CN105925721A - Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application - Google Patents
Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application Download PDFInfo
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- 238000004040 coloring Methods 0.000 title claims abstract description 31
- 239000003550 marker Substances 0.000 title claims abstract description 17
- 240000006413 Prunus persica var. persica Species 0.000 title 1
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- 244000144730 Amygdalus persica Species 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 235000013399 edible fruits Nutrition 0.000 claims description 42
- 240000005809 Prunus persica Species 0.000 claims description 27
- 238000012408 PCR amplification Methods 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 15
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Abstract
The invention discloses a single nucleotide polymorphism marker site, a primer and a kit for identifying the coloring property of peach epidermis and application. The single nucleotide polymorphism marker site is the 3473250th nucleotide of a third chromosome of a peach genome, and the nucleotide is T or G. The 3473250bp of the third chromosome of the peach genome is taken as a nucleotide polymorphism maker site which can be used for identification or assisting identification of the coloring property of peach epidermis, and is relatively high in accuracy rate when identifying the coloring property of peach epidermis. The SNPs accuracy rates of 221 single peach plants of 15 hybridized colonies to be tested are verified, the result shows that when the phenotypic characters of the hybridized colonies are predicted, and the accuracy rates of dominant phenotypes are relatively high, that is, the accuracy rates are increased to 61.29% from 8.06% when being compared with those of other research reported lately. The results show that when being used for detection, the single nucleotide polymorphism marker site disclosed by the invention has the advantages of simplicity, rapidness and low cost, and large-scale application on practical production can be achieved.
Description
Technical field
The present invention relates to for identifying that Peach fruits epidermis colours the single nucleotide polymorphism site of (red) character, primer, examination
Agent box and application, belong to biological technical field.
Background technology
Selection is one of most important link in breeding, and it refers to select satisfactory genotype in a colony, carries out
Follow-up cultivation.But in traditional breeding method, owing to being difficult to know the genotype of offspring, the foundation typically Phenotype therefore selected
And non genotype, this system of selection is usually effective for qualitative trait, but for quantitative trait, because its performance
Clear and definite corresponding relation is lacked between type and genotype, thus inefficient.Additionally, for the fruit tree with fruit properties as target
For, these character have its specific performance period, it usually needs spend the 3-5 virgin phase even more for a long time, thus choosing
The time selected is later.This is tall and big for those plant, occupation of land is many, the gardening of the crop of Growing season length, particularly fruit tree etc is made
Thing, it is clear that be the most disadvantageous.
The molecular marking technique based on DNA developed rapidly over nearly 20 years, i.e. " molecular marker assisted selection "
(marker-assisted selection, be abbreviated as MAS) provides brand-new approach to breeding.It is by analyzing and genes of interest
The genotype of closely linked molecular marker carries out breeding, thus reaches to improve the purpose of breeding efficiency.Yamamoto(2001)
Utilize AFLP (Amplified Fragment Length Polymorphism, amplified fragment length polymorphism), RAPD (random
Amplified polymorphic DNA, randomly amplified polymorphic DNA), SSR (Simple Sequence Repeats, simply
Repetitive sequence) and RFLP (Restriction Fragment Length Polymorphism, restricted enzyme fragment length is polymorphic
Property) labelling, fruit colour is positioned the 6th linkage group, the SSR marker of linkage distance (3.7cM) recently is UDP96-015 therewith.
There are two important premises owing to carrying out molecular marker assisted selection, are first to obtain labelling closely linked with objective trait,
I.e. set up the linkage relationship of target gene and molecular marker.Next to that the automatization of detection, due to molecular marker assisted selection requirement
Breeding population is detected on a large scale, thus require the method for detection want simple, quickly, low cost, comparison accurate, with reality
Now detect the automatization of process (including the extraction of DNA, the detection of molecular marker, data analysis etc.).However, it is possible to send out
The molecular marker used in a very long time the most in the 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. typically require after enzyme action or PCR by electrophoresis detection and genotyping result, very
Difficulty realizes this point.
