, saliva is No. 315 excellent, saliva is excellent 308, P02, Wan Nong No. tri- numbers, 9930, prince charming, local king cucumber, De Ruite 723, Hai Nuoyi Number, section, army 18, dark green section at, European No.1, strong fruit nodule 80, capital A008, rich U.S. 8, capital AK42, the rich melon king of U.S., capital The good glossy continuous cropping king of AK18, Dare LD-1, De Ruite F16, De Ruite Y8, ancient cooking vessel, dirty oil be bright 999, the positive A207 of saliva, green mound 8, Moral Rett 727, De Ruite 7876, De Ruite, Dare LD-3, glossy dark green prince, capital grind 118, converges and wins 15, the big Trivhosantnes Kirilouii Manim of cyanine, mini by 3 Number, saliva is prosperous 203, emerald green U.S. Fruit Cucumber, green emerald green precious, large nine, Tian Yi, odd sword 1, green excellent 188, spring happiness F1, strong non-irrigated nodule, Xing Yan No. four, Xing Yan bis-, dirty oil be bright 507, odd sword 2, Xinjin spring No. four, saliva are excellent 409, the sharp nova of grain rains U.S., 83-ms702, capital are ground Mini No. 2, Dai Duoxing, smart No. 2 green, CU003, dimension lucky tail Asia, CU001, stoke 9006, CU002, smart No. 5 green, green essence Spirit 3, the short cucumber 3966 of stingless fruit, 88-ms701, Ma Sha's cucumber pickling type, lucky miscellaneous mini, Bai Jingling 2, monarch excellent 66 are yellow Melon, De Ruite cucumber 136-7, Peking silicite ultrawhite leaf three, special Ji are No. four miscellaneous, No. 7 green exquisite, emerald green hat bright star, Tian Jiao China are ground in capital Southern type, auspicious light two, the green excellent cucumber of brothers, selected leaf three, grain rains beauty, grain rains dry land cucumber, with 52, De Ruite cucumber L14-2, Moral Rett cucumber GZ1601, De Ruite cucumber L14-5, De Ruite cucumber, South Korea cucumber 2-06-97-164, Sheng Feng 908, De Rui The close thorn cucumber of special 15-10, Jin Dongke moisten that 99 close thorn cucumber, middle peasant No. 106, middle peasant No. 27, saliva is No. 4 excellent, saliva is No. 11 excellent, saliva miscellaneous 2 Number, Zhongnong No.16, saliva is excellent 38, PI197088, H9, WI2757, true lemon, 1,364 2014.11 and Z298 2013.11.
CN201811634016.2A 2018-12-29 2018-12-29 Method for identifying authenticity of cucumber variety and special SNP primer combination thereof Active CN109517923B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811634016.2A CN109517923B (en) 2018-12-29 2018-12-29 Method for identifying authenticity of cucumber variety and special SNP primer combination thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811634016.2A CN109517923B (en) 2018-12-29 2018-12-29 Method for identifying authenticity of cucumber variety and special SNP primer combination thereof

Publications (2)

Publication Number Publication Date
CN109517923A true CN109517923A (en) 2019-03-26
CN109517923B CN109517923B (en) 2020-11-24

Family

ID=65798091

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811634016.2A Active CN109517923B (en) 2018-12-29 2018-12-29 Method for identifying authenticity of cucumber variety and special SNP primer combination thereof

Country Status (1)

