CN107630077A - It is a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique - Google Patents
It is a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique Download PDFInfo
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Abstract
The invention belongs to lichee Biotechnology in Genetic Breeding field, and in particular to a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique.Applicant develops pair of primers LcCeED F and LcCeED R according to the difference of two type LcFT1 genes, and based on capillary electrophoresis technique identification easily into flower lichee germ plasm resource.The method of the present invention can be on DNA level to easily identifying have the characteristics that method simplicity, efficiency high, qualification result are accurate and are not affected by the external environment, be with a wide range of applications in lichee breeding from now on into flower lichee germ plasm resource.
Description
Technical field
The invention belongs to lichee Biotechnology in Genetic Breeding field, and in particular to one kind based on capillary electrophoresis technique identification easily into
The method of flower lichee germ plasm resource.
Background technology
Lichee (Litchi chinensis Sonn.) is Sapindaceae lichee platymiscium, originating from China, cause and effect reality shape,
Excellent in color and it is nutritious and praise claim " south of the Five Ridges fruit king " have won fame both at home and abroad.Conventional knowhow shows that low temperature is that lichee is completed
The crucial external environment factor needed for floral induction, it was found that the Litchi Varieties (such as " March is red ", " brown hair litchi ") of only a few are only
Floral induction can just be completed by needing the low temperature of short period, easily into flower, thus be referred to as easily into flower Litchi Varieties；It is and most of
Litchi Varieties have to pass through the low temperature in whole winter (such as " osmanthus taste ", " glutinous rice wrapped in lotus leaves ", " purple good happiness ") floral induction could be completed,
Hardly possible is referred to as hardly possible into flower Litchi Varieties into flower.Research finds easily to be originating primarily from China's lichee production into flower Litchi Varieties
The northern area (such as Yunnan etc.) in area, and difficult southern area (such as Hainan that lichee producing region is originating primarily from into flower Litchi Varieties
Etc. ground).Conventional knowhow also shows：Winter low temperature deficiency is the main reason for lichee can not stablize into flower, especially for difficulty
It is even more so for into flower Litchi Varieties.
In lichee production, every year due into spend it is unstable caused by yield not have guarantee be numerous orchard workers face for a long time
The main bugbear faced.One of them is main reason is that difficult too high into flower Litchi Varieties proportion, and easily into flower lichee
Kind proportion is on the low side, and Yield of Litchi is influenceed very big by winter low temperature deficiency.A main cause of above phenomenon is caused to exist
In lack to easily into flower Litchi Varieties Molecular Identification, another main reason is that can meet commodity cultivation it is high-quality easily into
Flower Litchi Varieties are rare.Though have the easy into flower Litchi Varieties (as " March is red ") of minority at present, due to its poor quality by by
Gradually eliminate.Hardly possible is into flower Litchi Varieties (such as " osmanthus taste " and " glutinous rice wrapped in lotus leaves "), although being influenceed very greatly by winter low temperature deficiency into flower,
It is due to its high-quality by the pro-gaze of consumers in general, is gradually widely applied, but annual yield cannot
Ensure, such as 2017 due to by winter low temperature deficiency influenceed (warm winter), the yield underproduction 80% or so.Therefore, to easily into flower litchi
Branch kind Molecular Identification and it is high-quality easily into flower Litchi Varieties seed selection it is particularly important.
