CN109609678A - A kind of molecular labeling and application for predicting collard internal lobe color - Google Patents
A kind of molecular labeling and application for predicting collard internal lobe color Download PDFInfo
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- CN109609678A CN109609678A CN201910003917.XA CN201910003917A CN109609678A CN 109609678 A CN109609678 A CN 109609678A CN 201910003917 A CN201910003917 A CN 201910003917A CN 109609678 A CN109609678 A CN 109609678A
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- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 claims description 8
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- 229930002877 anthocyanin Natural products 0.000 claims description 6
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- 150000004636 anthocyanins Chemical class 0.000 claims description 6
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- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 5
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- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
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- 244000061458 Solanum melongena Species 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
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- 241000196324 Embryophyta Species 0.000 description 8
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- 241000692870 Inachis io Species 0.000 description 5
- 241000272201 Columbiformes Species 0.000 description 4
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 3
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Abstract
The molecular labeling and application that the present invention relates to a kind of for predicting collard internal lobe color, the molecular labeling has nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6, and the application method of the Molecular Prediction collard internal lobe color includes the following steps: that (1) extracts leaves genomic DNA；(2) PCR amplification, amplimer are the molecular labeling；(3) agarose gel electrophoresis detects；The present invention establishes a kind of application molecular labeling in the case where not being affected by temperature, the method for early prediction collard internal lobe color, is that collard internal lobe color identification and breeding of new variety have important scientific guidance meaning.
Description
Technical field
The invention belongs to plant molecular breeding fields, and in particular to a kind of for predicting the molecule of collard internal lobe color
Label and application.
Background technique
Collard (Brassica oleracea var.acephala) it is Cruciferae brassica plant, ornamental value
By force.
DNA molecular marker is a kind of mark for being able to detect and analyzing a certain section of specific DNA segment in Plant Genome
Note method.It has influenced not by environmental condition differentia influence, not by whether can gene or express, not by plant some fertility
Effect stepwise is not limited by tissue site when sampling, multiple advantages such as required DNA template quantity is few, is plant molecular marker
The important scientific basis of assisted selection.
InDel label refers to that insertion and deletion marks, and is a kind of molecular labeling developed based on genome sequence, is to wait
The length polymorphism variation generated on the gene loci of position due to nucleotides inserted or missing.InDel label polymorphism can lead to
It crosses the simple steps such as polymerase chain reaction (PCR) and agarose gel electrophoresis and achievees the purpose that Genotyping, have special
Property it is high, stability is good, detection method is simple the advantages that.
Collard internal lobe color is its important economic characters, however the internal lobe color of collard needs plant strain growth
It to certain scale of construction, and needs just show in low temperature environment induction, this leverages collard internal lobe color new varieties
Breeding process, have not yet to see through the method for Molecular Prediction collard internal lobe color.
Summary of the invention
To make up for the shortcomings of the above existing technologies, the present invention proposes a kind of for predicting point of collard internal lobe color
Genome sequence and collard internal lobe color are connected, are allowed to any by son label and application, the molecular labeling
Under the conditions of temperature, collard molecular mark system is established and improved to the internal lobe color of early prediction collard.
The invention proposes a kind of for predicting that the molecular labeling of collard internal lobe color, the molecular labeling have
Nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6.
The invention also provides the preparation methods of the molecular labeling of the prediction collard internal lobe color: specific steps packet
It includes as follows:
(1) collard is cultivated: with collard red internal lobe kind ' red dove ' for source material, using continuous autocopulation method,
Obtain red internal lobe self-mating system ' 42 is red '；With collard white internal lobe kind ' Bai Ou ' for source material, using continuous selfing
Method obtains white internal lobe self-mating system ' 1631 '；Red internal lobe self-mating system ' 42 is red ' and white internal lobe self-mating system ' 1631 ' are sowed
To the rosette state；
(2) internal lobe color collard variety genome DNA is extracted
Using improvement CTAB method, collard red internal lobe self-mating system ' 42 is red ' and white internal lobe self-mating system are extracted respectively
' 1631 ' genomic DNA, the specific method is as follows:
A. young leaflet tablet is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
It is quickly transferred in centrifuge tube；
B. CTAB Extraction buffer prepare: weigh 10 g of cetyl trimethylammonium bromide (CTAB), 16.38 g of sodium chloride,
Polyvinylpyrrolidone (PVP) 10g measures the 1mol/L Tris-Hcl (pH=8) of 20ml, the 1 mol/L ethylenediamine of 8ml
Tetraacethyl disodium (EDTA, pH=8), deionized water are settled to 200ml, 121 DEG C of high pressure sterilization 20 min, spare；
C. 65 DEG C of CTAB Extraction buffer is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
1 h of water-bath turns upside down every 10 min and mixes once；
D. chloroform-isoamyl alcohol solution (chloroform: isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, mixing of gently turning upside down
Afterwards in 12,000 r/min, room temperature is centrifuged 7 min；
E. in centrifuge tube the isopropanol of -20 DEG C of pre-coolings is added, it is heavy that mixing is placed on -20 DEG C of refrigerators in Aspirate supernatant after being centrifuged
Form sediment 1 h；
F. centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；75% ethyl alcohol is added, 12,000
It is centrifuged 7 min under r/min, abandons supernatant, rinsing is repeated twice；
G. centrifuge tube is uncapped and is placed in room temperature and dries, TE buffer is added, saved after being placed in 4 DEG C of 12 h of refrigerator back dissolving in -20 DEG C
It is spare；
(3) it synthesizesDFRFull length gene cloning primer:
According to NCBI(https: //www.ncbi.nlm.nih.gov/) in wild cabbage TO1000 genome reference sequences, according to
Anthocyanin accumulates path geneDFR(accession number XM_013753582.1) sequence, synthesis Anthocyanin accumulate path base
Because the full length sequence of DFR expands upstream primer F and downstream primer R；
The upstream primer F has nucleotide sequence shown in SEQ ID NO:3；Downstream primer R has shown in SEQ ID NO:4
Nucleotide sequence；
(4) PCR polymerase chain reaction expands:
A. PCR polymerase chain reaction system:
5 × Prime STAR GXL Buffer, 4.0 μ l, dNTP Mixture 1.6 μ l, primers F 1.0 μ l, primer R
1.0 2.0 μ l, Prime STAR GXL DNA Polymerase of μ l, DNA 0.4 μ l, 10 μ l of deionized water；
B. PCR polymerase chain reaction condition: 95 °C of 5 min of initial denaturation；98 °C of 10 s of denaturation, 60 °C of 15 s of annealing, 68 °C are prolonged
Stretch 90 s, 30 circulations；68 °C of 5 min of extension；4 °C save backup；
(5) agarose gel electrophoresis:
1% agarose gel electrophoresis of PCR product is detected, gel imaging system shoots electrophoresis photographs；
(6) amplified production recycle: referring to Shanghai bio-engineering corporation provide SanPrep pillar DNA plastic recovery kit into
Row recycling；
(7) it is sequenced: the PCR product of above-mentioned recycling is obtained with nucleotide shown in SEQ ID NO:1 and SEQ ID NO:2
Sequence, described that there is nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2 to be respectively designated as 42 is red and 1631；
(8) molecular labeling for predicting collard internal lobe color is synthesized
According to gained nucleotide sequence in step (7), two Oligonucleolide primers dfr1801F and dfr1801R are synthesized；It is described
For predicting the molecular labeling of collard internal lobe color；
The dfr1801F has nucleotide sequence shown in SEQ ID NO:5；The dfr1801R has SEQ ID NO:6 institute
The nucleotide sequence shown.
