CN112226433A - SNP (Single nucleotide polymorphism) site primer combination for identifying white bark pine germplasm resources and application - Google Patents
SNP (Single nucleotide polymorphism) site primer combination for identifying white bark pine germplasm resources and application Download PDFInfo
- Publication number
- CN112226433A CN112226433A CN202010899756.XA CN202010899756A CN112226433A CN 112226433 A CN112226433 A CN 112226433A CN 202010899756 A CN202010899756 A CN 202010899756A CN 112226433 A CN112226433 A CN 112226433A
- Authority
- CN
- China
- Prior art keywords
- snp
- seq
- site
- primer
- fragment
- 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
Links
- 241001236255 Pinus albicaulis Species 0.000 title claims abstract description 249
- 239000002773 nucleotide Substances 0.000 title claims abstract description 128
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 128
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000010276 construction Methods 0.000 claims abstract description 11
- 239000012634 fragment Substances 0.000 claims description 321
- 238000011144 upstream manufacturing Methods 0.000 claims description 321
- 241001236247 Pinus bungeana Species 0.000 claims description 106
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 235000005205 Pinus Nutrition 0.000 claims description 14
- 241000218602 Pinus <genus> Species 0.000 claims description 14
- 230000003321 amplification Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 238000012163 sequencing technique Methods 0.000 claims description 12
- 230000002860 competitive effect Effects 0.000 claims description 11
- 108700028369 Alleles Proteins 0.000 claims description 7
- 238000007844 allele-specific PCR Methods 0.000 claims description 7
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000007621 cluster analysis Methods 0.000 claims description 4
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 183
- 102000053602 DNA Human genes 0.000 abstract description 180
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 210000000056 organ Anatomy 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 18
- 230000002068 genetic effect Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 241000894007 species Species 0.000 description 8
- 238000004925 denaturation Methods 0.000 description 7
- 230000036425 denaturation Effects 0.000 description 7
- TWQHGBJNKVFWIU-UHFFFAOYSA-N 8-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-8-azaspiro[4.5]decane-7,9-dione Chemical compound C1C(=O)N(CCCCN2CCN(CC2)C=2N=C3C=CC=CC3=CC=2)C(=O)CC21CCCC2 TWQHGBJNKVFWIU-UHFFFAOYSA-N 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 5
- 235000011613 Pinus brutia Nutrition 0.000 description 5
- 241000018646 Pinus brutia Species 0.000 description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 235000018783 Dacrycarpus dacrydioides Nutrition 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 235000011201 Ginkgo Nutrition 0.000 description 1
- 244000194101 Ginkgo biloba Species 0.000 description 1
- 235000008100 Ginkgo biloba Nutrition 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 101000595467 Homo sapiens T-complex protein 1 subunit gamma Proteins 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 240000007320 Pinus strobus Species 0.000 description 1
- 235000008578 Pinus strobus Nutrition 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
- 102100023706 Steroid receptor RNA activator 1 Human genes 0.000 description 1
- 101710187693 Steroid receptor RNA activator 1 Proteins 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000007418 data mining Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000012253 re-sequencing analysis Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000001459 whitebark Nutrition 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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
- C12Q1/6869—Methods for sequencing
-
- 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)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a core SNP (single nucleotide polymorphism) site and primer combination for identifying the authenticity of white bark pine germplasm, a DNA (deoxyribonucleic acid) fingerprint database and database construction method based on the SNP site, and an identification method and application of the authenticity of white bark pine germplasm based on the SNP site. The SNP sites are selected from any 1 to 36 of the first to the third sixteenth SNP sites. The invention establishes a DNA fingerprint database for identifying the authenticity of the white bark pine germplasm based on high-throughput sequencing, can be used for carrying out early identification on the white bark pine germplasm in tissues or organs such as seeds, seedlings, leaves and the like, and provides technical support for white bark pine germplasm resource protection. The method provided by the invention can be used for identifying the germplasm of an unknown white bark pine and also can be used for identifying the authenticity of the germplasm of the known white bark pine. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.
Description
Technical Field
The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a core SNP (single nucleotide polymorphism) site and a primer combination for identifying pinus bungeana germplasm resources, a DNA (deoxyribonucleic acid) fingerprint database and database construction method based on the SNP site, and an identification method and application of the pinus bungeana germplasm resources based on the SNP site.
Background
White bark pine, named Pinus bungeana Zucc.ex Endl by Latin school, also named white bone pine, sanzhen pine, white ginkgo pine, tiger skin pine and coiled pine, is a unique local tree species in China and has a long cultivation history. It belongs to the species of needle-leaved trees in Pinus, and is naturally distributed in Shanxi, Shaanxi, Gansu, Henan, Sichuan, Hubei, etc. The pinus bungeana has high ecological value and economic value. It has strong cold resistance, drought resistance and barrenness resistance, excellent wood quality and beautiful tree shape. Has become an excellent tree species for the greening construction of ecological environment forests and urban gardens in China. Meanwhile, the white bark pine has the functions of resisting ozone, smoke dust and SO2Acting; the pollen rosin has high content and has health promotion effect. The latest research shows that the white bark pine has the strongest capacity of adsorbing PM2.5 in tested tree species, and the capacity of purifying the environment of the white bark pine can enable the white bark pine to become white bark pineThe star tree species for urban garden construction.
Due to the factors of ecological environment damage, artificial interference, difficult updating of the white bark pine and the like, the number of the white bark pine is small, natural groups of the white bark pine gradually degrade to a cliff or a mountain top, and the storage of diversity faces a great challenge. The pinus bungeana grows very slowly in the early stage and is difficult to mutate through long-term evolution, so that the pinus bungeana genetic resource is effectively protected. Most of pinus bungeana are sporadically distributed, the original germplasm resources are reduced, the diversity is relatively poor, and the identification work and protection of most of the germplasm resources are in a blank state at present. Therefore, a DNA rapid detection technology suitable for white bark pine germplasm resources is urgently needed to be developed for white bark pine genetic diversity, population genetic structure, germplasm resource identification and evaluation and the like, and the work of protecting and utilizing white bark pine germplasm resources and breeding improved varieties is promoted.
In recent years, people have studied the diversity of the pinus bungeana population at the molecular level, such as various molecular markers like AFLP, ISSR, SCAR, SRAP, STS and the like. SNP is a third-generation molecular marker, and has been widely regarded as the advantages of large quantity, wide distribution, stable heredity, and the like. With the continuous development of high-throughput sequencing technology and the continuous reduction of sequencing cost, more stable and efficient SNP sites can be mined based on analysis of pinus bungeana variant group information. At present, there is no report on identifying germplasm resources of pinus bungeana by using SNP sites. In scientific research and practice, a white bark pine DNA fingerprint database based on core SNP loci, a method for identifying white bark pine germplasm resources based on core SNP loci and application are required to be constructed.
Disclosure of Invention
The invention provides a core SNP (single nucleotide polymorphism) site and a primer combination for identifying white bark pine germplasm resources, a DNA (deoxyribonucleic acid) fingerprint database and a database construction method based on the SNP site, and an identification method and application of the white bark pine germplasm resources based on the SNP site.
The invention is realized by the following technical scheme:
identifying SNP sites of the white bark pine germplasm resources, wherein the SNP sites are selected from any 1 to 36 of the following first SNP site to the third sixteen SNP site: a first SNP site located in the genome of Pinus bungeana as shown in SEQ ID NO: 109, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence at position 21, wherein the nucleotide base of the site is T or C; a second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 110, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or T; and a third SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 111, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A; a fourth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 112, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or C; a fifth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 113 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G; and a sixth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 114, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or T; a seventh SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 115, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A; an eighth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 116, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A; a ninth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 117 at position 21 of the sequence, or at a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is G or C; a tenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 118, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G; an eleventh SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 119, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A; a twelfth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 120, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is G or T; a thirteenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 121, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or A; a fourteenth SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 122, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is A or T; a fifteenth SNP site, wherein the fifteenth SNP site is located in the white bark pine genome and shown as SEQ ID NO: 123, or the corresponding site on the homologous genome fragment among the germplasm resources of the 21 st site of the sequence, wherein the nucleotide base of the site is G or A; and a sixteenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 124, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or T; a seventeenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 125 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or A; an eighteenth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 126, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is C or G; a nineteenth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 127 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or A; a twentieth SNP site, wherein the second SNP site is located in the white bark pine genome and is shown as SEQ ID NO: 128 or the corresponding site on the homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or A; a twenty-first SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 129, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T; a twenty-second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 130, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or G; a twenty-third SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 131 or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is C or A; a twenty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 132, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T; a twenty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 133, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G; a twenty-sixth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 134 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T; a twenty-seventh SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 135, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or C; a twenty-eighth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 136, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G; a twenty-ninth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 137, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is T or G; and the thirtieth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 138, or a corresponding site on a homologous genomic fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G; a thirty-first SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 139 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T; and a thirty-second SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 140, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A; and a thirty-third SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 141 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or G; and a thirty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 142, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is T or C; and a thirty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 143 or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is a or G; and the thirty-sixth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 144, or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is T or G.
In some embodiments, the SNP site: the sequences of the first SNP locus and bases at the upstream and downstream are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 112 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 114 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a); the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%; the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity; the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%; the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 126 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%; the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 129 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of SEQ ID NO: 129, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 130 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 130, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 131 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 131, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 132 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO 132: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a); the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 133 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 133 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 134 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 134 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical; the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 135 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 135 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 136 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 136 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 137 or a genome fragment homologous to the germplasm resources thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 137, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 138 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 138, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 139 or a genome fragment homologous to a germplasm resource thereof, more preferably to the sequence set forth in SEQ ID NO: 139, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 140 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 140 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the thirteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 141 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 141, greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the thirty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 142 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 142 is greater than or equal to 95%, 96%, 97%, 98%, or 99%; the sequence of the fifteenth SNP locus and the bases at the upstream and downstream of the fifteenth SNP locus is SEQ ID NO: 143 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 143 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical; the sequences of the sixteenth SNP locus and bases on the sixteenth SNP locus are SEQ ID NO: 144 or a germplasm resource-homologous genomic fragment thereof, more preferably a fragment identical to the sequence set forth in SEQ ID NO: 144 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
The SNP primer group for identifying the white bark pine germplasm resources respectively amplifies the SNP loci comprises the following steps: a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a second eighteen SNP primer set for amplifying the second eighteen SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a third twelve SNP primer set for amplifying the third twelve SNP sites; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a thirty-fourth SNP primer set for amplifying the thirty-fourth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a thirty-sixth SNP primer set for amplifying the thirty-sixth SNP site.