Inventor is cultivated by Artificial facilities carries out the experiment of anti-season low temperature induction to lichee in summer, effectively demonstrates low
Temperature is to induce lichee into colored key environmental factors, blade and terminal bud development topical hypothermia Induction experiments to lichee, it was demonstrated that
The blade of lichee is the critical organ for experiencing low temperature induction signal, and for this, inventor carries out RNA-Seq to the blade of low-temperature treatment
With tiny RNA high flux deep sequencing.A lichee is wherein screened into key gene LcFT1 is spent by RNA-Seq, further
Research has shown that low temperature is to cause it into flower by inducing the expression of LcFT1 genes in Litchi Leaves.Further study show that
There are two types, i.e. low-temperature sensitive type and low temperature non-sensitive type in lichee LcFT1 genes, this is mainly by both promoter alkali
The difference of basic sequence causes, it was found that the lichee containing low-temperature sensitive type LcFT1 genes is easily into flower, i.e., LcFT1 genes are pure
Close low-temperature sensitive type or for heterozygous lichee easily into flower.Main reason is that low-temperature sensitive type LcFT1 gene pairs low temperature is non-
Often sensitive, seldom low temperature amount is with regard to that can induce its expression even in the time of warm winter, and then it is (related to cause lichee to be easy into flower
As a result with Promoter difference of LcFT1is a leading cause of natural variation of
Flowering timing in different litchi cultivars (Litchi chinensis Sonn.) are published in
plant science,2015, 241:128-137).Above result of study is also consistent with conventional knowhow, such as easily into flower litchi
Branch kind (such as " March is red ", " brown hair litchi ", " cv. Feizixiao ", " Lan Zhu ", " osmanthus morning litchi ") is all containing low-temperature sensitive type
The lichee of LcFT1 genes, they only need the low temperature of short period in winter just to complete floral induction, thus Yi Chenghua.And
It is difficult into flower Litchi Varieties (such as " Ma Guili ", " osmanthus taste ", " glutinous rice wrapped in lotus leaves ", " the good happiness of purple ") be all LcFT1 gene pures low temperature not
Responsive type lichee, the low temperature that they have to pass through whole winter can complete floral induction, need low temperature amount more, thus it is difficult into
Flower.Inventor develops pair of primers LcCeED-F and LcCeED-R according to the difference of two type LcFT1 gene promoters, and
Based on capillary electrophoresis technique identification easily into flower lichee germ plasm resource.
Capillary Electrophoresis (Capillary Electrophoresis, CE) is that the one kind to come out the 1980s is efficient
Liquid phase separation method, it is the product that classical electrophoretic techniques and modern Micro-Column Separation technology are combined, it is considered to be contemporary analysis science
Most active Some Questions To Be Researched.Capillary electrophoresis technique is because its separative efficiency is high, analyze speed is fast, amount of samples is few, easy
Be widely used in the fields such as molecular biology, medical science, pharmacy, macromolecule in the automation the features such as, especially in bioanalysis and
Wide application prospect is shown in life science correlative study.The present invention for it is high-quality from now on easily into flower Litchi Varieties seed selection and push away
Certain basis is extensively laid, is with a wide range of applications.
The content of the invention
The present invention is reflected according to the exploitation of the difference of two type LcFT1 genes of lichee is a kind of based on capillary electrophoresis technique first
Determine easily into the method for flower lichee germ plasm resource.
One of the object of the invention is to provide the invention theoretical foundation of the above method.
The two of the object of the invention are to provide the specific content of the invention of the above method.
The three of the object of the invention are to provide the specific detecting step of the above method.
In order to realize foregoing invention purpose, the present invention uses following technical scheme：
1. the present invention provides the invention theoretical foundation of the above method：Low temperature can induce floral genes in Litchi Leaves
LcFT1 expression and then it is caused two types to be present into flower, lichee LcFT1 genes, i.e. low-temperature sensitive type and low temperature is insensitive
Type, this is mainly caused by both differences of promoter base sequence, wherein including 4 small indels, further research
It was found that the lichee of the LcFT1 genes of type containing low-temperature sensitive is easily into flower.
2. the present invention provides the specific content of the invention of the above method：According to low-temperature sensitive type and low temperature non-sensitive type LcFT1
Be present small indels differences at 4 in gene promoter, set in rear 3 small indels diversity sequences both ends identical region
Count pair of primers LcCeED-F and LcCeED-R.It is 674bp to detect low-temperature sensitive type LcFT1 gene purpose fragments size, detection
Low temperature non-sensitive type LcFT1 gene purpose fragments size is 659bp.
LcCeED-F：5’-TTTGTTGGCGTTGTCGGTTCTAATTA-3’；
LcCeED-R：5’-TAGCTAAGCAGCCACAAACTCAAT-3’.
