CN104651515B - A kind of method building Camellia sinensis DNA fingerprinting - Google Patents
A kind of method building Camellia sinensis DNA fingerprinting Download PDFInfo
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- CN104651515B CN104651515B CN201510089500.1A CN201510089500A CN104651515B CN 104651515 B CN104651515 B CN 104651515B CN 201510089500 A CN201510089500 A CN 201510089500A CN 104651515 B CN104651515 B CN 104651515B
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Abstract
The present invention relates to a kind of method building Camellia sinensis DNA fingerprinting, belong to Folium Camelliae sinensis field of molecular biotechnology.The method uses ISSR molecular marker technology and planar bar code technology to build Camellia sinensis DNA fingerprinting, finger printing information is processed by planar bar code technology, changes into two-dimension code pattern.Build Camellia sinensis DNA fingerprinting by the method to embody with the form of two-dimension code pattern, have the advantages that information capacity is big, directly perceived, succinct, application is wide.Carry out being processed by planar bar code technology during building Camellia sinensis DNA fingerprinting, by substantial amounts of finger printing information, change into two-dimension code pattern.The DNA fingerprinting built can the DNA fingerprinting information of this Camellia sinensis sample of correct response, can utilize at field further genralrlization such as the market circulation of Folium Camelliae sinensis, cultivation managements again, and be applied to review System Construction and management.
Description
Technical field
The present invention relates to a kind of method building Camellia sinensis DNA fingerprinting, belong to Folium Camelliae sinensis field of molecular biotechnology.
Background technology
Camellia sinensis (Camellia sinensis(L.) O.Kuntze) belong to Theaceae Camellia tea subgenus tea kind plant, origin
In Southwestern China area, through long-term introducing a fine variety, propagating, China's growth of tea plant region from N 18 ° 30 '-35 ° 13 ', E94 °-122 °,
South is to edge, the torrid zone, and north is to warm temperate zone.China is the main original producton location of Camellia sinensis, and the most the artificial culture of more than 3000 year is gone through
History, through the artificial or natural breeding of thousand of years, defines very abundant Tea Germplasm.At present, in country's Camellia sinensis kind
Matter resource garden has preserved nearly 4000 parts of tea germplasms, and each province, Tea Germplasm garden, place, district also preserve a large amount of tea germplasm.This
Outward, the tea hill vast stretch of wooded country that China is wide also has the Camellia sinensis new germ plasm not much being collected preservation, be wherein no lack of some excellent precious
Dilute Camellia sinensis new germ plasm, has many excellent germplasms to fail by the most effective and utilizes in these resources.Therefore, by these germ plasm resource
Effectively classify according to sibship, kind matter breediness, finger printing difference etc. and identify, Tea Germplasm is ground
Study carefully, the innovation of kind particularly important.At present, the fast development of molecular biotechnology, DNA molecular marker technology is the most ripe,
It is widely used in multiple kinds of crops.
DNA molecular marker with determine biological heredity characteristic genomic DNA as object of study, to a certain degree represents biological
Inherited character, and the Character change planting matter is closely bound up with it.DNA molecular marker technology has, accurately, stability strong, polymorphic
Property the advantage such as high, good, the requirement that kind is quick and precisely identified and standard finger-print builds can be met.At present, DNA molecular marker
Technology is widely used in the analysis of genetic diversity of organism, DNA fingerprinting structure, Relationship iden-tification, cultivar identification, new
The aspects such as breed breeding.And DNA fingerprinting technology can reflect the difference of nucleotide sequence, detect the difference between individuality, mirror
Fixed specific genotype, it is possible to quickly and accurately differentiate different kind matter, apply wide.
DNA fingerprinting refers to that DNA sample specific molecule markers technical finesse demonstrates have the total of specific DNA fragments
Claim.DNA fingerprinting technology is the earliest for determining the identity of people, then entering along with biotechnology in criminal investigation or paternity test
Step and development, DNA fingerprinting technology is widely used on a lot of plants.At present, constructed dna fingerprint image it is usually used in
The molecular marker of spectrum has SSR, AFLP, SRAP, SCoT, RAPD, SNP and ISSR.The DNA fingerprint figure that different molecular labelling builds
Spectrum is respectively arranged with its feature.Wherein, most widely used general with ISSR, SSR, SRAP, RAPD, as early molecule labelling, also has certain answering
With.SNP, SCoT labelling is as third generation molecular marker, and at present application is extensive, and technology is not mature enough, and with other 2 kinds of labellings
Comparing, technical difficulty is higher, less with the application that this technology is main constructed dna finger printing.And SRAP molecular marker, during experiment
Between longer, experimental implementation is relatively complicated, and agents useful for same toxic and side effects is relatively big, bigger on experimenter's impact.ISSR is current
The analysis of genetic diversity and the DNA fingerprinting that can be used in high-volume germ plasm resource build optimal a kind of molecular marker.
