CN109337997B - Camellia polymorphism chloroplast genome microsatellite molecular marker primer and method for screening and discriminating kindred species - Google Patents
Camellia polymorphism chloroplast genome microsatellite molecular marker primer and method for screening and discriminating kindred species Download PDFInfo
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Abstract
The invention belongs to the technical field of forestry molecular biology, and particularly relates to a microsatellite molecular marker primer of a polymorphism chloroplast genome of camellia and a method for screening and discriminating related species. Designing a cpPSSR primer aiming at a specific microsatellite locus with variation among species based on the relative consistency of the microsatellite locus of a chloroplast DNA sequence of the camellia species; extracting DNA of different camellia species, mixing with primers, amplifying, and performing electrophoresis detection; finally obtaining 16 pairs of camellia polymorphism chloroplast genome microsatellite molecular marker primers. The method for screening the kindred species comprises the following steps: extracting DNA of Zhejiang red camellia and its kindred species, adopting polymorphism primer to make capillary electrophoresis separation, and according to PCR product fragment separation discriminating kindred species. The primer screening method provided by the invention is rapid and efficient, is widely suitable for other plant species with chloroplast genome sequence information, and the obtained polymorphic cpsR marker can be used for researches such as systematic classification of camellia plants, screening of related species, genetic diversity analysis and the like.
Description
Technical Field
The invention relates to the technical field of forestry molecule biology, in particular to a polymorphism primer of a camellia chloroplast microsatellite molecular marker, a screening method thereof and a method for screening related species.
Background
Camellia (Camellia L.) is the largest genus in the Camellia family, and a plurality of species in the genus have important economic value, for example, tea (c.sinensis) in the tea group is one of three major drinks in the world; the oil tea group has high oil content in seeds of various species and is edible, and the most notable of the oil tea group is the oil tea called oriental olive tree (c.oleifera); in addition, the red camellia group has great ornamental value for various species, such as international famous camellia (c. japonica), and also for both ornamental and oil, such as zhejiang red camellia (c. chekianggoleosa). The camellia has complex relationships among various species, rich and diverse tree species morphological variations and more centralized and common multiplicities, and the domestic classification of the tree species is mainly based on morphological characteristics and has larger divergence. The phylogenetic research belongs to the research focus in China at present.
Microsatellite sequences (cpSSR) can be developed into molecular markers widely distributed in Chloroplast genomes (cPDNA), the molecular markers not only have the advantages of codominance of SSR markers, high polymorphism, wide distribution and the like, but also inherit the characteristics of monogenetic, simple structure and relative conservation of the Chloroplast genomes, and the molecular markers are widely applied to germplasm resource identification, parental analysis, population evolution and phylogenetic analysis research at present, and especially have great application value in gene drift research of higher classification level and natural plant populations. Three general approaches are used to develop cpSSR molecular markers: 1) searching a known cpDNA sequence by using a gene library, and developing cpPSSR by combining a PCR technology; 2) obtaining a cpDNA sequence through sequencing, and developing the cpSR by combining a PCR technology; 3) using the versatility of the marker, the selection was made from cpsRs from species known to be closely related. The universal cpsSR marker of the related species obtained by the third method is easy to generate false positive amplification; the cpPSSR marker obtained by the first and second methods is real and reliable, but the cpDNA sequence of the species is required to be used as the marker development base. In recent years, with the improvement of sequencing technology and the great reduction of sequencing price, the work of chloroplast genome sequencing is greatly carried out, the whole chloroplast genome database of camellia is increasingly enriched, the development of cpsSR markers on the basis of cpDNA sequences is possible, and the research on the aspect is not reported at present.
In the process of implementing the invention, the inventor finds that the prior art has at least the following problems:
the traditional development of cpPSSR markers follows a general route of searching microsatellite loci by utilizing a cpDNA sequence, designing primers and screening polymorphic primers by a PCR test, wherein in the polymorphic primer screening step, because the polymorphic microsatellite loci need to be obtained by comparing between related species by the PCR amplification test, the time and the labor are consumed.
