CN108410967B - A kind of method of Rapid identification river camellia tradition famous-object - Google Patents

A kind of method of Rapid identification river camellia tradition famous-object Download PDF

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CN108410967B
CN108410967B CN201810200510.1A CN201810200510A CN108410967B CN 108410967 B CN108410967 B CN 108410967B CN 201810200510 A CN201810200510 A CN 201810200510A CN 108410967 B CN108410967 B CN 108410967B
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camellia
river
dna
seq
nucleotide sequence
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CN108410967A (en
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权俊萍
张绍林
韩国勇
刘家艳
刘宇
田波
沈薛洁
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Chongqing Nanshan Botanical Garden Management Department
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Chongqing Nanshan Botanical Garden Management Department
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Abstract

The present invention relates to a kind of methods of Rapid identification river camellia tradition famous-object, and the present invention provides 3 couples of SSR label primers P06, A54 and A80, and they are applied to cultivar identification.Silver staining detects after the present invention carries out PCR amplification and polyacrylamide gel electrophoresis using the DNA of 3 pairs of SSR primer pairs, 10 river camellia kinds.1, the SSR marker map specific band that the present invention obtains is clear, Unspccific bands are few, polymorphism is high.2,3 pairs of SSR primers that the present invention chooses can simultaneously all distinguish 10 river camellia kinds one by one, and identification kind is more.3, this method objectivity compared with Conventional wisdom discrimination method is stronger, more accurately.4, this method can save time and cost compared with the qualitative or quantitative comprehensive judgement method of observation data, provide more very wide application prospect to the identification of river camellia kind.

