CN111088387B - Cinnamomum camphora chloroplast whole genome PCR (polymerase chain reaction) amplification primer and application thereof - Google Patents
Cinnamomum camphora chloroplast whole genome PCR (polymerase chain reaction) amplification primer and application thereof Download PDFInfo
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Abstract
The invention discloses a cinnamomum camphora chloroplast whole genome PCR amplification primer and application thereof, belonging to the technical field of biological science. The application designs 26 pairs of specific camphor chloroplast whole genome PCR amplification primers, the nucleotide sequences of the primers are shown in SEQ ID NO.1-SEQ ID NO.52, the primers are applied to camphor chloroplast whole genome sequence PCR amplification, camphor variety identification and lauraceae chloroplast genome phylogeny research, amplification and Sanger sequencing of 5 camphor different chemical type individual chloroplast genomes are completed, and a phylogeny tree is constructed by utilizing 68 common chloroplast protein coding gene sequences of 19 lauraceae plants based on a Bayesian method. The primer has good repeatability, can specifically amplify the cinnamomum camphora chloroplast genome sequence, achieves the aim of rapid identification, and provides important basis for cinnamomum camphora variety identification, genetic relationship analysis and genetic evolution analysis.
Description
Technical Field
The invention belongs to the technical field of bioscience, and particularly relates to a camphor chloroplast whole genome PCR amplification primer and application thereof.
Background
Cinnamomum camphora (L.) Presl) is a tall tree of Lauraceae family, has important ecological, cultural and economic values, and is an important material and special economic tree species in China. According to the main chemical components in the leaves of cinnamomum camphora, people divide cinnamomum camphora into different chemical types. Li Xiwen et al, for the first time, classified the cinnamomum camphora into three types, i.e., cinnamomum camphora, also known as cinnamomum camphora (mainly containing camphor), cinnamomum camphora (mainly containing linalool), and cinnamomum camphora (mainly containing eucalyptol), and described the subtle differences in the morphology of the three types of cinnamomum camphora. In 1985, 164 cinnamomum camphora essential oil types are investigated by Long Guangyuan and the like by adopting chromatography, chromatography-mass spectrometry, infrared spectroscopy and other chemical methods, and the types of cinnamomum camphora include three chemical types of cinnamomum camphora, cinnamomum camphora and cinnamomum camphora, and the cinnamomum camphora also includes two types of cinnamomum camphora (mainly containing nerolidol and isobavarin).
The genetic variation is also called genetic diversity at the population level, in the 19 th century, mundler of Austria learns that phenotypic traits are used as genetic markers for genetic research for the first time in pea hybridization experiments, the genetic markers are greatly developed along with the continuous development of genetics and molecular biology, and the current genetic markers can be divided into four types, namely morphological markers (morphological markers), cytological markers (cytological markers), biochemical markers (biochemical markers) and DNA molecular markers (DNA molecular markers).
The morphological marker refers to the morphological characteristics that the plant can inherit, and can be measured and observed by naked eyes or instruments, such as the height, the flower color, the leaf type, the fruit color, the fruit type and the like of the plant, the physiological characteristics, the anatomical characteristics, the stress resistance and the like of the plant. However, the morphological markers cannot truly reflect the size of genetic diversity in some cases due to the small number of the markers, susceptibility to environmental influences, unstable genetic expression and the like.
The cytological markers mainly refer to karyotype and banding pattern analysis of chromosomes, and although some defects of morphological markers are overcome, a large amount of manpower and material resources are consumed, and the number of the markers is limited, so that the application of the cytological markers in genetic breeding is limited.
Biochemical markers are primarily referred to as isoenzyme markers. However, isozymes are gene expression products, which are easily affected by external factors, and when studying individuals with close relativity, they often fail to provide sufficient polymorphic markers, so that the study of genetic diversity using isozyme markers is still limited by certain conditions.
The DNA molecular marker can directly reflect the change of the gene, is not influenced by the environment, tissues and organs and development stages, and is widely applied in various fields. The most representative of them are the first generation of molecular marker Restriction Fragment Length Polymorphism (RFLP), the second generation of molecular marker Simple Sequence Repeat (SSR) and the third generation of molecular marker Single Nucleotide Polymorphism (SNP). However, the RFLP marker has the defects of small polymorphic site information amount, dependence on restriction endonuclease, high requirement on DNA quality, complex operation, need of radioactive isotope, high cost and the like, and the application is greatly limited. SSR markers are abundant, co-dominant markers separated in a Mendelian manner, and are detected by a PCR technology. However, the flanking sequences may differ from species to species, which results in poor versatility, and additionally, the phenomenon of homologies and heterohomologies of microsatellites may lead to erroneous results from studies performed solely on PCR product fragments, and the phenomenon of null alleles may lead to erroneous identification of microsatellite genotypes. SNP markers have the defects of limited information sites, high cost and the like.