The invention also provides the application methods of the molecular labeling of the prediction collard internal lobe color: specific steps
Include the following:
(1) extraction of different internal lobe color collard variety genome DNAs
A. collard young leaflet tablet is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, will be ground
Good powder is quickly transferred in centrifuge tube；
B. CTAB Extraction buffer prepare: weigh 10 g of cetyl trimethylammonium bromide (CTAB), 16.38 g of sodium chloride,
Polyvinylpyrrolidone (PVP) 10g measures the 1mol/L Tris-Hcl (pH=8) of 20ml, the 1 mol/L ethylenediamine of 8ml
Tetraacethyl disodium (EDTA, pH=8), deionized water are settled to 200ml, 121 DEG C of high pressure sterilization 20 min, spare；
C. 65 DEG C of CTAB Extraction buffer is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
1 h of water-bath turns upside down every 10 min and mixes once；
D. chloroform-isoamyl alcohol solution (chloroform: isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, mixing of gently turning upside down
Afterwards in 12,000 r/min, room temperature is centrifuged 7 min；
E. in centrifuge tube the isopropanol of -20 DEG C of pre-coolings is added, it is heavy that mixing is placed on -20 DEG C of refrigerators in Aspirate supernatant after being centrifuged
Form sediment 1 h；
F. centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；75% ethyl alcohol is added, 12,000
It is centrifuged 7 min under r/min, abandons supernatant, rinsing is repeated twice；
G. centrifuge tube is uncapped and is placed in room temperature and dries, TE buffer is added, saved after being placed in 4 DEG C of 12 h of refrigerator back dissolving in -20 DEG C
It is spare；
(2) PCR amplification primer is dfr1801F and dfr1801R；The dfr1801F has nucleosides shown in SEQ ID NO:5
Acid sequence；Dfr1801R has nucleotide sequence shown in SEQ ID NO:6；
(3) PCR amplification and electrophoresis detection:
A. PCR reaction system: 2 × EasyTaq PCR SuperMix, 51 0.5 μ l of μ l, dfr1801F of μ l, DNA,
0.5 μ l of dfr1801R, 3 μ l of deionized water；
B. PCR reaction condition: 95 °C of 5 min of initial denaturation；95 °C of denaturation 30s, 60 °C of annealing 30s, 72 °C of extension 30s, 30
Circulation；72 °C of extension 5min；4 °C of preservations；
C. agarose gel electrophoresis detects: 1% agarose gel electrophoresis of PCR product being detected, gel imaging system shooting electricity
Swimming photo, the specific item of the amplifiable 443bp out of the genomic DNA of the collard kind of tool pink, red or aubergine
The collard kind of band, redfree or white internal lobe is without specific band.
The beneficial effects of the present invention are:
A kind of application molecular labeling is established in the case where not being affected by temperature, the side of early prediction collard internal lobe color
Method is that collard internal lobe color identification and breeding of new variety have important scientific guidance meaning.
Detailed description of the invention
Fig. 1 is agarose gel electrophoresis figure in embodiment 2；
Fig. 2 is agarose gel electrophoresis figure in embodiment 3；
Fig. 3 is agarose gel electrophoresis figure in embodiment 4.
Specific embodiment
Agents useful for same can be commercially available by market in embodiment.
Embodiment 1 predicts the preparation method of the molecular labeling of collard internal lobe color
Specific steps include the following:
(1) collard is cultivated: with collard red internal lobe kind ' red dove ' for source material, using continuous autocopulation method,
Red internal lobe self-mating system ' 42 is red ' is obtained after 6 generations of continuous selfing；With collard white internal lobe kind ' Bai Ou ' for source material,
Using continuous autocopulation method, the continuous white internal lobe self-mating system ' 1631 ' of acquisition after being selfed for 6 generations；Red internal lobe self-mating system ' 42 is red ' with
White internal lobe self-mating system ' 1631 ' was sowed to the rosette state；
(2) internal lobe color collard variety genome DNA is extracted
Using improvement CTAB method, collard red internal lobe self-mating system ' 42 is red ' and white internal lobe self-mating system are extracted respectively
' 1631 ' genomic DNA, the specific method is as follows:
A. young leaflet tablet is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
It is quickly transferred in centrifuge tube；
B. CTAB Extraction buffer prepare: weigh 10 g of cetyl trimethylammonium bromide (CTAB), 16.38 g of sodium chloride,
Polyvinylpyrrolidone (PVP) 10g measures the 1mol/L Tris-Hcl (pH=8) of 20ml, the 1 mol/L ethylenediamine of 8ml
Tetraacethyl disodium (EDTA, pH=8), deionized water are settled to 200ml, 121 DEG C of high pressure sterilization 20 min, spare；
C. 65 DEG C of CTAB Extraction buffer is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
1 h of water-bath turns upside down every 10 min and mixes once；
D. chloroform-isoamyl alcohol solution (chloroform: isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, mixing of gently turning upside down
Afterwards in 12,000 r/min, room temperature is centrifuged 7 min；
E. in centrifuge tube the isopropanol of -20 DEG C of pre-coolings is added, it is heavy that mixing is placed on -20 DEG C of refrigerators in Aspirate supernatant after being centrifuged
Form sediment 1 h；
F. centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；75% ethyl alcohol is added, 12,000
It is centrifuged 7 min under r/min, abandons supernatant, rinsing is repeated twice；
G. centrifuge tube is uncapped and is placed in room temperature and dries, TE buffer is added, saved after being placed in 4 DEG C of 12 h of refrigerator back dissolving in -20 DEG C
It is spare；
(3) it synthesizesDFRFull length gene cloning primer:
According to NCBI(https: //www.ncbi.nlm.nih.gov/) in wild cabbage TO1000 genome reference sequences, according to
Anthocyanin accumulates path geneDFR(accession number XM_013753582.1) sequence, synthesis Anthocyanin accumulate path base
Because the full length sequence of DFR expands upstream primer F (SEQ ID NO:3) and downstream primer R(SEQ ID NO:4)；
Upstream primer F:ATGGTAGCTCACAAAGAGACT
Downstream primer R:CTAAGCACAGATCTGCTGTG
(4) PCR polymerase chain reaction expands:
A. PCR polymerase chain reaction system:
5 × Prime STAR GXL Buffer, 4.0 μ l, dNTP Mixture 1.6 μ l, primers F 1.0 μ l, primer R
1.0 2.0 μ l, Prime STAR GXL DNA Polymerase of μ l, DNA 0.4 μ l, 10 μ l of deionized water；
B. PCR polymerase chain reaction condition: 95 °C of 5 min of initial denaturation；98 °C of 10 s of denaturation, 60 °C of 15 s of annealing, 68 °C are prolonged
Stretch 90 s, 30 circulations；68 °C of 5 min of extension；4 °C save backup；
(5) agarose gel electrophoresis:
1% agarose gel electrophoresis of PCR product is detected, gel imaging system shoots electrophoresis photographs；
(6) amplified production recycle: referring to Shanghai bio-engineering corporation provide SanPrep pillar DNA plastic recovery kit into
Row recycling；
(7) it is sequenced:
By the PCR product of above-mentioned recycling, sequence (the SEQ ID for red internal lobe collard self-mating system ' 42 is red ' is respectively obtained
), and the sequence (SEQ ID NO:2) of white internal lobe collard self-mating system ' 1631 ' NO:1；
(8) molecular labeling for predicting collard internal lobe color is synthesized
According to gained nucleotide sequence in step (7), two Oligonucleolide primers dfr1801F(SEQ ID NO are designed and synthesized:
5) and dfr1801R(SEQ ID NO:6)；
Dfr1801F:CACTGTTCGCGATCCTGGTA
Dfr1801R:TCTTCGTACGGTCTTTGCCT
Embodiment 2: prediction collard red internal lobe kind ' red peacock ' internal lobe color.
Using the molecular labeling (dfr1801F for being used to predict collard internal lobe color described in the acquisition of embodiment 1
And dfr1801R) predicted, the specific method is as follows for implementation:
A. the cultivation of collard: sowing collard kind ' red peacock ' seed.
The hole tray for preparing 120 caves is gently compacted after filling up matrix, is soaked matrix with water, and one seed of every cave program request covers
Native 2cm, sowing can emerge for 3 days at a temperature of being placed on 25 degree；
The detection of B molecular labeling:
1. extracting the genomic DNA of collard red internal lobe kind ' red peacock ', specific method using improvement CTAB method
It is as follows:
(1) 0.15 g blade is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
End is quickly transferred in 2 mL centrifuge tubes；
(2) the CTAB extracting solution of 65 DEG C of 700 μ L is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
Middle 1 h of water-bath turns upside down every 10 min and mixes once；
(3) 700 μ L chloroform-isoamyl alcohol solution (chloroform and isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, gently up and down
In 12,000 r/min after being mixed by inversion, room temperature is centrifuged 7 min；
(4) 400 μ L supernatants are drawn in 1.5mL centrifuge tube after being centrifuged, the isopropanol of L -20 DEG C of 800 μ pre-coolings is added, are mixed
It is even to be placed on -20 DEG C of 1 h of refrigerators precipitating；
(5) centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；800 μ L75% ethyl alcohol are added,
It is centrifuged 7 min under 12,000 r/min, abandons supernatant, rinsing is repeated twice；
(6) centrifuge tube is uncapped and is placed in room temperature and dries, 50 μ L TE buffers are added, be placed in after 4 DEG C of 12 h of refrigerator back dissolving in-
20 DEG C save backup；
2. PCR amplification primer is dfr1801F and dfr1801R；The dfr1801F has nucleosides shown in SEQ ID NO:5
Acid sequence；Dfr1801R has nucleotide sequence shown in SEQ ID NO:6；；
Dfr1801F:CACTGTTCGCGATCCTGGTA
Dfr1801R:TCTTCGTACGGTCTTTGCCT
3. PCR polymerase chain reaction expands；
(1) PCR reaction system: 2 × EasyTaq PCR SuperMix, 51 0.5 μ l of μ l, dfr1801F of μ l, DNA,
0.5 μ l of dfr1801R, 3 μ l of deionized water；
(2) PCR reaction condition: 95 °C of 5 min of initial denaturation；95 °C of denaturation 30s, 60 °C of annealing 30s, 72 °C of extension 30s, 30
Circulation；72 °C of extension 5min；4 °C of preservations；
(3) agarose gel electrophoresis detects: 1% agarose gel electrophoresis of PCR product being detected, gel imaging system shooting electricity
Swimming photo, the results showed that, collard red internal lobe kind ' red peacock ' has shown the specific band (see figure 1) of 443bp；
C. the application of molecular labeling: by using the molecular labeling, the internal lobe color of collard kind ' red peacock ' is predicted
For red, gained internal lobe solid colour is cultivated with later period plant.
Embodiment 3: prediction collard white internal lobe kind ' pigeon ' internal lobe color
Using embodiment 1 obtain described in for predict collard internal lobe color molecular labeling (dfr1801F and
Dfr1801R it) is predicted, the specific method is as follows for implementation:
A. the cultivation of collard: sowing collard kind ' pigeon ' seed.
The hole tray for preparing 120 caves is gently compacted after filling up matrix, is soaked matrix with water, and one seed of every cave program request covers
Native 2cm, sowing can emerge for 3 days at a temperature of being placed on 25 degree.
The detection of B molecular labeling:
1. extracting the genomic DNA of collard red internal lobe kind ' pigeon ', specific method is such as using improvement CTAB method
Under:
(1) 0.15 g blade is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
End is quickly transferred in 2 mL centrifuge tubes；
(2) the CTAB extracting solution of 65 DEG C of 700 μ L is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
Middle 1 h of water-bath turns upside down every 10 min and mixes once；
(3) 700 μ L chloroform-isoamyl alcohol solution (volume ratio=24:1) are added into centrifuge tube, gently turn upside down mixing after in
12,000 r/min, room temperature are centrifuged 7 min；
(4) 400 μ L supernatants are drawn in 1.5mL centrifuge tube after being centrifuged, the isopropanol of L -20 DEG C of 800 μ pre-coolings is added, are mixed
It is even to be placed on -20 DEG C of 1 h of refrigerators precipitating；
(5) centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；800 μ L75% ethyl alcohol are added,
It is centrifuged 7 min under 12,000 r/min, abandons supernatant, rinsing is repeated twice；
(6) centrifuge tube is uncapped and is placed in room temperature and dries, 50 μ L TE buffers are added, are placed in after 4 DEG C of 12 h of refrigerator back dissolving in -20
It DEG C saves backup；
2. PCR amplification primer is dfr1801F and dfr1801R；The dfr1801F has nucleosides shown in SEQ ID NO:5
Acid sequence；Dfr1801R has nucleotide sequence shown in SEQ ID NO:6；
Dfr1801F:CACTGTTCGCGATCCTGGTA
Dfr1801R:TCTTCGTACGGTCTTTGCCT
3. PCR polymerase chain reaction expands；
(1) PCR reaction system: 2 × EasyTaq PCR SuperMix, 51 0.5 μ l of μ l, dfr1801F of μ l, DNA,
0.5 μ l of dfr1801R, 3 μ l of deionized water；
(2) PCR reaction condition: 95 °C of 5 min of initial denaturation；95 °C of denaturation 30s, 60 °C of annealing 30s, 72 °C of extension 30s, 30
Circulation；72 °C of extension 5min；4 °C of preservations；
(3) agarose gel electrophoresis detects: 1% agarose gel electrophoresis of PCR product being detected, gel imaging system shooting electricity
It swims photo, water is used to do template as blank control (CK), as a result ' 1631 ' the band (see figure 2) for not amplifying 443pb；
C. the application of the molecular labeling: by using the molecular labeling, predict that collard internal lobe kind ' pigeon ' internal lobe is not
Red cultivates gained internal lobe solid colour with later period plant.
Embodiment 4: prediction collard red internal lobe kind ' ibis ' internal lobe color
Using embodiment 1 obtain described in for predict collard internal lobe color molecular labeling (dfr1801F and
Dfr1801R it) is predicted, the specific method is as follows for implementation:
A. the cultivation of collard: sowing collard kind ' ibis ' seed.
The hole tray for preparing 120 caves is gently compacted after filling up matrix, is soaked matrix with water, and one seed of every cave program request covers
Native 2cm, sowing can emerge for 3 days at a temperature of being placed on 25 degree；
The detection of B molecular labeling:
1. extracting the genomic DNA of collard red internal lobe kind ' ibis ', specific method is such as using improvement CTAB method
Under:
(1) 0.15 g blade is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
End is quickly transferred in 2 mL centrifuge tubes；
(2) the CTAB extracting solution of 65 DEG C of 700 μ L is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
Middle 1 h of water-bath turns upside down every 10 min and mixes once；
(3) 700 μ L chloroform-isoamyl alcohol solution (chloroform and isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, gently up and down
In 12,000 r/min after being mixed by inversion, room temperature is centrifuged 7 min；
(4) 400 μ L supernatants are drawn in 1.5mL centrifuge tube after being centrifuged, the isopropanol of L -20 DEG C of 800 μ pre-coolings is added, are mixed
It is even to be placed on -20 DEG C of 1 h of refrigerators precipitating；
(5) centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；800 μ L75% ethyl alcohol are added,
It is centrifuged 7 min under 12,000 r/min, abandons supernatant, rinsing is repeated twice；
(6) centrifuge tube is uncapped and is placed in room temperature and dries, 50 μ L TE buffers are added, be placed in after 4 DEG C of 12 h of refrigerator back dissolving in-
20 DEG C save backup；
2. PCR amplification primer is dfr1801F and dfr1801R；The dfr1801F has nucleosides shown in SEQ ID NO:5
Acid sequence；Dfr1801R has nucleotide sequence shown in SEQ ID NO:6；
Dfr1801F:CACTGTTCGCGATCCTGGTA
Dfr1801R:TCTTCGTACGGTCTTTGCCT
3. PCR polymerase chain reaction expands；
(1) PCR reaction system: 2 × EasyTaq PCR SuperMix, 51 0.5 μ l of μ l, dfr1801F of μ l, DNA,
0.5 μ l of dfr1801R, 3 μ l of deionized water；
(2) PCR reaction condition: 95 °C of 5 min of initial denaturation；95 °C of denaturation 30s, 60 °C of annealing 30s, 72 °C of extension 30s, 30
Circulation；72 °C of extension 5min；4 °C of preservations；
(3) agarose gel electrophoresis detects: 1% agarose gel electrophoresis of PCR product being detected, gel imaging system shooting electricity
Swim photo, the results showed that, collard red internal lobe kind ' ibis ' it is dominant go out the specific band (see figure 3) of 443bp；
C. the application of molecular labeling: by using the molecular labeling, the internal lobe color for predicting collard kind ' ibis ' is
Red cultivates gained internal lobe solid colour with later period plant.
Sequence table
<110>Agricultural University Of Shenyang
<120>a kind of molecular labeling and application for predicting collard internal lobe color
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1579
<212> DNA
<213>collard (ornamental kale inbre d line Red 42)
<400> 1
atggtagctc acaaagagac cgtgtgcgta accggcgcat caggattcat tggttcatgg 60
ctcgtgatgc ggctactgga acgtggttac tttgtccgtg ccactgttcg cgatcctggt 120
acgtatctta caaactcgtt aatttctcct aagagtatat gttaatacgt atcactttgt 180
gtgttttaag taacttacga gttttcttgg cctgtaaagg aaatttgaag aaagtgcaac 240
atcttcttga tttgccaaac gcgaagacgc aactcacttt atggaaagcc gatttatctg 300
acgaaggaag ctacgatgac gccataaacg gatgcgacgg cgtttttcac atagctactc 360
ccatggattt tgaatccaag gatcccgagg tgagttatac tatgaacctt tttcttatta 420
catatcaatc ctacaagatt ttgttaaatg agtttgtttg aatcagaacg aagtgataaa 480
accaacagtg aatggagtgt tggggataat gaaagcatgt gataaggcaa agaccgtacg 540
aagaattgtg tttacttcgt ctgctggaac ggttaatgtt gaggaacacc agaaaaatgt 600
ctatgatgaa aacgattgga gtgatcttga ctttatcatg tccaagaaga tgacaggatg 660
ggtatatata ttaaggatca tatataaaaa attaacccga ggttgatctt cttcaaagta 720
atttatgttt ttgataaatt gttggcagat gtatttcatg tcgaaaacgt tagccgagaa 780
agcagcttgg gattacgcta aggaaaaagg aatagatttc attagtatta tcccgacatt 840
ggtgatcggt ccatttataa caacatctat gccgcctagc cttattaccg cgctctctcc 900
tatcactcgt gagtgagcct actttctaat ccctcttttt taactaagag gttaatttaa 960
aacggtaaaa atgttttagg taacgaggca cattactcca tcataagaca aggacagtat 1020
gtccacttgg acgacttatg caatgctcat atattcttgt acgaacaagc tgctgccaag 1080
ggacgttatg tttgttcctc tcacgatgca acgattcata ctatctccga gtttctcagg 1140
caaaaatatc cagaatataa cgtgccttca acgtaagatt tttatcatta ccggtttaag 1200
ctttttttgc atattcagtt taattttttt tttctgaata tgaactcttt ggaacaggtt 1260
tgaaggagtg gatgagaatc taaagagcat tatgttcagt tccaagaagc tgattgatat 1320
gggatttaac ttcaagtata gtctcgagga tatgttggtg gaatcgattg agacatgtcg 1380
tcaaaagggt tttctccctg tcactttacc ggaacatttg aaatctgagg acaaagttcc 1440
gggcagtgat gacaataagg agattaaaaa cggatctgca ggtttaactg atggtatggt 1500
agcttgtaag aagaccgaac cagggatggc cggcgagaaa gccgatagtc acatgtcggc 1560
acagcagatc tgtgcttag 1579
<210> 2
<211> 3857
<212> DNA
<213>collard (ornamental kale inbre d line Red 42)
<400> 2
atggtagctc acaaagagac cgtgtgcgta accggcgcat caggattcat tggttcatgg 60
ctcgtgatcc actttgtcat aagctgttct aagtatttgc agcttcaatc tcggatctaa 120
aacagccccc atagctagaa caagactgta ttcatcccaa tacttagaaa acttaactct 180
cattttctta gccatctctt tcattactgg atcatcacaa ctagcatact tcatcagtaa 240
gcattcaatt ttccacactt gtagaaaata tgcattagcc gttggatatc taacgcctga 300
gaagaacgtt gtaatagtgc tgaaaggctt cagaaactca caaattttct gccctcgatc 360
ccactcatcc tctgaaggca atgatttgta actcctgtca cactctttca aactagtgaa 420
tgcgtcacga aacttcagag ctctagcaag catatcataa gttgaattcc atctggttgg 480
tacatctaga gacagtccag ctccactcct aatccctaca ctctgaacac atgctgcaaa 540
tgcttctatc cttgatccag atgctttgac aaacttaaca ctctctcgga tattttccag 600
aagaccaaca gcaagttcta aaccttcttt cactatgaga ttcaaaatgt gtgcacagca 660
tctaacgtgg aagaacttcc catcacacaa caacccgttg ccgctagcca tttgaagtcg 720
atgcttgagt atcttctgca tactatcgtt ataagtagca ttatccaagg tcatagagaa 780
gacctttttc tctaatcccc actccttcaa acagctaata agtttgttag ccacttcttc 840
accggtatgt ggaggtttca actcactgaa gactagtatc ttgttgttca acttgaagct 900
ctcatcaaca tagtgagctg tcagacagat atatcccgtc atagtagtag aagctgtcca 960
tatatctgag gtaaaagata cccgaccctt gaattctgct aactctttct tcagcttctc 1020
tctttcttcc tcatatctct tatagacatc agctccagca gtttgtctag atatatgctg 1080
gcatttggga ttcaggtact tgtccctagc tctaaccttc tcatactcaa cgtatttgaa 1140
aggctgatca tggaaaataa tgatctcact gatcatatca cgatctactt tcggatcata 1200
ctcacgatca accacttgac ggaggctttt tgggacagat ttctaaatgc cgtttcatag 1260
aagatgttcc tgaggcagat tctgatacta acttcttatt acaatgaatg caacgactcc 1320
ttctttttcc atctgcctct actcctacta ctacgaaatg gttccaaaca agagactttg 1380
cgcgtttaga atgactcaca gtttcagttg cttgaacagg ttgagtttca ctttgtgctt 1440
gtccttgagt ttctgcttca tcatcatcat caacctccat ttgctcattt gcagcctcaa 1500
gagctgctag tgtgtcaagt gtttgtgaat ccatcctgga aaaaacatta aacaatcaga 1560
aaatttaagc tattattgaa tagcgtaacg gcacacatac gcacacataa aacaatatta 1620
ttgtttcagc acaagaccaa accaccactg ataatcaacc tgttaagcac aacaacaaga 1680
aagcccacaa tatgcaaacg taacggcaca catgcgcaca catagaacaa tgttgttgtt 1740
agcacaaaat caatcaaacc ctgagttcac gacctaaatt ttctctataa atggttgatt 1800
aaacttttat tttctttttc tctaacaaaa caaagtactt gatcgaacaa agtgattttt 1860
tacaatcatt gttgtccggg acaacttgac caaccaacca atgatctata gcacaaagac 1920
aaaacccact caagttcttc cagagagttt taagaacaaa caaaagcata gttcttacat 1980
tttatagaga gaacgaagag gaaggtaatc acgtacggac ttcttctggt aacactttga 2040
ctcctttcgt tggggcctaa accaattagg atagaatcga tggttagtca accagattct 2100
aaagcatgag aatcaaaaca caaaaacaca tttcgaaatc ttaccaatga acccaatcta 2160
gaatgaatcg tcgcaagaaa gcatgtctaa tttctagatc tgtgttttgt ggctgtggtt 2220
acgggagaaa caaaaatttg gggtaaaata taatgtaaaa cgacgtcgtt tcggttaatt 2280
ttttttttaa aaaaaaaacc atcgggtacc cgaagcccga tagtgtaaac ccgatagggt 2340
aataaacaaa acaagacccg cccaacaaaa acccgcgagt tttaaaatct aaaagtacgg 2400
gtttcgggtt tcaaatttca accgggcttc gggtacccta tgggtaaaaa cccattatta 2460
acatccctac ttacgagttt tcttggcctg taaaggaaat ttgaagaaag tgcaacatct 2520
tcttgatttg ccaaacgcga agacgcaact cactttatgg aaagccgatt tatctgacga 2580
aggaagctac gatgacgcca taaacggatg cgacggcgtt tttcacatag ctactcccat 2640
ggattttgaa tccaaggatc ccgaggtgag ttatactatg aacctttttc ttattacaca 2700
tcaatcctac aagattttgt taaatgagtt tgtttgaatc agaacgaagt gataaaacca 2760
acagtgaatg gagtgttggg gataatgaaa gcatgtgata aggcaaagac cgtacgaaga 2820
attgtgttta cttcgtctgc tggaacggtt aatgttgagg aacaccagaa aaatgtctat 2880
gatgaaaacg attggagtga tctcgacttt atcatgtcca agaagatgac aggatgggta 2940
tatatattaa ggatcatata taaaaaatta acccgaggtt gatcttcttc aaagtaattt 3000
atgtttttga taaattgttg gcagatgtat ttcatgtcga aaacgttagc cgagaaagca 3060
gcttgggatt acgctaagga aaaaggaata gatttcatta gtattatccc gacattggtg 3120
atcggtccat ttataacaac atctatgccg cctagcctta ttaccgcgct ctctcctatc 3180
actcgtgagt gagcctactt tctaatccct cttttttaac taagaggtta atttaaaacg 3240
gtaaaaatgt tttaggtaac gaggcacatt actccatcat aagacaagga cagtatgtcc 3300
acttggacga cttatgcaat gctcatatat tcttgtacga acaagctgct gccaagggac 3360
gttatgtttg ttcctctcac gatgcaacga ttcttactat ctccgagttt ctcaggcaaa 3420
aatatccaga atataacgtg ccttcaacgt aagattttta tcattaccgg tttaagcttt 3480
ttttccatat tcagtttaat tttttttttt ctgaatatga actctttgga aacaggtttg 3540
aaggagtgga tgagaatcta aagagcatta tgttcagttc caaaaagctg attgatatgg 3600
gatttaactt caagtatagt ctcgaggata tgttggtgga atcgattgag acatgtcgtc 3660
aaaagggttt tctccctgtc actttaccgg aacatttgaa atctgaggac aaagttccgg 3720
gcagtgatga caataaggag attaaaaacg gatctgcagg tttaactgat ggtatggtag 3780
cttgtaagaa gaccgaacca gggatggccg gcgagaaagc cgatagtcac atgtcggcac 3840
agcagatctg tgcttag 3857
<210> 3
<211> 21
<212> DNA
<213>artificial synthesized (artificial synthesis)
<400> 3
atggtagctc acaaagagac t 21
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized (artificial synthesis)
<400> 4
ctaagcacag atctgctgtg 20
<210> 5
<211> 20
<212> DNA
<213>artificial synthesized (artificial synthesis)
<400> 5
cactgttcgc gatcctggta 20
<210> 6
<211> 20
<212> DNA
<213>artificial synthesized (artificial synthesis)
<400> 6
tcttcgtacg gtctttgcct 20
Claims (3)
1. it is a kind of for predicting the molecular labeling of collard internal lobe color, it is characterized in that: the molecular labeling has SEQ ID
Nucleotide sequence shown in NO:5 and SEQ ID NO:6.