In some embodiments, the SNP primer set: in the first SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the second SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the third SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the fourth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the fifth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the seventh SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the eighth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the ninth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the tenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the eleventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the twelfth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the fourteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the fifteenth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the seventeenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the nineteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the twentieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; in the twenty-first SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the second twelve SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the twenty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the twenty-fifth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the twenty-sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; in the twenty-seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the twenty-ninth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the thirtieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the thirty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the sequence shown in SEQ ID NO: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the third twelve SNP primer groups, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 97. SEQ ID NO: 98. SEQ ID NO: 99, and preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; in the thirty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 100. SEQ ID NO: 101. SEQ ID NO: 102 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the fifteenth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 103. SEQ ID NO: 104. SEQ ID NO: 105 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 106. SEQ ID NO: 107. SEQ ID NO: 108 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM, HEX.
An SNP kit for identifying white bark pine germplasm resources is characterized in that: the SNP kit is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises: the SNP primer sets preferably comprise a first upstream primer, a second upstream primer and a downstream primer of each primer set, wherein the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer in the SNP primer sets in the system is 2:2: 5.
A white bark pine germplasm resource DNA fingerprint database based on SNP markers is characterized in that: the DNA fingerprint database includes: the genotype of the SNP locus of the standard white bark pine germplasm resource.
In some embodiments, the standard white bark pine germplasm resources are selected from the following 137 white bark pine germplasm resources: jing series No. 1, Jing series No. 11, Jing series No. 12, Jing series No. 13, Jing series No. 14, Jing series No. 15, Jing series No. 2, Jing series No. 3, Jing series No. 4, Jing series No. 5, Jing series No. 6, Jing series No. 7, Jing series No. 8, Jing series No. 9, Xian, Ruiwang, Jiuquan, Ganjin No. 1, Ganjin No. 10, Ganjin No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 5, Ganjin No. 6, Ganjin No. 7, Ganjin No. 8, Ganjin No. 9, Ganjin No. 1, Ganjin No. 10, Ganjin No. two No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 6, Ganjin No. 7, Ga, jin Yan No. 15, jin Yan No. 2, jin Yan No. 3, jin Yan No. 4, jin Yan No. 5, jin Yan No. 6, jin Yan No. 8, jin Yan No. 9, gan Tian No. 1, gan Tian No. 10, gan Tian No. 11, gan Tian No. 12, gan Tian No. 13, gan Tian No. 14, gan Tian No. 15, gan Tian No. 2, gan Tian No. 3, gan Tian No. 4, gan Tian No. 5, gan Tian No. 6, gan Tian No. 7, gan Tian No. 8, gan Tian No. 9, jin Xixiao No. 13, jin Xiao No. 14, jin Xiao No. 15, Shaanluo No. 1, Shaanluo No. 10, Shaanluo No. 11, Shaanluo No. 12, Shaanluo No. 13, Shaanluo No. 14, Shaanluo No. 15, Shaanluo No. 2, Shaanluo No. 3, Shaanluo No. 4, Shaanluo No. 5, Shaanluo blue No. 6, Shaanluo No. 7, Shaanluo blue No. 1, Shaanluo blue No. 11, Shaanluo blue No. 12, shandan No. 6, Shaanlan No. 8, Shaanlan No. 9, Shaanlong No. 1, Shaanlong No. 10, Shaanlong No. 11, Shaanlong No. 12, Shaanlong No. 13, Shaanlong No. 14, Shaanlong No. 15, Shaanlong No. 2, Shaanlong No. 3, Shaanlong No. 4, Shaanlong No. 5, Shaanlong No. 6, Shaanlong No. 7, Shaanlong No. 8, Shaanlong No. 9, Zheng No. 1, Zheng No. 10, Zheng No. 11, Zheng No. 12, Zheng No. 13, Zheng No. 14, Zheng No. 15, Zheng No. 2, Zheng No. 4, Zheng No. 5, Zheng No. 6, Zheng No. 7, Zheng No. 8, and Zheng No. 9.
The construction method of the DNA fingerprint database comprises the following steps: and (3) PCR reaction steps: performing competitive allele specific PCR amplification reaction on the standard pinus bungeana germplasm resource by adopting the PCR reaction system to obtain a PCR reaction product; SNP locus genotype obtaining step: detecting the PCR reaction product to obtain the genotype of the SNP locus; preferably, the detection is fluorescence signal detection or direct sequencing.
A detection method for identifying white bark pine germplasm resources comprises the following steps: the method comprises the following steps: detecting the genotype of the SNP locus of the pinus bungeana to be detected; step two: judging the germplasm resources of the pinus bungeana to be detected: if the number of the genotype of the pinus albus to be detected based on the 36 SNP sites and the number of the difference sites of a certain specified germplasm resource in the standard pinus albus germplasm resources in the database based on the genotype of the 36 SNP sites are 0-2, judging the pinus albus to be detected as a similar germplasm resource with the specified germplasm resource; if the number of the genotype of the pinus bungeana to be detected based on the 36 SNP sites and the number of the different sites of a certain specified germplasm resource in the standard pinus bungeana germplasm resources in the database based on the genotype of the 36 SNP sites are more than 2, judging the pinus bungeana to be detected and the specified germplasm resource as different pinus bungeana germplasm resources; preferably, the result of the determination is obtained from a cluster analysis.
The SNP locus, or the SNP primer combination, or the SNP kit, or the DNA fingerprint database obtained by the construction method, or the detection method is applied to the following X1 or X2: x1: identifying whether the germplasm resource of the white bark pine to be detected belongs to one of the standard white bark pine germplasm resources; x2: and identifying the germplasm resources of the white bark pine to be detected as the specific standard white bark pine germplasm resources.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention establishes a DNA fingerprint database for identifying the white bark pine germplasm resources based on high-throughput sequencing, can be used for carrying out early identification on the white bark pine germplasm resources in tissues or organs such as seeds, seedlings, leaves and the like, practically protects the rights and interests of breeders, and provides technical support for white bark pine germplasm resource protection.
2. The method provided by the invention can be used for identifying the germplasm resources of unknown white bark pine and also can be used for identifying the germplasm resources of known white bark pine. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.
3. The invention carries out big data mining on SNP of the white bark pine germplasm resources based on SLAF sequencing data of 137 white bark pine germplasm resources, provides candidate sites for the identification of the white bark pine germplasm resources, develops specific primers of the white bark pine germplasm resources by adopting an allele competitive specific PCR method, carries out high-throughput, low-cost and automatic rapid detection, and finally obtains the SNP genotype of the white bark pine germplasm resources.
Drawings
FIG. 1: the identification of 150 germplasm resources for the 36 SNPs and the random 36 SNPs in the invention in example 1 was performed. Wherein, the curve of the circular mark is the identification capability of identifying 150 white bark pine germplasm resources by 36 SNPs in the application; the curve marked by triangles is the identification capability of identifying 150 white bark pine germplasm resources by 36 randomly selected SNPs.
FIG. 2: is a schematic diagram of SNP typing results of a 96-well sample plate for detecting 137 test pinus bungeana germplasm resource genotypes by 36 SNP loci in example 2.
FIG. 3: a cluster map of 137 test pinus bungeana germplasm resources built on 36 SNP primer sets in example 2 was generated.
FIG. 4: a graph of the identification capability of identifying 137 test pinus bungeana germplasm resources for the 36 SNP sites in example 2.
Detailed Description
The definition of the invention is as follows:
identifying white bark pine germplasm resources: essentially, the method refers to the correspondence between white bark pine germplasm resources and genetic backgrounds thereof; in actual work, whether a certain germplasm resource for testing is similar to a germplasm resource of white bark pine in a white bark pine germplasm resource database refers to whether the germplasm resource for testing conforms to a file record (such as a germplasm resource specification, a label and the like).
Germplasm resource homologous genome fragment: refers to non-repetitive (single copy) homologous genomic fragments of the same segment of the same chromosome from different germplasm sources of a species, such as white bark pine, including but not limited to the 137 standard germplasm sources described herein. Non-duplicative means that the genome segment exists only at one genome position in one germplasm resource, can be homozygous or heterozygous for polyploidy, and the genome segment with high homology does not exist at other positions of the genome of one plant resource, but the genome segment with high homology generally exists in different germplasm resources, such as SEQ ID NO: 109 shows one of the homologous genomic fragments between pinus bungeana germplasm resources.
Corresponding sites on the homologous genomic fragments between germplasm resources: refers to a species of germplasm resources (such as, but not limited to, one of 137 standard germplasm resources, such as white pine), as a reference germplasm resource, which has a specific polymorphic genome segment, in which the base at a specific position (such as, for example, position 21 of the sequence shown in SEQ ID NO: 109) is highly polymorphic between different germplasm resources (such as, for example, position 21 of the sequence shown in SEQ ID NO: 109 is mainly T or C, and possibly other bases), and a sequence upstream and downstream thereof (such as, for example, positions 1-20 and 22-41 of the sequence shown in SEQ ID NO: 109) is highly conserved between different germplasm resources (this region may have small mutations, which are random and not diffused into the population, which has NO prevalence within one germplasm resource, ignoring the mutation and looking at the SNP, the SNP has a prevalence within the germplasm resources), the highly polymorphic base in the highly conserved genomic fragment is the corresponding site on the germplasm resources homologous genomic fragment among the germplasm resources homologous genomic fragments of the species.
The Sequencing of the 150 white bark pine germplasm resources is completed based on SLAF Sequencing (Specific-local Amplified Fragment Sequencing) in the early stage. Since the pinus bungeana genome is highly heterozygous and the genome is large, the scaffold level genome has only been published in recent years. The 150 germplasm resource re-sequencing analysis is based on a non-reference genome method, obtains SNP sites with high polymorphism, and is further verified by a KASPar platform. Due to the incompleteness of the reference genome, 5 out of 36 SNPs could be assigned to the scaffold of the genome. The invention constructs a white bark pine DNA fingerprint database based on the core SNP locus by utilizing the 36 SNPs, and a method for identifying white bark pine germplasm resources based on the core SNP locus and application thereof.
