CN109337997A - A kind of Camellia polymorphism Chloroplast gene microsatellite molecular marker primer and screening and the method for screening sibling species - Google Patents

Abstract

The invention belongs to forestry technical field of molecular biology, and in particular to a kind of Camellia polymorphism Chloroplast gene microsatellite molecular marker primer and screening and the method for screening sibling species.Based on camellia species chloroplast DNA sequence microsatellite locus relative uniformity, the specific microsatellite locus that there is variation for inter-species designs cpSSR primer；The DNA for extracting different camellia species, mixes with primer, expands, electrophoresis detection；Final acquisition Camellia polymorphism Chloroplast gene microsatellite molecular marker primer 16 is right.The method for screening sibling species includes: to extract the DNA of C.chekiangoleosa and its sibling species, carries out capillary electrophoresis separation using polymorphism primer, separates by PCR product segment and screen sibling species.Primer screening method provided by the invention is rapidly and efficiently, other plant species with Chloroplast gene sequence information are widely used in, the polymorphism cpSSR label of acquisition can be used for the research such as the genealogical classification of Camellia Plants, the examination of sibling species and analysis of genetic diversity.

Description

A kind of Camellia polymorphism Chloroplast gene microsatellite molecular marker primer and screening With the method for screening sibling species

Technical field

The present invention relates to forestry field of molecular biotechnology, in particular to a kind of Camellia Chloroplast Simple molecular labeling Polymorphism primer and its screening technique, screen sibling species method.

Background technique

Camellia (Camellia L.) is maximum category in Theaceae, and belonging to interior multiple species has important economic value, If the tea (C.sinensis) in tea group is one of big beverage in the world three；And the seed oil content of the multiple species of oil tea group it is very high and Can be edible, wherein foremost is the oil tea (C.oleifera) of referred to as east Chinese olive tree；In addition, many objects of Section Camellia Kind there is huge ornamental value, such as famous camellia (C.japonica) in the world, while both with using there are also ornamental and oil Species such as C.chekiangoleosa (C.chekiangoleosa).Each object interspecies relation of Camellia is complicated, and tree species morphological variation is abundant more Sample, while polyploidization phenomenon is more concentrated generally, mainly according to morphological feature, there is also larger for the domestic classification to such tree species Disagreement.Category systematic growth research is current domestic research hotspot.

Widely distributed microsatellite sequence in Chloroplast gene (Chloroplast genome DNA, cpDNA) (Chloroplast simple sequence repeats, cpSSR) can be developed into molecular labeling, not only have SSR marker Many advantages, such as codominance, high polymorphism, widely distributed property, while also inheriting Chloroplast gene monolepsis, structure letter Single, relatively conservative feature is now widely used for germplasm identification, parent's analysis, Swarm Evolution and Phylogenetic Analysis It is huge especially to study upper application value in the horizontal genetic drift between natural plants group of more high-class for research.CpSSR molecule mark Generally there are three types of methods for the development scheme of note: 1) utilizing cpDNA sequence known to gene library lookup, develop in conjunction with round pcr cpSSR；2) cpDNA sequence is obtained by sequencing, develops cpSSR in conjunction with round pcr；3) using the versatility of label, from known It is screened in the cpSSR of the closer species of affiliation.The acquisition of method three is the versatility cpSSR label of nearly edge species, often It is easy to appear false positive amplification；And the cpSSR label that the exploitation of method one and two obtains is true and reliable, but needs species itself CpDNA sequence is as marker development basis.In recent years, being greatly reduced for price is sequenced with the promotion of sequencing technologies, Ye Lv The work of body gene order-checking is largely carried out, and Camellia chloroplaset full-length genome database also increasingly enriches, in cpDNA sequence base It develops cpSSR label on plinth to be possibly realized, research has not been reported in this respect at present.

In the implementation of the present invention, the inventor finds that the existing technology has at least the following problems:

Traditional cpSSR marker development, it then follows utilize cpDNA sequence retrieval microsatellite locus, design primer passes through PCR again The general route of experiment sieving polymorphism primer, wherein polymorphism primer screens link, due to having the microsatellite locus of polymorphism It needs by being compared acquisition, time and effort consuming by PCR amplification test between sibling species.

