CN109338002A - One kind SNP marker relevant to watermelon pericarp background color and its application - Google Patents
One kind SNP marker relevant to watermelon pericarp background color and its application Download PDFInfo
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- CN109338002A CN109338002A CN201811338549.6A CN201811338549A CN109338002A CN 109338002 A CN109338002 A CN 109338002A CN 201811338549 A CN201811338549 A CN 201811338549A CN 109338002 A CN109338002 A CN 109338002A
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- watermelon
- background color
- pericarp background
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- pericarp
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- 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
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- 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
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- 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
Abstract
The invention discloses a kind of SNP marker relevant to watermelon pericarp background color and its applications.The SNP site is located at the 29880100th nucleotide of No. 8 chromosome of watermelon genome, which is C or G.The nucleotide polymorphisms markers site that the present invention obtains can be used in identifying watermelon pericarp background color.SNP marker of the invention is the SNP marker with watermelon pericarp background color close linkage that developed based on candidate gene.The identification of pericarp background color is carried out to 105 parts of Watermelon Germplasms using the SNP marker, its accuracy rate identified the watermelon of blackish green pericarp background color and green pericarp background color is 100% as the result is shown.The present invention needs not move through the identification of Watermelon Fruit maturity period, can quickly and accurately identify watermelon pericarp background color in seedling stage, and breeding efficiency can be improved, and shortens qualification time, Genetic identification and breeding suitable for watermelon.
Description
Technical field
The invention belongs to field of biotechnology, are related to one kind SNP marker relevant to watermelon pericarp background color and its answer
With.
Technical background
In traditional selection and use, because it is difficult to determine that the genotype of offspring, the foundation of selection are usually the table of plant
Show type rather than genotype, selects the time longer, and influence of the phenotype vulnerable to environmental factor, lead to the inaccuracy and effect of selection
Rate is lower.Molecular mark screens objective trait with being labeled as tool with target shape compact linkage molecule,
The screening for carrying out germ plasm resource with genotype by molecular labeling has the advantages that not interfered by environmental condition accurately, quickly,
The blindness for avoiding character determination during traditional breeding method, improves breeding efficiency.
SNP marker (single nucleotide polymorphisms, single nucleotide polymorphism) refers in gene
The change of the DNA sequence dna due to caused by the mutation of single nucleotide acid in group level.It widely exists on genome, there is height
Flux, simple, stabilization, sensitivity height etc. are specific, are the molecules of great development prospect in current molecular mark work
Label.Watermelon is a kind of important garden crop, and China is the big watermelon production and consumption big country of the first in the world, however in watermelon
In breeding process, available molecular labeling is less, and still based on traditional selection and use, efficiency is lower, it is difficult to be quickly obtained full
The new varieties of the sufficient market demand.Watermelon pericarp background color is the important appearance of watermelon and commodity property, in generation 30 or 40 years in last century
Just there is researcher to study this character, however there are most common green correlated traits as in watermelon pericarp background color
(green and blackish green), there is not yet having the research report in the molecular labeling of this character close linkage, therefore there are demand developments
The research of watermelon pericarp background color, exploitation can be used for the molecular labeling of watermelon breeding, improve breeding efficiency to shorten breeding process.
Summary of the invention
To solve the problems, such as to encounter during above-mentioned watermelon breeding, the present invention provides a kind of related to watermelon pericarp background color
SNP marker position.The present invention is the SNP molecule with watermelon pericarp background color close linkage that developed based on candidate gene
Label, can quickly and accurately identify watermelon pericarp background color using the SNP marker in seedling stage, amplification big with flux
The advantage that product is stable, detection is quick, can be improved the efficiency of watermelon pericarp background color selection and use.
The technical scheme to solve the above technical problems is that
A kind of SNP marker relevant to watermelon pericarp background color, the nucleotide sequence of the SNP marker such as SEQ ID
Shown in NO:1, it is SNP site which, which has held the 340th bit base, which is No. 8 chromosome of watermelon genome
29880100 nucleotide.
Further, the pericarp background color for the watermelon that the base of the SNP site is G be it is blackish green, the SNP site
The pericarp background color for the watermelon that base is C is green.
The present invention also provides above-mentioned SNP markers relevant to watermelon pericarp background color in watermelon molecular marker assisted selection
Application in breeding.