The chloroplast is a special organelle for photosynthesis of plants, provides a main source of energy for the plants, has a genome, and is applied to phylogenetic research, so that the chloroplast genome has many advantages: 1. the chloroplast genome is small and relatively conserved, and the complete sequence is easily obtained; 2. the chloroplast genome contains a large amount of nucleotide information, and the base substitution rate is moderate; 3. the evolution speed difference of the chloroplast coding region and the non-coding region is obvious, and the method is suitable for the phylogenetic research of different levels; 4. the chloroplast genome is all single copy genes except the inverted repeat region, and almost no paralog gene interference exists.
Disclosure of Invention
Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide a camphor chloroplast whole genome PCR amplification primer; the invention aims to solve another technical problem of providing the application of the cinnamomum camphora chloroplast whole genome PCR amplification primer.
In order to solve the technical problems, the invention adopts the technical scheme that:
the camphor chloroplast whole genome PCR amplification primer comprises 26 pairs of primers, and the nucleotide sequence of the primers is as follows:
the Cp1 upstream primer is shown as SEQ ID NO. 1; the Cp1 downstream primer is shown as SEQ ID NO. 2;
the Cp2 upstream primer is shown as SEQ ID NO. 3; the Cp2 downstream primer is shown as SEQ ID NO. 4;
the Cp3 upstream primer is shown as SEQ ID NO. 5; the Cp3 downstream primer is shown as SEQ ID NO. 6;
the Cp4 upstream primer is shown as SEQ ID NO. 7; the Cp4 downstream primer is shown as SEQ ID NO. 8;
the Cp5 upstream primer is shown as SEQ ID NO. 9; the Cp5 downstream primer is shown as SEQ ID NO. 10;
the Cp6 upstream primer is shown as SEQ ID NO. 11; the Cp6 downstream primer is shown as SEQ ID NO. 12;
the Cp7 upstream primer is shown as SEQ ID NO. 13; the Cp7 downstream primer is shown as SEQ ID NO. 14;
the Cp8 upstream primer is shown as SEQ ID NO. 15; the Cp8 downstream primer is shown as SEQ ID NO. 16;
the Cp9 upstream primer is shown as SEQ ID NO. 17; the Cp9 downstream primer is shown as SEQ ID NO. 18;
the Cp10 upstream primer is shown as SEQ ID NO. 19; the Cp10 downstream primer is shown as SEQ ID NO. 20;
the Cp11 upstream primer is shown as SEQ ID NO. 21; the Cp11 downstream primer is shown as SEQ ID NO. 22;
the Cp12 upstream primer is shown as SEQ ID NO. 23; the Cp12 downstream primer is shown as SEQ ID NO. 24;
the Cp13 upstream primer is shown as SEQ ID NO. 25; the Cp13 downstream primer is shown as SEQ ID NO. 26;
the Cp14 upstream primer is shown as SEQ ID NO. 27; the Cp14 downstream primer is shown as SEQ ID NO. 28;
the Cp15 upstream primer is shown as SEQ ID NO. 29; the Cp15 downstream primer is shown as SEQ ID NO. 30;
the Cp16 upstream primer is shown as SEQ ID NO. 31; the Cp16 downstream primer is shown as SEQ ID NO. 32;
the Cp17 upstream primer is shown as SEQ ID NO. 33; the Cp17 downstream primer is shown as SEQ ID NO. 34;
the Cp18 upstream primer is shown as SEQ ID NO. 35; the Cp18 downstream primer is shown as SEQ ID NO. 36;
the Cp19 upstream primer is shown as SEQ ID NO. 37; the Cp19 downstream primer is shown as SEQ ID NO. 38;
the Cp20 upstream primer is shown as SEQ ID NO. 39; the Cp20 downstream primer is shown as SEQ ID NO. 40;
the Cp21 upstream primer is shown as SEQ ID NO. 41; the Cp21 downstream primer is shown as SEQ ID NO. 42;
the Cp22 upstream primer is shown as SEQ ID NO. 43; the Cp22 downstream primer is shown as SEQ ID NO. 44;
the Cp23 upstream primer is shown as SEQ ID NO. 45; the Cp23 downstream primer is shown as SEQ ID NO. 46;
the Cp24 upstream primer is shown as SEQ ID NO. 47; the Cp24 downstream primer is shown as SEQ ID NO. 48;
the Cp25 upstream primer is shown as SEQ ID NO. 49; the Cp25 downstream primer is shown as SEQ ID NO. 50;
the Cp26 upstream primer is shown as SEQ ID NO. 51; the Cp26 downstream primer is shown as SEQ ID NO. 52.
