CN105925721B - For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character - Google Patents
For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character Download PDFInfo
- Publication number
- CN105925721B CN105925721B CN201610536612.1A CN201610536612A CN105925721B CN 105925721 B CN105925721 B CN 105925721B CN 201610536612 A CN201610536612 A CN 201610536612A CN 105925721 B CN105925721 B CN 105925721B
- Authority
- CN
- China
- Prior art keywords
- peach
- seq
- character
- primer
- pcr amplification
- 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.)
- Expired - Fee Related
Links
- 235000006040 Prunus persica var persica Nutrition 0.000 title claims abstract description 53
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 39
- 240000005809 Prunus persica Species 0.000 title claims abstract description 28
- 239000002773 nucleotide Substances 0.000 title claims abstract description 28
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 28
- 210000002615 epidermis Anatomy 0.000 title claims abstract description 27
- 238000004040 coloring Methods 0.000 title claims abstract description 19
- 244000144730 Amygdalus persica Species 0.000 claims abstract description 25
- 239000003550 marker Substances 0.000 claims abstract description 17
- 238000012408 PCR amplification Methods 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 238000009395 breeding Methods 0.000 claims description 9
- 230000001488 breeding effect Effects 0.000 claims description 8
- 238000003205 genotyping method Methods 0.000 claims description 8
- 239000003086 colorant Substances 0.000 claims description 7
- 239000003147 molecular marker Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000011033 desalting Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000000349 chromosome Anatomy 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 102000054765 polymorphisms of proteins Human genes 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 15
- 101100240528 Caenorhabditis elegans nhr-23 gene Proteins 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 238000000546 chi-square test Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010000178 IGF-I-IGFBP-3 complex Proteins 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229930014669 anthocyanidin Natural products 0.000 description 1
- 150000001452 anthocyanidin derivatives Chemical class 0.000 description 1
- 235000008758 anthocyanidins Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses for identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character.The single nucleotide polymorphism site is the 3473250th nucleotide of the 3rd chromosome of peach genome, which is T or G.The present invention can be used in identifying or assisting identification Peach fruits epidermis coloring character, the accuracy rate with higher when identifying Peach fruits epidermis coloring character using the 3473250bp of the 3rd chromosome of peach genome as nucleotide polymorphisms markers site.To 15 hybrid Populations, totally 221 parts of peach single plants to be measured carry out the verifying of SNPs accuracy rate to the present invention, the result shows that the present invention is in the phenotypic character prediction for carrying out hybrid Population, it is higher for dominant phenotype's accuracy rate, compared with other researchs reported recently, 61.29% is promoted to from 8.06%.This explanation, using mononucleotide marker site of the invention carry out detection have the advantages that it is simple, quick, at low cost, can be realized produce in apply on a large scale.
Description
Technical field
The present invention relates to be used to identify the single nucleotide polymorphism site of Peach fruits epidermis coloring (red) character, draw
Object, kit and application, belong to field of biotechnology.
Background technique
Selection is one of most important link in breeding, it, which refers to, selects satisfactory genotype in a group,
To carry out subsequent cultivation.But in traditional breeding method, due to being difficult to know that the genotype of offspring, the foundation of selection are usually
Phenotype and non genotype, this selection method are usually effective for qualitative character, but for quantitative character, because
Lack specific corresponding relationship between its Phenotype and genotype, thus it is inefficient.In addition, for using fruit properties as target
Fruit tree for, these characters have it specifically to show period, it usually needs spend the even more prolonged juvenile phase of 3-5,
Thus the time of selection is later.The crop that this is tall and big for those plant, land occupation is more, Growing season is long, especially fruit tree etc
Garden crop, it is clear that be very unfavorable.