3. a kind of identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique, comprise the following steps：
(1) DNA of lichee to be detected is extracted, is comprised the following steps that：
(a) blade material is cleaned and be put into the mortar of liquid nitrogen frozen, add a little insoluble PVP, liquid feeding nitrogen is ground into
Powder；
(b) take 0.5g powder to be transferred in 10mL centrifuge tubes and add the CTAB extract solutions of 65 DEG C of water-baths of 3mL, acutely shake
Swing, 65 DEG C of water-bath 30-60min, gently shaken once every 10min；
(c) isometric phenol is added：Chloroform：Isoamyl alcohol (V/V/V：25：24：1) gentle inversion mixes, 12000rpm centrifugations
10min；
(d) supernatant is taken, adds isometric chloroform：Isoamyl alcohol (V/V：24：1) gentle inversion mixes, 12000rpm centrifugations
10min；
(e) supernatant is taken, adds isometric chloroform：Isoamyl alcohol (V/V：24：1), gentle inversion mixes, 12000rpm centrifugations
10min, supernatant is taken, the isopropanol gentle inversion for adding 2/3V precoolings mixes, -20 DEG C of precipitation DNA 30min；
(f) 12000rpm centrifuges 20min, abandons supernatant, adds 70% ethanol washing precipitation 2 times；
(g) add 100-300 μ L TE buffer solutions, its quality, ultraviolet spectrometry light are detected by 0.8% agarose gel electrophoresis
Degree meter detects its concentration and purity, at last most DNA are diluted to 50ng/ μ L, it is standby to be put into -20 DEG C of refrigerators.
(2) PCR is expanded：Using lichee DNA to be detected as template, with sense primer LcCeED-F and anti-sense primer
LcCeED-R enters performing PCR amplification, and its standard PCR reaction systems are 25 μ L, and concrete composition is as follows：The μ L of DNA profiling 1, sense primer 1
The 9.5 μ L of μ L, ddH2O of μ L, 1 μ L, 2 × Taq PCR MasterMix (TIANGEN) of anti-sense primer 12.5.PCR specifically expands journey
Sequence is as follows：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulations, 72 DEG C extend
10min, 10 DEG C of stoppings.
(3) PCR primer capillary electrophoresis detection：1 μ L are taken to be added in each hole of 96 orifice plates from the PCR primer of amplification,
9 μ L deionized formamides, 1 μ L GS3730-500 molecular weight internal standards, 95 DEG C of pre-degeneration 5min, 4 DEG C of insulations are separately added into again
3000rpm centrifuges 1min after 10min, then carries out Capillary Electrophoresis 30min, voltage by ABI3730XL DNA analyses instrument
2kv, sample injection time 5 s, fluorescence 6-fam, initial data is collected with the softwares of ABI data collection 2.1, then used
GeneMarker V1.91Demo softwares are analyzed the initial data of collection, and system is by the position of each peak value and its swimming lane
GS3730-500 molecular weight internal standards compare determination clip size, generate image.
(4) easily into the identification and analysis of flower lichee germ plasm resource：Enter performing PCR as template using lichee DNA sample to be detected to expand
Increase, pcr amplification product shows that lichee to be detected is through capillary electrophoresis detection if there is single peak value at 674bp
LcFT1 gene pure low-temperature sensitives type is easily into flower lichee germ plasm resource；If occur two peaks simultaneously at 659bp and 674bp
Value then shows that lichee to be detected easily into spends lichee germplasm money for the heterozygosis of LcFT1 gene low-temperature sensitive types and low temperature non-sensitive type
Source；It is LcFT1 gene pure low temperature non-sensitive type lichee to show lichee to be detected if there is single peak value at 659bp
Germ plasm resource.
Compared with prior art, the present invention has the advantages that：
(1) it is provided by the present invention easily can be into the method for flower lichee germ plasm resource based on capillary electrophoresis technique identification
To easily being identified into flower lichee germ plasm resource on DNA molecular genetic level, tradition is overcome easily into flower lichee germ plasm resource table
Numerous shortcomings of type observation identification, such as：Phenotypic Observation identification can not carry out early stage identification in plantlet stage, by time restriction
Restrict；The shortcomings of Phenotypic Observation qualification result is very big by human factor and external environment influence, and qualification result reliability is not high.
(2) method of the invention, which is based on capillary electrophoresis technique, has that separative efficiency is high, analyze speed is fast, amount of samples
Less, it is easy to automation, the features such as qualification result is accurate, while not affected by environment, the advantages that selection target is clear and definite.
Brief description of the drawings
Fig. 1 is lichee low-temperature sensitive type LcFT1 gene promoters and low temperature non-sensitive type LcFT1 gene promoter sequence ratios
To figure；
Wherein, ' E ' is low-temperature sensitive type LcFT1 gene promoter sequences；' D ' is that low temperature non-sensitive type LcFT1 genes open
Promoter sequences；Black triangle be 4 existing for low-temperature sensitive type and low temperature non-sensitive type LcFT1 gene promoters at small
Indels differences；Black box is LcFT1 gene opens reading frame (ORF) initiation codon ATG.