SNPs labelling (single nucleotide polymorphisms, single nucleotide polymorphism) is primarily referred to as in genomic level
On by the DNA sequence polymorphism caused by the variation of single core thuja acid.It is widely distributed on genome, large number of, because of
This is readily possible to detect the labelling more chain relative to RFLP, RAPD, AFLP, SSR marker, and has height when detection
Flux, advantage simple, quick, highly sensitive, be by labelling most potential in molecular mark.In recent years,
Frett et al. (2014) utilizes SNPs chip to construct the linkage map comprising 1335 SNPs, at the 3rd Chromosome Identification
To the main effect QTL (Blush.Pp.ZC-3.1) of Folium Styracis Suberifoliae character, the most chain is labeled as SNP_IGA_341962 (Chr3:12836182
bp).Tuan et al. (2015), by checking order peach fruit ginned cotton pigment synthesis key gene PpMYB10, finds its equipotential base
There are 3 SNP between Yin, by comparing the typing of these 3 SNPs, find Chr3 (the 3rd chromosome): 12877863bp
When predicting fruit colour, there is higher accuracy rate.
But, existing distant, miscellaneous with the SSR marker distance objective gene mapping of the Peach fruits epidermis coloring linkage of characters
Hand over accuracy rate in the early stage qualification of offspring relatively low；And the existing SNPs labelling chain with objective trait is many identifies from chip
As a result, due to chip site less (less than 9000), therefore it cannot be guaranteed that the SNPs identified is to close most with objective trait
Connection or chain site.
Summary of the invention
In order to solve the problems referred to above, it is an object of the invention to provide the mononucleotide polymorphic for identifying Peach fruits epidermis coloring character
Property marker site, primer, test kit and application, this marker site has higher standard when identifying Peach fruits epidermis coloring character
Really rate.
To achieve these goals, the technical solution adopted in the present invention is:
For identifying the single nucleotide polymorphism site of Peach fruits epidermis coloring character, described single nucleotide polymorphism
Site is the 3473250th nucleotide of Fructus Persicae genome the 3rd chromosome, and this nucleotide is T or G.
For identifying the pcr amplification primer thing pair of Peach fruits epidermis coloring character, the forward primer of described primer centering is according to Fructus Persicae base
Because the 3473250th nucleotide and the upstream sequence thereof of group the 3rd chromosome are designed, the downstream primer of described primer centering is
The downstream sequence of the 3473250th nucleotide according to the 3rd chromosome is designed.
Described primer forms by two single strand dnas as shown in SEQ ID NO.2 and SEQ ID NO.3.
For identifying the Single base extension primer of Peach fruits epidermis coloring character, described Single base extension primer is such as SEQ ID
Nucleotide sequence shown in NO.4, SEQ ID NO.5 and SEQ ID NO.6.
For identify Peach fruits epidermis coloring character test kit, described test kit include described pcr amplification primer thing to and institute
The Single base extension primer stated.
The single nucleotide polymorphism of the 3473250th nucleotide of Fructus Persicae genome the 3rd chromosome is being identified or auxiliary qualification Peach fruits
Application in epidermis coloring character.
A kind of single nucleotide polymorphism site answering in terms of the coloring character molecular marker assisted selection breeding of Peach fruits epidermis
With.
Described single nucleotide polymorphism site is in the application identified or in auxiliary qualification Peach fruits epidermis coloring character.
The application in terms of the coloring character molecular marker assisted selection breeding of Peach fruits epidermis of a kind of test kit.
A kind of method utilizing single nucleotide polymorphism site to identify Peach fruits epidermis coloring character, comprises the following steps:
(1) PCR amplification: with the genomic DNA of Fructus Persicae to be measured as template, with pcr amplification primer thing to carrying out PCR amplification,
Obtain pcr amplification product；
(2) SAP reaction: preparation SAP reaction system, adds in the reaction system after step (1) pcr amplification reaction, removes
Remove unreacted dNTP in pcr amplification reaction；
(3) single base extension: preparation single base extension system, adds the reacted reactant of step (2) SAP
In system；
(4) gene type: the product completing single base extension carries out resin desalting processing, and point is on chip, by chip
Scanner scanning, detection is analyzed, and carries out gene type according to the molecular size range of different products.