Disclosure of Invention
The invention aims to rapidly screen and obtain a cpsSR marker with polymorphism on the same microsatellite locus in a chloroplast sequence of a related species by analyzing the variation of the microsatellite locus by utilizing a method for comparing chloroplast genomics aiming at the current situation that the number of polymorphic cpsSR primers which can be commonly used among related species of camellia is less. The invention also aims to solve the problem that false seedlings of Zhejiang red camellia are full of markets to influence the seedling production links of Zhejiang red camellia, and part of cpsSR polymorphic primers are used for screening Zhejiang red camellia and related species thereof. The planting area of the camellia japonica at Zhejiang is the fourth nationwide in camellia oleifera, has great economic value and occupies the leading position of the market, but the appearance difference between the camellia oleifera and related species at the seedling stage is small, and the camellia oleifera and related species are difficult to distinguish by using a traditional method, so that the method has very important practical significance.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides 16 pairs of polymorphism primers of camellia cpSSR molecular markers, and the primer sequences are shown in Table 4:
TABLE 4 polymorphic primers for Camellia cpSSR
The cpSSR molecular marker system is characterized by: a PCR reaction system and a PCR reaction program;
and (3) PCR reaction system: 10 ul comprises 50ng DNA template, 1.0 ul 10 XPCR Buffer, 2.5mmol/LMgcl2, 0.25mmol/L dNTP, upstream and downstream primers 0.5 ng/ul, 0.5U Taq DNA polymerase;
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; 94 ℃, 30s, Tm, 30s, 72 ℃, 30s, 25 cycles; finally, extension is carried out for 7min at 72 ℃ and storage is carried out at 4 ℃. The annealing temperature for each primer is detailed in Table 2. Detecting the PCR amplification product by 8% polyacrylamide gel.
The invention also provides a screening method of the polymorphism primer of the camellia chloroplast microsatellite molecular marker, which comprises the following steps:
(1) obtaining a chloroplast whole genome sequence of the camellia japonica thunbergii by an Miq250 sequencing technology, combining the existing chloroplast whole genome of camellia plants in a Genbank database, and analyzing the distribution characteristics of cpSSR of various species of camellia in chloroplast genome to show a relatively consistent rule by utilizing a chloroplast genomics comparison method;
(2) taking a chloroplast whole genome sequence of Zhejiang red camellia as a reference standard, quickly screening polymorphic microsatellite loci by comparing variation conditions of the same microsatellite loci on different related chloroplast genome sequences through blast, and developing corresponding cpSSR marker primers;
(3) extracting DNA of different camellia species, mixing with a cpsSR labeled primer, and carrying out amplification and electrophoresis detection; and obtaining 16 pairs of microsatellite molecular marker primers of the polymorphism chloroplast genome of the camellia, thereby quickly and effectively developing the cpSSR marker which is universal in the related species of the camellia and has certain polymorphism.
The invention also provides a method for screening Zhejiang red camellia and related species thereof by utilizing the polymorphic primer, which comprises the following steps:
1) extracting nucleus DNA of thick-leaf red camellia (C.crassima), sparkling red camellia (C.lucidissma), whole-edge red camellia (C.subentra) and southern mountain tea (C.semiserrata) of Zhejiang red camellia and its kindred;
2) mixing 8 pairs of primers in the polymorphism primers of the camellia cpSSR molecular marker with DNA of the Zhejiang red camellia and the allied species thereof, and carrying out PCR amplification to obtain an amplification product; the 8 pairs of primers are respectively numbered as Cc _ cPPSSR-04, Cc _ cPPSSR-12, Cc _ cPPSSR-15, Cc _ cPPSSR-40, Cc _ cPPSSR-49, Cc _ cPPSSR-71, Cc _ cPPSSR-74 and Cc _ cPPSSR-75, and are subjected to PCR amplification after 5' FAM modification, and are marked with red, and the sizes of the fragments are 70bp, 80bp, 100bp, 120bp, 160bp, 180bp, 200bp, 240bp, 280bp, 320bp, 360bp, 400bp, 450bp, 490bp and 500bp in sequence;
(3) performing the length polymorphism detection of the PCR product by adopting capillary electrophoresis on the PCR amplification product;