The molecular marking technique based on DNA developed rapidly in the past 20 years, i.e. " molecular marker assisted selection "
(marker-assisted selection, be abbreviated as MAS) provides brand-new approach to breeding.It passes through analysis and purpose
The genotype of the molecular labeling of gene close linkage carries out breeding, to achieve the purpose that improve breeding efficiency.Yamamoto
(2001) using AFLP (Amplified Fragment Length Polymorphism, amplified fragment length polymorphism),
RAPD (random amplified polymorphic DNA, randomly amplified polymorphic DNA), SSR (Simple Sequence
Repeats, simple repeated sequence) and RFLP (Restriction Fragment Length Polymorphism, it is restricted in
Enzyme cutting fragment length polymorphism) label, fruit colour is positioned into the 6th linkage group, therewith the SSR of linkage distance nearest (3.7cM)
Labeled as UDP96-015.It is that must obtain and target first due to carrying out molecular marker assisted selection there are two important premise
The label of character close linkage establishes the linkage relationship of target gene and molecular labeling.The automation followed by detected, due to
Molecular marker assisted selection requires to detect breeding population on a large scale, thus require the method for detection it is simple, it is quick, at
This is low, relatively more accurate, to realize the automatic of detection process (detection, data analysis of extraction, molecular labeling including DNA etc.)
Change.It has been found, however, that the molecular labeling used within a very long time in past, such as RFLP (Restriction
Fragment Length Polymorphism, restriction fragment length polymorphism), RAPD (random
Amplified polymorphic DNA, randomly amplified polymorphic DNA), AFLP and SSR etc. use after usually requiring digestion or PCR
Electrophoresis detection genotyping result is difficult to realize this point.
SNPs label (single nucleotide polymorphisms, single nucleotide polymorphism) is primarily referred to as in base
Because of DNA sequence polymorphism caused by a single nucleotide variation in group level.It is widely distributed in the genome, and quantity is many
It is more, therefore it is readily possible to detect the label more chain relative to RFLP, RAPD, AFLP, SSR marker, and there is height when detecting
Flux, simple, quickly, highly sensitive advantage, are to carry out label most potential in molecular mark.In recent years,
Frett et al. (2014) constructs the linkage map comprising 1335 SNPs using SNPs chip, in the 3rd Chromosome Identification to red
The main effect QTL (Blush.Pp.ZC-3.1) of skin character, most chain label be (Chr3:
12836182bp).Tuan et al. (2015) has found its etc. by synthesizing key gene PpMYB10 sequencing to peach pericarp anthocyanidin
There are 3 SNP between the gene of position, and by comparing the parting of this 3 SNPs, find Chr3 (the 3rd chromosome): 12877863bp is pre-
Survey accuracy rate with higher when fruit colour.
However, the SSR marker distance objective assignment of genes gene mapping distance of the existing and Peach fruits epidermis coloring linkage of characters is farther out,
Accuracy rate is lower in the early stage identification in generation after hybridization；And the existing SNPs label chain with objective trait reflects mostly from chip
It is fixed as a result, since chip site is less (being less than 9000), it cannot be guaranteed that the SNPs identified be with objective trait most
Association or chain site.
Summary of the invention
To solve the above-mentioned problems, the object of the present invention is to provide for identifying the monokaryon glycosides of Peach fruits epidermis coloring character
Sour polymorphism mark site, primer, kit and application, the marker site identify Peach fruits epidermis coloring character when have compared with
High accuracy rate.
To achieve the goals above, the technical scheme adopted by the invention is that:
For identifying the single nucleotide polymorphism site of Peach fruits epidermis coloring character, the mononucleotide polymorphic
Property marker site be the 3rd chromosome of peach genome the 3473250th nucleotide, the nucleotide be T or G.
For identifying the PCR amplification primer pair of Peach fruits epidermis coloring character, the upstream primer in the primer pair is root
It is designed according to the 3473250th nucleotide and its upstream sequence of the 3rd chromosome of peach genome, the downstream in the primer pair
Primer is designed according to the downstream sequence of the 3473250th nucleotide of the 3rd chromosome.
The primer pair is made of two single strand dnas as shown in SEQ ID NO.2 and SEQ ID NO.3.
For identifying that the Single base extension primer of Peach fruits epidermis coloring character, the Single base extension primer are such as SEQ
Nucleotide sequence shown in ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
For identifying that the kit of Peach fruits epidermis coloring character, the kit include the PCR amplification primer
To with the Single base extension primer.
