CN105734145A - Strawberry anthocyanin regulatory gene functional marker - Google Patents
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Abstract
The invention provides a strawberry anthocyanin regulatory gene functional marker and belongs to the fields of fruit tree breeding and molecular genetics. The strawberry anthocyanin regulatory gene functional marker is characterized in that one mononucleotide mutation exists in coding regions by carrying out sequence comparison on a strawberry anthocyanin regulatory gene MYB10 in red and yellow strawberries, a dCAPS marker Fv-Ry is designed according to difference of the mononucleotide, the dCAPS marker can distinguish yellow, heterozygous red and homozygous red strawberries, and the marker and strawberry fruit colour character are in cosegregation. By utilizing the functional marker, a material carrying an anthocyanin regulatory gene can be rapidly and accurately screened and identified from a large number of strawberry germplasm resources, molecular marker early selective breeding is carried out, and progress of strawberry fruit colour breeding is greatly accelerated.
Description
Technical field
The invention belongs to fruit breeding and molecular genetics field, and provide a kind of Fructus Fragariae Ananssae anthocyanidin regulator gene function
Labelling.
Background technology
Fructus Fragariae Ananssae (Fragaria × ananassaDuch.) is Rosaceae strawberry plants.Because of the color that it is bright-coloured or beautiful
Damp, higher nutritive value and the mouthfeel of deliciousness and standby welcomed by the people.The cultivated area of China Fructus Fragariae Ananssae is more than 2,000,000 at present
Mu, bright-coloured or beautiful color and luster can the market competitiveness of improving product, directly affect the economic benefit of strawberry cultivating person.But
It is that strawberry fruit color just can show at Strawberry ripening latter stage, has delayed breeding process greatly.Therefore to accelerate Fructus Fragariae Ananssae
Fruit color breeding speed, utilizes modern molecular biotechnology, carries out molecular mark the most necessary.At present, one
A little scholars have carried out genetic analysis and gene mapping to strawberry fruit anthocyanin accumulation related gene.As far back as nineteen sixty-five, right
The research of diploid forest Fructus Fragariae Ananssae (Fragaria vesca) finds, Fructus Pyracanthae character is to be controlled by chromosome C locus gene, and yellow
Fruit or Semen Ginkgo character are then to be controlled (Brown and Wareing 1965) by c locus gene.Williamson etc. are in nineteen ninety-five
By analyzing yellow fruit forest Fructus Fragariae Ananssae ' Yellow Wonder ' (YW) and Fructus Pyracanthae forest Fructus Fragariae Ananssae ' Baron Solemacher '
(BS) c site and shikimate dehydrogenase site close linkage (Williamson et al. are found during filial generation really color character
1995).Subsequently, Davis and Yu utilized BS and WC6(to take from the yellow fruit fraises des bois of the state of New Hampshire in 1997) hybridization
Informative population has the forest Fructus Fragariae Ananssae genetic linkage maps of 7 linkage groups, and genetic map shows the c position closed with yellow fruit form and aspect
Point location is at linkage group I bottom (Davis and Yu 1997).Calendar year 2001 Deng and Davis by location CHS, CHI, F3H,
Five structural genes of DFR and ANS and regulator gene RAN have found the linkage relationship of they and c site, result display c site
Gene and F3H gene close linkage, thus it is speculated that both are likely to same gene.But relevant the dividing of the most relevant Fructus Fragariae Ananssae color and luster
Sub-labelling is fewer, with strawberry fruit color and luster be divided into from molecular marker there is not been reported.