In a first aspect, the present invention provides a method for identifying a core SNP site (core SNP site is defined as a group of minimal SNP combinations capable of identifying a target germplasm resource and representing a genome-wide SNP as much as possible; the core site is distinguished from non-core sites by higher polymorphism of the core site and using minimal markers to distinguish the target germplasm resource), which is selected from any 1 to 36 of the following first to third sixteen SNP sites, as shown in Table 1:
a first SNP site located in the genome of Pinus bungeana as shown in SEQ ID NO: 109, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence at position 21, wherein the nucleotide base of the site is T or C;
a second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 110, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or T;
and a third SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 111, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a fourth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 112, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or C;
a fifth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 113 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
and a sixth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 114, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or T;
a seventh SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 115, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
an eighth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 116, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
a ninth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 117 at position 21 of the sequence, or at a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is G or C;
a tenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 118, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
an eleventh SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 119, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a twelfth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 120, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is G or T;
a thirteenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 121, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a fourteenth SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 122, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is A or T;
a fifteenth SNP site, wherein the fifteenth SNP site is located in the white bark pine genome and shown as SEQ ID NO: 123, or the corresponding site on the homologous genome fragment among the germplasm resources of the 21 st site of the sequence, wherein the nucleotide base of the site is G or A;
and a sixteenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 124, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or T;
a seventeenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 125 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or A;
an eighteenth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 126, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is C or G;
a nineteenth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 127 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or A;
a twentieth SNP site, wherein the second SNP site is located in the white bark pine genome and is shown as SEQ ID NO: 128 or the corresponding site on the homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or A;
a twenty-first SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 129, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T;
a twenty-second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 130, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or G;
a twenty-third SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 131 or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is C or A;
a twenty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 132, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T;
a twenty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 133, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a twenty-sixth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 134 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T;
a twenty-seventh SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 135, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or C;
a twenty-eighth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 136, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a twenty-ninth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 137, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is T or G;
and the thirtieth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 138, or a corresponding site on a homologous genomic fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a thirty-first SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 139 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T;
and a thirty-second SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 140, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
and a thirty-third SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 141 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or G;
and a thirty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 142, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is T or C;
and a thirty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 143 or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is a or G;
and the thirty-sixth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 144, or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is T or G.
The sequences of the first SNP locus and bases at the upstream and downstream are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 112 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 114 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a);
the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%;
the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%;
the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 126 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%;
the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 129 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of SEQ ID NO: 129, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 130 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 130, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 131 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 131, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 132 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO 132: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a);
the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 133 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 133 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 134 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 134 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 135 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 135 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 136 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 136 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 137 or a genome fragment homologous to the germplasm resources thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 137, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 138 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 138, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 139 or a genome fragment homologous to a germplasm resource thereof, more preferably to the sequence set forth in SEQ ID NO: 139, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 140 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 140 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 141 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 141, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 142 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 142 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the fifteenth SNP locus and the bases at the upstream and downstream of the fifteenth SNP locus is SEQ ID NO: 143 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 143 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the sixteenth SNP locus and bases on the sixteenth SNP locus are SEQ ID NO: 144 or a germplasm resource-homologous genomic fragment thereof, more preferably a fragment identical to the sequence set forth in SEQ ID NO: 144 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
In a second aspect, the present invention provides a combination of SNP primer sets for identifying pinus bungeana germplasm resources, the SNP primer sets comprising:
a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a second eighteen SNP primer set for amplifying the second eighteen SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a third twelve SNP primer set for amplifying the third twelve SNP sites; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a thirty-fourth SNP primer set for amplifying the thirty-fourth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a thirty-sixth SNP primer set for amplifying the thirty-sixth SNP site.
In some embodiments, the first SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) of the first SNP primer set, the downstream primer (R) of the first SNP primer set, are identical to SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the second SNP primer set, which includes a specific portion of the first upstream primer (F1) of the second SNP primer set, a specific portion of the second upstream primer (F2) of the second SNP primer set, and a downstream primer (R) of the second SNP primer set, are identical to SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the third SNP primer set, which includes a specific portion of the first upstream primer (F1) of the third SNP primer set, a specific portion of the second upstream primer (F2) of the third SNP primer set, and a downstream primer (R) of the third SNP primer set, are identical to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the fourth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fourth SNP primer set, are linked to SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the fifth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fifth SNP primer set, are linked to SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the sixth SNP primer set, including the specific portion of the first upstream primer (F1) of the sixth SNP primer set, the specific portion of the second upstream primer (F2) of the sixth SNP primer set, and the downstream primer (R) of the sixth SNP primer set, are linked to SEQ ID NOs: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the seventh SNP primer set, including the specific portion of the first upstream primer (F1) of the seventh SNP primer set, the specific portion of the second upstream primer (F2) of the seventh SNP primer set, and the downstream primer (R) of the seventh SNP primer set, are linked to SEQ ID NOs: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the eighth SNP primer set, including the specific portion of the first upstream primer (F1) of the eighth SNP primer set, the specific portion of the second upstream primer (F2) of the eighth SNP primer set, and the downstream primer (R) of the eighth SNP primer set, are linked to SEQ ID NOs: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the ninth SNP primer set, including the specific portion of the first upstream primer (F1) of the ninth SNP primer set, the specific portion of the second upstream primer (F2) of the ninth SNP primer set, and the downstream primer (R) of the ninth SNP primer set, are linked to SEQ ID NOs: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the tenth SNP primer set, including the specific portion of the first upstream primer (F1) of the tenth SNP primer set, the specific portion of the second upstream primer (F2) of the tenth SNP primer set, and the downstream primer (R) of the tenth SNP primer set, are linked to SEQ ID NOs: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the eleventh SNP primer set, including the specific portion of the first upstream primer (F1) of the eleventh SNP primer set, the specific portion of the second upstream primer (F2) of the eleventh SNP primer set, and the downstream primer (R) of the eleventh SNP primer set, are linked to SEQ ID NOs: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the twelfth SNP primer set, which includes a specific portion of the first upstream primer (F1) of the twelfth SNP primer set, a specific portion of the second upstream primer (F2) of the twelfth SNP primer set, and a downstream primer (R) of the twelfth SNP primer set, is identical to the sequence set shown in SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the thirteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the thirteenth SNP primer set, the specific portion of the second upstream primer (F2) of the thirteenth SNP primer set, the downstream primer (R) of the thirteenth SNP primer set, is identical to SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the fourteenth SNP primer set, which includes a specific portion of the first upstream primer (F1) of the fourteenth SNP primer set, a specific portion of the second upstream primer (F2) of the fourteenth SNP primer set, and a downstream primer (R) of the fourteenth SNP primer set, is identical to SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the fifteenth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fifteenth SNP primer set, are linked to SEQ ID NOs: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the sixteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the sixteenth SNP primer set, the specific portion of the second upstream primer (F2) of the sixteenth SNP primer set, and the downstream primer (R) of the sixteenth SNP primer set, are linked to SEQ ID NOs: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the seventeenth SNP primer set, including the specific portion of the first upstream primer (F1) of the seventeenth SNP primer set, the specific portion of the second upstream primer (F2) of the seventeenth SNP primer set, and the downstream primer (R) of the seventeenth SNP primer set, are linked to SEQ ID NOs: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the eighteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the eighteenth SNP primer set, the specific portion of the second upstream primer (F2) of the eighteenth SNP primer set, and the downstream primer (R) of the eighteenth SNP primer set, are linked to SEQ ID NOs: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the nineteenth SNP primer set, including the specific portion of the first upstream primer (F1) of the nineteenth SNP primer set, the specific portion of the second upstream primer (F2) of the nineteenth SNP primer set, and the downstream primer (R) of the nineteenth SNP primer set, are identical to SEQ ID NOs: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the twentieth SNP primer set, including the specific portion of the first upstream primer (F1) of the twentieth SNP primer set, the specific portion of the second upstream primer (F2) of the twentieth SNP primer set, and the downstream primer (R) of the twentieth SNP primer set, are linked to SEQ ID NOs: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity;
the twenty-first SNP primer set including a specific portion of the first upstream primer (F1) of the twenty-first SNP primer set, a specific portion of the second upstream primer (F2) of the twenty-first SNP primer set, a downstream primer (R) of the twenty-first SNP primer set, and a sequence listing corresponding to SEQ ID NOs: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the second twelve SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) of the second twelve SNP primer set, the downstream primer (R) of the second twelve SNP primer set, and the sequence numbers of SEQ ID NOs: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the thirteenth SNP primer set including a specific portion of the first upstream primer (F1) of the thirteenth SNP primer set, a specific portion of the second upstream primer (F2) of the thirteenth SNP primer set, and a downstream primer (R) of the thirteenth SNP primer set, are identical to SEQ ID NOs: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the twenty-fourth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-fourth SNP primer set, are identical to SEQ ID NOs: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the twenty-fifth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-fifth SNP primer set, are identical to SEQ ID NOs: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the twenty-sixth SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the twenty-sixth SNP primer set, respectively, have the same sequence as SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
the twenty-seventh SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the twenty-seventh SNP primer set, are identical to SEQ ID NOs: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the second eighteen SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), the downstream primer (R), of the second eighteen SNP primer set, is identical to SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the twenty-ninth SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the twenty-ninth SNP primer set, respectively, have the same sequence as SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the thirtieth SNP primer set comprises a specific part of the first upstream primer (F1) of the thirtieth SNP primer set, a specific part of the second upstream primer (F2) of the thirtieth SNP primer set and a downstream primer (R) of the thirtieth SNP primer set, which are respectively connected with the sequence numbers of SEQ ID NOs: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the thirty-first SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the thirty-first SNP primer set, are identical to SEQ ID NOs: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the third twelve SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) and the downstream primer (R) of the third twelve SNP primer set, respectively, are identical to SEQ ID NOs: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the thirteenth SNP primer set including a specific portion of the first upstream primer (F1), a specific portion of the second upstream primer (F2), and a downstream primer (R) of the thirteenth SNP primer set, respectively, overlap with SEQ ID NOs: 97. SEQ ID NO: 98. SEQ ID NO: 99, and preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity;
the thirty-fourth SNP primer set, which comprises a specific part of the first upstream primer (F1) of the thirty-fourth SNP primer set, a specific part of the second upstream primer (F2) of the thirty-fourth SNP primer set, and a downstream primer (R) of the thirty-fourth SNP primer set, is respectively identical to the sequence of SEQ ID NO: 100. SEQ ID NO: 101. SEQ ID NO: 102 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the fifteenth SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R) of the fifteenth SNP primer set, respectively, are identical to SEQ ID NOs: 103. SEQ ID NO: 104. SEQ ID NO: 105 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
the third sixteen SNP primer set including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R) of the third sixteen SNP primer set, respectively, have the same sequence as SEQ ID NOs: 106. SEQ ID NO: 107. SEQ ID NO: 108 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%.
In a preferred embodiment, the SNP primer combination is selected from one or more of the primer sets 01 to 36; the DNA sequence information of the primer groups 01-36 is shown in a sequence table SEQ ID: 108, see table 2.
In the primer group, the 5' end of the upstream primer can be provided with a fluorescent tag sequence for fluorescent PCR detection, the first upstream primer and the second upstream primer in each group of primers are connected with different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM and HEX, and further preferably, the first upstream primer in each group of primers is connected with FAM, and the second upstream primer is connected with HEX; for example, the fluorescence signal of FAM fluorescent tag sequence is blue, and the fluorescence signal of HEX fluorescent tag sequence is red.