(4) the method for obtaining the result of capillary electrophoresis comprises the following steps: the capillary electrophoresis results were analyzed using PeakScanner Software1.0 Software, and the band length (bp) of the amplification product was determined from the peak pattern. Calculating a Nei genetic similarity coefficient by adopting popgene software according to different haplotype statistical data; then, a genetic relationship clustering chart is constructed by using UPGMA (Unweighted Pair-group Method with Arithmetric means, Unweighted group average Method) in the SAHN program in NTSYS2.2 software (http:// www.exetersoftware.com/cat/ntsyspc. html);
(5) comparing the capillary electrophoresis result with the identification result in the table 1 to finish species discrimination;
TABLE 1 Zhejiang red camellia and identification results (unit bp) of related species thereof
Compared with the prior art, the invention has the beneficial effects that: the distribution characteristics of the cpsRs of various species of the camellia in the chloroplast genome are analyzed and compared to show a relatively consistent rule by bioinformatics, and the cpsRs with polymorphism can be rapidly screened and obtained by further comparing variation conditions on the same microsatellite loci of different species. Therefore, the invention provides a method for rapidly screening the camellia cpsSR polymorphism primer, and the obtained polymorphism cpsSR marker can be applied to the related research fields of genetic diversity analysis, germplasm resource identification, parent identification, phylogeny and the like. In addition, the cpPSSR polymorphic primer provided by the research can be used for screening Zhejiang red camellia and related species thereof, so that the phenomenon of 'fish eyes mixed with beads' in the seedling market is greatly avoided, and the method has very important significance.
Drawings
FIG. 1 is a silver-stained PAGE pattern of PCR amplification of a portion of Camellia species with Cc _ cpSSR-04 and Cc _ cpSSR-06;
FIG. 2 is a capillary electrophoresis test of Cc _ cpSSR-12 on Zhejiang camellia and its kindred species;
FIG. 3 is a phylogenetic tree diagram constructed by using ntsys2.2 software to calculate Nei genetic similarity coefficient matrix and UPGMA.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to specific embodiments below.
Example 1
Screening of polymorphism primers of camellia cpSSR molecular marker
1. Excavating and screening of chloroplast microsatellite molecular markers
(1) Obtaining and pretreating of Zhejiang red camellia cpDNA sequence
Firstly, Chloroplast suspension is obtained through a Chloroplast DNA Isolation Kit, then DNeasy plant Mini Kit is utilized to extract the Chloroplast DNA of the Zhejiang red camellia, a sequencing library is constructed, and a Chloroplast whole genome sequence is obtained through Illumina Miseq250 high-throughput sequencing reaction. After low-quality sequences are removed from sequencing data through screening, SOAPdenovo is used for framework splicing (the optimal Kmer is 31), meanwhile, Newbler and MITobim are used for auxiliary assembly, and finally, GapCloser is used for hole filling, so that a complete chloroplast complete gene sequence of the red camellia from Zhejiang is obtained (MG 431968).
(2) Screening of cpPSSR polymorphic primers
The whole genome sequences of Camellia sinensis (C.impressurervis, KF156835), Camellia crassipes (C.crapnelliana, KF753632), Cocia chinensis (C.cusperida, KF156833), Calophylla de-clathra (C.danzaiensis, KF156834), Cocia chinensis (C.luteoflora, KY626042), Camellia oleifera (C.oleeiera, JQ975031), Camellia sinensis five-column Yunnan (C.yunnanensis, KF 082838) and Camellia sinensis (C.sinnensis, KC 143chloroplast) were downloaded from GenBank public database (http:// www.ncbi.nlm.nih.gov /). The chloroplast whole genome of the Camellia plant was subjected to cpSSR excavation using MISA (http:/pgrc. ipk-gatersleen. de/MISA.) (Sahu J, sarah R, dehuryb, et al. mining for SSRs and FDMsfrom expressed sequence tags of Camellia sinensis [ J ]. Bioinformation, 2012, 8 (6): 260.), and the parameters for searching for the microsatellite repeat motifs were set to 10, 4, 3 for the minimum number of repeats of mononucleotides, dinucleotides, tetranucleotides, pentanucleotides and hexanucleotides, respectively, and the search criteria included perfect (perfect) and complex (complex) cpSSR. Performing blast alignment analysis on cpsR sequences obtained from chloroplast genomes of different plants, and selecting cpsR molecular markers with differences among species for designing and synthesizing cpsR polymorphic primers.