The PCR primer that Fig. 2 is primer LcCeED-F and LcCeED-R is identified by Capillary Electrophoresis easily into spends lichee germplasm
The collection of illustrative plates of resource；
Wherein, Fig. 2-A are LcFT1 gene pure low-temperature sensitives type easily into flower lichee germ plasm resource；Fig. 2-B are LcFT1 bases
Because of homozygous low temperature non-sensitive type lichee germ plasm resource；Fig. 2-C are the heterozygosis of LcFT1 gene low-temperature sensitive types and low temperature non-sensitive type
Easily into flower lichee germ plasm resource.
Embodiment
With reference to specific embodiment, make further details of elaboration to the present invention, but embodiments of the present invention are not
It is confined to the scope of embodiment expression.These embodiments are merely to illustrate the present invention, not for limitation the scope of the present invention.This
Outside, after present disclosure is read, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same
Sample falls within appended claims limited range of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
The material that uses, reagent etc., unless otherwise specified, are commercially obtained.
Embodiment 1：
It is a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique.
1. a kind of identified easily into the invention theoretical foundation for the method for spending lichee germ plasm resource based on capillary electrophoresis technique
It is characterized in that：Low temperature can induce floral genes LcFT1 expression in Litchi Leaves and then cause it into flower, litchi
There are two types, i.e. low-temperature sensitive type and low temperature non-sensitive type in branch LcFT1 genes, this is mainly by both promoter base sequences
The difference of row causes, wherein comprising 4 small indels, further study show that the litchi of the LcFT1 genes of type containing low-temperature sensitive
Branch is easily into flower.
2. easily comprised the following steps that into the identification of flower lichee germ plasm resource：
(1) 72 different lichee germ plasm resources are collected (such as：" March is red ", " cv. Feizixiao ", " Lan Zhu ", " osmanthus morning litchi ", " horse
Your litchi ", " osmanthus taste ", " glutinous rice wrapped in lotus leaves ", " purple good happiness " etc.), and their DNA is extracted, comprise the following steps that：(a) by blade material
Clean and be put into the mortar of liquid nitrogen frozen, add a little insoluble PVP, liquid feeding nitrogen grind into powder；(b) 0.5g powder is taken to shift
Into 10mL centrifuge tubes and the CTAB extract solutions of 65 DEG C of water-baths of 3mL are added, acutely concussion, 65 DEG C of water-bath 30-60min, every 10
Min gently shakes once；(c) isometric phenol is added：Chloroform：Isoamyl alcohol (V/V/V：25：24：1) gentle inversion mixes,
12000rpm centrifuges 10min；(d) supernatant is taken, adds isometric chloroform：Isoamyl alcohol (V/V：24：1) gentle inversion mixes,
12000rpm centrifuges 10min；(e) supernatant is taken, adds isometric chloroform：Isoamyl alcohol (V/V：24：1), gentle inversion mixes,
12000rpm centrifuges 10min, takes supernatant, and the isopropanol gentle inversion for adding 2/3V precoolings mixes, -20 DEG C of precipitation DNA 30
min；(f) 12000rpm centrifuges 20min, abandons supernatant, adds 70% ethanol washing precipitation 2 times；(g) plus 100-300 μ L TE are buffered
Liquid, its quality, its concentration of UV spectrophotometer measuring and purity, at last most DNA are detected by 0.8% agarose gel electrophoresis
50ng/ μ L are diluted to, it is standby to be put into -20 DEG C of refrigerators.
(2) design of primers：
As shown in figure 1, small at 4 be present according to according to low-temperature sensitive type and low temperature non-sensitive type LcFT1 gene promoters
Indels differences, rear 3 small indels diversity sequences both ends identical region design pair of primers LcCeED-F and
LcCeED-R.It is 674bp to detect low-temperature sensitive type LcFT1 gene purpose fragments size, detects low temperature non-sensitive type LcFT1 genes
Purpose fragment size is 659bp.
LcCeED-F：5’-TTTGTTGGCGTTGTCGGTTCTAATTA-3’；
LcCeED-R：5’-TAGCTAAGCAGCCACAAACTCAAT-3’.
(3) PCR is expanded：
Enter performing PCR amplification, its standard PCR reaction systems by template of the DNA of 72 lichee germ plasm resource to be detected respectively
It is as follows for 25 μ L, concrete composition：The μ L of DNA profiling 1, the μ L of sense primer 1, μ L, the 2 × Taq PCR MasterMix of anti-sense primer 1
(TIANGEN) 12.5 9.5 μ L of μ L, ddH2O.The specific amplification programs of PCR are as follows：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 57
DEG C annealing 30s, 72 DEG C extension 30s, 35 circulation, 72 DEG C extension 10min, 10 DEG C stopping.The PCR primer of amplification is through 1.5% concentration
Agarose electrophoresis detection.