2. it is according to claim 1 a kind of for predicting the molecular labeling of collard internal lobe color, it is characterized in that: described
Predict the preparation method of the molecular labeling of collard internal lobe color: specific steps include the following:
(1) collard is cultivated: with collard red internal lobe kind ' red dove ' for source material, using continuous autocopulation method,
Red internal lobe self-mating system ' 42 is red ' is obtained after 6 generations of continuous selfing；With collard white internal lobe kind ' Bai Ou ' for source material,
Using continuous autocopulation method, the continuous white internal lobe self-mating system ' 1631 ' of acquisition after being selfed for 6 generations；By red internal lobe self-mating system ' 42 is red '
It sows and is cultivated to the rosette state with white internal lobe self-mating system ' 1631 '；
(2) collard variety genome DNA is extracted
Using improvement CTAB method, collard red internal lobe red internal lobe self-mating system ' 42 is red ' and white internal lobe are extracted respectively certainly
The genomic DNA of system ' 1631 ' is handed over, the specific method is as follows:
A. young leaflet tablet is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
It is quickly transferred in centrifuge tube；
B. CTAB Extraction buffer prepare: weigh 10 g of cetyl trimethylammonium bromide (CTAB), 16.38 g of sodium chloride,
Polyvinylpyrrolidone (PVP) 10g measures the 1mol/L Tris-Hcl (pH=8) of 20ml, the 1 mol/L ethylenediamine of 8ml
Tetraacethyl disodium (EDTA, pH=8), deionized water are settled to 200ml, 121 DEG C of high pressure sterilization 20 min, spare；
C. 65 DEG C of CTAB Extraction buffer is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
1 h of water-bath turns upside down every 10 min and mixes once；
D. chloroform-isoamyl alcohol solution (chloroform: isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, mixing of gently turning upside down
Afterwards in 12,000 r/min, room temperature is centrifuged 7 min；
E. in centrifuge tube the isopropanol of -20 DEG C of pre-coolings is added, it is heavy that mixing is placed on -20 DEG C of refrigerators in Aspirate supernatant after being centrifuged
Form sediment 1 h；
F. centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；75% ethyl alcohol is added, 12,000
It is centrifuged 7 min under r/min, abandons supernatant, rinsing is repeated twice；
G. centrifuge tube is uncapped and is placed in room temperature and dries, TE buffer is added, saved after being placed in 4 DEG C of 12 h of refrigerator back dissolving in -20 DEG C
It is spare；
(3) it synthesizesDFRFull length gene cloning primer:
According to NCBI(https: //www.ncbi.nlm.nih.gov/) in wild cabbage TO1000 genome reference sequences, according to
Anthocyanin accumulates path geneDFR(accession number XM_013753582.1) sequence, synthesis Anthocyanin accumulate path base
Because the full length sequence of DFR expands upstream primer F and downstream primer R；
The upstream primer F has nucleotide sequence shown in SEQ ID NO:3；The downstream primer R has SEQ ID NO:4
Shown in nucleotide sequence；
(4) PCR polymerase chain reaction expands:
A. PCR polymerase chain reaction system:
5 × Prime STAR GXL Buffer, 4.0 μ l, dNTP Mixture 1.6 μ l, primers F 1.0 μ l, primer R
1.0 2.0 μ l, Prime STAR GXL DNA Polymerase of μ l, DNA 0.4 μ l, 10 μ l of deionized water；
B. PCR polymerase chain reaction condition: 95 °C of 5 min of initial denaturation；98 °C of 10 s of denaturation, 60 °C of 15 s of annealing, 68 °C are prolonged
Stretch 90 s, 30 circulations；68 °C of 5 min of extension；4 °C save backup；
(5) agarose gel electrophoresis:
1% agarose gel electrophoresis of PCR product is detected, gel imaging system shoots electrophoresis photographs；
(6) amplified production recycle: referring to Shanghai bio-engineering corporation provide SanPrep pillar DNA plastic recovery kit into
Row recycling；
(7) it is sequenced: the PCR product of above-mentioned recycling is obtained with nucleotide shown in SEQ ID NO:1 and SEQ ID NO:2
Sequence；
(8) molecular labeling for predicting collard internal lobe color is synthesized
There is nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2 according to gained in step (7), synthesize two widows
Nucleotide primer dfr1801F and dfr1801R；To be described for predicting the molecular labeling of collard internal lobe color；
The dfr1801F has nucleotide sequence shown in SEQ ID NO:5；The dfr1801R has SEQ ID NO:6 institute
The nucleotide sequence shown.