The present invention provides the PCR amplification primer for detecting above-mentioned SNP marker relevant to watermelon pericarp background color again
Right, the upstream primer of the primer pair is according to watermelon genome the 8th the 29880100th nucleotide of dyeing and upstream sequence
Column are designed, and the downstream primer of the primer pair is the 29880100th nucleosides according to No. 8 chromosome of watermelon genome
Acid and downstream sequence are designed.
Further, the primer pair is made of two single stranded DNAs, and upstream primer as shown in SEQ ID NO:2, draw by downstream
Object is as shown in SEQ ID NO:3.
The PCR amplification primer pair of above-mentioned SNP marker relevant to watermelon pericarp background color can be used for watermelon molecule mark
Remember in assisted selection.
Invention further provides for identifying that the kit of watermelon pericarp background color, the kit include above-mentioned for detecting
The PCR amplification primer pair of SNP marker relevant to watermelon pericarp background color.
The present invention also provides a kind of methods for identifying watermelon pericarp background color, comprising the following steps:
Step 1:DNA is extracted: extracting watermelon genomic DNA；
Step 2:PCR amplification: using watermelon genomic DNA to be measured as template, using above-mentioned primer pair to sample to be tested into
Row PCR amplification；
Step 3: connection conversion: after PCR product gel extraction, being attached conversion to competent cell, after coated plate, by what is grown
Bacterium colony is cultivated in LB liquid medium；
Step 4: Genotyping: bacterium solution is sequenced, and can determine whether pericarp background color, the position SNP according to the base of SNP site
Select the watermelon pericarp background color that base is G be it is blackish green, the watermelon pericarp background color that the base of SNP site is C be green.
Compared with prior art, the beneficial effects of the present invention are:
1, the present invention using full-length genome weight sequencing technologies (WGR) construct a watermelon dense genetic map, and navigate to
The relevant gene interval of pericarp background color (blackish green/green), realizes finely positioning by F2 group, based on candidate gene and obtains
With the SNP marker of watermelon pericarp background color close linkage, can rapidly be identified in plant seedling stage by the SNP marker
Watermelon pericarp background color out, it is easy to detect, accuracy rate is high, greatly improve the efficiency of breeding.
2, the PCR that the present invention is provided according to watermelon genome the 29880100th single nucleotide polymorphism of No. 8 chromosome
Amplimer pair, high specificity, expanding effect are stablized, and identification watermelon pericarp background color (blackish green/green) can be accurately used for.
3, the method provided by the present invention for identifying watermelon pericarp background color, can be used in each period to watermelon pericarp bottom
The identification of color, is not limited by developmental stage, and it is convenient to detect.
4, it is verified, is tied using the Watermelon Germplasm of SNP marker of the invention to 105 parts of different pericarp background colors
Fruit proves that the SNP marker is 100% to the accuracy that the Watermelon Germplasm of blackish green and green pericarp background color is identified,
Qualification result explanation, SNP marker provided by the invention and identification method have the advantages that efficient quick, accuracy rate are high, can
To be used for breeding work, breeding efficiency is improved.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless specifically indicated, the present invention uses reagent, method and apparatus is for the art conventional reagent, method and set
It is standby.
The acquisition in embodiment 1SNP molecular labeling site
The building of 1.1 dense genetic maps
Pass through continuous multi-generation certainly using the female parent (9904) of blackish green pericarp background color and the male parent (Handel) of green pericarp background color
Hand over building contain 126 single plants recombinant inbred lines, by full-length genome resurvey ordered pair parent and recombinant inbred lines into
Row sequencing constructs watermelon dense genetic map, detects altogether between parent using HighMap software development high density SNP
178762 are suitable for recombinant inbred lines and SNP marker of the depth not less than 4X.By bioinformatic analysis, obtain with
Between the relevant positioning area of watermelon pericarp background color in the region of No. 8 end of chromosome 2.07Mb of watermelon genome.
The acquisition in the site 1.2SNP
It is marked using the single nucleotide polymorphism design CAPS in positioning section, genotyping is carried out to segregating population F2, according to
Phenotype (blackish green pericarp background color or green) the screening recombination single plant of corresponding F2 single plant, finally obtains control watermelon pericarp background color
Candidate gene be located between 29869645 nucleotide and 29901009 nucleotide of No. 8 chromosome of watermelon genome.
Identifying 29880100 sites in the section is SNP site.
The method that embodiment 2 identifies watermelon pericarp background color using the SNP marker
2.1, the extraction of watermelon genomic DNA
Watermelon sample tissue DNA to be measured is extracted using conventional CTAB method, removal RNA pollution, DNA sample volume is 100 μ L.With purple
The OD of outer spectrophotometric determination DNA sample260/280Ratio, numerical value should obtain the DNA sample of high quality between 1.8~2.0
Product, concentration dilution to 100ng/ μ L.