Summary of the invention

The purpose of the present invention is at present can be general between Camellia sibling species polymorphism cpSSR primer less show Shape is passed through on the identical microsatellite locus in nearly edge species chloroplaset sequence using the method for comparing Chloroplast gene The variation of analysis microsatellite locus quickly screens acquisition, and there is the cpSSR of polymorphism to mark.It is another object of the present invention to solve Certainly C.chekiangoleosa vacation seedling, which floods market, influences its nursery production link problem, using part cpSSR polymorphism primer to Zhejiang Camellia and its sibling species are screened.C.chekiangoleosa cultivated area, according to the whole nation the 4th, has biggish warp in oil tea tree species Ji value, occupies the leading position in market, but its difference in appearance of Seedling Stage is smaller between sibling species, be difficult to using conventional method It differentiates, therefore is of great practical significance.

To achieve the goals above, the technical solution of the present invention is as follows:

The present invention provides the polymorphism primer of 16 pairs of Camellia cpSSR molecular labelings, and primer sequence is as shown in table 2:

The polymorphism primer of 2 Camellia cpSSR of table

The feature of cpSSR molecular labeling system includes: PCR reaction system and PCR response procedures；

PCR reaction system: including 50ng DNA profiling, 1.0 μ L 10X PCR Buffer, 2.5mmol/L in 10 μ l Mgcl2,0.25mmol/L dNTP, each 0.5ng/ μ L, 0.5U Taq archaeal dna polymerase of upstream and downstream primer；

PCR response procedures: 94 DEG C of initial denaturation 5min；94 DEG C, 30s, Tm, 30s, 72 DEG C, 30s, 25 circulations；Last 72 DEG C Extend 7min, 4 DEG C of preservations.See Table 3 for details for the corresponding annealing temperature of each primer.8% polyacrylamide gel detects PCR amplification and produces Object.

The present invention also provides a kind of screening techniques of the polymorphism primer of Camellia Chloroplast Simple molecular labeling:

(1) the chloroplaset whole genome sequence that C.chekiangoleosa is obtained by Miq250 sequencing technologies, in conjunction with Genbank number Have the chloroplaset full-length genome of Camellia Plants according to library, using Chloroplast gene method is compared, analyzes each object of Camellia More consistent rule is presented in Chloroplast gene distribution characteristics by kind cpSSR；

(2) using the chloroplaset whole genome sequence of C.chekiangoleosa as reference standard, different sibling species are compared by blast The variation situation of identical microsatellite locus in Chloroplast gene sequence, quickly screening has the microsatellite locus of polymorphism, and Develop corresponding cpSSR labeled primer；

(3) DNA for extracting different camellia species, mixes and is expanded with cpSSR labeled primer, electrophoresis detection；It obtains It is right to obtain Camellia polymorphism Chloroplast gene microsatellite molecular marker primer 16, to quickly and effectively develop close in Camellia General and cpSSR with certain polymorphism is marked in edge kind.

The present invention also provides a kind of method for screening C.chekiangoleosa and its sibling species using polymorphism primer, the methods The following steps are included:

1) thick leaf Camellia (C.crassissima), the flash of light Camellia of C.chekiangoleosa and its sibling species are extracted (C.lucidissima), the nucleus DNA of full edge Camellia (C.subintegra), South Mountain tea (C.semiserrata)；

2) by the polymorphism primer of the Camellia cpSSR molecular labeling 8 pairs of primers and the C.chekiangoleosa and The DNA of its sibling species mixes and carries out PCR amplification, obtains amplified production；The number of 8 pairs of primers is respectively Cc_cpSSR- 04、Cc_cpSSR-12、Cc_cpSSR-15、Cc_cpSSR-40、Cc_cpSSR-49、Cc_cpSSR-71、Cc_cpSSR-74、 Cc_cpSSR-75；

(3) Capillary Electrophoresis, Capillary Electrophoresis condition are used to pcr amplification product are as follows: primer synthesis is repaired using 5 ' FAM Decorations, red-label, clip size is respectively 70 80 100 120 160 180 200 240 280 320 360 400 450 490 500, carry out the detection of PCR product length polymorphism；