Summary of the invention
It is an object of the invention to for not enough present in above-mentioned technology, it is provided that a kind of Fructus Fragariae Ananssae anthocyanidin regulator gene function
Labelling, utilizes PCR, enzyme action and electrophoretic techniques effectively anthocyanidin regulator gene in strawberry fruit can be carried out genotype choosing
Select, can distinguish red and yellow strawberry fruit in seedling stage, thus eliminate non-targeted plant, substantially increase strawberry fruit face
Color breeding efficiency.Based on Fructus Fragariae Ananssae anthocyanidin regulator gene MYB10 at the red mononucleotide existed with coding region in yellow Fructus Fragariae Ananssae
Sudden change, devises dCAPS functional label Fv-RY according to this site, and this functional label is divided into Fructus Fragariae Ananssae anthocyanidin regulator gene
From.For utilizing this functional label to carry out strawberry fruit color molecule assisted Selection, accelerate breeding process and lay a good foundation.
The object of the present invention is achieved like this, it is characterized in that, the sequence analysis of (1) Fructus Fragariae Ananssae anthocyanidin regulator gene:
Fructus Fragariae Ananssae anthocyanidin regulator gene MYB10 only exists a mononucleotide difference, this nucleoside red in yellow fruit
Acid is positioned at the 35th nucleotide on first exon of MYB10 gene, is G in erythrocarpus, and is C Huang fruit.
(2) method of Fructus Fragariae Ananssae anthocyanidin regulator gene functional label:
Grass is developed according to the single nucleotide mutation that first, coding region in above-mentioned redness and yellow Fructus Fragariae Ananssae exon region exists
The functional label Fv-RY of certain kind of berries anthocyanidin regulator gene, the positive and negative primer sequence of this functional label Fv-RY is:
Forward primer (F): 5 ＇-TTCGGTGTGAGAAAAGGTGAAT-3 ＇
Downstream primer (R): 5 ＇-CTGTTTAAGCCTGCAAGTACAG-3 ＇
Functional label, after PCR amplification, enzyme action and electrophoresis, shows the fragment of 200 bp in erythrocarpus Fructus Fragariae Ananssae, and in yellow
Strawberry fruit shows the fragment of 180 bp.
(3) application process of Fv-RY functional label:
Primer sequence application in screening redness and yellow strawberry fruit, carries out PCR amplification, enzyme action and agarose the most respectively
Gel analysis.
PCR amplification system and response procedures be:
20 μ l PCR reaction systems are: template DNA 50 ng, Taq enzyme 1 U, the upstream and downstream primer each 0.8 of 10 μm ol
μ l, 0.3 μ l of 10 mM dNTPs, 2.0 μ l of 10 × PCR buffer, 1.2 μ l of 25 mM MgCl2, use
Sterile distilled water supplements reaction system to 20 μ l；
PCR response procedures is: first 95 DEG C of 3 min；Then 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 20 s, totally 33 are followed
Ring；Last 72 DEG C of 5 min, 4 DEG C of preservations.
Enzyme action system, time and temperature be:
20 μ l enzyme action systems are: PCR primer 5 μ l, restriction endonuclease HinfI 0.2 μ l, restriction endonuclease buffer 2 μ l, finally use nothing
Bacterium distilled water supplements reaction system to 20 μ l；The endonuclease reaction time is 3 hours, and temperature is 37 DEG C.Amplified production is at the fine jade of 3 %
Lipolysaccharide gel electrophoresis separates.
The invention has the beneficial effects as follows:
(1) functional label Fv-RY can screen the kind containing anthocyanidin regulator gene fast and accurately from strawberry germplasm
Matter resource；Can be used for strawberry fruit color and luster Marker-assisted selection breeding.Because the color of strawberry fruit until bloom the later stage could
Show, utilize this functional label Fv-RY can carry out genotype fast and accurately in seedling stage and select, highly shortened
The breeding time limit, improves breeding efficiency.
(2) functional label Fv-RY is directly to encode with anthocyanin accumulation regulator gene in yellow strawberry fruit according to red
The single nucleotide mutation exploitation in district forms, with strawberry fruit color trait be divided into from, the qualification of strawberry fruit color trait is tied
The molecular marker more chain with strawberry fruit color trait than other is more accurate.
(3) technical system i.e. allele characteristic round pcr have specificity high, reproducible, the most affected by environment and
The advantages such as low cost, it is possible to fast and accurately this Fructus Fragariae Ananssae anthocyanidin regulator gene is shifted in commercial variety and and other
Disease-resistant, the polymerization of high yield, adversity gene.