In a third aspect, the present invention provides a core SNP kit for identifying pinus bungeana germplasm resources, the kit being formulated as a competitive allele-specific PCR reaction system, the system preferably comprising: in the SNP primer sets, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer set in the system is 2:2: 5;
reagents, consumables and instruments in the reaction system are all provided by LGC company, including reagent dosage, usage and whole experimental steps are carried out according to the operation guide KASP user guide and manual (www.lgcgenomics.com) of LGC company, the KASPar reaction is carried out in 384-well plates (Part No. KBS-0750-001) or 96-well plates (Part No. KBS-0751-001), and the reaction system is 3ul or 1ul, as shown in the following table.
Table: KASP reaction system of 384-well plate or 96-well plate
Kits supplied by LGC company or otherwise having AS-PCR detection capability
The preparation method of the kit comprises the step of packaging each primer in any one primer group separately.
In a fourth aspect, the present invention provides the above DNA fingerprint database for white bark pine germplasm resources based on core SNP markers, where the DNA fingerprint database includes: the standard white bark pine germplasm resources have genotypes at the 36 SNP sites.
The standard white bark pine germplasm resources are selected from the following 137 white bark pine germplasm resources:
jing series No. 1, Jing series No. 11, Jing series No. 12, Jing series No. 13, Jing series No. 14, Jing series No. 15, Jing series No. 2, Jing series No. 3, Jing series No. 4, Jing series No. 5, Jing series No. 6, Jing series No. 7, Jing series No. 8, Jing series No. 9, Xian, Ruiwang, Jiuquan, Ganjin No. 1, Ganjin No. 10, Ganjin No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 5, Ganjin No. 6, Ganjin No. 7, Ganjin No. 8, Ganjin No. 9, Ganjin No. 1, Ganjin No. 10, Ganjin No. two No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 6, Ganjin No. 7, Ga, jin Yan No. 15, jin Yan No. 2, jin Yan No. 3, jin Yan No. 4, jin Yan No. 5, jin Yan No. 6, jin Yan No. 8, jin Yan No. 9, gan Tian No. 1, gan Tian No. 10, gan Tian No. 11, gan Tian No. 12, gan Tian No. 13, gan Tian No. 14, gan Tian No. 15, gan Tian No. 2, gan Tian No. 3, gan Tian No. 4, gan Tian No. 5, gan Tian No. 6, gan Tian No. 7, gan Tian No. 8, gan Tian No. 9, jin Xixiao No. 13, jin Xiao No. 14, jin Xiao No. 15, Shaanluo No. 1, Shaanluo No. 10, Shaanluo No. 11, Shaanluo No. 12, Shaanluo No. 13, Shaanluo No. 14, Shaanluo No. 15, Shaanluo No. 2, Shaanluo No. 3, Shaanluo No. 4, Shaanluo No. 5, Shaanluo blue No. 6, Shaanluo No. 7, Shaanluo blue No. 1, Shaanluo blue No. 11, Shaanluo blue No. 12, shandan No. 6, Shaanlan No. 8, Shaanlan No. 9, Shaanlong No. 1, Shaanlong No. 10, Shaanlong No. 11, Shaanlong No. 12, Shaanlong No. 13, Shaanlong No. 14, Shaanlong No. 15, Shaanlong No. 2, Shaanlong No. 3, Shaanlong No. 4, Shaanlong No. 5, Shaanlong No. 6, Shaanlong No. 7, Shaanlong No. 8, Shaanlong No. 9, Zheng No. 1, Zheng No. 10, Zheng No. 11, Zheng No. 12, Zheng No. 13, Zheng No. 14, Zheng No. 15, Zheng No. 2, Zheng No. 4, Zheng No. 5, Zheng No. 6, Zheng No. 7, Zheng No. 8, and Zheng No. 9.
In a fifth aspect, the invention provides a method for constructing the white bark pine germplasm resource DNA fingerprint database, which comprises the following steps:
s1: KASP reaction: carrying out competitive allele specific PCR amplification reaction on the standard pinus bungeana germplasm resource by adopting the reaction system to obtain a PCR reaction product;
the PCR reaction program is: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min, and continuous amplification for 26 cycles; final extension: 10min at 72 ℃. The resulting amplification product was stored at 4 ℃ before electrophoresis.
S2: SNP locus genotype acquisition: and detecting the PCR reaction product to obtain the genotype of the SNP locus.
The above detection method may be selected from: for fluorescent signal detection, direct sequencing and restriction enzyme digestion are carried out.
In a sixth aspect, the invention provides a resource detection method for identifying pinus bungeana germplasm resources, which comprises the following steps:
s1, detecting the genotype of the SNP locus of the pinus bungeana to be detected:
respectively taking the genomic DNA of pinus bungeana to be detected as a template, and respectively adopting the primer groups in the SNP primer combination to carry out competitive allele specific PCR amplification reaction to obtain PCR amplification products;
s2, judging the germplasm resources of the white bark pine to be detected: comparing the genotype of the SNP locus of the pinus bungeana to be detected with the DNA fingerprint database, obtaining and judging the result through cluster analysis, wherein the judgment standard is as follows:
if the number of the malposition points of the white bark pine germplasm resource to be detected and a standard white bark pine germplasm resource (to-be-detected white bark pine germplasm resource) is more than 2, the white bark pine germplasm resource to be detected and the standard white bark pine germplasm resource are judged to be different white bark pine germplasm resources; the greater the number of differential sites, the more distant the genetic relationship.
And if the number of the difference sites between the white bark pine germplasm resource to be detected and a standard white bark pine germplasm resource (the white bark pine germplasm resource to be detected) is 0-2, judging the white bark pine germplasm resource to be detected and the standard white bark pine germplasm resource as similar white bark pine germplasm resources.
In a seventh aspect, the invention provides the above SNP sites, SNP primer combinations, SNP kits, DNA fingerprint database detection methods, for use in X1 or X2:
x1: identifying whether the germplasm resource of the white bark pine to be detected belongs to one of the standard white bark pine germplasm resources;
x2: identifying the specific germplasm resource of the white bark pine to be detected as any one of the standard white bark pine germplasm resources;
the X1 and X2 belong to the application of identifying white bark pine germplasm resources, wherein the identification of the authenticity of the white bark pine germplasm resources is included.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.
Example 1
Acquisition of locus and primer combination for identifying white bark pine germplasm resources
Discovery of one, 36 core SNP sites
The invention adopts an Illumina sequencer, and based on SLAF Sequencing (Specific-local Amplified Fragment Sequencing) data of 150 white bark pine germplasm resources, 36 SNP sites are finally obtained. The 150 white bark pine germplasm resources come from different provinces in China, have high genetic diversity and extremely have genetic representativeness.
The SNP site screening criteria are as follows: (1) and (3) SLAF sequencing, wherein 20,911SNP is obtained based on a non-reference genome method, the SNP sites with MAF <0.05, deletion rate >0.3 and heterozygosity rate greater than 0.3 are removed, and 7,795SNP is obtained. (2) Extracting flanking sequences, removing 20bp of SNP positioned in the head and the tail of two reads, and intercepting 20-40bp of upstream and downstream according to actual conditions to obtain 3,843 SNP. (3) Designing a Kaspar platform primer, and designing 1,629SNP into the Kaspar primer. (4) In order to screen a representative group of SNPs, the correlation coefficient of the genetic distance calculated according to the candidate SNP locus and the genetic distance calculated according to the 7,795SNP, namely the Pearson, is more than 0.9; meanwhile, at least 1SNP genotype is different between any two germplasm resources. And (3) carrying out data processing by using java programming, and finally screening 36 SNP sites for identifying the white bark pine germplasm resources.
According to the invention, by utilizing blast software, 20bp sequences flanking the 36 SNPs are aligned to a reference genome at a scaffold level, and 5 SNPs in the 36 SNPs can be determined to the scaffold level. This is because the pinus bungeana genome is large and complex, and the assembled genome is incomplete. The 36 SNPs are obtained by double verification of SLAF sequencing and KASPar platform, and the determination is real and reliable. The basic information of the 36 SNP sites is detailed in Table 1.
TABLE 1.36 basic information of SNP sites
Evaluation of resource efficiency of 150 white bark pine germplasm resources by two or 36 SNPs
FIG. 1 shows that the circular marked curve is the identification ability of the 36 SNPs in the invention to identify 150 pinus bungeana germplasm resources; the curve marked by triangles is the identification capability of identifying 150 white bark pine germplasm resources by 36 randomly selected SNPs (selected from the 20,911 SNPs). By taking at least 2 SNP sites as a threshold value for sample identification, the 16 SNPs in the invention can identify 150 white bark pine germplasm resources by 100 percent, but all 36 SNPs in the invention can identify other germplasm resources for more; only 97.3% of the SNPs could be identified by a random 36 SNPs. Therefore, the identification capability of the 36 SNPs in the invention on 150 white bark pine germplasm resources is better.
Thirdly, obtaining of primer combination for identifying pinus bungeana germplasm resources
According to the 36 SNP loci discovered above, a primer combination which can represent genome-wide SNP and is suitable for identifying white bark pine germplasm resources by an allele competitive specific PCR (KASP) method is developed.
The SNP primer set consists of 48 primer sets, and the name of each primer set is shown in the table (Table 2). Each primer group consists of 3 primer sequences, comprises a first upstream primer, a second upstream primer and a downstream primer, and is used for amplifying one SNP site. The nucleotide sequences of the individual primers in the 48 primer sets are shown in Table 2. In columns 2-4 of table 2, the FAM fluorescent tag sequence (GAAGGTGACCAAGTTCATGCT) is single underlined, the HEX fluorescent tag sequence (GAAGGTCGGAGTCAACGGATT) is double underlined, and the sequence of the specific portion is not underlined.
Table 2: statistical table of SNP primer nucleic acid sequences of 36 SNP sites
Example 2
This example is a validation test of the SNP primer combination developed in example 1, and is also based on the construction of the DNA fingerprint database of pinus bungeana germplasm resources with the 36 core SNP markers.
The 137 pinus bungeana germplasm resources tested in this example are all common excellent germplasm resources, and the specific conditions are as follows in table 3:
table 3: source of germplasm resources
1. Obtaining of genome DNA of pinus bungeana germplasm resource to be tested
And respectively extracting 137 genome DNAs of the needle leaves of the pinus bungeana germplasm resource to be tested by adopting a CTAB method to obtain the genome DNAs of the pinus bungeana germplasm resource to be tested.
The CTAB method is specifically operated as follows:
(1) respectively picking up 137 needle leaves of pinus bungeana germplasm resource in the mature period, and placing the needle leaves in a freeze dryer (CoolSafe 55-4) for dehydration; adding Vc (vitamin C) powder of about 5mg, grinding and pulverizing.
(2) To the pulverized sample, 800. mu.L of CTAB extract (2 w/v% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1 w/v% PVP-40, 0.2 v/v% beta-mercaptoethanol) was added, shaken, mixed well and placed in a 65 ℃ water bath for 30min, during which shaking was carried out 3 to 5 times.