(4) PCR primer capillary electrophoresis detection：
Take 1 μ L to be added in each hole of 96 orifice plates from the PCR primer of amplification, then be separately added into 9 μ L deionization formyls
Amine, 1 μ L GS3730-500 molecular weight internal standards, 95 DEG C of pre-degeneration 5min, 4 DEG C are incubated 3000rpm centrifugation 1min after 10min, then
Capillary Electrophoresis 30min is carried out by ABI3730XL DNA analyses instrument, voltage 2kv, sample injection time 5s, fluorescence 6-fam, uses ABI
The softwares of data collection 2.1 collect initial data, then with GeneMarker V1.91Demo softwares to the original of collection
Data are analyzed, and the position of each peak value is compared and determines that fragment is big by system with GS3730-500 molecular weight internal standard in its swimming lane
It is small, generate image.
(5) easily into the identification and analysis of flower lichee germ plasm resource：
As shown in Fig. 2 entering performing PCR amplification by template of lichee DNA sample to be detected, pcr amplification product is through capillary
Electrophoresis detection, if occurring single peak value at 674bp, it has the sequence as shown in Seq ID NO.3, then shows to be detected
Lichee is LcFT1 gene pure low-temperature sensitives type easily into flower lichee germ plasm resource (Fig. 2-A)；
If occurring single peak value at 659bp, it has the sequence as shown in Seq ID NO.4, then shows to be detected
Lichee is LcFT1 gene pure low temperature non-sensitive type lichee germ plasm resources (Fig. 2-B)；If go out simultaneously at 659bp and 674bp
Existing two peak values then show that lichee to be detected easily into spends lichee for the heterozygosis of LcFT1 gene low-temperature sensitive types and low temperature non-sensitive type
Germ plasm resource (Fig. 2-C).
Analyze and identify and show more than, there are 29 in above-mentioned 72 lichee germ plasm resource easily into flower lichee germ plasm resource,
Wherein have 3 for LcFT1 gene pure low-temperature sensitives type easily into flower lichee germ plasm resource (i.e. " March is red ", " cv. Feizixiao " and " orchid
Bamboo "), have 26 for heterozygosis easily into flower lichee germ plasm resource (such as " osmanthus morning litchi ", " ridge is rich glutinous ", " uncle (mother's brother) emperor "), remaining 43
Individual is LcFT1 gene pure low temperature non-sensitive type lichee germ plasm resource (such as " osmanthus taste ", " glutinous rice wrapped in lotus leaves ", " purple good happiness ").
The qualification result of above lichee germ plasm resource will easily be laid well to be high-quality from now on into the seed selection of flower lichee new germ plasm
Basis.
Sequence table
<110>Guangxi Autonomous Region Academy of Agricultural Sciences's Horticultural Research Institute
<120>It is a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique
<130> ZYWS
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>Artificial sequence (a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique)
<400> 1
tttgttggcg ttgtcggttc taatta 26
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique)
<400> 2
tagctaagca gccacaaact caat 24
<210> 3
<211> 674
<212> DNA
<213>Artificial sequence (a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique)
<400> 3
tttgttggcg ttgtcggttc taattacgag cttaatattc ttatatactg agtattttta 60
tttatttttt cacatatggt tttagttaat taacattaat taattaatta attaattata 120
atttgacacg ttctacaata cacttggaat tgttaccatt aattttttaa taagttagac 180
ttaagggttg atacttaatt ttcaattcat aggttaacac cttagatttg atcaattaaa 240
acttaaatga ttaaggtttc aaatatatct atacatactc ctgtgcattg tagattgtgt 300
ctcgtatttt ctatgtaaaa agtttatttt tgcatggagt acagggatgg gatgctctgg 360
accggcttga gcataggctg ttccaatcag aatataagat agataggtcc accttcactt 420
tacaattcca ggtatcactc ttttgatcta gctaggaatc aagaggaaaa ataaaagcaa 480
attaaattca gaaaataaaa gtagcaccca cagattctat tttctagtga ggtggtacta 540
aagataaagt agaaccacag aacaaaaata tatatagctg tgctgcatgt cttgaaaatc 600
agcaatcaac caacaatatt tcagttgaat attcttcttt aacactacta attgagtttg 660
tggctgctta gcta 674
<210> 4
<211> 659
<212> DNA