Application ISSR molecular marker constructed dna finger printing, can obtain good effect on different plants.There is primer consumption
Less, the advantage that signature is many, effective information is big.
At present, the research in Camellia sinensis of DNA fingerprinting is few, with ISSR molecular marker technology and planar bar code technology
The research building Camellia sinensis DNA fingerprinting has no report.
Summary of the invention
A kind of method building Camellia sinensis DNA fingerprinting of offer is provided, builds Camellia sinensis DNA fingerprint by the method
Collection of illustrative plates embodies with the form of two-dimension code pattern, has the advantages that information capacity is big, directly perceived, succinct, application is wide.
The present invention uses ISSR molecular marker technology and planar bar code technology to build Camellia sinensis DNA fingerprinting, by finger printing
Information is processed by planar bar code technology, changes into two-dimension code pattern.
It is embodied as follows:
Use modified CTAB method extract for examination tea tree breed STb gene, by agarose gel electrophoresis detect its concentration and
Purity, and DNA sample is diluted to 100 ng/uL, utilize the primer of the ISSR reaction system after optimizing and screening to Camellia sinensis DNA
Sample carries out PCR amplification.Amplified production is carried out agarose gel electrophoresis, separates ISSR molecular marker, shoot electrophoresis picture.Root
According to the presence or absence of ISSR collection of illustrative plates band, pcr amplification product is carried out statistical analysis, electrophoresis pattern is converted into the former of " 0-1 " form
Beginning character string.Planar bar code technology is utilized to process original character string, by a coffin by black, white pixel in a matrix
Different distributions encode, original character string is transformed into two-dimension code pattern.
Described pcr amplification product is carried out statistical analysis, be the presence or absence according to ISSR collection of illustrative plates band, utilize manual method to unite
Meter tape reading, by the band occurred stable on electrophoretogram, no matter power is all entered as " 1 ", and same position is entered as " 0 " without band,
Input tape reading data, set up original character string.
ISSR reaction system after optimization is: in 20 μ L systems, Ex-Buffer 1 × Ex-Buffer, 1U/ L Ex-
Taq enzyme, 0.2mM/ L dNTPs, 0.5mM/ L ISSR primer, 2ng/ L DNA profiling.The primer such as SEQ ID of screening
Shown in NO.1-12.
The Camellia sinensis DNA fingerprinting built can pass through image input device or photoelectric scanning device automatically identifying and reading to realize letter
Breath automatically processes, and is just understood the finger print information of this tea tree breed by scanning two-dimension code pattern, thus differentiates different Camellia sinensis product
Kind.
It is an advantage of the current invention that:
Carry out being processed by planar bar code technology during building Camellia sinensis DNA fingerprinting, substantial amounts of finger printing is believed
Breath, changes into two-dimension code pattern.The DNA fingerprinting built can the DNA fingerprinting information of this Camellia sinensis sample of correct response,
Can utilize at the field such as the market circulation of Folium Camelliae sinensis, cultivation management further genralrlization again, and be applied to review System Construction and
Management.
Accompanying drawing explanation
Its amplified production, for Murrayae Folium Et Cacumen is carried out ISSR-PCR amplification with table one shown 12 primers, is added up by Fig. 1
Analyze, form original fingerprint information character string, and the DNA fingerprinting built with its original fingerprint information character string.
Fig. 2 is the fine jade that place, 48 parts of different Wuyi Mountains tea germplasm carries out ISSR-PCR amplification gained with primer UBC834
Sepharose electrophoresis pattern.
Detailed description of the invention
Embodiment 1
1, Camellia sinensis total DNA extraction
Utilize the CTAB method after improvement to extract for examination Camellia sinensis STb gene, detect its purity by agarose gel electrophoresis, pass through
The detection of trace dna analyzer for examination DNA sample at 260 nm, the light absorption value of 280 nm, 260/280 ratio, 260/230 ratio,
And sample concentration value, select the DNA sample that purity is good, concentration is high to be diluted to 100 ng/uL.