(3) Taking the sample out of the water bath, adding 700 mu L of chloroform isoamyl alcohol (volume ratio is 24:1), reversing and mixing uniformly, centrifuging at 12000rpm for 5min, and taking the supernatant and transferring to a new centrifuge tube. This procedure was repeated until the supernatant was clear.
(4) Adding 0.7 times volume of precooled isopropanol into the supernatant, reversing and mixing uniformly, and putting the uniformly mixed sample into a refrigerator at-20 ℃ for refrigeration for 30 min. The tube was removed, centrifuged at 12000rpm for 5min, the DNA was precipitated at the bottom of the tube and all liquid was decanted.
(5) At this time, the precipitate at the bottom of the tube is DNA, and then the precipitate obtained is washed by using 75% ethanol water solution; centrifuging at 12000rpm for 5min again to recover precipitate, and discarding liquid; and then the sample tube is dried in the air (or dried by a fume hood) to volatilize the residual alcohol.
The quality and concentration of the genome DNA of the pinus bungeana germplasm resource to be tested both need to meet the PCR requirement, and the standard of reaching the standard is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; detecting that the ratio of A260 to A280 is about 1.8 and the ratio of A260 to A230 is more than 1.8 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of the genome DNA of the pinus bungeana germplasm resource to be tested is 30-50 ng/mu L.
2. Respectively adopting genome DNAs of 137 pinus bungeana germplasm resources to be tested as templates, and respectively adopting 36 primer groups to carry out competitive allele specific PCR amplification so as to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer is 2:2: 5.
Reagents, consumables and instruments in the reaction system were provided by LGC company, including reagent amounts, usage and the whole experimental procedure were performed according to the LGC company's operating manual KASP user guide and manual (www.lgcgenomics.com), the KASPar reaction was performed in 384 well plates (Part No. KBS-0750-001) or 96 well plates (Part No. KBS-0751-001), and the reaction system was 3ul or 1ul, as shown in Table 4 below.
Table 4: KASP reaction system of 384-well plate or 96-well plate
Kits supplied by LGC company or otherwise having AS-PCR detection capability
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min, and continuous amplification for 26 cycles; final extension: 10min at 72 ℃. The resulting amplification product was stored at 4 ℃ before electrophoresis.
3. And (3) fluorescent signal detection: after the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, scanning and reading fluorescence values through FAM and HEX light beams of a microplate reader (reading values are observed when the FAM fluorescent label sequence is at 485nm of exciting light and at 520nm of emitting light, reading values are observed when the HEX fluorescent label sequence is at 528nm of exciting light and at 560nm of emitting light), and judging the genotypes of 137 pinus bungeana germplasm resources to be tested based on each SNP site according to the colors of fluorescence signals.
The specific judgment principle is as follows:
if a certain test pinus bungeana germplasm resource shows a blue fluorescent signal based on a certain SNP locus, the genotype of the test pinus bungeana germplasm resource based on the SNP locus is homozygous for the complementary base of the 1 st base at the 3' end of the first upstream primer for amplifying the SNP locus;
if the certain test pinus bungeana germplasm resource shows a red fluorescent signal based on a certain SNP locus, the genotype of the certain test pinus bungeana germplasm resource based on the SNP locus is homozygous for the complementary base of the 1 st base at the 3' end of the second upstream primer for amplifying the SNP locus;
if a certain test white bark pine germplasm resource shows a green fluorescent signal based on a certain SNP locus, the genotype of the test white bark pine germplasm resource based on the SNP locus is a heterozygote, one base is a complementary base of the 1 st base at the 3 'end of a first upstream primer for amplifying the SNP locus, and the other base is a complementary base of the 1 st base at the 3' end of a second upstream primer for amplifying the SNP locus.
In the 137 pinus albus germplasm resources, the genotype of each germplasm resource at each of the 36 SNP sites constitutes a pinus albus germplasm resource DNA fingerprint database based on the 36 core SNP markers, and the database can be used for identifying whether an unknown pinus albus germplasm resource belongs to the 137 test germplasm resources or specifically belongs to one of the 137 test germplasm resources.
If the fluorescence signal is weak after the PCR amplification is finished and affects data analysis, cycles (denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min and 5 cycles) can be added until the result is satisfactory.
Partial results are shown in FIG. 2. The result shows that each primer group can obtain good typing effect in the pinus bungeana germplasm resource to be tested. As shown, the fluorescence signal of PCR amplification product at 137 pinus bungeana germplasm resources (137 samples) for each SNP site clearly appears in 3 forms: 1) the aggregate appears blue in the sample near the X-axis, the genotype is the allele that joins the HEX fluorescent tag sequence; 2) the aggregate appears red in the sample near the Y-axis, and the genotype is the allele that joins the FAM fluorescent tag sequence; 3) samples on the X and Y axes are shown in green and the genotype is a heterozygote of the two alleles. There were also few samples with no fluorescence signal or no discrimination, showing pink color, and amplification products were not clearly typed, possibly due to poor DNA quality or too low a concentration. Therefore, the amplification effect of each primer is good, and the genotypes of 137 pinus bungeana germplasm resources to be tested can be obviously distinguished.
4. Cluster analysis
And (3) carrying out clustering analysis on 137 test white bark pine germplasm resources by utilizing MEGA7 software according to the genotypes of the 137 test white bark pine germplasm resources based on 36 SNP loci.
A cluster map of 137 test white bark pine germplasm resources built on 36 primer sets is shown in figure 3. The result shows that the 36 primer groups can completely distinguish 137 pinus bungeana germplasm resources to be tested in the table. Therefore, the SNP primer combination developed in the embodiment 1 can be applied to the construction of a white bark pine germplasm resource DNA fingerprint database and the identification of germplasm resource resources.
5. Evaluation of efficiency
The germplasm resource identification can adopt a sequential analysis mode to reduce the workload. The inventors of the present invention compared the relationship between the number of SNP markers (i.e., the number of primer sets) and the amount of germplasm resources for differentiating 137 Pinus bungeana from each other.
The experimental result shows (figure 4), at least 2 SNP sites are different to be used as germplasm resource identification threshold values, wherein 18 SNP markers can completely identify 137 white bark pine germplasm resources, the identification capability reaches 100%, and meanwhile, the other 18 SNP markers are used as extension sites to identify more white bark pine germplasm.
Example 3
The embodiment is a method for detecting whether a germplasm resource of a white bark pine to be detected belongs to the germplasm resources of 137 white bark pine to be detected, the germplasm resource of the white bark pine to be detected is unknown, and whether the germplasm resource of the white bark pine to be detected is one of the 137 germplasm resources is obtained by the detection method of the embodiment. The germplasm resources of the white bark pine to be detected in the embodiment are actually Jing series No. 20, and do not belong to the 137 white bark pine germplasm resources; under the condition that the white bark pine germplasm resource to be detected is known, the method provided by the invention is utilized to verify whether the white bark pine germplasm resource to be detected belongs to one of the 137. If the verification result is in accordance with the actual result, the method of the invention can be used for detecting whether the unknown white bark pine germplasm resource is one of the 137. The detection method of the embodiment comprises the following steps:
1. obtaining of genome DNA of pinus bungeana germplasm resource to be detected
The leaf of Jing series No. 20 of white bark pine germplasm resource to be tested is taken from needle leaf.
Replacing the leaves of the white bark pine germplasm resource to be tested with the leaves of the white bark pine germplasm resource to be tested according to the method in the step 1 in the embodiment 2, and obtaining the genome DNA of the white bark pine germplasm resource to be tested without changing other steps.
2. Configuration of SNP primer and PCR reaction system
Replacing the 'genome DNA of the white bark pine germplasm resource to be tested' with the 'genome DNA of the white bark pine germplasm resource to be tested' according to the method of the step 2 in the embodiment 2, carrying out competitive allele specific PCR reaction without changing other steps, and obtaining a PCR product of the white bark pine germplasm resource to be tested.
3. Fluorescence signal detection
Taking a PCR product of the white bark pine germplasm resource to be detected, judging the genotype of the white bark pine germplasm resource to be detected based on each site in the 48 SNP sites according to the color of the fluorescent signal, wherein the specific judgment principle is as described in the step 3 of the embodiment 2, and replacing the 'white bark pine germplasm resource to be detected' with the 'white bark pine germplasm resource to be detected'.
4. Specific germplasm resource judgment of to-be-detected white bark pine germplasm resource
The genotypes of the 36 SNP sites of the pinus bungeana germplasm resources to be detected are compared with the pinus bungeana germplasm resource DNA fingerprint database composed of 137 pinus bungeana germplasm resources to be detected in the embodiment 2, the number of the difference sites between the pinus bungeana germplasm resources to be detected and each pinus bungeana germplasm resource to be detected is counted, and then the following judgment is carried out:
if the number of the malposition points of the white bark pine germplasm resource to be detected and a standard white bark pine germplasm resource (to-be-detected white bark pine germplasm resource) is more than 2, the white bark pine germplasm resource to be detected and the standard white bark pine germplasm resource are judged to be different white bark pine germplasm resources; the greater the number of differential sites, the more distant the genetic relationship.
And if the number of the difference sites between the white bark pine germplasm resource to be detected and a standard white bark pine germplasm resource (the white bark pine germplasm resource to be detected) is 0-2, judging the white bark pine germplasm resource to be detected and the standard white bark pine germplasm resource as similar white bark pine germplasm resources.