Accompanying drawing illustrates:
Fig. 1 is Fructus Pyracanthae and yellow fruit Fructus Fragariae Ananssae anthocyanidin regulator gene MYB10 sequence alignment analysis figure
Fig. 2 is that Fv-RY is marked at redness, yellow Fructus Fragariae Ananssae parent and F thereof2The amplification figure of individual plant in colony
Fig. 3 is that Fv-RY is marked in different cultivation and fraises des bois kind, strain and carries out proof diagram.
Detailed description of the invention:
Below in conjunction with embodiment, the present invention will be further described
As shown in Figure 1, black triangle is red and mononucleotide difference position in yellow Fructus Fragariae Ananssae, underscore be functional label just
Site is designed to reverse primer.
As shown in Figure 2, swimming lane information: from left to right, 50 bp DNA ladder；1, yellow parent；2, red parent；3-
5, F2Isozygoty in colony erythrocarpus；6-8, F2Homozygous yellow fruit in colony；9-11, F2Heterozygosis erythrocarpus in colony.
From the figure 3, it may be seen that 1-12 point sample order be respectively as follows: ' gorgeous ', ' Fructus Persicae smoke ', ' large and beautiful ', ' beauty ', ' red beautiful ',
' Hawaii 4 ', ' sweet Charlie ', ' Ruegen ', ' beautiful ', ' Yellow Wonder ', ' strain 181 ', ' strain 29 '.
Below in conjunction with the example in detail present invention.Enforcement is for a better understanding of the present invention, but is not limited to the present invention.Below
Experimental technique in implementation is conventional method, and involved experiment reagent is routine biochemistry reagent.
Example 1 utilizes functional label Fv-RY to F2Segregating population Fructus Fragariae Ananssae individual plant carries out color identification in early days
(1) using CTAB method to extract the red and genomic DNA of yellow Strawberry seedlings, extraction step is as follows:
A. take 0.2g and remove the fresh leaflet tablet of master pulse, be positioned in the centrifuge tube of 2 mL, put into quick-freezing in liquid nitrogen, use 75% wine
The washed clean plastics pestle of seminal plasma is ground to rapidly powder；
B. add in centrifuge tube 600 μ L 65 DEG C preheating extract with CTAB liquid (CTAB 2%, Tris-HCl(PH8.0), 100
Mmol/L, EDTA 20 mmol/L, NaCl 1.4mol/L), mix rapidly；
Being put into by centrifuge tube the most subsequently in 65 DEG C of water-baths, centre is shaken once at interval of 8 for min minute, water-bath 30 min；
D. taking out centrifuge tube, add isopyknic chloroform, after shaking up 10 min, at 4 DEG C, 12000 r/min are centrifuged 10 min；
E. upper phase (about 600 μ L) is transferred in another centrifuge tube, add isopyknic chloroform, shake up 10 gently
Min, at 4 DEG C, 12000 r/min are centrifuged 10 min；
F. taking supernatant (about 400 μ L), add the dehydrated alcohol of 2 times of volume pre-coolings, mixing makes DNA unite gently ,-20 DEG C of conditions
Lower precipitation 30 min, under the conditions of 4 DEG C, 12000 r/min are centrifuged 10 min；
G. abandoning supernatant, add 500 μ l 75 % washing with alcohol and precipitate after 2 times, precipitate room temperature is dried；
H. add 500 μ L TE buffer solution DNA, add 0.3 μ L RNaseA (10 g/ L), after mixing slightly from