2, ISSR reaction system optimization and primer screening
Use the method that single-factor is analyzed, respectively with initial reaction system under specific reaction system and extender
Based on, do the comparative analysis of different parameters gradient, DNA profiling usage amount, reaction primer concentration, the Ex-Tag to PCR reaction
Each parameters such as enzyme dosage, dNTP concentration, Ex-Buffer concentration, primer annealing temperature and reaction primer carry out screening one by one and adjust
Whole, optimize and set up ISSR reaction system.ISSR-PCR reaction system after optimization is: in 20 μ L systems, Ex-Buffer is with 1
× Ex-Buffer, 1U/ L Ex-Taq enzyme, 0.2mM/ L dNTPs, 0.5mM/ L ISSR primer, 2ng/ L DNA profiling energy
Preferably carry out ISSR-PCR amplification.Above-mentioned System For Screening is utilized to go out band clear, the primer that polymorphism is good, primer sequence information
And annealing temperature (Tm) is as shown in table 1.ISSR reaction system and the primer of screening after optimization can expand Theaceae Flos Camelliae Japonicae simultaneously
Belong to tea kind plant.
12 primers that table 1 filters out
Note: R=(A, G), Y=(C, T)
3, ISSR-PCR amplification
Utilize the ISSR reaction system after optimizing with the 12 of screening primers respectively to place, 48 parts of different Wuyi Mountains Camellia sinensis
DNA sample carries out PCR amplification.PCR amplification program is 94 DEG C of 300s, 94 DEG C of 60s, Tm(annealing temperatures) 30s, 72 DEG C
120s, 39 circulations, 72 DEG C of 600s, 4 DEG C save backup.
4, amplification electrophoretic analysis
The amplified production obtained in step 3 is carried out on 1% agarose gel electrophoresis, separates the expansion of ISSR molecular marker
Increasing fragment, and shoot electrophoresis picture, accompanying drawing 2 is the amplification of primer UBC834.
5, the statistical analysis of pcr amplification product
According to the presence or absence of ISSR collection of illustrative plates band, each band (DNA fragmentation) in electrophoresis pattern is all as a molecule mark
Note, represents a pair binding site of this species gene and Primers complementary.Utilize manual method statistics tape reading, by steady on electrophoretogram
No matter power is all entered as " 1 " to make existing band, and same position is entered as " 0 " without band, by the amplification of 12 primers in table 1
Tape reading data input Excel form, sets up original fingerprint information character string.Character string is that string comprises 12 primer finger print informations
" 0-1 " binary digit character string.Original fingerprint information character string such as Murrayae Folium Et Cacumen: 101101000001000001000111
10101001100001110101100000110000010101011100000000000000001110100101001111111
111001100101010110100000011111111100010000000011101110010000000110。
6, Camellia sinensis DNA fingerprinting is built
Planar bar code technology is utilized to process original fingerprint information character string, by passing through black, white pixel at a coffin
Different distributions in a matrix encodes.On matrix element position, appearance side's point, round dot or other shape points represent that two enter
System " 1 ", occurs without an expression binary " 0 ", and the permutation and combination of point determines the finger printing that matrix two-dimensional code is comprised
Information.Original fingerprint information character string is transformed into the two-dimension code pattern i.e. DNA fingerprinting of this Camellia sinensis, structure
Camellia sinensis DNA fingerprinting can be automatically processed to realize information by image input device or photoelectric scanning device automatically identifying and reading, logical
The comparison crossing two-dimension code pattern can differentiate different tea tree breeds.