Identification is being identified or assisted to the single nucleotide polymorphism of 3473250th nucleotide of the 3rd chromosome of peach genome
Peach fruits epidermis colours the application in character.
A kind of single nucleotide polymorphism site colours character molecular marker assisted selection breeding side in Peach fruits epidermis
The application in face.
Identifying or assisting answering in identification Peach fruits epidermis coloring character in the single nucleotide polymorphism site
With.
A kind of application of kit in terms of Peach fruits epidermis colours the breeding of character molecular marker assisted selection.
A method of character, including following step are coloured using single nucleotide polymorphism site identification Peach fruits epidermis
It is rapid:
(1) PCR amplification: using the genomic DNA of peach to be measured as template, carrying out PCR amplification with PCR amplification primer pair, obtains
Pcr amplification product；
(2) SAP reacts: preparing SAP reaction system, in the reaction system after step (1) pcr amplification reaction is added, removes
Unreacted dNTP in pcr amplification reaction；
(3) single base extension: preparing single base extension system, the reactant after step (2) SAP reaction is added
In system；
(4) Genotyping: carrying out resin desalting processing for the product for completing single base extension, put on chip, by
Chip scanner scanning, tests and analyzes, and carries out Genotyping according to the molecular size range of different products.
The present invention screens to obtain the 3473250th nucleotide position nucleotide polymorphisms markers of the 3rd chromosome of peach genome
Site can be used in identifying or assisting identification Peach fruits epidermis coloring (red) character, colour (red) property in identification Peach fruits epidermis
Accuracy rate with higher when shape.
The present invention devises specific for the characteristic of the 3473250th nucleotide polymorphisms of the 3rd chromosome of peach genome
PCR primer amplification to and Single base extension primer, will complete single base extension product carry out resin desalting processing,
Point is scanned on chip by chip scanner, carries out MALDI-TOF Mass Spectrometer Method, and Typer4.0 software analyzes experimental data, root
Its genotypic results is obtained according to the molecular size range of different products.
To 15 hybrid Populations, totally 221 parts of peach single plants to be measured carry out the verifying of SNPs accuracy rate to the present invention, the results showed that, this
Invention is to accuracy rate with higher in dominant phenotype (50% or less red coloration of pericarp), up to 61% or more.This explanation, utilizes
Mononucleotide marker site of the invention carry out detection have the advantages that it is simple, quick, at low cost, can be realized produce in advise greatly
The application of mould.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
The acquisition of 1 SNPs marker site of embodiment
129 part peach kinds of the present invention to be obtained at random from institute, Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy peach Germplasm Resources
Matter is sample, extracts sample DNA using conventional CTAB method, and by 2000 sequenator of Illumina HiSeq to 129 parts of peach kinds
Matter carries out resurveying sequence, obtains 121Gb data, averagely covering peach genome 89.28%, and average sequencing depth is 4.21 × left and right.
According to the reads for the 50-150bp that sequencing obtains, genome v.1.0 (http://www.rosaceae.org/ is referred to peach
Node/355 it) is compared, identifies 4063377 SNPs, carried out using phenotypic character of these SNPs to 129 parts of germplasm complete
Genome association analysis identifies the 3473250bp for having significant associated SNPs to be located at the 3rd chromosome with peach fruit coloration
Place.
The method that embodiment 2 colours (red) character using SNP marker identification Peach fruits epidermis
1, DNA is extracted
The DNA of peach sample tissue to be measured is extracted using conventional CTAB method, removes RNA, and DNA sample total volume is not less than 15 μ
l.With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm, DNA content and OD are calculated260/280Ratio.
DNA sample purity OD260/280Value should be between 1.8-2.0, concentration dilution to 10ng/ μ l.
2, design primer
According to each 200bp sequence (the specific nucleotide sequence in left and right at the 3473250th of the 3rd chromosome of peach genome
It is shown in Table 1), design primer.
1 SNPs flanking sequence information of table
Wherein, K indicates T or G.
After primer is synthesized by biotech company, dilution PCR amplification upstream and downstream primer to final concentration is 0.5 μM.Dilution
Single base extension primer to 1,2,3 final concentration of extension primer is respectively 8 μM, 10 μM, 15 μM, spare.Primer sequence is shown in Table 2.