The application of the primer in the PCR amplification of the camphor chloroplast whole genome sequence comprises the following steps:
1) Extracting DNA of a cinnamomum camphora sample;
2) And (2) carrying out PCR amplification on the DNA extracted in the step 1) by using the primers.
Further, the PCR amplification system is: 1 mu L of each of the upstream primer and the downstream primer; 60ng of DNA;25 μ L PrimerSTAR Max Premix 2 ×; add ddH 2 O to 50 μ L as a whole.
Further, the PCR amplification procedure is as follows: pre-denaturation at 98 ℃ for 3min; denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 5s, wherein the three steps of denaturation, annealing and extension are circulated for 35 times; finally, extension is carried out for 4min at 72 ℃.
The application of the primer in identification of cinnamomum camphora varieties.
The application of the primer in research on development of a lauraceae chloroplast genome system.
The invention designs 26 pairs of specific PCR primers, completes the amplification and Sanger sequencing of chloroplast genomes of 5 cinnamomum camphora individuals with different chemotypes, and the genome sizes are 152,734bp of cinnamomum camphora, 152,733bp of cinnamomum camphora, 152,729bp of borneol camphor, 152,735bp of cinnamomum camphora and 152,723bp of cinnamomum camphora. Wherein the LSC length is 93,686-93, 693bp, the SSC length is 18,893-18, 898bp, and the IRs length is 20,074bp. And constructing a phylogenetic tree by using 68 common chloroplast protein coding gene sequences of 19 Lauraceae plants based on a Bayesian method, wherein the result shows that the cinnamomum camphora is relatively close to the relativity of the cinnamomum camphora to the Lindera glauca (Lindera glauca) of the Lindera, the Litsea glutinosa (Litsea glaucosa) of the Litsea and the Laurus nobilis (Laurus nobilis) of the Laurus.
Has the advantages that: compared with the prior art, the primer has good repeatability, can specifically amplify a cinnamomum camphora chloroplast genome sequence to achieve the purpose of rapid identification, and meanwhile, the chloroplast genome is taken as genetic material contained in chloroplast and is independent of organ genomes except nuclear genomes, and the sequence and the structure are highly conserved but still have certain changes, which provide important basis for cinnamomum camphora variety identification, genetic relationship analysis and genetic evolution analysis.
Drawings
FIG. 1 is a genome annotation circle of a Cinnamomum camphora chloroplast of Cinnamomum camphora;
FIG. 2 is a sliding window analysis chart of chloroplast genome of 5 chemotype individuals of Cinnamomum camphora;
FIG. 3 is a phylogenetic tree diagram of 19 genes encoding chloroplast genome proteins of Lauraceae plants (Liriodendron tulipifera and Pistacia weinmannifolia are used as the outer group).
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
The main test materials used in the following examples were: cinnamomum camphora type and Cinnamomum camphora type cinnamomum camphora are collected in Experimental forest of scientific research institute of forestry in Jiangxi province, and Cinnamomum camphora type, cinnamomum camphora type and Cinnamomum camphora type are collected in university campus of Nanjing forestry. Collecting 5-10 fresh leaves of each sample, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C.
Example 1
1. Extraction and detection of total DNA of cinnamomum camphora
The extraction of the total DNA of the cinnamomum camphora is carried out by adopting an improved CTAB method, and the specific operation steps are as follows:
1) Opening the water bath kettle, and preheating at 65 ℃;
2) Taking 10mL of a centrifuge tube, adding 4mL of CTAB lysate and 80 mu L of beta-mercaptoethanol, and putting the centrifuge tube into a water bath for preheating;
3) Taking about 2g of cinnamomum camphora leaves, putting the leaves into a mortar prepared in advance, introducing liquid nitrogen for grinding until the sample is in a uniform powder state, adding the powder into the centrifugal tube in the step 2), putting the centrifugal tube into a water bath kettle, carrying out water bath at 65 ℃ for 30min, and reversing and uniformly mixing the powder every 5min;
4) The centrifuge tube was removed and placed on ice to cool for three minutes, and then an equal volume of chloroform isoamyl alcohol mixture (chloroform: isoamyl alcohol =24: 1) Fully and uniformly mixing, putting into a centrifuge, and centrifuging for 10min at 4 ℃ and 12000 r;
5) Preparing a new 10mL centrifuge tube, sucking the supernatant into the new centrifuge tube by using a pipette, adding the chloroform isoamylol mixed solution with the same volume again, uniformly mixing, placing in a centrifuge, and centrifuging for 10min at 4 ℃ at 12000 r;
6) Respectively sucking 500 μ L of supernatant, subpackaging in 3 centrifuge tubes of 1.5mL, respectively adding 1mL of ethanol and 50 μ L of sodium acetate solution, reversing, mixing, placing in a refrigerator at-20 deg.C, standing for 2h, and waiting for floccule to precipitate; transferring the residual supernatant to a 2mL centrifuge tube, and placing the centrifuge tube in a refrigerator at-20 ℃ for later use;
7) Preparing new 1.5mL centrifuge tubes, respectively adding 1mL75% ethanol solution, picking floccules separated out from 3 centrifuge tubes into the new centrifuge tubes, vortexing for 1min, and centrifuging for 5min at 10000 r;
8) Carefully pouring off the ethanol solution, adding a new 1mL of 75% ethanol solution, and repeating the previous step;
9) Pouring off the ethanol solution again, and drying by using a vacuum drying instrument;
10 200 μ L of 1 XTE solution was added, dissolved for 3 hours, and centrifuged after being taken out, and the supernatant was the extracted DNA.
