CN111172312B

CN111172312B - PCR amplification primer for chloroplast genomes of bamboo cypress and long-leaf bamboo cypress and application of PCR amplification primer

Info

Publication number: CN111172312B
Application number: CN202010069452.0A
Authority: CN
Inventors: 祁浩然; 杨春霞; 胥猛
Original assignee: Nanjing Forestry University; Jiangxi Academy of Forestry
Current assignee: Nanjing Forestry University; Jiangxi Academy of Forestry
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2023-01-17
Anticipated expiration: 2040-01-20
Also published as: CN111172312A

Abstract

The invention discloses a chloroplast genome PCR amplification primer of bamboo cypress and bamboo cypress with long leaves and application thereof, belonging to the technical field of molecular biology. The application designs 29 PCR amplification primers for chloroplast genomes of specific bamboo cypresses and long-leaf bamboo cypresses, the nucleotide sequences of the primers are shown in SEQ ID No. 1-SEQ ID No.58, the average fragment of a product is about 3.7kb, sequencing and splicing verification are carried out by a Sanger sequencing method, the result is consistent with the result of high-throughput sequencing by extracting chloroplast DNA, and the obtained chloroplast genome sequence length of the bamboo cypress is 133724bp, and the chloroplast genome sequence of the long-leaf bamboo cypress is 133777bp. The primer has good repeatability, can specifically amplify chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress, and is favorable for researching the systematic evolution, the parental analysis, the variety identification, the systematic geography and the like of the bamboo cypress and the long-leaf bamboo cypress leaves through the chloroplast genomes.

Description

PCR amplification primer for chloroplast genomes of bamboo cypress and long-leaf bamboo cypress and application of PCR amplification primer

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a chloroplast genome PCR (polymerase chain reaction) amplification primer for bamboo cypress and long-leaf bamboo cypress and application thereof.

Background

Bamboo cypress (nagia nagi) and bamboo cypress (nagia fleurayi) are rare endangered plants of Podocarpaceae (Podocarpaceae) integrating ornamental, medicinal and ecological functions and are widely distributed in southern China, japan and the like. The podocarpus family is the most diverse group of morphologically differentiated conifers, and although the relationship between the origin and phylogeny of the conifers is well known, the phylogenetic position of the conifers is still controversial. Chloroplasts are important places for photosynthesis of green plants, serve as semi-autonomous organelles, have highly conserved structures and compositions of genomes, and are widely applied to researches on system evolution, parent analysis, variety identification, systematic geography and the like.

The development of DNA sequencing technology has great promotion effect on plant molecular biology. In 1977, the dideoxynucleotide end termination method invented by Sanger is the cornerstone of the first generation sequencing technology, and on the basis of the first generation sequencing technology, a first generation sequencing platform represented by ABI 3730xl is gradually developed, so that the chloroplast whole genome can be obtained by clone sequencing or an improved clone sequencing method. In 1986, the publication of the entire genome sequence of chloroplasts of tobacco (Nicotiana tabacum L.) and liverwort (Marchantia polymorpha L.) revealed the structural characteristics of chloroplasts for the first time. By 12 months 2019, chloroplast genome sequences of thousands of species have been published in GenBank, including the chloroplast whole genome of over 300 crops and forest trees, and still show a rapid development trend.

The chloroplast genome sequence used for species evolution research was firstly analyzed by single gene or gene interval nucleotide polymorphism, and then, a plurality of genes are developed to be analyzed in an overlapping way, but the joint analysis of a plurality of genes cannot solve the classification of some families with complex evolutionary relationships. Leebens-Mack et al propose to increase the data volume of basic analysis, i.e., increase the chloroplast whole genome sequence of species, in order to obtain a more accurate phylogenetic tree. Chloroplast genome contains a large amount of genetic information, the size of the chloroplast genome is moderate, sequencing is convenient, the nucleotide replacement rate of chloroplast DNA is moderate, and the molecular evolution speeds of a coding region and a non-coding region are obviously different, so that the chloroplast genome can be applied to different levels of systematic research. At present, the method based on sequence analysis is widely applied to research of chloroplast phylogenetic genomics and is consistent with the traditional molecular phylogenetic research method. In addition, the information of the chloroplast genome coding region can better distinguish the phylogenetic relationship of higher classification orders, and the sequence comparison of the chloroplast whole genome can better solve lower order taxonomic groups, such as subfamily and subordinate classification orders.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide PCR amplification primers of chloroplast genomes of bamboo cypress and long-leaf bamboo cypress; the invention also aims to solve the technical problem of providing the application of the chloroplast genome PCR amplification primers of the bamboo cypress and the long-leaf bamboo cypress.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the PCR amplification primers for chloroplast genomes of bamboo cypress and bamboo cypress comprise 29 pairs of primers, wherein nucleotide sequences of the 29 pairs of primers are as follows:

the cpl forward primer is shown as SEQ ID NO. 1; the cp1 reverse primer is shown as SEQ ID NO. 2;

the cp2 forward primer is shown as SEQ ID NO. 3; the cp2 reverse primer is shown as SEQ ID NO. 4;

the cp3 forward primer is shown as SEQ ID NO. 5; the cp3 reverse primer is shown as SEQ ID NO. 6;

the cp4 forward primer is shown as SEQ ID NO. 7; the cp4 reverse primer is shown as SEQ ID NO. 8;

the cp5 forward primer is shown as SEQ ID NO. 9; the cp5 reverse primer is shown as SEQ ID NO. 10;

the cp6 forward primer is shown as SEQ ID NO. 11; the cp6 reverse primer is shown as SEQ ID NO. 12;