The heart, 37 DEG C of insulation 30 min；
I. adding 3 mol/L NaAc solution 50 L and the dehydrated alcohol of 2 times of volume pre-coolings, mixing makes DNA unite gently, and-20
30 min are precipitated under the conditions of DEG C；
J. under the conditions of 4 DEG C, 12000 r/min are centrifuged 10min, abandon supernatant, and the ethanol purge of addition 75% precipitates 1～2 time
After, precipitate room temperature dries addition sterilizing 400-500 u L ddH2O dissolves use, or it is standby to add 75% ethanol-20 DEG C preservation
With；
(2) design of primers and PCR amplification
Find with sequence alignment analysis in yellow strawberry fruit, in red and yellow red according to Fructus Fragariae Ananssae anthocyanidin regulator gene
In strawberry fruit there is the difference of mononucleotide in first exon region, therefore devises dCAPs mark according to this nucleotide difference
Note Fv-RY:
Forward primer (F): 5 ＇-TTCGGTGTGAGAAAAGGTGAAT-3 ＇
Downstream primer (R): 5 ＇-CTGTTTAAGCCTGCAAGTACAG-3 ＇
Then redness and yellow Fructus Fragariae Ananssae F2 colony individual plant DNA to extract make template, according to following PCR amplification condition and program
It is analyzed:
1. 20 μ l PCR reaction systems are: template DNA 50 ng, Taq enzyme 1 U, the upstream and downstream primer of 10 μm ol is each
0.8 μ l, 0.3 μ l of 10 mM dNTPs, 2.0 μ l of 10 × PCR buffer, 1.2 μ l of 25 mM
MgCl2, supplement reaction system to 20 μ l with sterile distilled water；
2. PCR response procedures is: first 95 DEG C of 3 min；Then 95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 20 s, totally 30
Circulation；Last 72 DEG C of 5 min, 4 DEG C of preservations.
(3) endonuclease reaction of PCR primer
PCR primer will be appealed, be analyzed according to following PCR amplification condition and program:
20 μ l enzyme action systems are: PCR primer 5 μ l, restriction endonuclease HinfI 0.2 μ l, restriction endonuclease buffer 2 μ l, finally use nothing
Bacterium distilled water supplements reaction system to 20 μ l；The endonuclease reaction time is 3 hours, and temperature is 37 DEG C.
(4) detection of amplified production
Amplified production is detected by the agarose gel electrophoresis utilizing 3%, and electrophoretic buffer is 1 × TAE, electrophoresis 45 under 150V
Min, dyes with EB after electrophoresis, and takes a picture with gel imaging system, and genotype and phenotype are finally combined by result such as (figure two)
It is analyzed statistics.
Example 2 utilizes functional label Fv-RY to identify different colours strawberry cultivars, strain
(1) use CTAB method from different colours strawberry cultivars, strain young leaflet tablet extract genomic DNA, extraction step is such as
Shown in lower:
A. take 0.2g and remove the fresh leaflet tablet of master pulse, be positioned in the centrifuge tube of 2 mL, put into quick-freezing in liquid nitrogen, use 75% wine
The washed clean plastics pestle of seminal plasma is ground to rapidly powder；
B. add in centrifuge tube 600 μ L 65 DEG C preheating extract with CTAB liquid (CTAB 2%, Tris-HCl(PH8.0), 100
Mmol/L, EDTA 20 mmol/L, NaCl 1.4mol/L), mix rapidly；
Being put into by centrifuge tube the most subsequently in 65 DEG C of water-baths, centre is shaken once at interval of 8 for min minute, water-bath 30 min；