Country Link
CN (1) CN109517923B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111254215A (en) * 2020-04-07 2020-06-09 北京市农林科学院 Method for identifying purity of cucumber hybrid and SNP primer combination used by same
CN111411165A (en) * 2020-04-20 2020-07-14 北京市农林科学院 SNP (Single nucleotide polymorphism) site primer combination for identifying cucumber germplasm authenticity and application
CN112094938A (en) * 2020-09-29 2020-12-18 北京市农林科学院 Primer combination for identifying authenticity of cabbage variety
CN116751890A (en) * 2023-07-12 2023-09-15 北京市农林科学院 Method for identifying mitochondrial DNA fingerprint of cucumber variety and purity of hybrid and primer combination thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104846062A (en) * 2014-02-17 2015-08-19 天津市农业质量标准与检测技术研究所 Cucumber variety identification method based on SNP markers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104846062A (en) * 2014-02-17 2015-08-19 天津市农业质量标准与检测技术研究所 Cucumber variety identification method based on SNP markers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JIAN ZHANG ET AL.: "A new SNP genotyping technology Target SNP-seq and its application in genetic analysis of cucumber varieties", 《SCIENTIFIC REPORTS》 *
QINGZHEN WEI ET AL.: "An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment (SLAF) sequencing", 《BMC GENOMICS》 *
姚丹青 等: "基于SNP标记的黄瓜遗传多样性分析", 《上海农业学报》 *
姚丹青 等: "应用SNP标记高效鉴定黄瓜杂交种纯度", 《西北农业学报》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111254215A (en) * 2020-04-07 2020-06-09 北京市农林科学院 Method for identifying purity of cucumber hybrid and SNP primer combination used by same
CN111411165A (en) * 2020-04-20 2020-07-14 北京市农林科学院 SNP (Single nucleotide polymorphism) site primer combination for identifying cucumber germplasm authenticity and application
CN111411165B (en) * 2020-04-20 2021-04-27 北京市农林科学院 SNP (Single nucleotide polymorphism) site primer combination for identifying cucumber germplasm authenticity and application
CN112094938A (en) * 2020-09-29 2020-12-18 北京市农林科学院 Primer combination for identifying authenticity of cabbage variety
CN116751890A (en) * 2023-07-12 2023-09-15 北京市农林科学院 Method for identifying mitochondrial DNA fingerprint of cucumber variety and purity of hybrid and primer combination thereof
CN116751890B (en) * 2023-07-12 2024-02-13 北京市农林科学院 Method for identifying mitochondrial DNA fingerprint of cucumber variety and purity of hybrid and primer combination thereof

Also Published As

Publication number Publication date
CN109517923B (en) 2020-11-24

Similar Documents

Publication Publication Date Title
CN109517923A (en) A kind of method for identifying cucumber variety authenticity and its combination of dedicated SNP primer
CN109706261B (en) Method for identifying authenticity of watermelon variety and special SNP primer combination thereof
CN109666756B (en) Method for identifying authenticity of melon variety and special SNP primer combination thereof
CN111979350B (en) Method for identifying authenticity of cucurbita pepo variety
CN105969862A (en) Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers
CN111254215B (en) Method for identifying purity of cucumber hybrid and SNP primer combination used by same
CN106326962A (en) Corn two-dimensional code molecule identity card and construction method therefor
CN112877453B (en) Method for identifying authenticity of pepper variety
CN110195126B (en) SSR core primer group developed based on tartary buckwheat whole genome data and application thereof
CN112094939B (en) Method for identifying authenticity of eggplant variety and primer combination used by method
CN109295179A (en) A kind of method and its dedicated kit for screening different Zn contents and iron content wheat
CN107988413A (en) A kind of method for identifying cucumber variety authenticity and its special SSR primer sets
CN109811075A (en) A kind of method for identifying Chinese cabbage cultivar authenticity and its combination of dedicated SNP primer
CN104031912B (en) Nucleic acid, method for detecting transgenic rice B2A68 and derived line thereof, and kit and application thereof
CN116144819B (en) SNP molecular marker closely linked with main effect QTL of pumpkin pulp carotenoid and application of SNP molecular marker
CN110317898A (en) A kind of method for identifying cucumber variety authenticity and its combination of dedicated SSR primer
CN112094938B (en) Primer combination for identifying authenticity of cabbage variety
CN104946657B (en) Reference gene, its screening technique and the application of the stable expression of white backed planthopper different larval instar
CN108977570B (en) Method for identifying 28 good carrot varieties and special primer set thereof
US10450618B2 (en) Method for identifying variety of hop
Anderson et al. Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues
CN116622883B (en) Specific probe, primer, kit and method for identifying rice H23 transformant
CN116004897B (en) Method for identifying different species in chloranthus
CN117802262B (en) SNP molecular marker closely linked with pumpkin soluble sugar character and application thereof
CN113817861B (en) KASP (KASP-related fragment thereof) mark related to isoorientin content of corn and application of KASP mark

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant