CN106191276A - A kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock - Google Patents
A kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock Download PDFInfo
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Abstract
The present invention relates to a kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock.With Apple Leaves DNA to be detected as masterplate, using two pairs of oligodeoxynucleotide sequence C o1 and Co2 as primer pair, the DNA molecular marker mutually chain with columnar gene is carried out PCR simultaneously and react amplification, amplified production is carried out electrophoresis detection, in the amplified production electrophoresis result of 2 pairs of primers, at least 1 pair of primer has the DNA specific band that described DNA molecular marker is corresponding, then illustrate that the apple nursery stock of correspondence is columnar apple；The amplified production electrophoresis result of 2 pairs of primers without corresponding DNA specific band, is then all plain edition Fructus Mali pumilae.Utilize the inventive method can easily and fast, the column type type that accurately distinguishes out in 12 years green apple nursery stocks, be greatly improved breeding efficiency, accelerate breeding process；Simultaneously, moreover it is possible to simply and quickly identify true and false columnar apple kind nursery stock, it is to avoid the dispute that false nursery stock brings.
Description
Technical field:
The present invention relates to a kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock, belong to molecular genetic
Breeding technical field.
Background technology:
It is short that columnar apple (Columnar apple) has internode, and axillary bud sprouting is substantial amounts of brachyplast, little or raw without side
Extend the feature of young sprout, in natural cordon, be both different from plain edition, also different from general Spur Type or dwarf-type.This
Tree-shaped is compact, pruning rate is extremely light, is especially suitable for high dense planting and mechanization management.Promote this tree-shaped and will greatly save labour force
And production cost, meet the demand for development of Fructus Mali pumilae Labor-saving cultivation.Research shows, Fructus Mali pumilae Columnar character is by dominant single-gene
(Co) qualitative trait controlled.So, columnar apple is the preferable genetic resources of fruit tree Plant-type Breeding.But columnar apple is with common
Type Fructus Mali pumilae is not easily distinguishable at Seedling Stage, and Morphological Identification can only wait it to grow into a certain degree, relies on tree-like and formalness
Make a distinction, the longest, waste time and energy.
Along with hereditism and the development of molecular biology, people recognize and utilize genetic marker to carry out assisted Selection
(Marker-assisted selection, MAS), can be greatly enhanced efficiency of selection, reduces the blindness of breeding process.
Labelling breeding (Marker breeding) is to utilize and the closely linked genetic marker of objective trait gene, enters objective trait
The technology that line trace selects.It is to utilize to carry out indirect selections with the closely linked molecular marker of objective trait gene, is
To objective trait in the selection of molecular level, the most affected by environment, select reliable results；It is being polymerized the same of favo(u)rable target character
Time, can be selected by genetic background during backcrossing and gradually oozing, reduce Linkage drag, accelerate breeding process.Relevant with breeding
Genetic marker mainly have four types: morphology labelling (Morphological Markers), cytological marker
(Cytological Markers), biochemical marker (Biochemical Markers) and molecular marker (Molecular
Markers)。
In recent years, the plant genetics and breeding that develops into of molecular biology provides a kind of new technique hands based on DNA variation
Section, i.e. molecular marking technique.Compared with other labelling, DNA molecular marker has following main feature: one is can be to each
Educate the individuality in period, organ, even cell to detect, whether do not limited with gene expression by environment；Two is that quantity is the most, throughout
Whole genome；Three is that polymorphism is high, utilizes a large amount of primer, probe can complete the analysis of covering gene group；Four is to show as
Property, the most not affecting objective trait expresses, with bad character without necessary connection；Five is that many is labeled as codominance, it is possible to identify
Homozygous genotype and heterozygous genotypes, it is provided that complete hereditary information.
Summary of the invention:
It is an object of the invention to overcome the raw columnar apple hybrid Seedling of 1-2 and columnar apple kind Seedling Stage in form
The defect distinguishing and distinguishing it is difficult to, it is provided that a kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock, i.e. on
One and the control Fructus Mali pumilae closely linked DNA molecular marker of columnar gene Co, utilize round pcr to detect the side of this molecular marker
Method.
In order to realize foregoing invention purpose, with the 2 of the present invention pairs of primers in columnar apple filial generation segregating population
Column type and plain edition be individual and column type and plain edition apple variety carry out the effectiveness of verification mark.With primer, Co1 is carried out
PCR amplification can amplify the DNA specific fragment of a 585bp in columnar apple；Use Co2 primer to carrying out PCR amplification, energy
The DNA specific fragment of a 319bp is amplified in columnar apple；Primer to Co1, primer to Co2 in plain edition Fructus Mali pumilae all
Can not expand any band.By utilizing this molecular marker that Fructus Mali pumilae Columnar character is detected, to 1 year to filial generation
Raw seedling on purpose selects, and eliminates Non-target traits early, is greatly improved breeding efficiency, utilizes the present invention to carry
The high accuracy differentiating Seedling Stage columnar apple kind, the producing and selling for columnar apple nursery stock provide technological guidance.
Provided by the present invention for identifying that differentiation columnar apple and medium-sized molecular marker primer are to Co1, Co2, Co1
With Co2 two, primer at least can be amplified in columnar apple 1 specific band, in plain edition Fructus Mali pumilae, 2 pairs of primers all expand
Can not increase band, wherein, the deoxyribonucleotide sequence of Co1 is by primer:
Forward primer Co1-F:GATGCGAGAATTAACTAGCACAC
Downstream primer Co1-R:GAATTGTTGTATGCGTTTTTCC
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 585bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGG
TATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAAC
AAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAA
CAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATA
ATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAA
ACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTA
AATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAAT
CTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
The deoxyribonucleotide sequence of Co2 is by primer:
Co2-F:TCTACTCCTCTTTTGCCTTGG
Co2-R:ACTTCGAATTCACTCGTCTTT
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 319bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCT
TTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTT
TTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTT
ATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAA
AGACGAGTGAATTCGAAGT
The inventive method operates in accordance with the following steps:
One, CTAB method is used to extract apple genome DNA
1, take 2ml centrifuge tube, add 1.2ml CTAB (cetyl trimethylammonium bromide) and 50 μ L beta-mercaptoethanols, and
Preheat in 65 DEG C of water-baths, take the Apple Leaves that about 0.5g children is tender, add liquid nitrogen and quickly grind to form fine powder, proceed to above-mentioned dress
Have in the centrifuge tube of CTAB, concussion mixing, it is placed in water-bath 30min in 65 DEG C of water-baths, period repeatedly reverse mixing；
2, taking-up is cooled to room temperature, adds chloroform, and reverse mixing, static gently, 4 DEG C, and 12000rpm is centrifuged, and takes 1000 μ L
Supernatant is in new 2ml centrifuge tube；
3, add Tris (trishydroxymethylaminomethane) the saturated phenol of supernatant 1/2 volume, overturn mixing, static, then add
Enter the chloroform of supernatant 1/2 volume, reverse mixing, static, 4 DEG C, 12000rpm is centrifuged, take 800 μ L of supernatant liquid to new 2ml from
In heart pipe；
4, add chloroform, reverse mixing, static, 4 DEG C, 12000rpm is centrifuged, take 600 μ L of supernatant liquid to new 1.5ml from
In heart pipe；
5, the dehydrated alcohol of supernatant 1/10 and the sodium acetate of 1/20 are added ,-20 DEG C of static 30min, 4 DEG C, 12000rpm
Centrifugal, supernatant is moved on in the centrifuge tube of new 1.5ml；
6, adding isopyknic isopropanol ,-20 DEG C static 2 hours, 4 DEG C, and 12000rpm is centrifuged, and abandons supernatant, retains precipitation；
7, the washes of absolute alcohol adding 1ml 75% precipitates once, then utilizes washes of absolute alcohol once, treats ethanol
After volatilization is dry, add 50 distilled water (Distillation-Distillation H aseptic for μ L2O, ddH2O) dissolve, take 5 μ
LDNA utilizes concentration and the quality of the agarose gel electrophoresis detection DNA of 1%；
Two, PCR amplification
PCR amplification is carried out with the Fructus Mali pumilae DNA extracted for masterplate, with inspection first with Fructus Mali pumilae house keeper's gene M dActin primer
Survey the quality of DNA, get rid of the test error caused because of the reason of DNA, then recycle primer to Co1 and Co2 with the Herba Marsileae Quadrifoliae extracted
Really DNA is that masterplate carries out PCR amplification, and the primer sequence of Fructus Mali pumilae MdActin gene used is:
MdActin-F:AAGATTTGGCATCACACGTTC
MdActin-R:TGGATGGCAACATACATAGCA
PCR reaction employing 12 μ L systems:
PCR response procedures:
Three, amplified production is carried out electrophoresis detection
After PCR reaction terminates, will product carry out 1.5% agarose gel electrophoresis, check different testing sample DNA
The DNA band produced in PCR reaction；
Wherein, the deoxyribonucleotide sequence of Co1 is by described primer:
Forward primer Co1-F:GATGCGAGAATTAACTAGCACAC
Downstream primer Co1-R:GAATTGTTGTATGCGTTTTTCC
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 585bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGG
TATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAAC
AAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAA
CAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATA
ATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAA
ACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTA
AATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAAT
CTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
The deoxyribonucleotide sequence of Co2 is by described primer:
Co2-F:TCTACTCCTCTTTTGCCTTGG
Co2-R:ACTTCGAATTCACTCGTCTTT
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 319bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCT
TTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTT
TTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTT
ATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAA
AGACGAGTGAATTCGAAGT
PCR result shows: can amplify DNA specific band with Co1 and Co2 primer in columnar apple, and commonly
Type Fructus Mali pumilae can not expand DNA specific band.
The inventive method is with Apple Leaves DNA to be detected as masterplate, with two pairs of oligodeoxynucleotide sequence C o1
With Co2 as primer pair, the DNA molecular marker mutually chain with columnar gene is carried out PCR simultaneously and react amplification, to amplified production
Carrying out electrophoresis detection, in the amplified production electrophoresis result of 2 pairs of primers, at least 1 pair of primer has the DNA that described DNA molecular marker is corresponding
Specific band, then illustrate that the apple nursery stock of correspondence is columnar apple；The amplified production electrophoresis result of 2 pairs of primers is all without correspondence
DNA specific band, then be plain edition Fructus Mali pumilae.
The inventive method can reach 88% to column type Apple Materials discrimination power；Plain edition Apple Materials discrimination power is reached
100%.
Utilize DNA molecular marker, can effectively carry out assisted Selection in early days in seedling stage, eliminate non-targeted early individual.
The inventive method, with Apple Leaves for examination material, utilizes molecular marker primer disclosed by the invention, can be at Seedling Stage to filial generation
Apple seedling carry out molecular marker assisted selection, it is to avoid Seedling Stage morphology is not easy the defect identified, is greatly improved and educates
Plant efficiency, accelerate breeding process.Meanwhile, column type and plain edition apple nursery stock can be distinguished quickly and easily, easily and fast, accurate
Really distinguish the column type type in 1-2 green apple nursery stock, it is to avoid the dispute that false nursery stock brings.
Accompanying drawing illustrates:
Fig. 1 is that 56 examination materials utilize MdActin primer pair amplifies product band characteristic pattern；
Fig. 2 is Co1 and Co2 primer pair amplifies product band characteristic pattern in 25 columnar apple；
Fig. 3 is Co1 and Co2 primer pair amplifies product band characteristic pattern in 31 plain edition Fructus Mali pumilaes.
Detailed description of the invention:
With specific embodiment, the inventive method is further elaborated below in conjunction with the accompanying drawings.
Embodiment 1,
The present embodiment is to be saved in 25 columnar apple types and 31 of Qingdao Agricultural University's Jiangzhou agricultural science and technology Demonstration Garden
Individual plain edition Fructus Mali pumilae type is examination material, and 25 column type types include 23 of ' Fuji ' Fructus Mali pumilae and columnar apple first familiar generation colony
Column type hybrid is individual, 2 columnar apple kinds ' Shandong adds No. 4 ' and ' the Wei Saike rising sun '；31 plain edition Fructus Mali pumilae types include ' rich
Scholar ' Fructus Mali pumilae is individual with 29 plain edition hybrids of columnar apple first familiar generation colony, 2 plain edition kinds ' Fuji ' and ' rising sun '.
Concrete operation step is as follows:
One, CTAB method is used to extract apple genome DNA
1, take the centrifuge tube of 2ml, add 1.2ml CTAB and 50 μ L beta-mercaptoethanols, and preheat in 65 DEG C of water-baths,
Take the Apple Leaves that about 0.5g children is tender, add liquid nitrogen and quickly grind to form fine powder, proceed to above-mentioned equipped with in the centrifuge tube of CTAB, shake
Swing mixing, be placed in water-bath 30min in 65 DEG C of water-baths, period repeatedly reverse mixing；
2, taking-up is cooled to room temperature, adds 700 μ L chloroforms, gently reverse mixing, static 5min, and 4 DEG C, 12000rpm is centrifuged
15min, takes 1000 μ L of supernatant liquid in new 2ml centrifuge tube；
3, add the saturated phenol of Tris of supernatant 1/2 volume, reverse mixing, static 5min, add supernatant 1/2 volume
Chloroform, reverse mixing, static 5min, 4 DEG C, 12000rpm is centrifuged 15min, takes 800 μ L of supernatant liquid to new 2ml centrifuge tube
In；
4, adding 800 μ L chloroforms, reverse mixing, static 5min, 4 DEG C, 12000rpm is centrifuged 15min, takes 600 μ L of supernatant liquid
To new 1.5ml centrifuge tube；
5, the dehydrated alcohol of supernatant 1/10 and the sodium acetate of 1/20 are added ,-20 DEG C of static 30min, 4 DEG C, 12000rpm
Centrifugal 15min, moves on to supernatant in the centrifuge tube of new 1.5ml；
6, adding isopyknic isopropanol ,-20 DEG C static 2 hours, and 4 DEG C, 12000rpm is centrifuged 15min, abandons supernatant, retains
Precipitation；
7, the washes of absolute alcohol adding 1ml 75% precipitates once, then utilizes washes of absolute alcohol once, treats ethanol
After volatilization is dry, add 50 ddH aseptic for μ L2O dissolves.Take the concentration that 5 μ LDNA utilize the agarose gel electrophoresis detection DNA of 1%
And quality；
Two, PCR amplification:
PCR amplification is carried out with the Fructus Mali pumilae DNA extracted for masterplate, with inspection first with Fructus Mali pumilae house keeper's gene M dActin primer
Survey the quality of DNA, get rid of the test error caused because of the reason of DNA, then recycle primer to Co1 and Co2 with the Herba Marsileae Quadrifoliae extracted
Really DNA is that masterplate carries out PCR amplification, and the primer sequence of Fructus Mali pumilae MdActin gene used is:
MdActin-F:AAGATTTGGCATCACACGTTC
MdActin-R:TGGATGGCAACATACATAGCA
PCR reaction employing 12 μ L systems:
PCR response procedures:
Three, amplified production is carried out electrophoresis detection
After PCR reaction terminates, will product carry out 1.5% agarose gel electrophoresis, check different testing sample DNA
The DNA band produced in PCR reaction.
Wherein, the deoxyribonucleotide sequence of Co1 is by described primer:
Forward primer Co1-F:GATGCGAGAATTAACTAGCACAC
Downstream primer Co1-R:GAATTGTTGTATGCGTTTTTCC
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 585bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:1:
GATGCGAGAATTAACTAGCACACAAATTAAACCCTCTTTTTGTCAATTGTAGTATAAGTATAAGTAGGG
TATCGTTCTAGGCCGGGGATTAGGAGGGCTTGCTAAAACCTCTTAAAAACATAAAAACAAAGTTAAAAATATTAAAC
AAGACTCAAGGACACAAAACTAGGCTAAAAACTCTAATAATTCGAAACACACTTAAAATGACTCAAAATAATAAAAA
CAATCAAAATAGACACTAGGAATTGAATGGACGGAAATTAAATTAAAAGACTAACAATAAAGAAAACTAACTAAATA
ATATAATTTAATAATGGGTGGGTGTTTGGTTTGATGAAAAGTAAATTAAACTTAATTAAATTACAGAATTGACAAAA
ACATAAAATTAAGGTGAAAGGATAAATGACGGACTAGCTAGAGGGTTCTTCTCCACACATGACACATATGCAACCTA
AATTGATTTTCAGTTGTTCTTTCAATAAATTGTGAATCTCAATACTCCAGATTAACCGTGAACAGCACTTTTTTAAT
CTTCAAGTTTTCCTTAAGTTATTGAATTGGACGGAAAAACGCATACAACAATTC
The deoxyribonucleotide sequence of Co2 is by described primer:
Co2-F:TCTACTCCTCTTTTGCCTTGG
Co2-R:ACTTCGAATTCACTCGTCTTT
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 319bp in columnar apple, should
The DNA sequence of fragment is as shown in following SEQ ID NO:2:
TCTACTCCTCTTTTGCCTTGGATTCTTTCCTTTCTTTGTCCAACTCTTTCTTTCCACCGTTTTCTTTCT
TTTTTTTTTCTACAAAACAAAACCATCATGATGATGTTTCAAACATCATCACGACTTATCATTATTAAATATAATTT
TTAATTTAATTTAAAAGCTGATCTACACAGACAGTTTTGACGAATTTATTACATAAAATTTCACTTGTTCTATTTTT
ATTTTCTTTGCATAACAAATCCTATAAACACAAAAATAACGTAAATAGCTCAAAAATATAAGGAACTAACTAAGAAA
AGACGAGTGAATTCGAAGT
The inventive method carries out PCR amplification first with Fructus Mali pumilae house keeper's gene M dActin primer to 56 examination materials, and result shows
Show that all examination materials can amplify purpose band, illustrate that extracted DNA may be used for subsequent experimental, as it is shown in figure 1, Fig. 1
In, M:DNA maker；1-23: columnar apple in F-1 hybrids；24: columnar apple ' Shandong adds No. 4 ', 25: columnar apple ' Wei Sai
Gram rising sun '；Plain edition Fructus Mali pumilae in 26-54 F-1 hybrids；55: plain edition Fructus Mali pumilae ' Fuji '；56: plain edition Fructus Mali pumilae ' rising sun '；Then profit
With primer, Co1 and Co2 is carried out PCR amplification to 56 examination materials.If the swimming lane of DNA to be detected has Co1 primer to 585bp or Co2
The DNA specific band of the 319bp of primer pair, then can interpolate that this sample belongs to columnar apple, if not having Co1 and Co2 primer
To corresponding DNA specific band, then can interpolate that this sample belongs to plain edition Fructus Mali pumilae.Result shows 23 column type Hybrids F1
In Dai, only 3 material use Co1 and Co2 primer is to amplifying corresponding specific band, as in figure 2 it is shown, in Fig. 2,
M:DNA maker；1-23: columnar apple in F-1 hybrids；24: columnar apple ' Shandong adds No. 4 ', 25: columnar apple ' Wei Saike
The rising sun '；A is the primer amplified production band to Co1；B is the primer amplified production band to Co2.29 plain edition F-1 hybrids
It is unable to utilize Co1 and Co2 primer pair amplifies to go out corresponding specific band, as it is shown on figure 3, in Fig. 3, M:DNA maker；
1-29: plain edition Fructus Mali pumilae in F-1 hybrids；30: plain edition Fructus Mali pumilae ' Fuji '；31: plain edition Fructus Mali pumilae ' rising sun '.
The present embodiment is also to column type apple variety ' the Wei Saike rising sun ', ' Shandong adds No. four ' and plain edition Fructus Mali pumilae ' rising sun ' and ' richness
Scholar ' detected, PCR result shows: with Co1 and Co2 primer in columnar apple ' the Wei Saike rising sun ' with in ' Shandong adds No. four '
All can amplify DNA specific band, as shown in Figure 2；And in plain edition Fructus Mali pumilae ' rising sun ' and ' Fuji ' amplification not go out DNA special
Property band, as shown in Figure 3.
The inventive method can distinguish 22 to 25 columnar apple materials, and discrimination power reaches 88%；To 31 plain editions
Apple Materials can distinguish 31, and discrimination power reaches 100%.
Sequence table
SEQ ID NO:1:
GATGCGAGAA TTAACTAGCA CACAAATTAA ACCCTCTTTT TGTCAATTGT AGTATAAGTA 60
TAAGTAGGGT ATCGTTCTAG GCCGGGGATT AGGAGGGCTT GCTAAAACCT CTTAAAAACA 120
TAAAAACAAA GTTAAAAATA TTAAACAAGA CTCAAGGACA CAAAACTAGG CTAAAAACTC 180
TAATAATTCG AAACACACTT AAAATGACTC AAAATAATAA AAACAATCAA AATAGACACT 240
AGGAATTGAA TGGACGGAAA TTAAATTAAA AGACTAACAA TAAAGAAAAC TAACTAAATA 300
ATATAATTTA ATAATGGGTG GGTGTTTGGT TTGATGAAAA GTAAATTAAA CTTAATTAAA 360
TTACAGAATT GACAAAAACA TAAAATTAAG GTGAAAGGAT AAATGACGGA CTAGCTAGAG 420
GGTTCTTCTC CACACATGAC ACATATGCAA CCTAAATTGA TTTTCAGTTG TTCTTTCAAT 480
AAATTGTGAA TCTCAATACT CCAGATTAAC CGTGAACAGC ACTTTTTTAA TCTTCAAGTT 540
TTCCTTAAGT TATTGAATTG GACGGAAAAA CGCATACAAC AATTC 585
SEQ ID NO:2:
TCTACTCCTC TTTTGCCTTG GATTCTTTCC TTTCTTTGTC CAACTCTTTC TTTCCACCGT 60
TTTCTTTCTT TTTTTTTTCT ACAAAACAAA ACCATCATGA TGATGTTTCA AACATCATCA 120
CGACTTATCA TTATTAAATA TAATTTTTAA TTTAATTTAA AAGCTGATCT ACACAGACAG 180
TTTTGACGAA TTTATTACAT AAAATTTCAC TTGTTCTATT TTTATTTTCT TTGCATAACA 240
AATCCTATAA ACACAAAAAT AACGTAAATA GCTCAAAAAT ATAAGGAACT AACTAAGAAA 300
AGACGAGTGA ATTCGAAGT 319
Claims (2)
1. one kind utilizes the method that DNA molecular marker quickly distinguishes columnar apple nursery stock, it is characterised in that grasp in accordance with the following steps
Make: the first step, use CTAB method to extract apple genome DNA: to take 2ml centrifuge tube, add 1.2ml cetyl trimethyl bromination
Ammonium CTAB and 50 μ L beta-mercaptoethanols, and preheat in 65 DEG C of water-baths, take the Apple Leaves that about 0.5g children is tender, add liquid nitrogen
Quickly grind to form fine powder, proceed to above-mentioned equipped with in the centrifuge tube of CTAB, concussion mixing, it is placed in water-bath 30min in 65 DEG C of water-baths,
Period reverse mixing；Taking-up is cooled to room temperature, adds chloroform, and reverse mixing, static gently, 4 DEG C, and 12000rpm is centrifuged, and takes
1000 μ L of supernatant liquid are in new 2ml centrifuge tube；Add the saturated phenol of trishydroxymethylaminomethane of supernatant 1/2 volume, reverse
Mixing, static, add the chloroform of supernatant 1/2 volume, reverse mixing, static, 4 DEG C, 12000rpm is centrifuged, and takes on 800 μ L
Clear liquid is in new 2ml centrifuge tube；Adding chloroform, reverse mixing, static, 4 DEG C, 12000rpm is centrifuged, and takes 600 μ L of supernatant liquid extremely
In new 1.5ml centrifuge tube；The dehydrated alcohol of addition supernatant 1/10 and the sodium acetate of 1/20 ,-20 DEG C of static 30min, 4 DEG C,
12000rpm is centrifuged, and is moved on to by supernatant in the centrifuge tube of new 1.5ml；Adding isopyknic isopropanol ,-20 DEG C are static 2 hours,
4 DEG C, 12000rpm is centrifuged, and abandons supernatant, retains precipitation；The washes of absolute alcohol adding 1ml 75% precipitates once, then utilizes
Washes of absolute alcohol once, after ethanol volatilization is dry, adds 50 distilled water ddH aseptic for μ L2O dissolves, and takes 5 μ LDNA and utilizes 1%
The agarose gel electrophoresis detection concentration of DNA and quality；Second step, PCR expands: first with Fructus Mali pumilae house-keeping gene
MdActin primer carries out PCR amplification with the Fructus Mali pumilae DNA extracted for masterplate, to detect the quality of DNA, gets rid of because the reason of DNA is drawn
The test error risen, then recycling primer carries out PCR amplification, used Herba Marsileae Quadrifoliae with the Fructus Mali pumilae DNA extracted for masterplate to Co1 and Co2
Really the primer sequence of MdActin gene is:
MdActin-F:AAGATTTGGCATCACACGTTC
MdActin-R:TGGATGGCAACATACATAGCA
PCR reaction employing 12 μ L systems:
PCR response procedures:
3rd step, amplified production is carried out electrophoresis detection: PCR reaction terminate after, will product carry out 1.5% agarose gel
Electrophoresis, checks the DNA band produced in different testing sample DNA PCR reaction；
Wherein, the deoxyribonucleotide sequence of Co1 is by described primer:
Forward primer Co1-F:GATGCGAGAATTAACTAGCACAC
Downstream primer Co1-R:GAATTGTTGTATGCGTTTTTCC
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 585bp, this fragment in columnar apple
DNA sequence as shown in SEQ ID NO:1 in sequence table；
The deoxyribonucleotide sequence of Co2 is by described primer:
Co2-F:TCTACTCCTCTTTTGCCTTGG
Co2-R:ACTTCGAATTCACTCGTCTTT
Use this primer to carry out PCR amplification, be only capable of amplifying the DNA specific fragment of a 319bp, this fragment in columnar apple
DNA sequence as shown in SEQ ID NO:2 in sequence table.
A kind of method utilizing DNA molecular marker quickly to distinguish columnar apple nursery stock the most according to claim 1, its feature
It is in the amplified production electrophoresis result of 2 pairs of primers that at least 1 pair of primer has the DNA specific band that described DNA molecular marker is corresponding,
Then corresponding apple nursery stock is columnar apple；The amplified production electrophoresis result of 2 pairs of primers is all without corresponding DNA specific band, then
For plain edition Fructus Mali pumilae；Column type Apple Materials discrimination power is reached 88%；Plain edition Apple Materials discrimination power is reached 100%.
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| CN112830962A (en) * | 2021-03-10 | 2021-05-25 | 中国农业大学 | Apple pillar type candidate gene Co41 related protein and coding gene and application thereof |
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| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112830962A (en) * | 2021-03-10 | 2021-05-25 | 中国农业大学 | Apple pillar type candidate gene Co41 related protein and coding gene and application thereof |
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