<213>Artificial sequence (a kind of to be identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique)
<400> 4
tttgttggcg ttgtcggttc taattacgag cttaatgctc atatataatg agtattttta 60
tttatttttt catatattgt tttagttaat taacattaat taattaatta taattcgaca 120
cattctacaa tacacttgga attgttacca ttaatttttt taataagtta gacttaaggg 180
ctgatactta attttcaatt cataggtcaa caccttagat ctgattaatt aaaaattaaa 240
tgattaaggt ttcaaatata tctatactcc agtgcattgt agattgtgtc tcgtattttc 300
taagtaaaaa gtttatttat gcatggagta cagggatggg atgctctgga ccggcttgag 360
cataggctgt tccaatcaga atataagata gataggtcca ccttcacttt acaattccag 420
gtatcactat tttgatctag ctaggaatca agaggaaaag taaaagcaaa attcagaaaa 480
taaaagtagc acccacatat tctattttct agtgaggtgg tactaaagat aaagtagaac 540
cacagaacaa aaatatatat agctgtgctg catgtcttga aaatcagcaa tcaaccaaca 600
atatttcagt tgattattct tctttaacac tactaattga gtttgtggct gcttagcta 659
Claims (4)
1. a kind of identified easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique, it is characterised in that including as follows
Step：
(1) DNA of lichee to be detected is extracted；
(2) PCR is expanded：Using lichee DNA to be detected as template, entered with sense primer LcCeED-F and anti-sense primer LcCeED-R
Performing PCR expands；
(3) PCR primer capillary electrophoresis detection：1 μ L PCR primer is taken to be separately added into 9 μ L deionized formamide and 1 μ L
GS3730-500 molecular weight internal standards, 95 DEG C of pre-degeneration 5min, 4 DEG C are incubated 10min again, then pass through ABI3730XL DNA analyses
Instrument carries out Capillary Electrophoresis, then carries out analysis generation collection of illustrative plates with GeneMarker V1.91 Demo softwares；
(4) easily into the identification and analysis of flower lichee germ plasm resource：Enter performing PCR amplification by template of lichee DNA sample to be detected,
Pcr amplification product is through capillary electrophoresis detection, if occurring single peak value at 674bp, it is LcFT1 to show lichee to be detected
Gene pure low-temperature sensitive type is easily into flower lichee germ plasm resource；If occurring two peak values simultaneously at 659bp and 674bp,
Show that lichee to be detected easily into spends lichee germ plasm resource for the heterozygosis of LcFT1 gene low-temperature sensitive types and low temperature non-sensitive type；Such as
There is single peak value at 659bp in fruit, then shows that lichee to be detected provides for LcFT1 gene pure low temperature non-sensitive type lichee germplasm
Source.
2. according to claim 1 identify that it is special easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique
Sign is that a pair of upstream and downstream primers LcCeED-F and LcCeED-R of design enter performing PCR amplification, the 674bp of amplification in step (4)
It has sequence as shown in Seq ID NO.3 to purpose band, for detecting LcFT1 gene low-temperature sensitive type promoters；Amplification
659bp purpose bands its there is sequence as shown in Seq ID NO.4, opened for detecting LcFT1 gene low temperature non-sensitive types
Mover.
3. according to claim 1 identify that it is special easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique
Sign is that totally 25 μ L, concrete composition are as follows for the reaction system of the PCR amplifications described in step (2)：2×Taq PCR
The μ L of MasterMix (TIANGEN) 12.5, the μ L of DNA profiling 1, each 1 μ L of upstream and downstream primer, the μ L of water 9.5.
4. according to claim 3 identify that it is special easily into the method for flower lichee germ plasm resource based on capillary electrophoresis technique
Sign is that described sense primer is the sequence shown in Seq ID NO.1, and described anti-sense primer is shown in Seq ID NO.2
Sequence.
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| CN107012243A (en) * | 2017-05-10 | 2017-08-04 | 广西壮族自治区农业科学院园艺研究所 | A kind of molecular labeling for identifying the true hybrid of lichee and its application |
Non-Patent Citations (1)
| Title |
|---|
| 杜娟编: "《医学细胞与分子生物学理论与技术》", 31 December 2012 * |
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Application publication date: 20180126 |