2.2, design of primers
According to No. 8 the 29880100th bit base of chromosome of watermelon genome or so each 400bp primers.Specific nucleosides
Acid sequence is shown in that sequence table SEQ ID NO:1, primer sequence are shown in Table 1.
1 primer sequence of table
2.3 PCR reaction systems
PCR is shown in reaction system such as table 2.
2 PCR reaction system of table
| Reagent | Volume (μ L) |
| 2×PCR Mix | 25 |
| Upstream primer | 2 |
| Downstream primer | 2 |
| DNA profiling | 2 |
| Deionized water | 19 |
| Total volume | 50 |
PCR amplification program are as follows: 94 DEG C of 1.5min, 94 DEG C of 20sec, 57 DEG C of 20sec, 72 DEG C of 50sec, 35 circulations；72℃
5min；4 DEG C of preservations.
2.4 connection conversions
Ago-Gel recycles pcr amplification product, the PCR product that recycling obtains is connected on pTOPO carrier, linked system is such as
The following table 3.
3 glue recovery product linked system of table
| Reagent | It is added volume (μ L) |
| 10×Enhancer | 0.5 |
| ptopO | 0.5 |
| PCR recovery product | 4.0 |
| Total volume | 5.0 |
Then sample is mixed, (20 DEG C~30 DEG C) of room temperature connect 5 minutes.
Connection product obtained above is converted into competent cell, taking bacterium solution coated plate, (culture medium contains ampicillin
50~100 μ g/ml), 37 DEG C are incubated overnight, and the bacterium colony grown is cultivated in LB liquid medium, and bacterium solution sequencing later can obtain
To the base sequence of SNP marker, pericarp background color may determine that according to the genotype of SNP site.The base of SNP site is G,
Then pericarp background color is blackish green；The base of SNP site is C, then pericarp background color is green.
Embodiment 3 identifies 105 parts of Watermelon Germplasms using SNP marker
Using the Watermelon Germplasm of 105 parts of different pericarp background colors in national western muskmelon mid-term library as material, SNP of the present invention is utilized
Molecular labeling identifies pericarp background color.Specific identification method is referring to the method in above-described embodiment 2.On qualification result shows
It states 105 parts of watermelon material phenotypes to comply fully with genotype results, illustrates that SNP marker of the invention can accurately identify west
The blackish green and green pericarp background color of melon, accuracy rate 100%.
31 parts of the watermelon of blackish green pericarp background color in 105 parts of germ plasm resources, 74 parts of the watermelon of green pericarp background color, specific product
Kind and phenotype see the table below 4, wherein ink indicates blackish green, green expression green.
The pericarp background color of 4 105 parts of Watermelon Germplasms of table
It, can be in conclusion SNP marker provided by the invention can quickly and accurately identify watermelon pericarp background color
Breeding efficiency is improved, qualification time, Genetic identification and breeding suitable for watermelon are shortened.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120>a kind of SNP marker relevant to watermelon pericarp background color and its application
<130>nothing
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 667
<212> DNA
<213>watermelon (Citrullus lanatus)
<220>
<221> variation
<222> (340)..(340)
<223>n=g or c
<400> 1
aaggccataa ccagatgcga ctgggacaac gatgttgttg atcatccatt ccttgcatag 60
aggaagttca ggagaaacaa gcaaaataag taggggcttt agaggtaaac actgagtaga 120
gactctgcta aatttgaagt gtgcaccaca cataaaaaat ttaggccttc acacaaaagt 180
acctgaatag cagcagtagt cggttgtgta gtcttgttaa agtcgagaat tgatacctta 240
ctacctggaa agaccacttg aattttgtaa ctgaatgacc atgttaacaa tcaagaagga 300
ccaaaaaaaa aaaacaaaaa ccttctttta gtactctacn catctcctct aatgctctcc 360
gtttatccac cacatttcgt agaccgtagc ccattgtaat ggcatcgaaa gagccgtcag 420
gaaatggcaa attgagtgca tcaccctcca cccaccttca ctttggcagc agcgttatta 480
ggttgttaat ttcacaaatc aattaccaat tacaattctc actgaagcaa aactgtatcg 540
tgaaatacac agaggaatgc ttcaatattt tcaaaatagt agaggccaac acattaattg 600
ttcaagttta gtaatgctca ctcaatgttg tcatagcagg agttggagag ggaacgttgg 660
cgagaag 667
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaggccataa ccagatgcga 20
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cttctcgcca acgttccct 19
Claims (8)
1. a kind of SNP marker relevant to watermelon pericarp background color, which is characterized in that the nucleosides of the SNP marker
Acid sequence is as shown in SEQ ID NO:1, and it is SNP site which, which has held the 340th bit base, which is watermelon gene
Group 29880100 nucleotide of No. 8 chromosome.