Description

A kind of method of Rapid identification river camellia tradition famous-object
Technical field
The present invention relates to Germplasm Identification technologies, establish a kind of side quick and precisely identified for river camellia tradition famous-object Method belongs to technical field of molecular biology.
Background technique
Camellia is Theaceae Theaceae Camellia Camellia plant, Chinese top ten traditional flowers, world's rare flower One of, it is high ornamental value Landscape Trees more important on the south Southwestern China and the Yangtze river basin.Camellia cultivation history is long, Many kinds are mostly that trophosome variation obtains, and interracial morphological difference is mostly more subtle, even some kinds are professional Personnel are also difficult to explicitly be distinguish from phenotypic character, bring to preserving seed, exchange, breed breeding and production aspect Very big puzzlement.Especially in recent years with the gradually rise of camellia seedling industry, seedling market circulation is increasingly frequent, occurs one A bit due to kind mistake caused by nursery stock dispute, it is especially especially prominent in the transaction of some rare kinds of tradition.Establish quickly, Efficiently, accurate camellia famous-object identification technology can become the exchange of protection excellent variety, research and the important technology branch promoted and applied Support.
River camellia is important one in the cultivation monoid greatly of camellia five, is gone through in the cultivation that Ba-Shu area has more than 2000 years History, there is many outstanding ancient traditional famous-objects, including white foreign piece, river agate, kermes squama, Zijin hat, goldentop is bright red, seven hearts are red, Paeonia szechuanica Fang, liquor-saturated poplar prince wife, Chongqing are red etc..Nanshan Mountain, Chongqing botanical garden has the unique river camellia germ plasm resource base in the world, receives Collection child care has more than ancient kind 100 of river camellia.
The identification of nursery stock kind, first is that can be rule of thumb subject to from mode of appearance by experienced professional technician Determine, this often will cause subjectivity bring mistake；Second is that by the way that observation data are qualitative or quantitative comprehensive judgement, but often Need the data observation of a Growing season or some particular growth developmental stage (flower, fruit, seed) collect, camellia seeds seedling from It is seeded into bloom and generally requires 5 years or so time, therefore lack timeliness and accuracy, and phenotypic character is vulnerable to cultivation management With the influence of environmental condition；Third is that being identified by DNA molecular marker finger-print, have immediately, efficiently, intuitively, accurately The characteristics of.
With the development of molecular biology, various DNA molecular marker technologies continue to bring out, and detection technique is gradually perfect, is Realize camellia kind early stage, quickly and precise Identification provide new way SSR (Simple sequence repeats, simply Sequence repeats) it is that the molecular marking technique for being widely used in all kinds of plant researchs in recent years has without predicting species gene group sequence Have the advantages that experimental implementation is simple, stability is good, rich polymorphism, is carrying out Genetic Diversity analysis, Germplasm Identification, something lost The building of linkage map, the searching of gene linkage label and the assignment of genes gene mapping and comparative genomics research etc. is passed to have obtained very well Application, the research in camellia at present primarily focuses on the discussion in terms of the affiliation to certain class resource of Camellia material.It will Research in terms of the technology is used for the Germplasm Identification of river camellia place famous-object has not been reported.
Summary of the invention
The purpose of the present invention is to provide a kind of method of Rapid identification river camellia tradition famous-object, this method utilizes SSR points Sub- marking fingerprint identifies camellia kind, can 10 kinds of river camellia tradition famous-objects of Rapid identification.
A kind of method of Rapid identification river camellia tradition famous-object of the invention, SSR label primer used are respectively as follows: P06 Its downstream as shown in SEQ ID NO.2 as nucleotide sequence upstream primer as shown in SEQ ID NO.1 and nucleotide sequence Primer composition, A54 it as nucleotide sequence upstream primer as shown in SEQ ID NO.3 and nucleotide sequence such as SEQ ID The composition of downstream primer shown in NO.4, A80, it is as nucleotide sequence upstream primer and nucleotide as shown in SEQ ID NO.5 Sequence downstream primer as shown in SEQ ID NO.6 composition.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of method of Rapid identification river camellia tradition famous-object, which is characterized in that this method comprises the steps of
(1) sample: the adult healthy of sample child care in Nanshan Mountain, Chongqing botanical garden country camellia germplasm resource bank is planted Strain includes the traditional famous-objects of river camellia 10 altogether, is respectively as follows: that white foreign piece, river agate, kermes squama, Zijin hat, goldentop be bright red, seven hearts Red, paeonia szechuanica Fang, liquor-saturated poplar prince wife, Chongqing is red, white jade piece (Nanshan Mountain, Chongqing botanical garden country camellia germplasm resource bank is commercially available)；
(2) DNA is extracted: extracting camellia genomic DNA using the CTAB method of improvement；
(3) the river camellia genomic DNA extracted using step (2) is template, by 3 couples of SSR label primers P06, A54 and A80 For PCR amplification, PCR uses 20 μ L amplification systems:
50ng template DNA, 2.5mmolL-1Mg2+、0.05mmol·L-1dNTPs、0.2μmol·L-1Primer and 0.5UTaqDNA polymerase；
PCR amplification condition: 94 DEG C of 5min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 38 circulations, 72 DEG C of last extensions The amplified production of 5min, 4 DEG C of preservations after having expanded, PCR carry out electrophoresis；
(4) DNA fingerprinting after electrophoresis is counted, " 0 ", " 1 " matrix table are established, if two Differences bit number of points >= 2, then determine two kinds for different cultivars.
It is that the present invention obtains the utility model has the advantages that