The result shows that the number of the different sites of the white bark pine germplasm resource to be detected and 137 white bark pine germplasm resources to be detected is more than 6 on 36 SNP sites, so that the white bark pine germplasm resource to be detected does not belong to any one of the 137 white bark pine germplasm resources to be detected, namely the white bark pine germplasm resource to be detected is not one of the 137 white bark pine germplasm resources to be detected. The result is consistent with the reality that the germplasm resource of the white bark pine to be tested is Jing series No. 1, and is not one of the 137 white bark pine germplasm resources to be tested.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> forestry research institute of China forestry science research institute
<120> SNP locus primer combination for identifying white bark pine germplasm resources and application
<130> C1CNCN200904
<141> 2020-07-30
<150> 202010757560.7
<151> 2020-07-31
<160> 144
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 1
cagtacagaa atagagacaa aagtccta 28
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 2
agtacagaaa tagagacaaa agtcctg 27
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 3
atagtaaatt ctttgtgctt catgtggaga t 31
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 4
actcagaaaa cccttgtcaa acttaag 27
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 5
cactcagaaa acccttgtca aacttaat 28
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 6
gagtggtggg ataaattcac aagatagtat 30
<210> 7
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 7
cctctcaaaa ggagcttatg aacttc 26
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 8
tcctctcaaa aggagcttat gaacttt 27
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 9
gttgaggggc tccgagtgga att 23
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 10
gcttgcgggt tcgaaggtga at 22
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 11
cttgcgggtt cgaaggtgaa g 21
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 12
gatcactgac agagctgtta atggttta 28
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 13
tgtgaagaaa aaggatggag cgctt 25
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 14
tgaagaaaaa ggatggagcg ctc 23
<210> 15
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 15
ggtcacttta tttaatttcc tgtaatcaat ataaa 35
<210> 16
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 16
tccattagaa cttcttggac tagatc 26
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 17
ctccattaga acttcttgga ctagata 27
<210> 18
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 18
aagtggaagt tgaccaagaa gtaaatcaat t 31
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 19
tcaaccttat aactagggtt tc 22
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 20
tcaaccttat aactagggtt tt 22
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 21
cacaggatca agtgaaaaac acttcatgta 30
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 22
tgtaagcaat agagtccact acctc 25
<210> 23
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 23
ctgtaagcaa tagagtccac tacctt 26
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 24
ggaaactgga ggaaggagga tgttaat 27
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 25
cctagttgaa cacggttggc tg 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 26
cctagttgaa cacggttggc tc 22
<210> 27
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 27
tgtgaaaaga tcatacattt agtatgcgct t 31
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 28
tctgccttgc aactcatgat ctcta 25
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 29
tgccttgcaa ctcatgatct ctg 23
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 30
tcccctgata tagcaaatac attgagaatt 30
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 31
ggaaaaggag gccagttagt gg 22
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 32
aaggaaaagg aggccagtta gtga 24
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 33
accccttggt ggcctccaca at 22
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 34
ctagtgccta gttcactcgc tc 22
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 35
tctagtgcct agttcactcg cta 23
<210> 36
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 36
tcactcgcaa acagatgggt cgatt 25
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 37
gcggtgtgct aggataatac acc 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 38
gcggtgtgct aggataatac act 23
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 39
tcggtaagcc cggtgcaacc ta 22
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 40
caccgccatt gaaatatgtg caagt 25
<210> 41
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 41
caccgccatt gaaatatgtg caaga 25
<210> 42
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 42
catacataga ttttgcacca aaaaaagaaa gttt 34
<210> 43
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 43
gagtgagtgc acaaaatcgg ataac 25
<210> 44
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 44
tgagtgagtg cacaaaatcg gataat 26
<210> 45
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 45
ccttctacag gttcctgtaa ctaattcta 29
<210> 46
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 46
tataggattt tccataggct cctgc 25
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 47
tataggattt tccataggct cctgt 25
<210> 48
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 48
gtcttccgtt ggtccacatc cttaa 25
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 49
caagaagggc agcaccaatc ag 22
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 50
atacaagaag ggcagcacca atcat 25
<210> 51
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 51
cagaggatga cagagggaat atgcaa 26
<210> 52
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 52
ccaggttacc aacccttgag ac 22
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 53
ccaggttacc aacccttgag ag 22
<210> 54
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 54
gatcaactca cagtacgaat caacaaaatt t 31
<210> 55
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 55
ctttctccat taagggggag tgtt 24
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 56
ctttctccat taagggggag tgta 24
<210> 57
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 57
gaatctctct taaaataaat ctctccatta tctt 34
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 58
ctcacactct ctaggccaag c 21
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 59
cctcacactc tctaggccaa ga 22
<210> 60
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 60
ggtttaacac ctcctccttc tatcctt 27
<210> 61
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 61
cacctggaga ctgtagtcaa acg 23
<210> 62
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 62
tacacctgga gactgtagtc aaaca 25
<210> 63
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 63
gtatctttct gattaatgga tgtagagtga t 31
<210> 64
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 64
gtttgggagc atcatatcat ctcgt 25
<210> 65
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 65
tttgggagca tcatatcatc tcgg 24
<210> 66
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 66
atcgaaaact aagacaacag gaaaaaaaga ttta 34
<210> 67
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 67
ccgatgaggt gcttcatctc aac 23
<210> 68
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 68
atccgatgag gtgcttcatc tcaaa 25
<210> 69
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 69
cttggttcga gttccaatag aaggaattt 29
<210> 70
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 70
gccacatggt aaatctcacc tcg 23
<210> 71
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 71
ttgccacatg gtaaatctca cctca 25
<210> 72
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 72
aacctttata ataaacatct cccatcctct t 31
<210> 73
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 73
caacttacat atccccctgc tatca 25
<210> 74
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 74
aacttacata tccccctgct atcg 24
<210> 75
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 75
gataattgca agaaaaaggg aagattacat caa 33
<210> 76
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 76
gatattatga ggccttaact cttacttc 28
<210> 77
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 77
ttgatattat gaggccttaa ctcttactta 30
<210> 78
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 78
ccattcccac caacttaaac tcaaagta 28
<210> 79
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 79
gttttttgtc tagatatttc aactcacatc 30
<210> 80
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 80
gttttttgtc tagatatttc aactcacatg 30
<210> 81
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 81
ttgtgtggat atttgtttat gagtctctat atat 34
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 82
cacaagattc agctgcactt tccta 25
<210> 83
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 83
acaagattca gctgcacttt cctg 24
<210> 84
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 84
ccaattttgc ttggatcagt gtgagata 28
<210> 85
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 85
atcccttcct tatccggtat ctcta 25
<210> 86
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 86
cccttcctta tccggtatct ctc 23
<210> 87
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 87
cgtgtgaagc agtagatgcc caaat 25
<210> 88
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 88
ctaggctaga ggaagctagg cta 23
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 89
taggctagag gaagctaggc tg 22
<210> 90
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 90
caattctact ctagtctcgt gtagctta 28
<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 91
ggccctatac ttggcccaaa ac 22
<210> 92
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 92
gggccctata cttggcccaa aaa 23
<210> 93
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 93
atgttgagtc cattgaagcc tagatcaa 28
<210> 94
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 94
ggatgctaca ggctagtgac tg 22
<210> 95
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 95
atggatgcta caggctagtg acta 24
<210> 96
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 96
catcatggag atgaggctag accatt 26
<210> 97
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 97
agtgacatct tgttcctcta tgctg 25
<210> 98
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 98
agtgacatct tgttcctcta tgctc 25
<210> 99
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 99
atacatcttg tgttaggaag aagtgaagaa t 31
<210> 100
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 100
gcccaatgta acctagcgca ct 22
<210> 101
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 101
cccaatgtaa cctagcgcac c 21
<210> 102
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 102
gtgcgctggg ataattcagc acat 24
<210> 103
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 103
tgcctcatgt ctaagaagga tgcaa 25
<210> 104
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 104
cctcatgtct aagaaggatg cag 23
<210> 105
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 105
gattgtagga agatatcaac tccctcttta 30
<210> 106
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 106
tttgtgggtg caagggaatc tcata 25
<210> 107
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 107
gtgggtgcaa gggaatctca tc 22
<210> 108
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 108
catgtcaaag gatgtggagg ccat 24
<210> 109
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 109
gcttcatgtg gagatcatgc taggactttt gtctctattt c 41
<210> 110
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 110
aaaacccttg tcaaacttaa gaatactatc ttgtgaattt a 41
<210> 111
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 111
tggaattcca ttacaaccct gaagttcata agctcctttt g 41
<210> 112
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 112
agctgttaat ggtttacact attcaccttc gaacccgcaa g 41
<210> 113
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 113
ctgtaatcaa tataaagtct aagcgctcca tcctttttct t 41
<210> 114
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 114
aagaagtaaa tcaattgaaa gatctagtcc aagaagttct a 41
<210> 115
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 115
aacacttcat gtacatactc gaaaccctag ttataaggtt g 41
<210> 116
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 116
aggaggatgt taattcagtt gaggtagtgg actctattgc t 41
<210> 117
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 117
ctagttgaac acggttggct ggagacactc aagcgcatac t 41
<210> 118
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 118
ccttgcaact catgatctct acctttagaa ttctcaatgt a 41
<210> 119
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 119
gaaaaggagg ccagttagtg gattgtggag gccaccaagg g 41
<210> 120
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 120
cgcaaacaga tgggtcgatt gagcgagtga actaggcact a 41
<210> 121
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 121
gcaacctagc gcacctgtgc ggtgtattat cctagcacac c 41
<210> 122
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 122
caccaaaaaa agaaagtttg acttgcacat atttcaatgg c 41
<210> 123
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 123
ggttcctgta actaattcta gttatccgat tttgtgcact c 41
<210> 124
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 124
ggattttcca taggctcctg cttaaggatg tggaccaacg g 41
<210> 125
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 125
tgacagaggg aatatgcaac ctgattggtg ctgcccttct t 41
<210> 126
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 126
caggttacca acccttgaga caaattaaaa ttttgttgat t 41
<210> 127
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 127
tctccattaa gggggagtgt tgaagataat ggagagattt a 41
<210> 128
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 128
ctcacactct ctaggccaag ctcctccaaa ggatagaagg a 41
<210> 129
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 129
tggatgtaga gtgatctctc cgtttgacta cagtctccag g 41
<210> 130
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 130
gggagcatca tatcatctcg ttaaatcttt ttttcctgtt g 41
<210> 131
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 131
gatgaggtgc ttcatctcaa caaattcctt ctattggaac t 41
<210> 132
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 132
tcccatcctc ttctcagcac cgaggtgaga tttaccatgt g 41
<210> 133
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 133
ttacatatcc ccctgctatc aagttgatgt aatcttccct t 41
<210> 134
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 134
taaactcaaa gtagcgaaga gaagtaagag ttaaggcctc a 41
<210> 135
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 135
tttatgagtc tctatatata gatgtgagtt gaaatatcta g 41
<210> 136
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 136
agattcagct gcactttcct aactatctca cactgatcca a 41
<210> 137
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 137
gtagatgccc aaatcccctc tagagatacc ggataaggaa g 41
<210> 138
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 138
aggctagagg aagctaggct agggtaagct acacgagact a 41
<210> 139
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 139
tccattgaag cctagatcaa gttttgggcc aagtataggg c 41
<210> 140
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 140
gatgctacag gctagtgact ggatgtggcc aatggtctag c 41
<210> 141
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 141
gaagaagtga agaatgttcg cagcatagag gaacaagatg t 41
<210> 142
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 142
cccaatgtaa cctagcgcac tgatgtgctg aattatccca g 41
<210> 143
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 143
tcatgtctaa gaaggatgca agataaagag ggagttgata t 41
<210> 144
<211> 41
<212> DNA
<213> Pinus bungeana
<400> 144
tgtggaggcc atcgcacaga tatgagattc ccttgcaccc a 41
Claims (10)
1. Identifying SNP sites of the white bark pine germplasm resources, wherein the SNP sites are selected from any 1 to 36 of the following first SNP site to the third sixteen SNP site:
a first SNP site located in the genome of Pinus bungeana as shown in SEQ ID NO: 109, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence at position 21, wherein the nucleotide base of the site is T or C;
a second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 110, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or T;
and a third SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 111, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a fourth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 112, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or C;
a fifth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 113 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
and a sixth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 114, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or T;
a seventh SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 115, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
an eighth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 116, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
a ninth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 117 at position 21 of the sequence, or at a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is G or C;
a tenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 118, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
an eleventh SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 119, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a twelfth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 120, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is G or T;
a thirteenth SNP site, wherein the second SNP site is located in the genome of white bark pine as shown in SEQ ID NO: 121, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is G or A;
a fourteenth SNP site, wherein the second SNP site is located in the genome of Pinus bungeana as shown in SEQ ID NO: 122, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is A or T;
a fifteenth SNP site, wherein the fifteenth SNP site is located in the white bark pine genome and shown as SEQ ID NO: 123, or the corresponding site on the homologous genome fragment among the germplasm resources of the 21 st site of the sequence, wherein the nucleotide base of the site is G or A;
and a sixteenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 124, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or T;
a seventeenth SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 125 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or A;
an eighteenth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 126, 21 st position of the sequence or corresponding position on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the position is C or G;
a nineteenth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 127 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or A;
a twentieth SNP site, wherein the second SNP site is located in the white bark pine genome and is shown as SEQ ID NO: 128 or the corresponding site on the homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is C or A;
a twenty-first SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 129, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T;
a twenty-second SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 130, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is T or G;
a twenty-third SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 131 or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is C or A;
a twenty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 132, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or T;
a twenty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 133, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a twenty-sixth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 134 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T;
a twenty-seventh SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 135, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or C;
a twenty-eighth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 136, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a twenty-ninth SNP site, wherein the second SNP site is located in a white bark pine genome as shown in SEQ ID NO: 137, or a corresponding site on a homologous genome fragment among germplasm resources of the sequence, wherein the nucleotide base of the site is T or G;
and the thirtieth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 138, or a corresponding site on a homologous genomic fragment among germplasm resources thereof, wherein the nucleotide base of the site is A or G;
a thirty-first SNP site, wherein the second SNP site is located in a white bark pine genome and shown as SEQ ID NO: 139 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or T;
and a thirty-second SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 140, position 21 of the sequence, or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is G or A;
and a thirty-third SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 141 or a corresponding site on a homologous genome fragment among germplasm resources thereof, wherein the nucleotide base of the site is C or G;
and a thirty-fourth SNP site, wherein the second SNP site is located in a white bark pine genome and has a nucleotide sequence shown as SEQ ID NO: 142, or the corresponding site on the homologous genome fragment among the germplasm resources of the sequence, wherein the nucleotide base of the site is T or C;
and a thirty-fifth SNP site, wherein the second SNP site is located in a white bark pine genome and is shown as SEQ ID NO: 143 or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is a or G;
and the thirty-sixth SNP site is positioned in the white bark pine genome and shown as SEQ ID NO: 144, or a corresponding site on a homologous genomic fragment between germplasm resources thereof, wherein the nucleotide base of the site is T or G.