11 Taking a small amount of extracted total DNA of the cinnamomum camphora, and detecting the quality and concentration of a DNA sample by using NanoDrop 2000c (ThermoFisher scientific); meanwhile, the quality of the DNA is detected by using 1% agarose gel electrophoresis, and whether the degradation of the DNA is caused or not is detected by observing whether the bands are dispersed or not.
12 DNA was diluted to 30 ng/. Mu.L and stored at-20 ℃.
2. Cinnamomum camphora chloroplast genome PCR amplification, sequencing and assembly
In this example, 26 pairs of primers were designed and synthesized by Nanjing Kingsler Biotechnology Ltd, and the specific primer sequences are shown in Table 1.
TABLE 1 Cinnamomum camphora chloroplast amplification primers
Product amplification was performed using PrimeSTAR MAX DNA Polymerase (Takara, dalibao bioengineering, inc.). The PCR reaction total system is specifically as follows: 1 mu L of each of the upstream primer and the downstream primer; 2. Mu.L of DNA (30 ng/. Mu.L); 25 μ L PrimerSTAR Max Premix (2 ×); add ddH 2 O to total 50. Mu.L. The PCR amplification procedure was: pre-denaturation at 98 ℃ for 3min; denaturation at 94 ℃, annealing at 10s,55 ℃, elongation at 5s, and elongation at 72 ℃ for 5s for 35 cycles; extension at 72 ℃ for 4min.
The band size and dispersion of the PCR product of the cinnamomum camphora chloroplast genome were detected by 1% agarose gel electrophoresis, and the PCR product was purified by AxyPrep-PCR clean kit, and all the purified DNA products were subjected to Sanger sequencing by ABI 3730 sequencer (done by Nanjing Kingsler Biotech Co., ltd.). According to the existing cinnamomum camphora chloroplast genome (NC-035882.1) as a reference genome, sequence assembly is carried out by using software CAP3 and manual proofreading is carried out.
Example 2
1. Cinnamomum camphora chloroplast genome structure
Taking an existing cinnamomum camphora chloroplast GenoMe (NC-035882.1) as a reference GenoMe, performing gene annotation on the cinnamomum camphora chloroplast GenoMe by using Dual Organillar GenoMe annotor (DOGMA) (default parameters), and manually determining the positions of a start codon and a stop codon; all tRNAs were identified by the software tRNAscan-SE v2.0 (http:// lowelab. Ucsc. Edu/tRNAscan-SE /) and supplemented with manual confirmation; a map of the Cinnamomum camphora chloroplast Genome circle was drawn by the software Organella Genome DRAW 1.2 (https:// chlorobox.
26 chloroplast genome amplification primers are utilized to carry out PCR amplification, cloning and sequencing on the chloroplast genomes of 5 cinnamomum camphora of different chemical individuals, such as cinnamomum camphora, cinnamomum camphora and the like, so that 5 complete chloroplast genomes are obtained in total, namely cinnamomum camphora (152734 bp), cinnamomum camphora (152733 bp), cinnamomum camphora (152729 bp), cinnamomum camphora (152735 bp) and cinnamomum camphora (152723 bp). The cinnamomum camphora chloroplast genome is a typical tetrad structure and comprises a long single copy sequence (LSC), a short single copy sequence (SSC) and two Inverted Repeats (IRs). The base composition and length of the cinnamomum camphora chloroplast genome of 5 chemotypes of individuals are basically consistent, and fig. 1 shows an annotated circular diagram of the cinnamomum camphora chloroplast genome of a cinnamomum camphora type. The length of the long single copy sequence is 93686-93693 bp, the length of the short single copy sequence is 18893-18898 bp, the lengths of the two inverted repeat regions are 20074bp except that the IRb of the isosafrole is 20075bp because of one more base T, and the specific table is shown in table 2.
