CN107858448A - For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach flower powder fertility character - Google Patents
For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach flower powder fertility character Download PDFInfo
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Abstract
The invention discloses single nucleotide polymorphism site, primer pair, kit and the application for identifying peach flower powder fertility character, described single nucleotide polymorphism site is the 2 of the chromosome of peach genome the 6th, 116,368 nucleotides, the nucleotides are C or T.The present invention is compared by the sequencing to a large amount of peach kind quality samples, 4063377 SNPs are identified, whole-genome association are carried out to the powder fertility character of 129 parts of germplasm using these SNPs, identify has the SNP significantly associated to be located at the 2nd of the 6th chromosome with peach powder fertility, at 116,368bp.And specific PCR primer amplification pair and Single base extension primer are devised, the product for completing single base extension is handled and analyzed, its genotypic results is obtained according to the molecular size range of different products.Carrying out detection using the mononucleotide marker site of the present invention has the advantages of simple, quick, cost is low, can realize and be applied on a large scale in production.
Description
Technical field
The present invention relates to the single nucleotide polymorphism site for identifying peach flower powder fertility character, primer pair, reagent
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,
To carry out follow-up cultivation.But in traditional breeding method, due to being difficult the genotype for knowing offspring, therefore the foundation selected is typically
Phenotype and non genotype, this system of selection are usually effective for qualitative character, but for quantitative character, because
Lack clear and definite corresponding relation 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 phases of 3-5,
Thus the time of selection is later.This is for the crop that those plant are tall and big, occupation of land is more, Growing season is grown, particularly 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, so as to reach the purpose for improving breeding efficiency.Yamamoto
(2001) using AFLP (Amplified Fragment Length Polymorphism, AFLP),
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) mark, fruit colour is positioned into the 6th linkage group, therewith the SSR of linkage distance nearest (3.7cM)
Labeled as UDP96-015.There are two important premises due to carrying out molecular marker assisted selection, be that must obtain and target first
The mark of character close linkage, that is, establish the linkage relationship of target gene and molecular labeling.Next to that the automation of detection, due to
Molecular marker assisted selection requirement is detected on a large scale to breeding population, thus require detection method it is simple, quickly, into
This is low, relatively more accurate, to realize the automatic of detection process (including DNA extraction, the detection of molecular labeling, data analysis 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, it is difficult to realize this point.
SNPs marks (single nucleotide polymorphisms, SNP) are primarily referred to as in base
Because in group level by the DNA sequence polymorphism caused by the variation of single nucleotide acid.It is widely distributed on genome, and quantity is many
It is more, therefore be readily possible to detect relative to the more chain mark of RFLP, RAPD, AFLP, SSR marker, and there is height in detection
The advantages of flux, simple, quick, high sensitivity, be to carry out mark most potential in molecular mark.Chaparro
(1994) find that pollen fertility character there may be two genes, researcher is had found the 2 of the fertile germplasm White Glory of pollen
In individual hybrid Population, offspring's segregation ratio does not meet 3:1, i.e. the germplasm may contain the peach flower powder sterile gene with reporting before
(Ps) another gene Ps2 of equipotential, the latter and weeping branch and white flower character are chain.Dirlewanger (1998) is utilized
FerjalouJalousia × Fantasia F2Colony, Ps is positioned at the chromosome of peach the 6th, nearest RFLP is labeled as
FG40.Jun (2004) utilizes Yumyeong × Baekhyang F1Colony, point of pollen fertility using RAPD marker developments
Son mark.
Because pollen fertility character before marks labeled as RAPD, the mark can not be carried out in genome random distribution
Chromosome mapping, it is impossible to determine whether it is located at nearer physical distance with target gene.And RFLP marks before are due to behaviour
Make complex steps, seldom applied in the current technology.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide the mononucleotide for identifying peach flower powder fertility character
Polymorphism mark site, primer pair, kit and application, the marker site is when identifying peach flower powder fertility character with higher
Accuracy rate.
To achieve these goals, the technical solution adopted in the present invention is：
For identifying the single nucleotide polymorphism site of peach flower powder fertility character, described SNP mark
Note site is the 2nd, 116,368 nucleotides of the chromosome of peach genome the 6th, and the nucleotides is C or T.