2 primer sequence of table
3, PCR reaction system and Mass ARRAY analysis
PCR, SAP, single base extension are carried out according to SEQUENOM-iPLEX Standard Operating Procedure, key step is such as
Under:
(1) pcr amplification reaction
Firstly, preparing PCR amplification system, it is specifically shown in Table 3.
3 Sequenom MassArray system gene parting of table expands PCR reactive component
Wherein, Primer Mix is that 0.5 μ l is respectively added in PCR amplification upstream primer and downstream primer.
Mentioned reagent is transferred in PCR pipe or plate,
PCR amplification program is as follows: 94 DEG C of 15min；94 DEG C of 20s, 56 DEG C of 30s, 72 DEG C of 1min, totally 45 recycle；72℃
3min；4 DEG C of preservations.
1% Ago-Gel is configured, electrophoresis is carried out to the product after PCR reaction, if band is bright and single, is carried out
Follow-up test.
(2) SAP (shrimp alkaline phosphotase) reacts
Unexhausted dNTP is removed using the reaction, first preparation reaction system, be shown in Table 4:
4 SAP enzymatic reaction related reagent formulation components of table
The 2 μ l of above-mentioned solution prepared is added in the PCR pipe for completing PCR reaction or plate.It is anti-that SAP is carried out by following procedure
It answers.
SAP response procedures: 37 DEG C of 40min；85℃5min；4 DEG C of preservations.
(3) single base extension
Single base extension system is prepared first, is shown in Table 5:
5 extension related reagent component of table
Wherein, iPLEX Extend Primer Mix is the mix primer of Single base extension primer 1,2,3, primer 1,2,3
Addition concentration be respectively 8 μM, 10 μM, 15 μM, the additional amount of Single base extension primer 1,2,3 is identical, totally 0.94 μ l.
Then by the 2 μ l of solution prepared the PCR pipe being added to after SAP reaction or plate, extension is carried out, program is such as
Under: 94 DEG C of 30s；94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s, 5 circulations), totally 40 recycle；72℃3min；4 DEG C of preservations.
The product for completing extension is subjected to resin desalting processing, puts on chip, is scanned by chip scanner, into
Row MALDI-TOF Mass Spectrometer Method, Typer4.0 software analyze experimental data, obtain its base according to the molecular size range of different products
Because of genotyping result.When complete extension molecular weight of product be 6021 or so when, Chr3:3,473,250bp place for homozygosis position
Point, parting G/G, the phenotype of corresponding germplasm are 50% or more pericarp red coloration.When molecular weight is 6060.9 or so, Chr3:3,
It is homozygous site, parting T/T at 473,250bp；When molecular weight is about 6040.9, Chr3:3 is heterozygosis position at 473,250bp
Point, parting G/T；The corresponding germplasm phenotype of both partings of T/T and G/T is 50% or less red coloration of pericarp.
3 15 hybrid Populations of embodiment are verified using the Blind Test that fruit coloration association SNP marker carries out phenotypic character
1, the selection of experimental material
Using the conventional Peach cultivars planted in Zhengzhou fruit tree research institute resource garden as experimental material, therefrom chooses and investigated phenotype
15 hybrid Populations of character totally 221 parts of single plants, are specifically shown in Table 6.
Title and group size information of the table 6 for examination hybrid Population
2, the identification method of fruit coloration association SNP marker is utilized
Using the 3473250th of the 3rd chromosome of peach genome of the present invention as nucleotide polymorphisms markers site, to 15
Totally 221 parts of peach single plant fruit colorations carry out Blind Test identification, method of the specific identification method referring to embodiment 2 to hybrid Population.Together
When, using the peach site gene SNP s of existing external report as control, analysis is compared with the site SNPs of the invention.
3, genotyping result compares the predictive ability of phenotype in hybrid Population
From qualification result as can be seen that being associated what analysis obtained with about 9,000 SNPs of utilization of current external report
1 SNPs identified is sequenced in most chain SNPs (Chr3:12836182bp) and the existing candidate gene to fruit coloration character
(Chr3:12877863bp) it compares, the Chi-square test P value in the site SNPs of the present invention is minimum, i.e. conspicuousness highest (table 7).