TABLE 2 chloroplast genome length of different chemotype individuals
| Type (B) | Total length of the track | LSC(bp) | SSC(bp) | IR(bp) |
| Nao zhu type | 152734 | 93693 | 18893 | 20074 |
| Cinnamomum camphora type | 152733 | 93687 | 18898 | 20074 |
| Borneol camphor type | 152729 | 93686 | 18895 | 20074 |
| Iso camphor type | 152735 | 93693 | 18893 | 20075/20074 |
| Cinnamomum camphora type | 152723 | 93682 | 18893 | 20074 |
Note: LCS: a long single copy sequence; SSC: a short single copy sequence; IR: inverted repeat region
The GC content of the cinnamomum camphora chloroplast is obviously less than the AT content, taking the cinnamomum camphora as an example (Table 3); wherein the GC content of the inverted repeat region is 44.43% at most, the long single-copy sequence is 37.96%, and the GC content of the short single-copy sequence is 33.92% at least. The GC content of the coding region is only 39.02 percent which is obviously less than that of the non-coding region, and the GC content of the tRNAs and the rRNAs regions is higher and is more than 50 percent.
TABLE 3 Cinnamomum camphora chloroplast genome characterization
| Name (R) | Size(bp) | Content of C% | Content of G% | GC content% |
| The entire chloroplast genome | 152734 | 19.95 | 19.21 | 39.16 |
| Long single copy sequence | 93693 | 19.38 | 18.58 | 37.96 |
| Short single copy sequence | 18893 | 18.02 | 15.90 | 33.92 |
| Inverted repeat region | 20074 | 21.05 | 23.38 | 44.43 |
| Protein coding gene | 72685 | 18.32 | 20.70 | 39.02 |
| rRNAs | 9032 | 23.60 | 31.59 | 55.19 |
| tRNAs | 2662 | 23.63 | 29.53 | 53.16 |
Adopting Dual Organinella GenoMe annotor (DOGMA) software to perform gene annotation on chloroplast genomes of 5 chemical individuals, wherein the gene annotation results of the 5 chloroplast genomes are completely consistent, and 115 functional genes are obtained by annotating each GenoMe, including 81 protein coding genes, 30 tRNA genes and 4 rRNA genes; there were 3 protein-encoding genes, 6 tRNA genes, and 4 rRNA genes in the IR region, which were two copies, thus 128 genes in total.
Most of the genes in the chloroplast genome of cinnamomum camphora do not contain introns, and only 12 genes contain introns, wherein clpP and ycf3 contain two introns, and the rps12 gene is a trans-splicing gene, and the 5' exon is located in a long single-copy sequence region, and the other two exons are located in an inverted repeat region.
2. Chloroplast genome sequence difference analysis of cinnamomum camphora of different chemotype individuals
And (3) comparing camphor chloroplast genomes of different chemotypes of individuals by using sequence multiple alignment software MAFFT. Sliding window analysis of chloroplast genomes of these individuals was performed using DNAsp V6 software with parameters set to: the window length is 600bp, and the sliding step size is 200bp.
Through sequence alignment, the chloroplast genome of 5 chemotypes of individuals is found to have smaller difference. The DNAsp software is used for carrying out sliding window analysis on the variation degree of different segments of the whole genomes of the cinnamomum camphora chloroplasts of different types (figure 2), the sequence nucleotide polymorphism (Pi) value of each 600bp range of the chloroplast genomes of 5 cinnamomum camphora individuals is between 0.00067 and 0.008, and the variation level is very low. Overall, the level of variation in the LSC region was higher, and the level of variation in the SSC and IRs regions was lower. The regions with relatively large variations are mainly trnK (UUU) -rps16, atpF-atpH, rpoC1, trnE (UUC) -trnT (GGU), trnfM (CAU), ycf3 and clpP, wherein the region with the largest variation is trnK (UU) -rps16. The most part of the variant region is located in the non-coding region, and the coding region has less variation.