2. Design and synthesis of camellia cpSSR polymorphic primer
Based on the obtained chloroplast microsatellite molecule sequence, Primer design software Primer Premier 5.0(http:// www.premierbiosoft.com/Primer design /) is adopted to carry out Primer design on conserved sequences at two sides of the cpPSSR site obtained by screening. The main parameters for primer design are: the length of the primer is 16-24 bp, the GC content is 30-60%, the annealing temperature of the primer is 40-60 ℃, the annealing temperature difference of the upstream primer and the downstream primer is within 2 ℃, the length of the expected product is 100-500 bp, and secondary structures are avoided as much as possible. The primers of the cpSSR excavated and developed by using the whole chloroplast genome of the camellia plant are 80 pairs, and are numbered according to Cc _ cpSSR1-80, and the primers are synthesized by Shanghai biological engineering Co.
PCR amplification is carried out by adopting total genomic DNA of a plurality of camellia closely related species. Separating the amplification product by polyacrylamide gel electrophoresis: carrying out electrophoretic separation in a vertical electrophoresis tank (DYCZ-32 type electrophoresis tank (Beijing six one)) by using 8% polyacrylamide gel (10mL of perfusion solution containing 4.2g of urea, 1.25mL of 10 XTBE, 70 μ l of APS, 10 μ l of TEMED and 2.0mL of 40% glue (acrylamide: methylene acrylamide is 39: 1(w/w)), using 50bp Marker (Tiangen) as a standard molecular weight, carrying out data identification on each point after the electrophoresis product is subjected to silver staining, wherein the silver staining detection step comprises the steps of adding 4 μ l of 6 × loading buffer solution into 10 μ l of PCR product, loading 1 μ l of sample loading hole and carrying out constant voltage electrophoresis for 1-2 h at 150V, after the electrophoresis is finished, using fixing solution (10% ethanol, 0.5% acetic acid (V/V) and using deionized water to fix for 10min, rinsing for 2 times with deionized water for 1min each time, and then using silver staining solution (0.15% silver nitrate solution (w/V)) to stain for 8min, rinsing with deionized water for 2 times, each for 2 min; finally, the strips were developed with developer (1.5% NaOH, 0.00756% NaBO4, 0.75% formaldehyde (w/v)) until the strips were clear and recorded by digital photography. The differences of the amplification products were comparatively verified to screen the cpsSR primer 16 pair having polymorphisms in the Camellia allium species. The partial detection results are shown in figure 1 (note: M: 50bp DNA Ladder; 1-18: PCR amplification of partial camellia species, 1 is white flower Zhejiang red camellia, 2 is Japanese ramuli red camellia, 3 is Mao Caligur red camellia, 4 is big fruit red camellia, 5 is Mao Zi red camellia, 6 is false polydentate red camellia, 7 is Yunnan camellia, 8 is camellia, 9 is hongkong red camellia, 10 is white flower southern camellia, 11 is long tail red camellia, 12 is thick leaf red camellia, 13 is polydentate red camellia, 14 is Zhejiang red camellia, 15 is rhododendron, 16 is whole-edge red camellia, 17 is southern camellia, and 18 is camellia).
The 16 pairs of polymorphism primers of the camellia cpSSR molecular marker provided by the invention are specifically shown in table 2:
TABLE 2 annealing temperature of polymorphic primers of Camellia cpSSR
(note: wherein F represents a forward direction and R represents a reverse direction.)