Present invention screening obtains the nucleotide polymorphisms markers site, the 3473250th nucleotide position of Fructus Persicae genome the 3rd chromosome,
Can be used in identifying or auxiliary qualification Peach fruits epidermis coloring (red) character, when identifying Peach fruits epidermis coloring (red) character
There is higher accuracy rate.
The present invention is directed to the characteristic of the 3473250th nucleotide polymorphisms of Fructus Persicae genome the 3rd chromosome, devise specific PCR
Primer amplification to and Single base extension primer, by complete single base extension product carry out resin desalting processing, point is at core
On sheet, chip scanner scan, carry out MALDI-TOF Mass Spectrometer Method, Typer4.0 software analysis experimental data, according to
The molecular size range of different products obtains its genotypic results.
The present invention carries out the checking of SNPs accuracy rate to 15 hybrid Populations totally 221 parts of Fructus Persicae individual plants to be measured, and result shows, this
Bright have higher accuracy rate, up to more than 61% in dominant phenotype's (peel less than 50% red coloration).This explanation, utilizes
The mononucleotide marker site of the present invention carries out detecting and has simple, quick, the advantage of low cost, it is possible to advise greatly in realizing producing
The application of mould.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.
The acquisition of embodiment 1 SNPs marker site
The present invention is with 129 parts of Fructus Persicae kind matter obtaining at random from Zhengzhou fruit tree research institute of Chinese Academy of Agricultural Sciences Fructus Persicae Germplasm Resources as sample
Product, are used conventional CTAB method to extract sample DNA, and are entered 129 parts of Fructus Persicae kind matter by Illumina HiSeq 2000 sequenator
Row is resurveyed sequence, obtains 121Gb data, the average Fructus Persicae genome 89.28% that covers, and the average order-checking degree of depth is 4.21 × left and right.According to
The reads of the 50-150bp that order-checking obtains, enters with reference to genome v.1.0 (http://www.rosaceae.org/node/355) with Fructus Persicae
Row comparison, identifies 4063377 SNPs, utilizes these SNPs that the phenotypic character of 129 parts of kind matter is carried out full-length genome pass
Connection is analyzed, and identifies the SNPs significantly associated that has with Fructus Persicae fruit coloration and is positioned at the 3473250bp of the 3rd chromosome.
Embodiment 2 utilizes the method that SNP marker identifies Peach fruits epidermis coloring (red) character
1, DNA extraction
Using conventional CTAB method to extract the DNA of Fructus Persicae sample tissue to be measured, remove RNA, DNA sample cumulative volume is not less than 15 μ l.
By ultraviolet light photometric determination DNA sample OD value at 260nm, 280nm, calculate DNA content and OD260/280's
Ratio.DNA sample purity OD260/280Value should be between 1.8-2.0, concentration dilution to 10ng/ μ l.
2, design primer
At according to Fructus Persicae genome the 3rd chromosome the 3473250th, (concrete nucleotide sequence is shown in Table left and right each 200bp sequence
1), design primer.
Table 1SNPs flanking sequence information
Wherein, K represents T or G.
After primer is synthesized by biotech company, dilution PCR amplification upstream and downstream primer to final concentration is 0.5 μM.The single alkali of dilution
Base extends primer and is respectively 8 μMs, 10 μMs, 15 μMs to extending primer 1,2,3 final concentration, standby.Primer sequence is shown in Table 2.
Table 2 primer sequence
3, PCR reaction system and Mass ARRAY analyze
Carrying out PCR, SAP, single base extension according to SEQUENOM-iPLEX Standard Operating Procedure, key step is such as
Under:
(1) pcr amplification reaction
First, prepare PCR amplification system, be specifically shown in Table 3.
Table 3Sequenom MassArray system gene typing amplification PCR reactive component
Wherein, Primer Mix is that PCR expands forward primer and downstream primer respectively adds 0.5 μ l.
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 circulations；72℃3min；
4 DEG C of preservations.
Configuring 1% agarose gel, product reacted to PCR carries out electrophoresis, if band is bright and single, then after carrying out
Continuous test.