Described peach flower powder fertility is that pollen is fertile or pollen sterility.
For identifying the pcr amplification primer thing pair of peach flower powder fertility character, the sense primer in the primer pair is according to peach
The 2nd, 116,368 nucleotides and its upstream sequence of the chromosome of genome the 6th are designed, and the downstream in the primer pair is drawn
Thing is designed according to the downstream sequence of the 2,116,368th nucleotides of the chromosome of peach genome the 6th.
The nucleotide sequence of described sense primer is as shown in SEQ ID NO.2, the nucleotide sequence such as SEQ of anti-sense primer
Shown in ID NO.3.
For identifying the Single base extension primer of peach flower powder fertility character, the 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 identifying the kit of peach flower powder fertility character, including pcr amplification primer thing pair and Single base extension primer.
A kind of application of single nucleotide polymorphism site in identifying or aiding in identification peach flower powder fertility character.
A kind of single nucleotide polymorphism site is in terms of peach flower powder fertility trait molecular marker assisted selection
Using.
A kind of application of kit in terms of assisted selection.
A kind of method that peach flower powder fertility character is identified using single nucleotide polymorphism site, is comprised the following steps：
(1) PCR is expanded：Using the genomic DNA of peach to be measured as template, expanded, obtained to entering performing PCR with pcr amplification primer thing
Pcr amplification product；
(2) SAP reacts：SAP reaction systems are prepared, adds into the reaction system after step (1) pcr amplification reaction, removes
Remove unreacted dNTP in pcr amplification reaction；
(3) single base extension：Single base extension system is prepared, is added to the reacted reactions of step (2) SAP
In system；
(4) Genotyping：The product for completing single base extension is subjected to resin desalting processing, put on chip, by
Chip scanner scans, detection and analysis, carries out Genotyping according to the molecular size range of different products, predicts peach flower powder fertility
Shape.
The present invention is compared by the sequencing to a large amount of peach kind quality samples, is identified 4063377 SNPs, is utilized these SNPs
Whole-genome association is carried out to the powder fertility character of 129 parts of germplasm, identify has the SNP significantly associated with peach flower powder fertility
At the 2,116,368bp of the 6th chromosome.
The present invention is directed to the characteristic of the 2nd, 116,368 nucleotide polymorphisms of the 6th chromosome of peach genome second edition,
Specific PCR primer amplification pair and Single base extension primer are devised, the product tree of single base extension will be completed
Fat desalting processing, put on chip, scanned by chip scanner, carry out MALDI-TOF Mass Spectrometer Methods, Typer4.0 software analysis
Experimental data, its genotypic results is obtained according to the molecular size range of different products.
The present invention carries out the checking of SNP accuracys rate to 201 parts of natural population's materials to be measured, the results showed that, the present invention is to aobvious
Property phenotype (pollen is fertile) in there is higher accuracy rate, up to 97.79% or so；To accurate in recessive phenotype's (pollen sterility)
Rate can also reach 60% or so.This explanation, detection is carried out with simple, fast using the mononucleotide marker site of the present invention
Speed, the advantages of cost is low, it can realize and be applied on a large scale in production.
Embodiment
The embodiment of the present invention is described in further detail with reference to embodiments.
The acquisition of the SNPs marker sites of embodiment 1
129 part peach kinds of the present invention to be obtained at random from institute of Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy peach Germplasm Resources
Matter is sample, and sample DNA is extracted using conventional CTAB methods, and by the sequenators of Illumina HiSeq 2000 to 129 parts of peach kinds
Matter carries out resurveying sequence, obtains 121Gb data, averagely covers peach genome 89.28%, average sequencing depth is 4.21 × left and right.
The 50-150bp obtained according to sequencing reads, with peach reference gene group second edition (http://www.rosaceae.org/
Node/355) it is compared, identifies 4063377 SNPs.Pollen fertility character based on above-mentioned SNPs to 129 parts of germplasm
Whole-genome association is carried out, identifies and is located at the 2nd of the 6th chromosome with the notable association SNP of peach flower powder fertility character,
At 116,368bp.
The method that embodiment 2 identifies peach flower powder fertility character using SNP marker
1st, DNA is extracted
The DNA of peach sample tissue to be measured is extracted using conventional CTAB methods, removes RNA, DNA sample cumulative 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.