Secondly, identifying the genetic relationship of camellia plants and screening Zhejiang red camellia and related species thereof by using polymorphism primers of camellia cpsSR molecular markers
The genetic relationship of Zhejiang red camellia and related species thereof can be identified according to the result while the Camellia japonica and the related species thereof are screened by utilizing the polymorphism primer labeled by the cpsSR molecule of the Camellia, wherein the specific source information of the sample is shown in Table 3.
TABLE 3 sample information for 5 closely related species of Camellia
The process of identifying the kindred species and the kindred relationship is as follows:
(1) and (3) PCR amplification: the PCR program was performed using ABI9700(Applied Biosystems, Carlsbad, California, USA). Through optimized screening, a cpPSSR marker system with a wider spectrum in camellia species is constructed. And (3) PCR reaction system: 10 ul comprises 50ng DNA template, 1.0 ul 10 XPCR Buffer, 2.5mmol/L Mgcl2, 0.25mmol/L dNTP, upstream and downstream primers 0.5 ng/ul, 0.5U Taq DNA polymerase; the PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 94 ℃, 30s, Tm, 30s, 72 ℃, 30s, 25 cycles; finally, extension is carried out for 7min at 72 ℃ and storage is carried out at 4 ℃. The annealing temperature for each primer is detailed in Table 2.
(2) 8 pairs of primers Cc _ cPPSSR-04, Cc _ cPPSSR-12, Cc _ cPPSSR-15, Cc _ cPPSSR-40, Cc _ cPPSSR-49, Cc _ cPPSSR-71, Cc _ cPPSSR-74 and Cc _ cPPSSR-75 with polymorphism are initially selected for polyacrylamide gel electrophoresis detection.
(3) Further capillary electrophoresis validation was performed. The primers were synthesized by Shanghai Czeri bioengineering, Inc., modified with 5' FAM, and labeled with red color, with fragment sizes of 70bp, 80bp, 100bp, 120160bp, 180bp, 200bp, 240bp, 280bp, 320bp, 360bp, 400bp, 450bp, 490bp, and 500 respectively, and PCR product length polymorphism detection was performed with ABI 3730 analyzer. The results of the partial detection are shown in FIG. 2.
(4) Data processing: after capillary electrophoresis, the results were read using Peak Scanner Software V1.0(https:// www.thermofisher.com/order/catalog/product/4381867), and the range of amplified fragments of the cpPSSR molecular marker polymorphic primers in each species was recorded, expressed as A-B, and the number is the length of the amplified band in bp. According to the result of capillary electrophoresis, the identification of Zhejiang camellia and its kindred species can be completed by comparing the identification results provided in Table 1.
TABLE 1 Zhejiang red camellia and identification results (unit bp) of related species thereof
(5) Calculating a Nei genetic similarity coefficient by adopting popgene software according to different haplotype statistical data; then, a genetic relationship clustering chart is constructed by using UPGMA (Unweighted Pair-group Method with Arithmetric means) in the SAHN program in NTSYS2.2 software (http:// www.exetersoftware.com/cat/ntsyspc. html), and the chart is shown in FIG. 3.
The results show that:
1) capillary electrophoresis detection, 8 pairs of polymorphism primers of the cpsSR molecular markers are amplified in DNA samples of Zhejiang red camellia and its kindred species to obtain clear polymorphism bands, polymorphism amplification is obtained in samples of different geographical populations of Zhejiang red camellia, the sizes of amplified fragments are counted, Cc _ cpsSR-12 can identify southern camellia at 378-one time, Cc _ cpsSR-15 can identify thick-leaf red camellia at 262bp at one time, and other 8 markers are combined in pairs to distinguish whole red camellia from Zhejiang red camellia.
2) The UPGMA-based cluster map shows (FIG. 3) that the genetic relationship among Zhejiang red camellia, sparkling red camellia, thick-leaf red camellia, whole-edge red camellia and Nanshan camellia to be tested is different, and at the position of Nei genetic similarity coefficient of 0.72, 5 species can be divided into two major categories, namely, Guangfu red camellia subgroup (Zhejiang red camellia, sparkling red camellia, thick-leaf red camellia and whole-edge red camellia) and Dianthus subgroup (Nanshan camellia). Furthermore, according to the Nei genetic similarity coefficient, the relationship between 5 species is shown as follows: the southern camellia is more than the whole-margin red camellia is more than the thick-leaf red camellia is more than the Zhejiang red camellia/the sparkling red camellia, wherein the Zhejiang red camellia and the sparkling red camellia have close relationship, and the method meets the conclusion that the two are merged into one of the Zhejiang red camellia in the previous research.