D. taking out centrifuge tube, add isopyknic chloroform, after shaking up 10 min, at 4 DEG C, 12000 r/min are centrifuged 10 min；
E. upper phase (about 600 μ L) is transferred in another centrifuge tube, add isopyknic chloroform, shake up 10 gently
Min, at 4 DEG C, 12000 r/min are centrifuged 10 min；
F. taking supernatant (about 400 μ L), add the dehydrated alcohol of 2 times of volume pre-coolings, mixing makes DNA unite gently ,-20 DEG C of conditions
Lower precipitation 30 min, under the conditions of 4 DEG C, 12000 r/min are centrifuged 10 min；
G. abandoning supernatant, add 500 μ l 75 % washing with alcohol and precipitate after 2 times, precipitate room temperature is dried；
H. add 500 μ L TE buffer solution DNA, add 0.3 μ L RNaseA (10 g/ L), after mixing slightly from
The heart, 37 DEG C of insulation 30 min；
I. adding 3 mol/L NaAc solution 50 L and the dehydrated alcohol of 2 times of volume pre-coolings, mixing makes DNA unite gently, and-20
30 min are precipitated under the conditions of DEG C；
J. under the conditions of 4 DEG C, 12000 r/min are centrifuged 10min, abandon supernatant, and the ethanol purge of addition 75% precipitates 1～2 time
After, precipitate room temperature dries addition sterilizing 400-500 u L ddH2O dissolves use, or it is standby to add 75% ethanol-20 DEG C preservation
With；
(2) design of primers and PCR amplification
Fructus Fragariae Ananssae anthocyanidin regulator gene finds with sequence alignment analysis in yellow strawberry fruit, red and yellow Fructus Fragariae Ananssae red
In fruit there is the difference of single core thuja acid in first exon region, therefore devises dCAPs labelling according to this nucleotide difference
Fv-RW
Forward primer (F): 5 ＇-TTCGGTGTGAGAAAAGGTGAAT-3 ＇
Downstream primer (R): 5 ＇-CTGTTTAAGCCTGCAAGTACAG-3 ＇
Then to extract different cultivars, strain Fructus Fragariae Ananssae DNA makees template, is analyzed according to following PCR amplification condition and program:
1. 20 μ l PCR reaction systems are: template DNA 50 ng, Taq enzyme 1 U, the upstream and downstream primer of 10 μm ol
Each 0.8 μ l, 0.3 μ l of 10 mM dNTPs, 2.0 μ l of 10 × PCR buffer, 1.2 μ l of 25 mM
MgCl2, supplement reaction system to 20 μ l with sterile distilled water；
2. PCR response procedures is: first 95 DEG C of 3 min；Then 95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 20 s, totally 30
Individual circulation；Last 72 DEG C of 5 min, 4 DEG C of preservations.
(3) endonuclease reaction of PCR primer
PCR primer will be appealed, be analyzed according to following PCR amplification condition and program:
20 μ l enzyme action systems are: PCR primer 5 μ l, restriction endonuclease HinfI 0.2 μ l, restriction endonuclease buffer 2 μ l, finally use nothing
Bacterium distilled water supplements reaction system to 20 μ l；The endonuclease reaction time is 3 hours, and temperature is 37 DEG C.
(4) detection of amplified production
Amplified production is detected by the agarose gel electrophoresis utilizing 3%, and electrophoretic buffer is 1 × TAE, electrophoresis 45 under 150V
Min, dyes with EB after electrophoresis, and takes a picture with gel imaging system, and genotype and phenotype are finally combined by result such as (figure three)
It is analyzed statistics.