<110>University Of Agriculture and Forestry In Fujian
<120>a kind of method building Camellia sinensis DNA fingerprinting
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>artificial sequence
<400> 1
tgtgtgtgtg tgtgtgg 17
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>y=c or t
<400> 2
agagagagag agagagyt 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>y=c or t
<400> 3
agagagagag agagagya 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>y=c or t
<400> 4
gagagagaga gagagayt 18
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>y=c or t
<400> 5
gagagagaga gagagayc 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>r=a or g
<400> 6
ctctctctct ctctctrg 18
<210> 7
<211> 18
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (17)..(17)
<223>r=a or g
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tctctctctc tctctcrt 18
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<213>artificial sequence
<220>
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<222> (17)..(17)
<223>r=a or g
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tctctctctc tctctcrg 18
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ccgccgccgc cgccgccg 18
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tagatctgat atctgaattc cc 22
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agagttggta gctgttgatc 20
<210> 12
<211> 21
<212> DNA
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<400> 12
acttccccac aggttaacac a 21
Claims (3)
1. the method building Camellia sinensis DNA fingerprinting, it is characterised in that: use ISSR molecular marker technology and Quick Response Code skill
Art builds Camellia sinensis DNA fingerprinting, finger printing information is processed by planar bar code technology, changes into two-dimension code pattern, described
Method is embodied as follows:
Use modified CTAB method to extract the STb gene for examination tea tree breed, detect its concentration and purity by agarose gel electrophoresis,
And DNA sample is diluted to 100 ng/ μ L, utilize the primer of the ISSR reaction system after optimizing and screening to Camellia sinensis DNA sample
Carry out PCR amplification, amplified production is carried out agarose gel electrophoresis, separate ISSR molecular marker, shoot electrophoresis picture；According to
The presence or absence of ISSR collection of illustrative plates band, carries out statistical analysis to PCR amplified production, and electrophoresis pattern is converted into the former of " 0-1 " form
Beginning character string, utilizes planar bar code technology to process original character string, original character string is transformed into two-dimension code pattern；
The primer of described screening is as shown in SEQ ID NO.1-12.
The method of structure Camellia sinensis DNA fingerprinting the most according to claim 1, it is characterised in that: described to PCR amplification
Product carries out statistical analysis, is the presence or absence according to ISSR collection of illustrative plates band, utilizes manual method statistics tape reading, by steady on electrophoretogram
Making existing band, no matter power is all entered as " 1 ", and same position is entered as " 0 " without band, input tape reading data, sets up former
Beginning character string.
The method of structure Camellia sinensis DNA finger printing the most according to claim 1, it is characterised in that: the ISSR after optimization is anti-
The system is answered to be: in 20 μ L systems, Ex-Buffer 1 × Ex-Buffer, 1U/ μ L Ex-Taq enzyme, 0.2 mM/ μ L
DNTPs, 0.5 mM/ μ L ISSR primer, 2ng/ μ L DNA template.
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| CN105486684A (en) * | 2016-01-13 | 2016-04-13 | 广东产品质量监督检验研究院 | Non-destructive testing method for mahogany furniture wood varieties based on network platform |
| CN106294728A (en) * | 2016-08-08 | 2017-01-04 | 江勇 | The construction method of androphile's information bank and system thereof |
| CN107180165B (en) * | 2017-05-27 | 2020-11-03 | 云南省农业科学院质量标准与检测技术研究所 | Method for establishing Yunrui series corn variety DNA molecular label |
| CN107190082B (en) * | 2017-07-14 | 2018-03-23 | 中国农业科学院茶叶研究所 | For differentiating the molecular specificity labeled primers and its discrimination method of national improved tea variety ' meeting frost ' |
| CN107760797B (en) * | 2017-11-01 | 2020-10-09 | 上海市农业科学院 | Eggplant variety DNA fingerprint spectrum and establishing method and application thereof |
| CN114507747B (en) * | 2021-09-16 | 2022-11-15 | 贵州省烟草科学研究院 | Tobacco SNP marker developed based on whole genome re-sequencing and KASP technology and application thereof |
| CN115896333B (en) * | 2022-11-24 | 2024-06-07 | 安徽农业大学 | Method for identifying Jinyu No. 1 tea tree strain by utilizing SSR fingerprint |
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Non-Patent Citations (4)
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| 14 个黄‘山种’茶树单株的DNA指纹图谱构建;潘璐璐 等;《西南农业学报》;20121231;第25卷(第5期);1.2、1.3节 * |
| 20个云南无性系茶树良种的DNA指纹图谱构建;孙本英 等;《热带作物学报》;20111231;第32卷(第4期);1.2节 * |
| 孙本英 等.20个云南无性系茶树良种的DNA指纹图谱构建.《热带作物学报》.2011,第32卷(第4期), * |
| 潘璐璐 等.14 个黄‘山种’茶树单株的DNA指纹图谱构建.《西南农业学报》.2012,第25卷(第5期), * |
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