3. Phylogenetic analysis of chloroplast genome in Lauraceae
The genome sequences of 18 lauraceae chloroplasts were downloaded by NCBI (National Center for Biotechnology Information), litsea Litsea (Litsea glaucosa), lindera glauca (Lindera glauca) of the genus Cinnamomum, laurus nobilis (Laurus nobilis), alodoriphylus hancei (Alodoriphylus hunensis) of the genus Elaeagnus, alodoraphne hunensis (Alodoriphylus gracilis), alsemiaquinone (Alodoriphylus gracilis), bai Nan (Phebouranha) of the genus Machilus, phoebe omeiensis (Phoeby longensis), phoebe bournei (Phoebe bournei), phoebe zhenni (Phoeby rosea), phoebe zhennine (Phormiana officinalis), phormiana sinensis (Machilus sylvestris) of the genus), machilus sylvestris (Machilus sylvestris 5263), machilus sylvestris (Machilus). Finding out their common protein coding genes, using software mrboyes v3.2.6 to build tree, adopting jModelTest to screen optimum nucleic acid substitution model, GTR + I + G, running one million generations, sampling frequency is sampling every 100 generations.
In order to confirm the phylogenetic position of cinnamomum camphora in lauraceae, 68 genes encoding proteins were found in 19 chloroplast genomes of lauraceae plants, and phylogenetic trees were constructed using DNA sequences of tulip tree and sightseeing wood of magnolia through mrbayes v3.2.6 software (fig. 3). All branches of the phylogenetic tree have high support rate (bootstrap value), except that 99% of one node, the other nodes are 100%,19 Lauraceae plants show clear systemic relationship and can be divided into 4 branches. The rootless vine is used as the only liana of the lauraceae family and is independently used as one branch; the cangs and cangs Jiang Xin of the new cinnamomum genus are clustered into one branch; the relativity of cinnamomum camphora and cinnamomum zeylanicum in cinnamomum genus to litsea cubeba in lindera genus, litsea glutinosa in litsea genus and laurel in laurel genus is relatively close and one branch; the genus Plumbum, machilus and avocado form one branch.
It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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<212> DNA
<213> Cp5R(artificial)
<400> 10
atggccgtga tctgtcatta c 21
<210> 11
<211> 24
<212> DNA
<213> Cp6F(artificial)
<400> 11
gttccatctt aatctaccat atct 24
<210> 12
<211> 25
<212> DNA
<213> Cp6R(artificial)
<400> 12
ggataaacaa acccattctt cttca 25
<210> 13
<211> 22
<212> DNA
<213> Cp7F(artificial)
<400> 13
cgtggctgtg ttggtgtatt ct 22
<210> 14
<211> 24
<212> DNA
<213> Cp7R(artificial)
<400> 14
gatcataacc gctacctaca ttcc 24
<210> 15
<211> 23
<212> DNA
<213> Cp8F(artificial)
<400> 15
atgggatccg tagtaggcga gaa 23
<210> 16
<211> 23
<212> DNA
<213> Cp8R(artificial)
<400> 16
cgacacaatc aaacccgaac gaa 23
<210> 17
<211> 23
<212> DNA
<213> Cp9F(artificial)
<400> 17
<210> 18
<211> 18
<212> DNA
<213> Cp9R(artificial)
<400> 18
caagaacgga ctaacagg 18
<210> 19
<211> 22
<212> DNA
<213> Cp10F(artificial)
<400> 19
atcgtttcta tggttacttc tt 22
<210> 20
<211> 22
<212> DNA
<213> Cp10R(artificial)
<400> 20
cgtatgaaat cccccaataa ct 22
<210> 21
<211> 18
<212> DNA
<213> Cp11F(artificial)
<400> 21
agcggatcca tttgcaac 18
<210> 22
<211> 23
<212> DNA
<213> Cp11R(artificial)
<400> 22
taggtaggcg tcctgtagta aga 23
<210> 23
<211> 19
<212> DNA
<213> Cp12F(artificial)
<400> 23
ttcggcttta ctaatgacc 19
<210> 24
<211> 22
<212> DNA
<213> Cp12R(artificial)
<400> 24
acaggagcga aacaatcaac ct 22
<210> 25
<211> 24
<212> DNA
<213> Cp13F(artificial)
<400> 25
gcttcactgg ggtcattgta catc 24
<210> 26
<211> 24
<212> DNA
<213> Cp13R(artificial)
<400> 26
cagcaacaag aagaaggaaa agaa 24
<210> 27
<211> 22
<212> DNA
<213> Cp14F(artificial)
<400> 27