2nd, primer is designed
According to each 150bp sequences (the specific core in left and right at the 2,116,368th of the 6th chromosome of peach genome second edition
1) nucleotide sequence is shown in Table, design primer.
The SNP flanking sequence information of table 1
Wherein, Y represents C or T.
After primer is synthesized by biotech company, dilution PCR amplification upstream and downstream primer to concentration is 0.5 μM.Dilution is single
Base extension primer to the concentration of extension primer 1,2,3 is respectively 8 μM, 10 μM, 15 μM, standby.Primer sequence is shown in Table 2.
The primer sequence of table 2
3rd, PCR reaction systems and Mass ARRAY analyses
Enter performing PCR, SAP, single base extension according to SEQUENOM-iPLEX Standard Operating Procedure, key step is such as
Under：
(1) pcr amplification reaction
First, PCR amplification system is prepared, is specifically shown in Table 3.
The Sequenom MassArray system genes parting of table 3 expands PCR reaction systems
Wherein, Primer Mix are that the sense primer of 0.5 μM of concentration and anti-sense primer respectively add 0.5 μ l.
Mentioned reagent is transferred in PCR pipe or plate,
PCR amplification programs are as follows：94℃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.
1% Ago-Gel is configured, electrophoresis is carried out to the reacted products of PCR, if band is bright and single, carried out
Follow-up test.
(2) SAP reacts
Unexhausted dNTP is removed using the reaction, reaction system is prepared first, is shown in Table 4：
The SAP enzymatic reaction systems of table 4
The μ l of above-mentioned solution 2 prepared are added in the PCR pipe for completing PCR reactions or plate.It is anti-that SAP is carried out by following program
Should.
SAP response procedures：37℃40min；85℃5min；4 DEG C of preservations.
(3) single base extension
Single base extension system is prepared first, is shown in Table 5：
The single base extension system of table 5
Wherein, iPLEX Extend Primer Mix be Single base extension primer 1,2,3 mix primer, primer 1,2,3
Concentration be respectively 8 μM, 10 μM, 15 μM, the addition of Single base extension primer 1,2,3 is identical, totally 0.94 μ l.
Then the μ l of solution 2 prepared are added in the reacted PCR pipes of SAP or plate, carry out extension, program is such as
Under：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.
The product for completing extension is subjected to resin desalting processing, puts on chip, is scanned by chip scanner, entered
Row MALDI-TOF Mass Spectrometer Methods, Typer4.0 software analysis experimental datas, its base is obtained according to the molecular size range of different products
Because of genotyping result.When the molecular weight of product for completing extension is 6000.9 or so, Chr6：It is homozygosis at 2,116,368bp
Site, parting C/C, the pollen fertility phenotype of corresponding germplasm is infertility.When molecular weight is 6080.9 or so, Chr6：2,116,
It is homozygous site at 368bp, parting T/T；When molecular weight is 6040.9 or so, Chr6：It is heterozygosis position at 2,116,368bp
Point, parting C/T；Germplasm pollen fertility phenotype is fertile corresponding to both partings of T/T and C/T.
The Blind Test that the Fen Tao natural populations of embodiment 3 201 carry out phenotypic character using pollen fertility association SNP marker is verified
1st, the selection of experiment material
Using the 201 parts of common peach germplasm natural populations preserved in Zhengzhou fruit tree research institute resource garden as experiment material, wherein
20 parts, fertile 181 parts of infertility.
2nd, the authentication method of SNP marker is associated using pollen fertility
It is right with the 2nd, 116,368 of the chromosome of peach genome the 6th of the present invention as nucleotide polymorphisms markers site
201 parts of germplasm carry out Blind Test identification, method of the specific authentication method with reference to embodiment 2.
3rd, predictive ability (table 6) of the genotyping result to phenotype in natural population
From qualification result as can be seen that in 181 parts of fertile germplasm of pollen, only 4 partings are C/C, with expected results
It is not inconsistent, other are T/T or C/T partings, consistent with expected results, and the fertile phenotype accuracy rate of pollen is 97.79%；20
In part pollen sterility germplasm, it is C/C to have 12 partings, consistent with expected results, but it is T/T to have 7 partings, and 1 parting is C/
T, inconsistent with expected results, the phenotype accuracy rate of pollen sterility is 60%.