The present invention carries out PCR amplification and polyacrylamide gel electrophoresis using the DNA of 3 pairs of SSR primer pair river camellia kinds Silver staining detects afterwards.1, the SSR marker map specific band that the present invention obtains is clear, Unspccific bands are few, polymorphism is high.2, this hair 3 pairs of SSR primers of bright selection can simultaneously all distinguish 10 river camellia kinds one by one, and identification kind is more.3, this method Objectivity is stronger compared with Conventional wisdom discrimination method, more accurately.4, this method and observation data are qualitative or quantitative synthesis Determination method is compared, and can be saved time and cost, before more very wide application is provided to the identification of river camellia kind Scape.
Detailed description of the invention
Fig. 1: 10 river camellia kind DNA electrophoretograms.
Fig. 2: P06 primer SSR-PCR AFLP system is wherein: M:50bp gradient DNA molecular quality standard；1-10 material number It is respectively as follows: that white foreign piece, river agate, kermes squama, Zijin hat, goldentop is bright red, seven hearts are red, paeonia szechuanica Fang, liquor-saturated poplar prince wife, Chongqing are red, white jade Piece.
Fig. 3: A54 primer SSR-PCR AFLP system, in which: M:50bp gradient DNA molecular quality standard；1-10 material is compiled It number is respectively white foreign piece, river agate, kermes squama, Zijin hat, goldentop is bright red, seven hearts are red, paeonia szechuanica Fang, liquor-saturated poplar prince wife, Chongqing are red, white jade Piece.
Fig. 4: A80 primer SSR-PCR AFLP system, in which: M:50bp gradient DNA molecular quality standard；1-10 material is compiled Number it is respectively as follows: that white foreign piece, river agate, kermes squama, Zijin hat, goldentop is bright red, seven hearts are red, paeonia szechuanica Fang, liquor-saturated poplar prince wife, Chongqing are red, white Beautiful piece.
Specific embodiment
Embodiment 1
A kind of method of Rapid identification river camellia tradition famous-object, it is followed the steps below:
(1) sample: sample buys the adult healthy from child care in Nanshan Mountain, Chongqing botanical garden country camellia germplasm resource bank Plant includes the traditional famous-objects of river camellia 10 altogether, is that white foreign piece, white jade piece, river agate, kermes squama, Zijin hat, goldentop are big respectively It is red, seven hearts are red, tree peony tea, liquor-saturated poplar prince wife, Chongqing are red, the new hair tender leaf in tree crown middle and upper part periphery is selected after the florescence in May, each Kind chooses 3 single plant materials, separately sampled, and a part is directly extracted for fresh sample, after a part of blade is put into discoloration silica gel It is stand-by that -80 DEG C of refrigerators are deposited in sealing.
(2) DNA is extracted: camellia genomic DNA is extracted using the CTAB method of improvement, be added in 2%CTAB extracting solution etc. 3% beta -mercaptoethanol of volume, and with isometric chloroform: isoamyl alcohol (24:1) mixed liquor extracts twice, with 1.5% agarose electricity Swimming detection DNA purity and content are stored after being diluted to 30~50ng. μ l-1 for the DNA TE of examination using λ DNA concentration as reference Stand-by in 4 DEG C, undiluted -20 DEG C of DNA stoste preservations, the DNA electrophoretogram of 10 kinds of extraction is shown in Fig. 1.
(3) PCR amplification and electrophoresis: (2) DNA is extracted: use the CTAB method extraction camellia genomic DNA of improvement for template, 3 couples of SSR label primers P06, A54 and A80 are used for PCR amplification, the primer is synthesized by Shanghai Sheng Gong bio-engineering corporation, PCR uses 20 μ L amplification systems:
50ng template DNA, 2.5mmolL-1Mg2+、0.05mmol·L-1dNTPs、0.2μmol·L-1Primer and 0.5UTaqDNA polymerase；
PCR amplification condition: 94 DEG C of 5min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 38 circulations, 72 DEG C of last extensions 5min；
The amplified production of PCR carries out double vertical panel native polyacrylamide gel electrophoresis (8%), electrophoresis sample-adding amount 1.25 μ L, electrophoresis tank voltage 240V, electrophoresis time 2.5h.2 μ l bromophenol blues dyeing: 10% ethyl alcohol and 0.5% ice is carried out after electrophoresis Acetic acid mixed solution fixes l2~15min；2g·L-1AgNO3Aqueous solution silver staining l5~20min；Developing solution is used after washing (15gL-1NaOH, 0.5% formaldehyde, 0.02gL-1Na2S2O3) colour developing, until band is clear, in gel imaging system It photographs to record.
(4) DNA fingerprinting (see Fig. 2, Fig. 3, Fig. 4) after statistics amplification compares different cultivars and expands in same position The banding pattern and migration distance of segment have band to be denoted as " 1 " at same position, and no band is denoted as " 0 ", and building forms " 1 " " 0 " matrix, Determine two kinds for different cultivars if two Differences bit number of points >=2 according to map amplification situation and matrix result. Primer P06 amplified band label the results are shown in Table 1, and primer A54 amplified band label the results are shown in Table 2, primer A80 amplified band label It the results are shown in Table 3.
As shown in table 1 and Fig. 2: removing between No. 2 (river agates) and No. 3 (kermes squama) kinds, No. 4 (Zijin hats) and No. 5 (goldentops It is bright red) there is 2 or more difference number of sites between kind outside indifference, between other 6 kinds, it can distinguish each other.
It is as shown in Table 2 and Fig. 3: except No. 1 (white ocean piece), No. 2 (river agate), No. 3 (kermes squama), No. 4 (Zijin hat) kinds Outside, there is 2 or more difference number of sites between other 6 kinds, can distinguish each other.
As shown in table 3 and fig. 4: in addition to No. 1 (white ocean piece), No. 10 (white jade piece) kinds, there is 2 between other 8 kinds A above difference number of sites, can distinguish each other.
Table 1
Table 2
Table 3
Sequence table
<110>at Nanshan Mountain, Chongqing arboretum management
<120>a kind of method of Rapid identification river camellia tradition famous-object
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence ()
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cagggttgca agaagtaccg 20
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<211> 20
<212> DNA
<213>artificial sequence ()
<400> 2
atcaaccgta tgggcaaaag 20
<210> 3
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 3
ttttggttgc ctcgcctcc 19
<210> 4
<211> 21
<212> DNA
<213>artificial sequence ()
<400> 4
tgcttccctc taggtccctc c 21
<210> 5
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<212> DNA
<213>artificial sequence ()
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gctaatgata gaccatctgc tcct 24
<210> 6
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 6
ggccatgctc tcaatagtag aact 24

Claims (2)

1. a kind of method of Rapid identification river camellia tradition famous-object, it is characterised in that:
This method comprises the steps of
(1) it samples: the adult healthy plant of sample child care in Nanshan Mountain, Chongqing botanical garden country camellia germplasm resource bank, altogether Including the traditional famous-objects of river camellia 10, it is respectively as follows: that white foreign piece, river agate, kermes squama, Zijin hat, goldentop is bright red, seven hearts are red, river Tree peony, liquor-saturated poplar prince wife, Chongqing is red, white jade piece；
(2) DNA is extracted: extracting camellia genomic DNA；
(3) 3 pairs of SSR label primers are used for PCR amplification and electrophoresis as template by the river camellia genomic DNA extracted using step (2) ；
(4) DNA fingerprinting after electrophoresis is counted, " 0 ", " 1 " matrix table are established, if two Differences bit number of points >=2, Determine two kinds for different cultivars；
3 pairs of SSR label primers are respectively P06, A54 and A80, and the P06 is by nucleotide sequence such as SEQ ID NO.1 institute Upstream primer and the nucleotide sequence downstream primer as shown in SEQ ID the NO.2 composition shown, the A54 is by nucleotide sequence The upstream primer as shown in SEQ ID NO.3 and the nucleotide sequence downstream primer as shown in SEQ ID NO.4 composition, it is described A80 as nucleotide sequence upstream primer as shown in SEQ ID NO.5 and nucleotide sequence as shown in SEQ ID NO.6 under Swim primer composition.
2. a kind of method of Rapid identification river camellia tradition famous-object as described in claim 1, it is characterised in that: the PCR is anti- 20 μ L amplification systems should be used:
50ng template DNA, 2.5 mmolL-1 Mg2+、0.05mmol·L-1 dNTPs、0.2µmol·L-1Primer and
0.5U Taq DNA polymerase；
The PCR amplification condition: 94 DEG C of 5min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 38 circulations, 72 DEG C are finally prolonged 5min is stretched, 4 DEG C of preservations after having expanded.
CN201810200510.1A 2018-03-12 2018-03-12 A kind of method of Rapid identification river camellia tradition famous-object Expired - Fee Related CN108410967B (en)

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CN110343778B (en) * 2019-04-16 2023-08-22 中国林业科学研究院亚热带林业研究所 Polymorphic marker primer of camellia variety spring Jiang Gongxia and camellia variety identification method

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CN105018635A (en) * 2015-08-18 2015-11-04 大连民族大学 Molecular recognition method for rootstock-scion combination and selection in efficient old tea-oil tree and camellia grafting

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Publication number Priority date Publication date Assignee Title
CN105018635A (en) * 2015-08-18 2015-11-04 大连民族大学 Molecular recognition method for rootstock-scion combination and selection in efficient old tea-oil tree and camellia grafting

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Genetic relationships in a germplasm collection of Camellia japonica and Camellia oleifera using SSR analysis;Y. Zhao et al.;《Genetics and Molecular Research》;20170216;第16卷(第1期);MATERIAL AND METHODS、表1、表2 *
川山茶ISSR-PCR及SSR-PCR优化体系建立及比较;刘家艳 等;《江苏林业科技》;20161031;第43卷(第5期);摘要、第1.3.3节 *
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