gataagtgag agttcgagta ag 22
<210> 28
<211> 22
<212> DNA
<213> Cp14R(artificial)
<400> 28
cacaacgacg caattatcag gg 22
<210> 29
<211> 20
<212> DNA
<213> Cp15F(artificial)
<400> 29
<210> 30
<211> 20
<212> DNA
<213> Cp15R(artificial)
<400> 30
<210> 31
<211> 20
<212> DNA
<213> Cp16F(artificial)
<400> 31
<210> 32
<211> 25
<212> DNA
<213> Cp16R(artificial)
<400> 32
ctttttaccg atttcttcta ttcca 25
<210> 33
<211> 23
<212> DNA
<213> Cp17F(artificial)
<400> 33
aagaatgctt gcctagagtg tat 23
<210> 34
<211> 18
<212> DNA
<213> Cp17R(artificial)
<400> 34
tagatcagca gcgactcc 18
<210> 35
<211> 23
<212> DNA
<213> Cp18F(artificial)
<400> 35
ttcacagagg caataacggt aaa 23
<210> 36
<211> 22
<212> DNA
<213> Cp18R(artificial)
<400> 36
ggccacaaat aaatctaagg ac 22
<210> 37
<211> 25
<212> DNA
<213> Cp19F(artificial)
<400> 37
ttctttcttt cctcaatcct atcca 25
<210> 38
<211> 25
<212> DNA
<213> Cp19R(artificial)
<400> 38
cattaagaga aaaatagaac ccccc 25
<210> 39
<211> 19
<212> DNA
<213> Cp20F(artificial)
<400> 39
cgggattcat cactacttt 19
<210> 40
<211> 23
<212> DNA
<213> Cp20R(artificial)
<400> 40
cccaacccta ttgacatagt aac 23
<210> 41
<211> 21
<212> DNA
<213> Cp21F(artificial)
<400> 41
cccggtagaa atagatttag c 21
<210> 42
<211> 23
<212> DNA
<213> Cp21R(artificial)
<400> 42
attccgtcaa ttacccgtct atc 23
<210> 43
<211> 25
<212> DNA
<213> Cp22F(artificial)
<400> 43
gcgtctattt tatatgggtc tgtta 25
<210> 44
<211> 23
<212> DNA
<213> Cp22R(artificial)
<400> 44
atattcctgt actacccctt gtt 23
<210> 45
<211> 23
<212> DNA
<213> Cp23F(artificial)
<400> 45
ccgagttcct tagagagagt tgt 23
<210> 46
<211> 23
<212> DNA
<213> Cp23R(artificial)
<400> 46
<210> 47
<211> 21
<212> DNA
<213> Cp24F(artificial)
<400> 47
accttgttac gacttcactc c 21
<210> 48
<211> 23
<212> DNA
<213> Cp24R(artificial)
<400> 48
agcaccacat aaaaacattc ctc 23
<210> 49
<211> 23
<212> DNA
<213> Cp25F(artificial)
<400> 49
<210> 50
<211> 23
<212> DNA
<213> Cp25R(artificial)
<400> 50
cgtttatcaa agaatgactc tgg 23
<210> 51
<211> 23
<212> DNA
<213> Cp26F(artificial)
<400> 51
cgaggtttgt gaataagtga ttg 23
<210> 52
<211> 23
<212> DNA
<213> Cp26R(artificial)
<400> 52
accactgaaa atgaatctgc taa 23
Claims (6)
1. The camphor chloroplast whole genome PCR amplification primer is characterized by comprising 26 pairs of primers, wherein the primer nucleotide sequence is as follows:
the Cp1 upstream primer is shown as SEQ ID NO. 1; the Cp1 downstream primer is shown as SEQ ID NO. 2;
the Cp2 upstream primer is shown as SEQ ID NO. 3; the Cp2 downstream primer is shown as SEQ ID NO. 4;
the Cp3 upstream primer is shown as SEQ ID NO. 5; the Cp3 downstream primer is shown as SEQ ID NO. 6;
the Cp4 upstream primer is shown as SEQ ID NO. 7; the Cp4 downstream primer is shown as SEQ ID NO. 8;
the Cp5 upstream primer is shown as SEQ ID NO. 9; the Cp5 downstream primer is shown as SEQ ID NO. 10;
the Cp6 upstream primer is shown as SEQ ID NO. 11; the Cp6 downstream primer is shown as SEQ ID NO. 12;
the Cp7 upstream primer is shown as SEQ ID NO. 13; the Cp7 downstream primer is shown as SEQ ID NO. 14;
the Cp8 upstream primer is shown as SEQ ID NO. 15; the Cp8 downstream primer is shown as SEQ ID NO. 16;
the Cp9 upstream primer is shown as SEQ ID NO. 17; the Cp9 downstream primer is shown as SEQ ID NO. 18;
the Cp10 upstream primer is shown as SEQ ID NO. 19; the Cp10 downstream primer is shown as SEQ ID NO. 20;
the Cp11 upstream primer is shown as SEQ ID NO. 21; the Cp11 downstream primer is shown as SEQ ID NO. 22;
the Cp12 upstream primer is shown as SEQ ID NO. 23; the Cp12 downstream primer is shown as SEQ ID NO. 24;
the Cp13 upstream primer is shown as SEQ ID NO. 25; the Cp13 downstream primer is shown as SEQ ID NO. 26;