(2) SAP (shrimp alkaline phosphotase) reaction
Utilize this reaction to remove unexhausted dNTP, first prepare reaction system, be shown in Table 4:
Table 4SAP enzymatic reaction related reagent formulation components
The above-mentioned solution 2 μ l prepared is joined in PCR pipe or the plate of PCR reaction.SAP is carried out anti-by following program
Should.
SAP response procedures: 37 DEG C of 40min；85℃5min；4 DEG C of preservations.
(3) single base extension
First preparation single base extension system, is shown in Table 5:
Table 5 extension related reagent component
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 μMs, 10 μMs, 15 μMs, the addition of Single base extension primer 1,2,3 is identical, totally 0.94 μ l.
Then being joined in the reacted PCR pipe of SAP or plate by the solution 2 μ l prepared, carry out extension, program is as follows:
94℃30s；94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s, 5 circulations), totally 40 circulations；72℃3min；4 DEG C of preservations.
By complete extension product carry out resin desalting processing, point, on chip, is scanned by chip scanner, carries out
MALDI-TOF Mass Spectrometer Method, Typer4.0 software analysis experimental data, obtain its base according to the molecular size range of different products
Because of genotyping result.When the molecular weight of product completing extension is about 6021, Chr3:3, for position of isozygotying at 473,250bp
Point, typing is G/G, and the corresponding phenotype planting matter is more than 50% peel red coloration.When molecular weight is about 6060.9, Chr3:
For site of isozygotying at 3,473,250bp, typing is T/T；When molecular weight is about 6040.9, Chr3:3, is miscellaneous at 473,250bp
Closing site, typing is G/T；The kind matter phenotype that T/T with G/T both typings are corresponding is peel less than 50% red coloration.
3 15 hybrid Populations of embodiment utilize fruit coloration association SNP marker to carry out the Blind Test checking of phenotypic character
1, the selection of experiment material
In Zhengzhou fruit tree research institute resource garden, the conventional Peach cultivars of plantation is as experiment material, therefrom chooses and investigated phenotypic character
15 hybrid Population totally 221 parts of individual plants, are specifically shown in Table 6.
Table 6 is for trying title and the group size information of hybrid Population
2, the authentication method of fruit coloration association SNP marker is utilized
Using the 3473250th of Fructus Persicae genome the 3rd chromosome of the present invention as nucleotide polymorphisms markers site, to 15 hybridization
Colony's totally 221 parts of Fructus Persicae individual plant fruit colorations carry out Blind Test qualification, and concrete authentication method is with reference to the method for embodiment 2.Meanwhile,
Using the Fructus Persicae gene SNP s site of existing external report as comparison, compare analysis with the SNPs site of the present invention.
3, in hybrid Population, the predictive ability of phenotype is compared by genotyping result
From qualification result it can be seen that be associated analyzing connecting most of obtaining with about 9,000 SNPs of utilization of at present external report
Lock SNPs (Chr3:12836182bp) and existing 1 SNPs that the candidate gene order-checking of fruit coloration character is identified
(Chr3:12877863bp) comparing, the Chi-square test P value in SNPs site of the present invention is minimum, i.e. significance the highest (table 7).
Table 7 different fruit coloration association or chain SNPs are in the genotyping result of 15 hybrid Populations and Chi-square test
Integrated comparative, its accuracy rate is as shown in table 8, it can be seen that the present invention carry out hybrid Population phenotypic character predict time,
Higher for dominant phenotype's accuracy rate, compared with other researchs of report recently, it is promoted to 61.29% from 8.06%.
The SNPs that table 8 the present invention relates to phenotypic predictions ability (accuracy rate) after identifying 15 hybrid Populations compares
In sum, after the SNPs site of the present invention can help realization to utilize Fructus Persicae seedling DNA early prediction Fructus Persicae to become age, peel
The purpose of color character, the method is to utilize in more than 4,000,000 SNPs that sequence obtains that resurvey whole-genome association to be closed most
The site of connection thus realize, due to original SNPs substantial amounts, thus ensure that the connective marker relatedness obtained is high.
Claims (10)
1. for identifying the single nucleotide polymorphism site of Peach fruits epidermis coloring character, it is characterised in that described monokaryon
Nucleotide polymorphism marker site is the 3473250th nucleotide of Fructus Persicae genome the 3rd chromosome, and this nucleotide is T or G.