The invention utilizes the polymorphism primer of the camellia cpSSR molecular marker to identify the genetic relationship of the camellia plants, and utilizes the fact that the cpSSR molecular marker has the characteristics of codominance, high variation, polymorphism and the like of a nuclear DNA microsatellite molecular marker, and has the characteristics of monophilicity inheritance and relative conservation of chloroplast, so that the chloroplast microsatellite molecular marker has the characteristics of simple structure, multiple copies, small molecular weight and the like, and further the method for identifying the genetic relationship of the camellia plants provided by the invention can comprehensively reflect the genetic relationship between the camellia plants. In addition, the cpsR and the cell nucleus SSR molecular marker can be comprehensively used, so that the genetic relationship between the cytoplasm and the cell nucleus of a species can be further comprehensively, accurately and objectively reflected.
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<213> Artificial sequence (Artificial sequence)
<400>33
Claims (4)
1. The composition of the polymorphic primers of the camellia chloroplast microsatellite molecular marker is characterized by comprising 9 pairs of polymorphic primers of the camellia cpSSR molecular marker, wherein the primer information is as follows:
the forward primer of Cc _ cpSSR-04 is shown as SEQ ID NO.1 in the sequence table,
the reverse primer of Cc _ cpSSR-04 is shown as SEQ ID NO.2 in the sequence table;
the forward primer of Cc _ cpSSR-06 is shown as SEQ ID NO.3 in the sequence table,
the reverse primer of Cc _ cpSSR-06 is shown as SEQ ID NO.4 in the sequence table;
the forward primer of Cc _ cpSSR-12 is shown as SEQ ID NO.5 in the sequence table,
the reverse primer of Cc _ cpSSR-12 is shown as SEQ ID NO.6 in the sequence table;
the forward primer of Cc _ cpSSR-15 is shown as SEQ ID NO.9 in the sequence table,
the reverse primer of Cc _ cpSSR-15 is shown as SEQ ID NO.10 in the sequence table;
the forward primer of Cc _ cpSSR-40 is shown as SEQ ID NO.19 in the sequence table,
the reverse primer of Cc _ cpSSR-40 is shown as SEQ ID NO.20 in the sequence table;
the forward primer of Cc _ cpSSR-49 is shown as SEQ ID NO.21 in the sequence table,
the reverse primer of Cc _ cpSSR-49 is shown as SEQ ID NO.22 in the sequence table;
the forward primer of Cc _ cpSSR-71 is shown as SEQ ID NO.27 in the sequence table,
the reverse primer of Cc _ cpSSR-71 is shown as SEQ ID NO.28 in the sequence table;
the forward primer of Cc _ cpSSR-74 is shown as SEQ ID NO.29 in the sequence table,
the reverse primer of Cc _ cpSSR-74 is shown as SEQ ID NO.30 in the sequence table;
the forward primer of Cc _ cpSSR-75 is shown as SEQ ID NO.31 in the sequence table,
the reverse primer of Cc _ cpSSR-75 is shown as SEQ ID NO.32 in the sequence table.
2. The Camellia chloroplast microsatellite molecular marker of claim 1The polymorphic primer composition of (1), wherein the system for PCR amplification of the polymorphic primer is: 50ng DNA template, 1.0. mu.L 10 XPCR Buffer, 2.5mmol/L MgCl20.25mmol/L dNTP, 0.5 ng/muL of each of the upstream primer and the downstream primer, 0.5U Taq DNA polymerase, and the total volume of the PCR amplification system is 10 muL; the procedure for PCR amplification of the polymorphic primers is: pre-denaturation at 94 ℃ for 5 min; performing denaturation at 94 ℃ for 30s, annealing at 52-63 ℃ for 30s, annealing at 72 ℃ for 30s, and extending for 25 cycles; extending for 7min at 72 ℃, and storing at 4 ℃.