the Cp14 upstream primer is shown as SEQ ID NO. 27; the Cp14 downstream primer is shown as SEQ ID NO. 28;
the Cp15 upstream primer is shown as SEQ ID NO. 29; the Cp15 downstream primer is shown as SEQ ID NO. 30;
the Cp16 upstream primer is shown as SEQ ID NO. 31; the Cp16 downstream primer is shown as SEQ ID NO. 32;
the Cp17 upstream primer is shown as SEQ ID NO. 33; the Cp17 downstream primer is shown as SEQ ID NO. 34;
the Cp18 upstream primer is shown as SEQ ID NO. 35; the Cp18 downstream primer is shown as SEQ ID NO. 36;
the Cp19 upstream primer is shown as SEQ ID NO. 37; the Cp19 downstream primer is shown as SEQ ID NO. 38;
the Cp20 upstream primer is shown as SEQ ID NO. 39; the Cp20 downstream primer is shown as SEQ ID NO. 40;
the Cp21 upstream primer is shown as SEQ ID NO. 41; the Cp21 downstream primer is shown as SEQ ID NO. 42;
the Cp22 upstream primer is shown as SEQ ID NO. 43; the Cp22 downstream primer is shown as SEQ ID NO. 44;
the Cp23 upstream primer is shown as SEQ ID NO. 45; the Cp23 downstream primer is shown as SEQ ID NO. 46;
the Cp24 upstream primer is shown as SEQ ID NO. 47; the Cp24 downstream primer is shown as SEQ ID NO. 48;
the Cp25 upstream primer is shown as SEQ ID NO. 49; the Cp25 downstream primer is shown as SEQ ID NO. 50;
the Cp26 upstream primer is shown as SEQ ID NO. 51; the Cp26 downstream primer is shown as SEQ ID NO. 52.
2. The application of the primer of claim 1 in the PCR amplification of the cinnamomum camphora chloroplast whole genome sequence, which is characterized by comprising the following steps:
1) Extracting DNA of a cinnamomum camphora sample;
2) Performing PCR amplification on the DNA extracted in step 1) using the primers of claim 1.
3. The use according to claim 2, wherein the PCR amplification system is: 1 mu L of each of the upstream primer and the downstream primer; 60ng of DNA;25 μ L PrimerSTAR Max Premix 2 ×; add ddH 2 O to total 50. Mu.L.
4. The use according to claim 2, wherein the PCR amplification procedure is: pre-denaturation at 98 ℃ for 3min; denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 5s, wherein the three steps of denaturation, annealing and extension are circulated for 35 times; finally, extension is carried out for 4min at 72 ℃.
5. The use of the primer of claim 1 in the identification of Cinnamomum camphora species.
6. The use of the primer of claim 1 in a research study on the development of the genome system of chloroplast of Lauraceae.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841991A (en) * | 2018-07-25 | 2018-11-20 | 西南林业大学 | Poplar chloroplast full-length genome PCR amplification primer and its application |
| CN110195123A (en) * | 2019-06-20 | 2019-09-03 | 杭州师范大学 | Ku Zhi cpSSR labeled primer and its application are developed based on Chloroplast gene sequence |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841991A (en) * | 2018-07-25 | 2018-11-20 | 西南林业大学 | Poplar chloroplast full-length genome PCR amplification primer and its application |
| CN110195123A (en) * | 2019-06-20 | 2019-09-03 | 杭州师范大学 | Ku Zhi cpSSR labeled primer and its application are developed based on Chloroplast gene sequence |
Non-Patent Citations (3)
| Title |
|---|
| "Development and characterization of chloroplast microsatellite markers for Pinus massoniana and their application in Pinus (Pinaceae) species";Zhouxian Ni 等;《Journal of Genetics》;20180525;第97卷;第e53-55页，表1 * |
| "The re-sequencing of complete chloroplast genome of Cinnamomum camphora (Lauraceae) from Quanzhou, China";Meng-Yuan Qiu 等;《Mitochondrial DNA B Resour》;20200114;第5卷(第1期);第520页 * |
| 臭柏叶绿体基因组结构与系统进化分析;路东晔等;《西北植物学报》;20180815(第08期);全文 * |
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