2. one kind according to claim 1 SNP marker relevant to watermelon pericarp background color, which is characterized in that described
SNP site base be G watermelon pericarp background color be it is blackish green, the base of the SNP site is the pericarp bottom of the watermelon of C
Color is green.
3. SNP marker relevant to watermelon pericarp background color of any of claims 1 or 2 is in watermelon molecular marker assisted selection
Application in breeding.
4. the PCR amplification primer pair for detecting as claimed in claim 1 or 22 SNP markers relevant to watermelon pericarp background color,
It is characterized in that, the upstream primer of the primer pair is according to the 29880100th nucleotide of watermelon genome the 8th dyeing
And upstream sequence is designed, the downstream primer of the primer pair is according to No. 8 chromosome of watermelon genome
29880100 nucleotide and downstream sequence are designed.
5. according to claim 4 for detecting the PCR amplification primer of SNP marker relevant to watermelon pericarp background color
It is right, which is characterized in that the primer pair is made of two single stranded DNAs, and upstream primer is as shown in SEQ ID NO:2, downstream primer
As shown in SEQ ID NO:3.
6. described in claim 4 or 5 for detecting the PCR amplification primer of SNP marker relevant to watermelon pericarp background color
To the application in watermelon molecular marker assisted selection breeding.
7. for identifying the kit of watermelon pericarp background color, which is characterized in that the kit includes described in claim 4 or 5
Primer pair.
8. a kind of method for identifying watermelon pericarp background color, which comprises the following steps:
Step 1:DNA is extracted: extracting watermelon genomic DNA；
Step 2:PCR amplification: using watermelon genomic DNA to be measured as template, primer pair described in claim 4 or 5 is utilized
PCR amplification is carried out to sample to be tested；
Step 3: connection conversion: after PCR product gel extraction, being attached conversion to competent cell, after coated plate, by what is grown
Bacterium colony is cultivated in LB liquid medium；
Step 4: Genotyping: bacterium solution is sequenced, and can determine whether pericarp background color, the position SNP according to the base of SNP site
Select the watermelon pericarp background color that base is G be it is blackish green, the watermelon pericarp background color that the base of SNP site is C be green.
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| CN201811338549.6A CN109338002B (en) | 2018-11-12 | 2018-11-12 | SNP molecular marker related to watermelon peel background color and application thereof |
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| CN201811338549.6A CN109338002B (en) | 2018-11-12 | 2018-11-12 | SNP molecular marker related to watermelon peel background color and application thereof |
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| CN109338002A true CN109338002A (en) | 2019-02-15 |
| CN109338002B CN109338002B (en) | 2021-04-20 |
Family
ID=65314646
Family Applications (1)
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Cited By (3)
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| CN111549172A (en) * | 2020-06-12 | 2020-08-18 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN111647666A (en) * | 2020-06-28 | 2020-09-11 | 云南中烟工业有限责任公司 | Primer group, application, kit and method for detecting SNP (single nucleotide polymorphism) sites related to human watermelon preference |
| CN114457182A (en) * | 2022-02-21 | 2022-05-10 | 广东省农业科学院蔬菜研究所 | SNP molecular marker related to color of towel gourd peel and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111549172A (en) * | 2020-06-12 | 2020-08-18 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN111549172B (en) * | 2020-06-12 | 2023-02-28 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN111647666A (en) * | 2020-06-28 | 2020-09-11 | 云南中烟工业有限责任公司 | Primer group, application, kit and method for detecting SNP (single nucleotide polymorphism) sites related to human watermelon preference |
| CN111647666B (en) * | 2020-06-28 | 2023-09-22 | 云南中烟工业有限责任公司 | Primer group, application, kit and method for detecting SNP locus related to human watermelon preference |
| CN114457182A (en) * | 2022-02-21 | 2022-05-10 | 广东省农业科学院蔬菜研究所 | SNP molecular marker related to color of towel gourd peel and application thereof |
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