2. The SNP site according to claim 1, wherein:
the sequences of the first SNP locus and bases at the upstream and downstream are SEQ ID NO: 109 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 109 by greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 110 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 110 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 111 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 111 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 112 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 112 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 113 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 113 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 114 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 114 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 115 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 115, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 116 or a germplasm resource homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 116 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the ninth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 117 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence set forth in SEQ ID NO: 117 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the tenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 118 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a);
the sequence of the eleventh SNP site and bases on the eleventh SNP site is SEQ ID NO: 119 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 119, or greater than 95%, 96%, 97%, 98%, or 99%;
the sequence of the twelfth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 120 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 120, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the thirteenth SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 121 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 121, greater than or equal to 95%, 96%, 97%, 98%, or 99% identity;
the sequence of the fourteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 122 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 122 is greater than or equal to 95%, 96%, 97%, 98% or 99%;
the sequence of the fifteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 123 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 123 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the sequence of the sixteenth SNP locus and bases on the sixteenth SNP locus is SEQ ID NO: 124 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 124, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the seventeenth SNP site and the upstream and downstream bases thereof is SEQ ID NO: 125 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 125, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the eighteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 126 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 126, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the nineteenth SNP site and the sequences of bases on the nineteenth SNP site are SEQ ID NO: 127 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 127, 95%, 96%, 97%, 98% or 99%;
the twenty-second SNP site and the sequences of bases on the twenty-second SNP site are SEQ ID NO: 128 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 128 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the twenty-first SNP locus and the sequences of bases on the twenty-first SNP locus and bases on the twenty-first SNP locus are SEQ ID NO: 129 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of SEQ ID NO: 129, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the second twelve SNP locus and the base sequences of the second twelve SNP locus are SEQ ID NO: 130 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 130, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-third SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 131 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 131, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the twenty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 132 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO 132: greater than or equal to 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of (a);
the twenty-fifth SNP locus and the sequences of bases on the twenty-fifth SNP locus and the upstream and downstream of the twenty-fifth SNP locus are SEQ ID NO: 133 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 133 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-sixth SNP locus and the sequences of bases on the twenty-sixth SNP locus and bases on the twenty-sixth SNP locus are SEQ ID NO: 134 or a germplasm resource homologous genome fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 134 is greater than or equal to 95%, 96%, 97%, 98%, or 99% identical;
the twenty-seventh SNP locus and the sequences of bases on the twenty-seventh SNP locus and the upstream and downstream of the twenty-seventh SNP locus are SEQ ID NO: 135 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 135 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the twenty-eight SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 136 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of SEQ ID NO: 136 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the twenty-ninth SNP locus and the sequences of bases on the twenty-ninth SNP locus are SEQ ID NO: 137 or a genome fragment homologous to the germplasm resources thereof, more preferably a fragment identical to the sequence of SEQ ID NO: 137, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the thirty-third SNP site and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 138 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 138, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirty-first SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 139 or a genome fragment homologous to a germplasm resource thereof, more preferably to the sequence set forth in SEQ ID NO: 139, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequences of the third twelve SNP loci and bases at the upper and lower ends of the third twelve SNP loci are SEQ ID NO: 140 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 140 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirteenth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 141 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 141, greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the thirty-fourth SNP locus and the upstream and downstream bases thereof is SEQ ID NO: 142 or a germplasm resource homologous genomic fragment thereof, more preferably a fragment of the sequence of SEQ ID NO: 142 is greater than or equal to 95%, 96%, 97%, 98%, or 99%;
the sequence of the fifteenth SNP locus and the bases at the upstream and downstream of the fifteenth SNP locus is SEQ ID NO: 143 or a germplasm resource-homologous genomic fragment thereof, more preferably to the sequence set forth in SEQ ID NO: 143 is greater than or equal to 95%, 96%, 97%, 98% or 99% identical;
the sequences of the sixteenth SNP locus and bases on the sixteenth SNP locus are SEQ ID NO: 144 or a germplasm resource-homologous genomic fragment thereof, more preferably a fragment identical to the sequence set forth in SEQ ID NO: 144 is greater than or equal to 95%, 96%, 97%, 98% or 99%.
3. An SNP primer set for identifying pinus bungeana germplasm resources, the SNP primer set being used for respectively amplifying the SNP sites as set forth in claim 1, the SNP primer set comprising:
a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site; a ninth SNP primer set for amplifying the ninth SNP site; a tenth SNP primer set for amplifying the tenth SNP site; an eleventh SNP primer set for amplifying the eleventh SNP site; a twelfth SNP primer set for amplifying the twelfth SNP site; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a fourteenth SNP primer set for amplifying the fourteenth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a sixteenth SNP primer set for amplifying the sixteenth SNP site; a seventeenth SNP primer set for amplifying the seventeenth SNP site; an eighteenth SNP primer set for amplifying the eighteenth SNP site; a nineteenth SNP primer set for amplifying the nineteenth SNP site; a twentieth SNP primer set for amplifying the twentieth SNP site; a twenty-first SNP primer set for amplifying the twenty-first SNP site; a second twelve SNP primer set for amplifying the second twelve SNP sites; a twenty-third SNP primer set for amplifying the twenty-third SNP site; a twenty-fourth SNP primer set for amplifying the twenty-fourth SNP site; a twenty-fifth SNP primer set for amplifying the twenty-fifth SNP site; a twenty-sixth SNP primer set for amplifying the twenty-sixth SNP site; a twenty-seventh SNP primer set for amplifying the twenty-seventh SNP site; a second eighteen SNP primer set for amplifying the second eighteen SNP site; a twenty-ninth SNP primer set for amplifying the twenty-ninth SNP site; a thirtieth SNP primer set for amplifying the thirtieth SNP site; a thirty-first SNP primer set for amplifying the thirty-first SNP site; a third twelve SNP primer set for amplifying the third twelve SNP sites; a thirteenth SNP primer set for amplifying the thirteenth SNP site; a thirty-fourth SNP primer set for amplifying the thirty-fourth SNP site; a fifteenth SNP primer set for amplifying the fifteenth SNP site; a thirty-sixth SNP primer set for amplifying the thirty-sixth SNP site.
4. The SNP primer set according to claim 3, wherein:
in the first SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the second SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the third SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the fourth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the fifth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the seventh SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the eighth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the ninth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the tenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 30 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the eleventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 33, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the twelfth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 34. SEQ ID NO: 35. SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39, greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the fourteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the fifteenth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 43. SEQ ID NO: 44. SEQ ID NO: 45 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the seventeenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 52. SEQ ID NO: 53. SEQ ID NO: 54 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the nineteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 57 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the twentieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 58. SEQ ID NO: 59. SEQ ID NO: 60, preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity;
in the twenty-first SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 61. SEQ ID NO: 62. SEQ ID NO: 63 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the second twelve SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the twenty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the twenty-fifth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the twenty-sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;
in the twenty-seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the eighteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the twenty-ninth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the thirtieth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the primers shown in SEQ ID NO: 88. SEQ ID NO: 89. SEQ ID NO: 90 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the thirty-first SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with the sequence shown in SEQ ID NO: 91. SEQ ID NO: 92. SEQ ID NO: 93 greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the third twelve SNP primer groups, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 96 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the thirteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively identical to SEQ ID NO: 97. SEQ ID NO: 98. SEQ ID NO: 99, and preferably 100%, or greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity;
in the thirty-fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequence shown in SEQ ID NO: 100. SEQ ID NO: 101. SEQ ID NO: 102 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the fifteenth SNP primer set, the specific portion of the first upstream primer, the specific portion of the second upstream primer, and the downstream primer are respectively identical to SEQ ID NOs: 103. SEQ ID NO: 104. SEQ ID NO: 105 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
in the sixteenth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively connected with SEQ ID NO: 106. SEQ ID NO: 107. SEQ ID NO: 108 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;
preferably, the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM, HEX.