The pollen fertility of table 6 associates or chain SNP genotyping results on 201 parts of natural population's germplasm
In summary, SNP site of the invention can help to realize using peach seedling DNA early predictions peach into pollen after age
Fertility character purpose, this method is using resurveying more than 4,000,000 SNPs that sequence obtains, and combines whole-genome association
The site most associated is so as to realization, due to original SNPs substantial amounts, so as to ensure that obtained connective marker association
Property it is high, effectively increase the accuracy predicted using SNPs.
The optimal embodiment of the present invention is the foregoing is only, for those skilled in the art, the present invention can have
Various modifications and variations.Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., all should
Within protection scope of the present invention.
Sequence table
<110>Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120>For identifying single nucleotide polymorphism site, primer pair, kit and the application of peach flower powder fertility character
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ctaatagact tcttctgatt cttcaaggac ytgctaaggt caaatagtga aatgccactg 180
tgaaagaaat gatccctgtg aatatttcca ttgtggaaga agtgctaatt aaaaaatcac 240
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Claims (10)
1. the single nucleotide polymorphism site for identifying peach flower powder fertility character, it is characterised in that described monokaryon glycosides
Sour polymorphism mark site is the 2nd, 116,368 nucleotides of the chromosome of peach genome the 6th, and the nucleotides is C or T.
2. the single nucleotide polymorphism site according to claim 1 for being used to identify peach flower powder fertility character, it is special
Sign is, described peach flower powder fertility is that pollen is fertile or pollen sterility.
3. the pcr amplification primer thing pair for identifying peach flower powder fertility character, it is characterised in that the sense primer in the primer pair
It is to be designed according to the 2nd, 116,368 nucleotides and its upstream sequence of the chromosome of peach genome the 6th, in the primer pair
Anti-sense primer be to be designed according to the downstream sequence of the 2,116,368th nucleotides of the chromosome of peach genome the 6th.
4. the pcr amplification primer thing pair according to claim 3 for being used to identify peach flower powder fertility character, it is characterised in that institute
The nucleotide sequence for the sense primer stated is as shown in SEQ ID NO.2, the nucleotide sequence such as SEQ ID NO.3 institutes of anti-sense primer
Show.
5. for identifying the Single base extension primer of peach flower powder fertility character, it is characterised in that the Single base extension primer is
Nucleotide sequence as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
6. the kit for identifying peach flower powder fertility character, it is characterised in that described kit includes claim 3 or 4
Single base extension primer described in described pcr amplification primer thing pair and claim 5.
7. identification peach flower powder fertility is being identified or aided in a kind of single nucleotide polymorphism site as claimed in claim 1
Application in shape.
8. a kind of single nucleotide polymorphism site as claimed in claim 1 aids in peach flower powder fertility trait molecular marker
Application in terms of selection and use.
A kind of 9. application of the kit in terms of assisted selection as claimed in claim 6.
10. a kind of method that peach flower powder fertility character is identified using single nucleotide polymorphism site described in claim 1, its
It is characterised by, comprises the following steps：
(1) PCR is expanded：Using the genomic DNA of peach to be measured as template, expanded with pcr amplification primer thing to entering performing PCR, obtain PCR and expand
Increase production thing；
(2) SAP reacts：SAP reaction systems are prepared, are added into the reaction system after step (1) pcr amplification reaction, remove PCR
Unreacted dNTP in amplified reaction；
(3) single base extension：Single base extension system is prepared, is added to the reacted reaction systems of step (2) SAP
In；
(4) Genotyping：The product for completing single base extension is subjected to resin desalting processing, put on chip, by chip
Scanner scanning, detection and analysis, Genotyping is carried out according to the molecular size range of different products, predicts peach flower powder fertility character.
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| CN117248072A (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) marker related to fertility gene of peach pollen and application of KASP marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117248072A (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) marker related to fertility gene of peach pollen and application of KASP marker |
| CN117248072B (en) * | 2023-11-04 | 2024-08-27 | 中国农业科学院郑州果树研究所 | KASP (KASP-related antigen) marker related to fertility gene of peach pollen and application of KASP marker |
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