2. for identifying the pcr amplification primer thing pair of Peach fruits epidermis coloring character, it is characterised in that described primer centering upper
Trip primer is the 3473250th nucleotide according to Fructus Persicae genome the 3rd chromosome and upstream sequence is designed, described primer
The downstream primer of centering is that the downstream sequence of the 3473250th nucleotide according to the 3rd chromosome is designed.
Pcr amplification primer thing pair for identifying Peach fruits epidermis coloring character the most according to claim 1, its feature exists
In, described primer forms by two single strand dnas as shown in SEQ ID NO.2 and SEQ ID NO.3.
4. for identifying the Single base extension primer of Peach fruits epidermis coloring character, it is characterised in that described Single base extension primer
For the nucleotide sequence as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
5. for identifying the test kit of Peach fruits epidermis coloring character, it is characterised in that described test kit includes claim 2
Or the pcr amplification primer thing described in 3 to and claim 4 described in Single base extension primer.
6. the single nucleotide polymorphism of the 3473250th nucleotide of Fructus Persicae genome the 3rd chromosome is being identified or auxiliary qualification peach fruit
Application in real epidermis coloring character.
7. the single nucleotide polymorphism site described in a claim 1 is in Peach fruits epidermis coloring trait molecular marker auxiliary
Application in terms of selection and use.
Single nucleotide polymorphism site the most according to claim 7 is in Peach fruits epidermis coloring trait molecular marker auxiliary
Application in terms of selection and use, it is characterised in that described single nucleotide polymorphism site is being identified or auxiliary qualification peach fruit
Application in real epidermis coloring character.
9. the answering in terms of the coloring character molecular marker assisted selection breeding of Peach fruits epidermis of the test kit described in a claim 5
With.
10. the side of Peach fruits epidermis coloring character is identified in the single nucleotide polymorphism site that a kind utilizes described in claim 1
Method, it is characterised in that comprise the following steps:
(1) PCR amplification: with the genomic DNA of Fructus Persicae to be measured as template, with pcr amplification primer thing to carrying out PCR amplification,
Obtain pcr amplification product；
(2) SAP reaction: preparation SAP reaction system, adds in the reaction system after step (1) pcr amplification reaction；
(3) single base extension: preparation single base extension system, adds the reacted reactant of step (2) SAP
In system；
(4) gene type: the product completing single base extension carries out resin desalting processing, and point is on chip, by chip
Scanner scanning, detection is analyzed, and carries out gene type according to the molecular size range of different products.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636081A (en) * | 2016-12-01 | 2017-05-10 | 上海市农业科学院 | SNP molecular marker relating to peach tree bleeding disease resistance |
| CN107488659A (en) * | 2017-09-08 | 2017-12-19 | 华中农业大学 | A kind of sequence related to orange peel reddish yellow color trait and its application |
| CN107858447A (en) * | 2017-12-22 | 2018-03-30 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach blossom single-lobe/polyphyll character |
| CN107881256A (en) * | 2017-12-22 | 2018-04-06 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of Peach fruits hardship benevolence/sweet tea benevolence character |
| CN107893125A (en) * | 2017-12-22 | 2018-04-10 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach blossom bell type/rose type 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 |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636081A (en) * | 2016-12-01 | 2017-05-10 | 上海市农业科学院 | SNP molecular marker relating to peach tree bleeding disease resistance |
| CN106636081B (en) * | 2016-12-01 | 2019-06-11 | 上海市农业科学院 | One SNP marker relevant to peach gummosis resistance |
| CN107488659A (en) * | 2017-09-08 | 2017-12-19 | 华中农业大学 | A kind of sequence related to orange peel reddish yellow color trait and its application |
| 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 |
| CN107858447A (en) * | 2017-12-22 | 2018-03-30 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach blossom single-lobe/polyphyll character |
| CN107881256A (en) * | 2017-12-22 | 2018-04-06 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of Peach fruits hardship benevolence/sweet tea benevolence character |
| CN107893125A (en) * | 2017-12-22 | 2018-04-10 | 中国农业科学院郑州果树研究所 | For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach blossom bell type/rose type character |
| 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 |
| 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 |
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