5. An SNP kit for identifying white bark pine germplasm resources is characterized in that: the SNP kit is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises:
the SNP primer set according to claim 3 or 4,
preferably, in the SNP primer sets, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer set in the system is 2:2: 5.
6. A white bark pine germplasm resource DNA fingerprint database based on SNP markers is characterized in that: the DNA fingerprint database includes: the genotype of the SNP site of claim 1 of a standard white bark pine germplasm resource.
7. The DNA fingerprint database of claim 6, wherein: the standard white bark pine germplasm resources are selected from the following 137 white bark pine germplasm resources:
jing series No. 1, Jing series No. 11, Jing series No. 12, Jing series No. 13, Jing series No. 14, Jing series No. 15, Jing series No. 2, Jing series No. 3, Jing series No. 4, Jing series No. 5, Jing series No. 6, Jing series No. 7, Jing series No. 8, Jing series No. 9, Xian, Ruiwang, Jiuquan, Ganjin No. 1, Ganjin No. 10, Ganjin No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 5, Ganjin No. 6, Ganjin No. 7, Ganjin No. 8, Ganjin No. 9, Ganjin No. 1, Ganjin No. 10, Ganjin No. two No. 11, Ganjin No. 12, Ganjin No. 13, Ganjin No. 14, Ganjin No. 15, Ganjin No. 2, Ganjin No. 3, Ganjin No. 4, Ganjin No. 6, Ganjin No. 7, Ga, jin Yan No. 15, jin Yan No. 2, jin Yan No. 3, jin Yan No. 4, jin Yan No. 5, jin Yan No. 6, jin Yan No. 8, jin Yan No. 9, gan Tian No. 1, gan Tian No. 10, gan Tian No. 11, gan Tian No. 12, gan Tian No. 13, gan Tian No. 14, gan Tian No. 15, gan Tian No. 2, gan Tian No. 3, gan Tian No. 4, gan Tian No. 5, gan Tian No. 6, gan Tian No. 7, gan Tian No. 8, gan Tian No. 9, jin Xixiao No. 13, jin Xiao No. 14, jin Xiao No. 15, Shaanluo No. 1, Shaanluo No. 10, Shaanluo No. 11, Shaanluo No. 12, Shaanluo No. 13, Shaanluo No. 14, Shaanluo No. 15, Shaanluo No. 2, Shaanluo No. 3, Shaanluo No. 4, Shaanluo No. 5, Shaanluo blue No. 6, Shaanluo No. 7, Shaanluo blue No. 1, Shaanluo blue No. 11, Shaanluo blue No. 12, shandan No. 6, Shaanlan No. 8, Shaanlan No. 9, Shaanlong No. 1, Shaanlong No. 10, Shaanlong No. 11, Shaanlong No. 12, Shaanlong No. 13, Shaanlong No. 14, Shaanlong No. 15, Shaanlong No. 2, Shaanlong No. 3, Shaanlong No. 4, Shaanlong No. 5, Shaanlong No. 6, Shaanlong No. 7, Shaanlong No. 8, Shaanlong No. 9, Zheng No. 1, Zheng No. 10, Zheng No. 11, Zheng No. 12, Zheng No. 13, Zheng No. 14, Zheng No. 15, Zheng No. 2, Zheng No. 4, Zheng No. 5, Zheng No. 6, Zheng No. 7, Zheng No. 8, and Zheng No. 9.
8. The method of constructing a DNA fingerprint database according to claim 6, wherein: the construction method comprises the following steps:
and (3) PCR reaction steps: performing competitive allele-specific PCR amplification reaction on a standard pinus bungeana germplasm resource by using the PCR reaction system of claim 5 to obtain a PCR reaction product;
SNP locus genotype obtaining step: detecting the PCR reaction product to obtain the genotype of the SNP locus;
preferably, the detection is fluorescence signal detection or direct sequencing.
9. A detection method for identifying white bark pine germplasm resources is characterized by comprising the following steps: the detection method comprises the following steps:
the method comprises the following steps: detecting the genotype of the SNP locus of the pinus bungeana to be detected;
step two: judging the germplasm resources of the pinus bungeana to be detected:
if the number of the genotype of the pinus albus to be detected based on the 36 SNP sites and the number of the different sites of a certain specified germplasm resource in the standard pinus albus germplasm resources in the database according to claim 6 or 7 based on the genotype of the 36 SNP sites are 0-2, judging the pinus albus to be detected as a germplasm resource similar to the specified germplasm resource;
if the number of the difference sites of the pinus albus to be detected based on the genotypes of the 36 SNP sites and a certain specified germplasm resource in the standard pinus albus germplasm resources in the database according to claim 6 or 7 based on the genotypes of the 36 SNP sites is more than 2, judging the pinus albus to be detected as a pinus albus germplasm resource different from the specified germplasm resource;
preferably, the result of the determination is obtained from a cluster analysis.
10. The SNP site according to claim 1 or 2, or the SNP primer combination according to claim 3 or 4, or the SNP kit according to claim 5, or the DNA fingerprint database according to claim 6 or 7, or the DNA fingerprint database obtained by the construction method according to claim 8, or the detection method according to claim 9, wherein the SNP site is used in the following X1 or X2:
x1: identifying whether the germplasm resource of the white bark pine to be detected belongs to one of the standard white bark pine germplasm resources;
x2: and identifying the germplasm resources of the white bark pine to be detected as the specific standard white bark pine germplasm resources.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010757560 | 2020-07-31 | ||
| CN2020107575607 | 2020-07-31 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112226433A true CN112226433A (en) | 2021-01-15 |
| CN112226433B CN112226433B (en) | 2022-02-11 |
Family
ID=74115793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010899756.XA Expired - Fee Related CN112226433B (en) | 2020-07-31 | 2020-08-31 | SNP (Single nucleotide polymorphism) site primer combination for identifying white bark pine germplasm resources and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112226433B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116072229A (en) * | 2022-09-16 | 2023-05-05 | 中国林业科学研究院林业研究所 | Germplasm resource database for olive variety identification and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676649A (en) * | 2012-02-29 | 2012-09-19 | 西北大学 | Pinus bungeana paternal line identification method |
| KR20130136264A (en) * | 2012-06-04 | 2013-12-12 | 충북대학교 산학협력단 | Specific primers for pines distinction, analysis kit using its primers, and pines distinciton method using thereof |
| CN105850518A (en) * | 2016-04-19 | 2016-08-17 | 中国林业科学研究院林业研究所 | Method for increasing grafting propagation survival rate of pinus bungeana variations |
| CN108875300A (en) * | 2018-06-04 | 2018-11-23 | 河南农业大学 | A kind of method and application adapting to potentiality using landscape genomics assessment species |
-
2020
- 2020-08-31 CN CN202010899756.XA patent/CN112226433B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676649A (en) * | 2012-02-29 | 2012-09-19 | 西北大学 | Pinus bungeana paternal line identification method |
| KR20130136264A (en) * | 2012-06-04 | 2013-12-12 | 충북대학교 산학협력단 | Specific primers for pines distinction, analysis kit using its primers, and pines distinciton method using thereof |
| CN105850518A (en) * | 2016-04-19 | 2016-08-17 | 中国林业科学研究院林业研究所 | Method for increasing grafting propagation survival rate of pinus bungeana variations |
| CN108875300A (en) * | 2018-06-04 | 2018-11-23 | 河南农业大学 | A kind of method and application adapting to potentiality using landscape genomics assessment species |
Non-Patent Citations (2)
| Title |
|---|
| 张开慧等: "白皮松及其遗传多样性研究进展", 《陕西农业科学》 * |
| 赵罕等: "白皮松天然群体遗传多样性的EST-SSR分析", 《林业科学研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116072229A (en) * | 2022-09-16 | 2023-05-05 | 中国林业科学研究院林业研究所 | Germplasm resource database for olive variety identification and construction method and application thereof |
| CN116072229B (en) * | 2022-09-16 | 2023-10-20 | 中国林业科学研究院林业研究所 | Germplasm resource database for olive variety identification and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112226433B (en) | 2022-02-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111270004B (en) | Method for identifying authenticity of pepper variety and special SSR primer combination thereof | |
| CN108103235B (en) | SNP molecular marker and primer for identifying cold resistance of apple rootstock and application of SNP molecular marker and primer | |
| WO2019047074A1 (en) | Snp molecular marker combination for rice genotyping, and application thereof | |
| CN113584216B (en) | Development and application of KASP marker of wheat grain weight gene TaCYP78A16 | |
| US20200407806A1 (en) | Snp molecular marker tightly linked to weeping trait of mei and detection method and use thereof | |
| CN107090495B (en) | Molecular marker related to long shape of neck of millet and detection primer and application thereof | |
| CN111235302B (en) | Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof | |
| CN107586857B (en) | Nucleic acid, kit and method for rapidly identifying red and black hair color genes of pigs | |
| CN115678979A (en) | Pineapple liquid phase chip and application thereof | |
| CN112029890B (en) | SNP (Single nucleotide polymorphism) site primer combination for identifying melon germplasm authenticity and application | |
| CN112226433B (en) | SNP (Single nucleotide polymorphism) site primer combination for identifying white bark pine germplasm resources and application | |
| CN108220473B (en) | Identification of maize S-type cytoplasmic male sterile material by using chloroplast InDel marker | |
| CN110878376B (en) | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof | |
| CN116590453B (en) | SNP molecular marker related to dwarf trait of lotus plant and application thereof | |
| CN112063744A (en) | KASP molecular marker for detecting rice blast resistance gene Pita of rice and detection method | |
| CN112080497B (en) | SNP (Single nucleotide polymorphism) site primer combination for identifying watermelon germplasm authenticity and application | |
| CN113637790B (en) | KASP molecular marker of stripe rust resistance gene YrAS2388R, primer, kit and application | |
| CN111235300B (en) | Method for identifying authenticity of cabbage variety and special SSR primer combination thereof | |
| CN111235301B (en) | Method for identifying authenticity of bitter gourd varieties and special SSR primer combination thereof | |
| CN115141893A (en) | Molecular marker group containing 7 molecular markers and used for predicting dry matter content of kiwi fruit, application and kit thereof | |
| He et al. | Construction of fingerprint of Michelia germplasm by fluorescent SSR markers | |
| CN111270003B (en) | Method for identifying authenticity of carrot variety and special SSR primer combination thereof | |
| CN111411165B (en) | SNP (Single nucleotide polymorphism) site primer combination for identifying cucumber germplasm authenticity and application | |
| CN117925886B (en) | SNP molecular marker related to side-by-side load character and application | |
| LU503449B1 (en) | Two PARMS-SNP Molecular Markers for Identifying Resistant Gene VrTAF5 of Vigna radiata (Linn.) Wilczek Cercospora Leaf Spot Disease |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220211 |