(4) method of Capillary Electrophoresis result is obtained are as follows: analyze hair using 1.0 software of PeakScanner Software Cons electrophoresis is as a result, determine the band length (bp) of the amplified production according to peak figure.By different haplotype statistical data, use Popgene software calculates Nei genetic similarity index；Recycling NTSYS2.2 software (http: // Www.exetersoftware.com/cat/ntsyspc/ntsyspc.html the UPGMA in SAHN program in) (Unweighted Pair-group Method with Arithmetic means, non-weighting group average method), building heredity Relationship dendrogram；

(5) qualification result of Capillary Electrophoresis result and table 1 is compared, completes species and screens；

The qualification result of 1 C.chekiangoleosa of table and its sibling species

(note: numerical value is amplified fragments product, unit bp in table)

Compared with prior art, the beneficial effects of the present invention are: comparing each species of Camellia using bioinformatic analysis More consistent rule is presented in Chloroplast gene distribution characteristics by cpSSR, and by further comparing, different plant species are identical micro- to be defended Variation situation on championship point can quickly screen the cpSSR label for obtaining tool polymorphism.So the present invention provides a kind of quick Camellia cpSSR polymorphism primer method is screened, while the polymorphism cpSSR label obtained can be applied to genetic diversity point The Related Research Domains such as analysis, germplasm identification, parent's identification, systematic growth.In addition, the cpSSR polymorphism that this research provides The phenomenon that primer can be used for screening C.chekiangoleosa and its sibling species, avoid on nursery stock markets " mixing the genuine with the fictitious " significantly, has ten Divide important meaning.

Detailed description of the invention

Fig. 1 is that Cc_cpSSR-04 and Cc_cpSSR-06 schemes the silver staining PAGE of the PCR amplification of part camellia species；

Fig. 2 is capillary electrophoresis detection figure of the Cc_cpSSR-12 to C.chekiangoleosa and its sibling species；

Fig. 3 is Nei genetic similarty coefficient matrix to be calculated using ntsys2.2 software, then sent out with the system of UPGMA building Educate tree graph.

Specific embodiment

To make the object, technical solutions and advantages of the present invention clearer, the present invention is made combined with specific embodiments below Further it is described in detail.

Embodiment 1

One, the screening of the polymorphism primer of Camellia cpSSR molecular labeling

1, the excavation and screening of Chloroplast Simple molecular labeling

(1) acquisition and pretreatment of C.chekiangoleosa cpDNA sequence

Chloroplast Suspension is obtained by Chloroplast DNA Isolation kit first, recycles DNeasy Plant Mini Kit extracts C.chekiangoleosa chloroplast DNA, constructs sequencing library, high-throughput through Illumina Miseq250 Sequencing reaction obtains chloroplaset whole genome sequence.Sequencing data through screening remove low quality sequence after using SOAPdenovo into Row skeleton splicing (optimal Kmer is 31), while Newbler is utilized, MITObim auxiliary assembling is finally mended using GapCloser Hole finally obtains complete C.chekiangoleosa chloroplaset complete genome sequence (MG431968).

(2) screening of cpSSR polymorphism primer

(http://www.ncbi.nlm.nih.gov/) downloads Camellia Camellia nitidissima group from GenBank public database Concave veins Camellia nitidissima (C.impressinervis, KF156835), rough fruit tea group C. crapnelliana (C.crapnelliana, KF753632), Sect. Theopsis point Camellia fraternal (C.cuspidata, KF156833), the bald tea in bald tea group Danzhai (C.danzaiensis, KF156834), the small small chrysanthemum tea of chrysanthemum tea group (C.luteoflora, KY626042), oil tea group oil tea (C.oleifera, JQ975031), real five column Yunnan camellia (C.yunnanensis, KF156838) of fruit tea group and tea group tea The chloroplaset whole genome sequence of (C.sinensis, KC143082).Use MISA (http://pgrc.ipk- Gatersleben.de/misa/) (Sahu J, Sarmah R, Dehury B, et al.Mining for SSRs and FDMs From expressed sequence tags of Camellia sinensis [J] .Bioinformation, 2012,8 (6): cpSSR excavation 260.) being carried out to the chloroplaset full-length genome of the Camellia Plants, retrieval microsatellite repeats the ginseng of motif Number is set as the minimum number of repetition of mononucleotide, dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotide Respectively 10,4,3,3,3,3, search criteria include perfect type (perfect) and compound (compond) cpSSR.To different plants The cpSSR sequence obtained in object Chloroplast gene carries out blast and compares analysis, selects the cpSSR having differences between species Molecular labeling is used for the design and synthesis of cpSSR polymorphism primer.

2, the design and synthesis of Camellia cpSSR polymorphism primer

Chloroplast Simple molecular sequences based on above-mentioned acquisition, using primer-design software Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign/) two flank of the site cpSSR resulting to above-mentioned screening Conserved sequence carries out design of primers.The major parameter of design of primers are as follows: primer length be 16~24bp, G/C content 30%~ 60%, 40~60 DEG C of primer annealing temperature, upstream and downstream primer annealing temperature difference is within 2 DEG C, it is contemplated that and product length 100~ 500bp avoids secondary structure as far as possible.The cpSSR primer for excavating and developing using Camellia Plants chloroplaset full-length genome totally 80 Right, primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.

PCR amplification is carried out using multiple Camellia sibling species total genomic dnas.Amplified production uses polyacrylamide gel Electrophoretic separation: using 8% polyacrylamide gel (urea containing 4.2g, 1.25ml 10 × TBE, 70 μ l in 10mL encapsulating liquid APS, 10 40% glue (acrylamide: methene acrylamide=39: 1 (w/w)) of μ l TEMED, 2.0ml, in Vertial electrophorestic tank It is separated by electrophoresis in (DYCZ-32 type electrophoresis tank (Beijing 6 1)), 50bp Marker (Tiangeng) is used as standard molecular weight, electricity Product of swimming carries out data interpretation to everybody point after silver staining.Silver staining detecting step are as follows: 10 μ l PCR products add 4 μ 6 × loadings of l slow Fliud flushing, 1 1~2h of μ l, 150V constant pressure electrophoresis of loading wells loading；Electrophoresis finishes, and uses fixer (10% ethyl alcohol, 0.5% acetic acid (v/v), solvent is deionized water) 10min is fixed, it is rinsed 2 times with deionized water, each 1min；Silver staining liquid (0.15% nitre is used again Sour silver solution (w/v)) dyeing 8min, deionized water rinsing 2 times, each 2min；Finally with developer solution (1.5%NaOH, 0.00756%NaBO4,0.75% formaldehyde (w/v)) develop, digital photographing record clear to band.Compare verifying amplified production Difference has the cpSSR primer 16 of polymorphism right to filter out in Camellia sibling species.Partial detection see Fig. 1 (note: M:50bp DNA Ladder；1~18: part camellia species PCR amplification, No. 1 is white flower C.chekiangoleosa, and No. 2 from stamen Red Hill Tea, No. 3 are hair stamen Camellia, and No. 4 are big arnotto camellia, and No. 5 are hair seed Camellia, and No. 6 are Camellia apolyodonta, and No. 7 are Yunnan Camellia, No. 8 are camellia, and No. 9 are Hong Kong Camellia, and No. 10 are white flower South Mountain tea, and No. 11 are long-tail Camellia, and No. 12 red for thick leaf Camellia, No. 13 be Camellia Polyodonta How Ex Hu, No. 14 be C.chekiangoleosa, No. 15 be camellia azalea, No. 16 be full edge Camellia, No. 17 For South Mountain tea, No. 18 are oil tea).

The polymorphism primer of 16 pairs of Camellias cpSSR molecular labeling provided by the invention is referring specifically to table 2:

The annealing temperature of the polymorphism primer of 2 Camellia cpSSR of table

(note: wherein F indicates positive, and R indicates reversed.)

Two, the polymorphism primer identification Camellia Plants affiliation of Camellia cpSSR molecular labeling and examination Zhejiang are red Camellia and its sibling species

While screening C.chekiangoleosa and its sibling species using the polymorphism primer of Camellia cpSSR molecular labeling, Its affiliation can be identified according to its result, wherein sample specifying information is as shown in table 3.

The sample message of 35 sibling species of Camellia of table

Sibling species are screened and the process of Relationship iden- tification is as follows:

(1) it PCR amplification: is held using ABI9700 (Applied Biosystems, Carlsbad, California, USA) Row PCR program.Optimized screening, constructing one, more the cpSSR of wide spectrum marks system in camellia species.PCR reactant System: including 50ng DNA profiling, 1.0 μ L 10X PCR Buffer, 2.5mmol/L Mgcl2,0.25mmol/L in 10 μ l DNTP, each 0.5ng/ μ L, 0.5U Taq archaeal dna polymerase of upstream and downstream primer；PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C, 30s, Tm, 30s, 72 DEG C, 30s, 25 circulations；Last 72 DEG C of extensions 7min, 4 DEG C of preservations.The corresponding annealing temperature of each primer See Table 3 for details.

(2) 8 pairs of primers of polymorphism, Cc_cpSSR-04, Cc_ are provided in polyacrylamide gel electrophoresis detection primary election cpSSR-12、Cc_cpSSR-15、Cc_cpSSR-40、Cc_cpSSR-49、Cc_cpSSR-71、Cc_cpSSR-74、Cc_ cpSSR-75。

(3) further progress Capillary Electrophoresis is verified.Primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd, uses 5 ' FAM modification, model ROX-500-ILS, red-label, clip size is respectively 70 80 100 120 160 180 200 240 280 320 360 400 450 490 500, PCR product length polymorphism detection is carried out with 3730 analyzer of ABI.Portion Point testing result is shown in Fig. 2.

(4) data processing: after Capillary Electrophoresis, using Peak Scanner Software V1.0 (https: // Www.thermofisher.com/order/catalog/product/4381867 it) reads as a result, by the cpSSR molecule mark Note polymorphism primer range of amplified fragments in each species is recorded, and is indicated with the form of A-B, and number is amplified band Length, unit bp.According to Capillary Electrophoresis comparison table 1 provide qualification result, can be completed C.chekiangoleosa and The identification of its sibling species.

The qualification result of 1 C.chekiangoleosa of table and its sibling species (numerical value is amplified fragments product, unit bp in table)

(5) different haplotype statistical data are pressed, Nei genetic similarity index is calculated using popgene software；It recycles SAHN in NTSYS2.2 software (http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html) (Unweighted Pair-group Method with Arithmetic means, non-set of weights are average by UPGMA in program Method), genetic affinity dendrogram is constructed, sees Fig. 3.

The result shows that:

1) capillary electrophoresis detection, the polymorphism primer of 8 pairs of cpSSR molecular labelings is in C.chekiangoleosa and its sibling species Amplification obtains clearly polymorphic bands in DNA sample, and obtains in the sample of C.chekiangoleosa Different population Polymorphism amplification, counts amplified fragments size, and Cc_cpSSR-12 can disposably identify South Mountain tea, Cc_ at 378-379bp CpSSR-15 can disposably identify thick leaf Camellia at 262bp, and another 8 labels combination of two can be by full edge Camellia and Zhejiang River Camellia distinguishes.

2) dendrogram based on UPGMA shows (Fig. 3), for the C.chekiangoleosa of examination, flash of light Camellia, thick leaf Camellia, There are difference for affiliation between full edge Camellia, South Mountain tea, and at Nei genetic similarity index 0.72,5 species can be divided into Two major classes, i.e. light fruit tea subgroup (C.chekiangoleosa, flash of light Camellia, thick leaf Camellia, full edge Camellia) and Yunnan camellia subgroup (South Mountain tea).In addition, according to Nei genetic similarity index, show the affiliations of 5 species from the distant to the near as South Mountain Tea > full edge Camellia > thickness leaf Camellia > C.chekiangoleosa/flash of light Camellia, wherein C.chekiangoleosa and flash of light Camellia Affiliation is extremely close, meets judgement of forefathers' research by the two merger for C.chekiangoleosa one kind.

The present invention carries out the mirror of Camellia Plants affiliation using the polymorphism primer of Camellia cpSSR molecular labeling It is fixed, and codominance, height variation and polymorphism etc. using cpSSR molecular labeling with nucleus DNA microsatellite molecular marker Feature, and have the characteristics that the monolepsis of chloroplaset, guard relatively, so that Chloroplast Simple molecular labeling has structure letter Singly, the features such as multicopy and molecular weight are small, so that the method for Camellia Plants Relationship iden- tification provided by the invention It more can comprehensively react the affiliation between Camellia Plants.In addition, can also be by cpSSR and nucleus SSR molecule mark Remember integrated use, can further comprehensively, it is accurate, objectively respond Species Cell matter and endonuclear affiliation.

Sequence table

<110>Jiangxi Provincial Academy Of Forestry

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Claims (5)

1. a kind of polymorphism primer of Camellia Chloroplast Simple molecular labeling, which is characterized in that including 16 pairs of Camellias The polymorphism primer of cpSSR molecular labeling, primer information are as follows:

The forward primer of Cc_cpSSR-04 as shown in SEQ ID NO.1 in sequence table,

The reverse primer of Cc_cpSSR-04 is as shown in SEQ ID NO.2 in sequence table；

The forward primer of Cc_cpSSR-06 as shown in SEQ ID NO.3 in sequence table,

The reverse primer of Cc_cpSSR-06 is as shown in SEQ ID NO.4 in sequence table；

The forward primer of Cc_cpSSR-12 as shown in SEQ ID NO.5 in sequence table,

The reverse primer of Cc_cpSSR-12 is as shown in SEQ ID NO.6 in sequence table；

The forward primer of Cc_cpSSR-13 as shown in SEQ ID NO.7 in sequence table,

The reverse primer of Cc_cpSSR-13 is as shown in SEQ ID NO.8 in sequence table；

The forward primer of Cc_cpSSR-15 as shown in SEQ ID NO.9 in sequence table,

The reverse primer of Cc_cpSSR-15 is as shown in SEQ ID NO.10 in sequence table；

The forward primer of Cc_cpSSR-20 as shown in SEQ ID NO.11 in sequence table,

The reverse primer of Cc_cpSSR-20 is as shown in SEQ ID NO.12 in sequence table；

The forward primer of Cc_cpSSR-22 as shown in SEQ ID NO.13 in sequence table,

The reverse primer of Cc_cpSSR-22 is as shown in SEQ ID NO.14 in sequence table；

The forward primer of Cc_cpSSR-24 as shown in SEQ ID NO.15 in sequence table,

The reverse primer of Cc_cpSSR-24 is as shown in SEQ ID NO.16 in sequence table；

The forward primer of Cc_cpSSR-37 as shown in SEQ ID NO.17 in sequence table,

The reverse primer of Cc_cpSSR-37 is as shown in SEQ ID NO.18 in sequence table；

The forward primer of Cc_cpSSR-40 as shown in SEQ ID NO.19 in sequence table,

The reverse primer of Cc_cpSSR-40 is as shown in SEQ ID NO.20 in sequence table；

The forward primer of Cc_cpSSR-49 as shown in SEQ ID NO.21 in sequence table,

The reverse primer of Cc_cpSSR-49 is as shown in SEQ ID NO.22 in sequence table；

The forward primer of Cc_cpSSR-64 as shown in SEQ ID NO.23 in sequence table,

The reverse primer of Cc_cpSSR-64 is as shown in SEQ ID NO.24 in sequence table；

The forward primer of Cc_cpSSR-65 as shown in SEQ ID NO.25 in sequence table,

The reverse primer of Cc_cpSSR-65 is as shown in SEQ ID NO.26 in sequence table；

The forward primer of Cc_cpSSR-71 as shown in SEQ ID NO.27 in sequence table,

The reverse primer of Cc_cpSSR-71 is as shown in SEQ ID NO.28 in sequence table；

The forward primer of Cc_cpSSR-74 as shown in SEQ ID NO.29 in sequence table,

The reverse primer of Cc_cpSSR-74 is as shown in SEQ ID NO.30 in sequence table；

The forward primer of Cc_cpSSR-75 as shown in SEQ ID NO.31 in sequence table,

The reverse primer of Cc_cpSSR-75 is as shown in SEQ ID NO.32 in sequence table.