The genotyping result and Chi-square test of the different fruit coloration associations of table 7 or chain SNPs in 15 hybrid Populations
It is comprehensively compared, accuracy rate is as shown in table 8, it can be seen that the present invention is in the phenotypic character prediction for carrying out hybrid Population
When, it is higher for dominant phenotype's accuracy rate, compared with other researchs reported recently, 61.29% is promoted to from 8.06%.
Phenotypic predictions ability (accuracy rate) of the SNPs of the present invention of table 8 after identifying 15 hybrid Populations compares
In conclusion the site SNPs of the invention can help to realize using peach seedling DNA early prediction peach into age consequence
Skin colours the purpose of character, and this method is obtained using resurveying whole-genome association in 4,000,000 or more the SNPs that sequence obtains
Most associated site is to realization, due to original SNPs substantial amounts, so that the connective marker relevance that ensure that is high.
Claims (6)
1. for identifying the SNP marker of Peach fruits epidermis coloring character, which is characterized in that its sequence such as SEQ ID NO.1
Shown, wherein K is T or G.
2. a kind of reagent for being used to identify Peach fruits epidermis coloring character based on the preparation of SNP marker described in claim 1
Box, which is characterized in that the kit includes PCR amplification primer pair and Single base extension primer；
The PCR amplification primer pair is made of two single strand dnas as shown in SEQ ID NO.2 and SEQ ID NO.3；
The Single base extension primer is the nucleotides sequence as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6
Column.
3. the application in identification Peach fruits epidermis coloring character is being identified or assisted to SNP marker described in claim 1.
4. SNP marker described in claim 1 is in terms of Peach fruits epidermis colours the breeding of character molecular marker assisted selection
Using.
5. a kind of kit as claimed in claim 2 is in terms of Peach fruits epidermis colours the breeding of character molecular marker assisted selection
Using.
6. a kind of method using the identification Peach fruits epidermis coloring character of SNP marker described in claim 1, feature exist
In, comprising the following steps:
(1) PCR amplification: using the genomic DNA of peach to be measured as template, PCR amplification is carried out with PCR amplification primer pair, PCR is obtained and expands
Increase production object；
(2) SAP reacts: SAP reaction system is prepared, in the reaction system after step (1) pcr amplification reaction is added；
(3) single base extension: utilizing Single base extension primer, prepares single base extension system, is added step (2)
In reaction system after SAP reaction；
(4) Genotyping: the product for completing single base extension is subjected to resin desalting processing, is put on chip, by chip
Scanner scanning tests and analyzes, and carries out Genotyping according to the molecular size range of different products；
The PCR amplification primer pair is made of two single strand dnas as shown in SEQ ID NO.2 and SEQ ID NO.3；
The Single base extension primer is the nucleotides sequence as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6
Column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610536612.1A CN105925721B (en) | 2016-07-09 | 2016-07-09 | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610536612.1A CN105925721B (en) | 2016-07-09 | 2016-07-09 | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105925721A CN105925721A (en) | 2016-09-07 |
| CN105925721B true CN105925721B (en) | 2019-05-24 |
Family
ID=56827230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610536612.1A Expired - Fee Related CN105925721B (en) | 2016-07-09 | 2016-07-09 | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105925721B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636081B (en) * | 2016-12-01 | 2019-06-11 | 上海市农业科学院 | One SNP marker relevant to peach gummosis resistance |
| CN107488659B (en) * | 2017-09-08 | 2019-12-24 | 华中农业大学 | Sequence related to red and yellow color characters of citrus peels and application thereof |
| CN107881256B (en) * | 2017-12-22 | 2020-11-13 | 中国农业科学院郑州果树研究所 | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying bitter kernel/sweet kernel characteristics of peach fruit |
| CN107893125B (en) * | 2017-12-22 | 2021-03-09 | 中国农业科学院郑州果树研究所 | Single nucleotide polymorphism marker locus, primer pair, kit and application for identifying peach blossom bell/rose type trait |