3. The composition of the polymorphic primers of camellia cpssR for screening camellia red camellia and related species thereof in Zhejiang comprises 8 pairs of polymorphic primers labeled by camellia cpssR molecules, wherein the 8 pairs of primers are respectively numbered Cc _ cpsSR-04, Cc _ cpsSR-12, Cc _ cpsSR-15, Cc _ cpsSR-40, Cc _ cpsSR-49, Cc _ cpsSR-71, Cc _ cpsSR-74 and Cc _ cpsSR-75, and the primer information is as follows:
the forward primer of Cc _ cpSSR-04 is shown as SEQ ID NO.1 in the sequence table,
the reverse primer of Cc _ cpSSR-04 is shown as SEQ ID NO.2 in the sequence table;
the forward primer of Cc _ cpSSR-12 is shown as SEQ ID NO.5 in the sequence table,
the reverse primer of Cc _ cpSSR-12 is shown as SEQ ID NO.6 in the sequence table;
the forward primer of Cc _ cpSSR-15 is shown as SEQ ID NO.9 in the sequence table,
the reverse primer of Cc _ cpSSR-15 is shown as SEQ ID NO.10 in the sequence table;
the forward primer of Cc _ cpSSR-40 is shown as SEQ ID NO.19 in the sequence table,
the reverse primer of Cc _ cpSSR-40 is shown as SEQ ID NO.20 in the sequence table;
the forward primer of Cc _ cpSSR-49 is shown as SEQ ID NO.21 in the sequence table,
the reverse primer of Cc _ cpSSR-49 is shown as SEQ ID NO.22 in the sequence table;
the forward primer of Cc _ cpSSR-71 is shown as SEQ ID NO.27 in the sequence table,
the reverse primer of Cc _ cpSSR-71 is shown as SEQ ID NO.28 in the sequence table;
the forward primer of Cc _ cpSSR-74 is shown as SEQ ID NO.29 in the sequence table,
the reverse primer of Cc _ cpSSR-74 is shown as SEQ ID NO.30 in the sequence table;
the forward primer of Cc _ cpSSR-75 is shown as SEQ ID NO.31 in the sequence table,
the reverse primer of Cc _ cpSSR-75 is shown as SEQ ID NO.32 in the sequence table.
4. A method for screening Zhejiang camellia and related species thereof by using the composition of the polymorphic primers of camellia cpsSR of claim 3, comprising the following steps:
(1) extracting the nuclear DNA of thick-leaf camellia, sparkling camellia, whole-edge camellia and southern camellia from Zhejiang camellia and its kindred seeds;
(2) mixing 8 pairs of primers in the polymorphism primers of the camellia cpSSR molecular marker with DNA of the Zhejiang red camellia and the allied species thereof, and carrying out PCR amplification to obtain an amplification product; the 8 pairs of primers are numbered Cc _ cPPSSR-04, Cc _ cPPSSR-12, Cc _ cPPSSR-15, Cc _ cPPSSR-40, Cc _ cPPSSR-49, Cc _ cPPSSR-71, Cc _ cPPSSR-74 and Cc _ cPPSSR-75 respectively, are modified by 5' FAM and then are subjected to PCR amplification and are marked in red;
(3) performing the length polymorphism detection of the PCR product by adopting capillary electrophoresis on the PCR amplification product;
(4) the method for obtaining the result of capillary electrophoresis comprises the following steps: analyzing the capillary electrophoresis result by using PeakScanner software1.0 software, and determining the band length of the amplification product according to a peak diagram; calculating a Nei genetic similarity coefficient by adopting popgene software according to different haplotype statistical data; constructing a genetic relationship cluster map by using UPGMA in a SAHN program in NTSYS2.2 software;
(5) comparing the capillary electrophoresis result with the identification result in the table 1 to finish species discrimination;
TABLE 1 Zhejiang red camellia and identification results of its kindred species, unit bp
。
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