2. a kind of polymorphism primer of Camellia Chloroplast Simple molecular labeling according to claim 1, feature exist In the system of the PCR amplification of the polymorphism primer are as follows: 50ng DNA profiling, 1.0 μ L 10X PCR Buffer, 2.5mmol/ L Mgcl2,0.25mmol/L dNTP, each 0.5ng/ μ L, 0.5U Taq archaeal dna polymerase of upstream and downstream primer, the PCR amplification The total volume of system is 10 μ l；The program of the PCR amplification of the polymorphism primer are as follows: 94 DEG C, initial denaturation 5min；94 DEG C, denaturation 30s, 52~63 DEG C, anneal 30s, 72 DEG C, extends 30s, 25 circulations；72 DEG C, extend 7min, 4 DEG C of preservations.

3. a kind of screening technique of the polymorphism primer of Camellia Chloroplast Simple molecular labeling, which is characterized in that the side Method includes:

(1) the chloroplaset whole genome sequence that C.chekiangoleosa is obtained by Miq250 sequencing technologies, in conjunction with Genbank database The chloroplaset full-length genome of existing Camellia Plants, using Chloroplast gene method is compared, analysis obtains each object of Camellia Consistency is presented in the regularity of distribution of the kind cpSSR in Chloroplast gene；

(2) using the chloroplaset whole genome sequence of C.chekiangoleosa as reference standard, it is green that different sibling species leaves are compared by blast The variation situation of identical microsatellite locus on body genome sequence, quickly screening has the microsatellite locus of polymorphism, and develops Corresponding cpSSR labeled primer；

(3) DNA for extracting different camellia species, mixes and is expanded with cpSSR labeled primer, electrophoresis detection；Obtain mountain Camellia polymorphism Chloroplast gene microsatellite molecular marker primer 16 is right.

4. a kind of polymorphism primer for the Camellia cpSSR for screening C.chekiangoleosa and its sibling species, which is characterized in that including 8 To the polymorphism primer of Camellia cpSSR molecular labeling, the number of 8 pairs of primers is respectively Cc_cpSSR-04, Cc_ cpSSR-12、Cc_cpSSR-15、Cc_cpSSR-40、Cc_cpSSR-49、Cc_cpSSR-71、Cc_cpSSR-74、Cc_ cpSSR-75。

5. a kind of polymorphism primer using Camellia cpSSR described in claim 4 screens C.chekiangoleosa and its sibling species Method, which comprises the following steps:

(1) extract the thick leaf Camellias of C.chekiangoleosa and its sibling species, flash of light Camellia, full edge Camellia, South Mountain tea it is thin Nuclear DNA；

(2) by the 8 pairs of primers and the C.chekiangoleosa in the polymorphism primer of the Camellia cpSSR molecular labeling and its closely The DNA of edge kind mixes and carries out PCR amplification, obtains amplified production；The number of 8 pairs of primers be respectively Cc_cpSSR-04, Cc_cpSSR-12、Cc_cpSSR-15、Cc_cpSSR-40、Cc_cpSSR-49、Cc_cpSSR-71、Cc_cpSSR-74、Cc_ cpSSR-75；

(3) Capillary Electrophoresis, Capillary Electrophoresis condition are used to pcr amplification product are as follows: primer synthesis is modified using 5 ' FAM, red Color marker, clip size are respectively 7080100120160180200240280320360400450490500, carry out PCR product Length polymorphism detection；

(4) method of Capillary Electrophoresis result is obtained are as follows: analyze capillary using 1.0 software of PeakScanner Software Electrophoresis result determines the band length of the amplified production according to peak figure；By different haplotype statistical data, using popgene Software calculates Nei genetic similarity index；The UPGMA in the SAHN program in NTSYS2.2 software is recycled, heredity is constructed and closes It is dendrogram；

(5) qualification result of Capillary Electrophoresis result and table 1 is compared, completes species and screens；

The qualification result of 1 C.chekiangoleosa of table and its sibling species