| CN107858447B (en) * | 2017-12-22 | 2021-02-05 | 中国农业科学院郑州果树研究所 | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying peach blossom single-petal/double-petal character |
| CN113265411A (en) * | 2021-05-28 | 2021-08-17 | 中国农业科学院郑州果树研究所 | Gene for controlling peach blossom/fruit peel color and application thereof |
| CN117248073A (en) * | 2023-11-04 | 2023-12-19 | 中国农业科学院郑州果树研究所 | KASP (KASP-related antigen) mark related to pure color gene of peach peel and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531724A (en) * | 2015-02-06 | 2015-04-22 | 中国科学院武汉植物园 | Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof |
-
2016
- 2016-07-09 CN CN201610536612.1A patent/CN105925721B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531724A (en) * | 2015-02-06 | 2015-04-22 | 中国科学院武汉植物园 | Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Mapping quantitative trait loci associated with blush in peach;Frett et al;《Tree Genetics & Genomes》;20140110;全文 * |
| The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype;Tuan et al;《BioMed Central Plant Biology》;20151118;第8-9页，第11页 * |
| 桃性状遗传评价和分子标记技术研究;俞明亮;《中国优秀博硕士学位论文全文数据库农业科技辑》;中国学术期刊（光盘版）电子杂志社;20050615(第2期);全文 * |
| 蟠桃新品种‘中蟠桃10号’的选育;陈昌文等;《果树学报》;20150331;第32卷(第2期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105925721A (en) | 2016-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105925721B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character | |
| CN106191240B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermal hair character | |
| CN106048042B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits color traits | |
| D’hoop et al. | Population structure and linkage disequilibrium unravelled in tetraploid potato | |
| CN112080582B (en) | KASP molecular marker closely linked with major QTL locus of wheat spike length and application thereof | |
| CN110438252B (en) | Molecular marker closely linked with male and female sex of spinach and application thereof | |
| CN112513298B (en) | Mononucleotide polymorphism marker set for distinguishing genetic background or variety of local chicken and application thereof | |
| CN107858447B (en) | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying peach blossom single-petal/double-petal character | |
| KR20180077873A (en) | SNP markers for selection of marker-assisted backcross in watermelon | |
| CN107881256A (en) | For identifying single nucleotide polymorphism site, primer pair, kit and the application of Peach fruits hardship benevolence/sweet tea benevolence character | |
| CN109609687B (en) | KASP marker primer combination for detecting watermelon fusarium wilt resistance and application thereof | |
| CN110205396B (en) | SNP marker closely linked with irregular stripe character of cucumber fruit and application thereof | |
| CN109280709A (en) | One kind molecular labeling relevant to pig growth and reproductive trait and application | |
| CN113249510B (en) | Method for identifying authenticity of lettuce hybrid and KASP primer combination used by method | |
| CN104789648B (en) | Identify molecular labeling and its application of the section haplotypes of rice CMS restoring genes Rf 1 | |
| CN108866233A (en) | For identifying marker site, primer pair, kit and application of the peach to disease-resistant/susceptible character of Meloidogyne incognita | |
| CN105695454B (en) | A kind of molecular labeling and its identification method for identifying sesame genie male sterile line | |
| CN104762298B (en) | A kind of rice seedling resistant gene of salt qST11 and its molecule labelling method | |
| CN109439788B (en) | KASP molecular marker closely linked with major gene locus of wheat plant height and application thereof | |
| CN114736979B (en) | Molecular marker closely linked with watermelon complete leaf shape gene ClLL and application thereof | |
| CN114480718B (en) | Primer group and detection kit for rice high temperature resistant genotyping based on KASP technology and application of primer group and detection kit | |
| CN108841983A (en) | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development | |
| CN105483281B (en) | It is a kind of to be used to identify glutinous No. 1 SNP marker of five firework of waxy corn Shanghai and its identification method | |
| CN107858448B (en) | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying peach pollen fertility character | |
| CN114480721A (en) | Method for identifying whether melon variety to be detected is thin-skin melon or thick-skin melon and special SNP primer combination thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into 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 |
Granted publication date: 20190524 |