3. it is according to claim 1 a kind of for predicting the molecular labeling of collard internal lobe color, it is characterized in that: described
Predict the application method of the molecular labeling of collard internal lobe color: specific steps include the following:
(1) extraction of different internal lobe color collard variety genome DNAs
A. young leaflet tablet is weighed in mortar, is fully ground under liquid nitrogen frozen protective condition to powdered, by the powder of milled
It is quickly transferred in centrifuge tube；
B. CTAB Extraction buffer prepare: weigh 10 g of cetyl trimethylammonium bromide (CTAB), 16.38 g of sodium chloride,
Polyvinylpyrrolidone (PVP) 10g measures the 1mol/L Tris-Hcl (pH=8) of 20ml, the 1 mol/L ethylenediamine of 8ml
Tetraacethyl disodium (EDTA, pH=8), deionized water are settled to 200ml, 121 DEG C of high pressure sterilization 20 min, spare；
C. 65 DEG C of CTAB Extraction buffer is added into centrifuge tube, centrifuge tube is placed in 65 DEG C of water-baths after mixing well
1 h of water-bath turns upside down every 10 min and mixes once；
D. chloroform-isoamyl alcohol solution (chloroform: isoamyl alcohol volume ratio=24:1) is added into centrifuge tube, mixing of gently turning upside down
Afterwards in 12,000 r/min, room temperature is centrifuged 7 min；
E. in centrifuge tube the isopropanol of -20 DEG C of pre-coolings is added, it is heavy that mixing is placed on -20 DEG C of refrigerators in Aspirate supernatant after being centrifuged
Form sediment 1 h；
F. centrifuge tube is centrifuged 7 min in 12,000 r/min at room temperature, abandons supernatant；75% ethyl alcohol is added, 12,000
It is centrifuged 7 min under r/min, abandons supernatant, rinsing is repeated twice；
G. centrifuge tube is uncapped and is placed in room temperature and dries, TE buffer is added, saved after being placed in 4 DEG C of 12 h of refrigerator back dissolving in -20 DEG C
It is spare；
(2) PCR amplification primer is dfr1801F and dfr1801R；The dfr1801F has nucleosides shown in SEQ ID NO:5
Acid sequence；Dfr1801R has nucleotide sequence shown in SEQ ID NO:6；
(3) PCR amplification and electrophoresis detection:
A. PCR reaction system: 2 × EasyTaq PCR SuperMix, 51 0.5 μ l of μ l, dfr1801F of μ l, DNA,
0.5 μ l of dfr1801R, 3 μ l of deionized water；
B. PCR reaction condition: 95 °C of 5 min of initial denaturation；95 °C of denaturation 30s, 60 °C of annealing 30s, 72 °C of extension 30s, 30
Circulation；72 °C of extension 5min；4 °C of preservations；
C. agarose gel electrophoresis detects: 1% agarose gel electrophoresis of PCR product being detected, gel imaging system shooting electricity
Swimming photo, the specific item of the amplifiable 443bp out of the genomic DNA of the collard kind of tool pink, red or aubergine
Band, the collard kind of white internal lobe is without specific band.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910003917.XA CN109609678B (en) | 2019-01-03 | 2019-01-03 | Molecular marker for predicting color of collard inner leaves and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910003917.XA CN109609678B (en) | 2019-01-03 | 2019-01-03 | Molecular marker for predicting color of collard inner leaves and application |
Publications (2)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321240A (en) * | 2020-02-19 | 2020-06-23 | 沈阳农业大学 | Molecular marker for predicting collard leaf margin character and application thereof |
| CN113215303A (en) * | 2021-06-16 | 2021-08-06 | 沈阳农业大学 | Molecular marker of collard epidermis waxy character and distinguishing method thereof |
| CN117248052A (en) * | 2023-08-31 | 2023-12-19 | 中国农业科学院蔬菜花卉研究所 | Molecular marker and primer group related to ornamental collard color and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1656228A (en) * | 2002-05-29 | 2005-08-17 | 阿雷萨生物检测公司 | Reporter system for plants |
| CN103789433A (en) * | 2014-01-23 | 2014-05-14 | 黑龙江省农业科学院耕作栽培研究所 | Specific molecular marker for detecting structural gene IPS1 of rice spikes and detection method thereof |
| CN103882143A (en) * | 2014-04-10 | 2014-06-25 | 山东农业大学 | Detection primers for molecular markers in close linkage with major QTL of wheat ear length and application of detection primers |
| KR20150012338A (en) * | 2013-07-25 | 2015-02-04 | 서울대학교산학협력단 | CRTISO1 gene for discriminating variety of Chinese Cabbage accumulating lycopene of high content and representing orange color, molecular marker for verifying the gene and uses thereof |
| CN104846081A (en) * | 2015-04-27 | 2015-08-19 | 沈阳农业大学 | SSR labels of brassica oleracea red leaf gene Re and application thereof |
-
2019
- 2019-01-03 CN CN201910003917.XA patent/CN109609678B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1656228A (en) * | 2002-05-29 | 2005-08-17 | 阿雷萨生物检测公司 | Reporter system for plants |
| KR20150012338A (en) * | 2013-07-25 | 2015-02-04 | 서울대학교산학협력단 | CRTISO1 gene for discriminating variety of Chinese Cabbage accumulating lycopene of high content and representing orange color, molecular marker for verifying the gene and uses thereof |
| CN103789433A (en) * | 2014-01-23 | 2014-05-14 | 黑龙江省农业科学院耕作栽培研究所 | Specific molecular marker for detecting structural gene IPS1 of rice spikes and detection method thereof |
| CN103882143A (en) * | 2014-04-10 | 2014-06-25 | 山东农业大学 | Detection primers for molecular markers in close linkage with major QTL of wheat ear length and application of detection primers |
| CN104846081A (en) * | 2015-04-27 | 2015-08-19 | 沈阳农业大学 | SSR labels of brassica oleracea red leaf gene Re and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIN JEON等: "Transcriptome analysis and metabolic profiling of green and red kale (Brassica oleracea var. acephala) seedlings", 《FOOD CHEMISTRY》 * |
| 张彬等: "羽衣甘蓝花青素合成途径结构基因的表达特性", 《山西农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321240A (en) * | 2020-02-19 | 2020-06-23 | 沈阳农业大学 | Molecular marker for predicting collard leaf margin character and application thereof |
| CN111321240B (en) * | 2020-02-19 | 2023-03-28 | 沈阳农业大学 | Molecular marker for predicting collard leaf margin character and application thereof |
| CN113215303A (en) * | 2021-06-16 | 2021-08-06 | 沈阳农业大学 | Molecular marker of collard epidermis waxy character and distinguishing method thereof |
| CN117248052A (en) * | 2023-08-31 | 2023-12-19 | 中国农业科学院蔬菜花卉研究所 | Molecular marker and primer group related to ornamental collard color and application |
| CN117248052B (en) * | 2023-08-31 | 2024-02-27 | 中国农业科学院蔬菜花卉研究所 | Molecular marker and primer group related to ornamental collard color and application |
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