Claims (1)
1. a Fructus Fragariae Ananssae anthocyanidin regulator gene functional label, is characterized in that, specifically comprises the following steps that (1) Fructus Fragariae Ananssae anthocyanidin regulates
The sequence analysis of gene:
Fructus Fragariae Ananssae anthocyanidin regulator gene MYB10 only exists a mononucleotide difference, this nucleoside red in yellow fruit
Acid is positioned at the 35th nucleotide on first exon of MYB10 gene, is G in erythrocarpus, and is C Huang fruit；
(2) method of Fructus Fragariae Ananssae anthocyanidin regulator gene functional label:
Grass is developed according to the single nucleotide mutation that first, coding region in above-mentioned redness and yellow Fructus Fragariae Ananssae exon region exists
The functional label of certain kind of berries anthocyanidin regulator gene, the positive and negative primer sequence of this functional label is:
Forward primer (F): 5 ＇-TTCGGTGTGAGAAAAGGTGAAT-3 ＇
Downstream primer (R): 5 ＇-CTGTTTAAGCCTGCAAGTACAG-3 ＇
This functional label, after PCR amplification, enzyme action and electrophoresis, shows the fragment of 200 bp in erythrocarpus Fructus Fragariae Ananssae, and at Huang
Color strawberry fruit shows the fragment of 180 bp；
(3) application process of functional label
The application in screening redness and yellow strawberry fruit of the described primer sequence, carries out PCR amplification, enzyme action and agar respectively
Sugar gel analysis；
PCR amplification system and response procedures be:
20 μ l PCR reaction systems are: template DNA 50 ng, Taq enzyme 1 U, the upstream and downstream primer each 0.8 of 10 μm ol
μ l, 0.3 μ l of 10 mM dNTPs, 2.0 μ l of 10 × PCR buffer, 1.2 μ l of 25 mM MgCl2, use
Sterile distilled water supplements reaction system to 20 μ l；
PCR response procedures is: first 95 DEG C of 3 min；Then 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 20 s, totally 33 are followed
Ring；Last 72 DEG C of 5 min, 4 DEG C of preservations；
Enzyme action system, time and temperature be:
20 μ l enzyme action systems are: PCR primer 5 μ l, restriction endonuclease HinfI 0.2 μ l, restriction endonuclease buffer 2 μ l, finally use nothing
Bacterium distilled water supplements reaction system to 20 μ l；The endonuclease reaction time is 3 hours, and temperature is 37 DEG C, and amplified production is at the fine jade of 3 %
Lipolysaccharide gel electrophoresis separates；
(4) according to the method for above-mentioned Fructus Fragariae Ananssae anthocyanidin regulator gene functional label, it is characterised in that can be used for strawberry fruit face
Color carries out assisted Selection in early days, accelerates breeding process.
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Cited By (4)
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| CN110491444A (en) * | 2019-08-13 | 2019-11-22 | 西北农林科技大学 | A kind of development approach and application of the molecular labeling of quick screening apple red meat character |
| CN111139310A (en) * | 2020-01-03 | 2020-05-12 | 沈阳农业大学 | Indel marker for strawberry fruit color character assisted breeding and application |
| CN113186330A (en) * | 2021-04-25 | 2021-07-30 | 东北林业大学 | Primer pair, kit and method for predicting existence of anthocyanin in open-field chrysanthemum color |
| CN113186196A (en) * | 2021-03-04 | 2021-07-30 | 江苏省农业科学院 | Strawberry MYB10AG-insert gene and application thereof |
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| JP2006254710A (en) * | 2005-03-15 | 2006-09-28 | Tochigi Prefecture | Primer set for strawberry variety identification and method for strawberry variety identification using the same |
| CN102533747A (en) * | 2012-02-13 | 2012-07-04 | 东北农业大学 | Single nucleotide polymorphisms (SNP) locus linked to sporisorium-reiliana-resistance-associated gene, molecular marker LSdCAP4 based on same and use of same |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110491444A (en) * | 2019-08-13 | 2019-11-22 | 西北农林科技大学 | A kind of development approach and application of the molecular labeling of quick screening apple red meat character |
| CN111139310A (en) * | 2020-01-03 | 2020-05-12 | 沈阳农业大学 | Indel marker for strawberry fruit color character assisted breeding and application |
| CN111139310B (en) * | 2020-01-03 | 2023-05-23 | 沈阳农业大学 | Indel marker for strawberry fruit color character assisted breeding and application |
| CN113186196A (en) * | 2021-03-04 | 2021-07-30 | 江苏省农业科学院 | Strawberry MYB10AG-insert gene and application thereof |
| CN113186196B (en) * | 2021-03-04 | 2023-09-19 | 江苏省农业科学院 | Strawberry MYB10 AG-insert Gene and application thereof |
| CN113186330A (en) * | 2021-04-25 | 2021-07-30 | 东北林业大学 | Primer pair, kit and method for predicting existence of anthocyanin in open-field chrysanthemum color |
| CN113186330B (en) * | 2021-04-25 | 2022-05-06 | 东北林业大学 | Primer pair, kit and method for predicting existence of anthocyanin in open-field chrysanthemum color |
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