the cp7 forward primer is shown as SEQ ID NO. 13; the cp7 reverse primer is shown as SEQ ID NO. 14;

the cp8 forward primer is shown as SEQ ID NO. 15; the cp8 reverse primer is shown as SEQ ID NO. 16;

the cp9 forward primer is shown as SEQ ID NO. 17; the cp9 reverse primer is shown as SEQ ID NO. 18;

the cp10 forward primer is shown as SEQ ID NO. 19; the cp10 reverse primer is shown as SEQ ID NO. 20;

the cp11 forward primer is shown as SEQ ID NO. 21; the cp11 reverse primer is shown as SEQ ID NO. 22;

the cp12 forward primer is shown as SEQ ID NO. 23; the cp12 reverse primer is shown as SEQ ID NO. 24;

the cp13 forward primer is shown as SEQ ID NO. 25; the cp13 reverse primer is shown as SEQ ID NO. 26;

the cp14 forward primer is shown as SEQ ID NO. 27; the cp14 reverse primer is shown as SEQ ID NO. 28;

the cp15 forward primer is shown as SEQ ID NO. 29; the cp15 reverse primer is shown as SEQ ID NO. 30;

the cp16 forward primer is shown as SEQ ID NO. 31; the cp16 reverse primer is shown as SEQ ID NO. 32;

the cp17 forward primer is shown as SEQ ID NO. 33; the cp17 reverse primer is shown as SEQ ID NO. 34;

the cp18 forward primer is shown as SEQ ID NO. 35; the cp18 reverse primer is shown as SEQ ID NO. 36;

the cp19 forward primer is shown as SEQ ID NO. 37; the cp19 reverse primer is shown as SEQ ID NO. 38;

the cp20 forward primer is shown as SEQ ID NO. 39; the cp20 reverse primer is shown as SEQ ID NO. 40;

the cp21 forward primer is shown as SEQ ID NO. 41; the cp21 reverse primer is shown as SEQ ID NO. 42;

the cp22 forward primer is shown as SEQ ID NO. 43; the cp22 reverse primer is shown as SEQ ID NO. 44;

the cp23 forward primer is shown as SEQ ID NO. 45; the cp23 reverse primer is shown as SEQ ID NO. 46;

the cp24 forward primer is shown as SEQ ID NO. 47; the cp24 reverse primer is shown as SEQ ID NO. 48;

the cp25 forward primer is shown as SEQ ID NO. 49; the cp25 reverse primer is shown as SEQ ID NO. 50;

the cp26 forward primer is shown as SEQ ID NO. 51; the cp26 reverse primer is shown as SEQ ID NO. 52;

the cp27 forward primer is shown as SEQ ID NO. 53; the cp27 reverse primer is shown as SEQ ID NO. 54;

the cp28 forward primer is shown as SEQ ID NO. 55; the cp28 reverse primer is shown as SEQ ID NO. 56;

the cp29 forward primer is shown as SEQ ID NO. 57; the cp29 reverse primer is shown as SEQ ID NO. 58.

The primer is applied to PCR amplification of chloroplast genomes of bamboo cypress and long-leaf bamboo cypress.

Further, the application comprises the following steps:

1) Extracting the total DNA of the bamboo cypress or the long-leaf bamboo cypress or the DNA of a chloroplast genome;

2) Constructing a PCR amplification system by using the primer of claim 1, and carrying out PCR amplification on the DNA extracted in the step 1).

Further, the PCR amplification system is as follows: 100ng DNA, 10. Mu.M forward Primer, 10. Mu.M reverse Primer, 25. Mu.L Primer STAR Max Premix (2X), ddH ₂ And O is supplemented to 50 mu L.

Further, the procedure of PCR amplification is: 3min at 98 ℃; 10s at 98 ℃, 5s at 55 ℃, 5s/kb at 72 ℃ and 32 cycles; 4min at 72 ℃;4 ℃ forever.

The primer is applied to researching the phylogeny, parent analysis, variety identification or phylogeny of the bamboo cypress or the long-leaf bamboo cypress.

Compared with the prior art, the invention has the beneficial effects that:

the chloroplast genome amplification primers of the bamboo cypress and the long-leaf bamboo cypress designed by the invention are used for sequence amplification of the chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress, the average fragment of the product is about 3.7kb, sequencing and splicing verification are carried out by a Sanger sequencing method, the result is consistent with the result of high-throughput sequencing by extracting chloroplast DNA, the length of the chloroplast genome sequence of the bamboo cypress is 133724bp, and the chloroplast genome sequence of the long-leaf bamboo cypress is 133777bp. The primer has good repeatability, can specifically amplify the chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress, can be used for quickly amplifying the chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress, and is beneficial to the researches on the systematic evolution, the parental analysis, the variety identification, the systematic geography and the like of the bamboo cypress and the long-leaf bamboo cypress through the PCR amplification of the chloroplast genomes.

Drawings

FIG. 1 is a annotated ring diagram of the chloroplast genomes of Bambusa vulgaris and Bambusa vulgaris; note: the outer gene is transcribed clockwise, the inner gene is transcribed anticlockwise, and each color represents the same gene; the dotted dark and light grey parts inside the loop represent the GC and AT content of the chloroplast genome, respectively;

fig. 2 is a phylogenetic tree (bayesian) based on the whole genome sequence of chloroplasts of 21 species, note: the Bayesian posterior probability is displayed on each node branch.

Detailed Description

The invention is further described with reference to specific examples.

The main test materials used in the following examples were: bamboo cypress (nagelia nagi) and bamboo cypress (nagelia fleuryi) materials were from the jiangxi institute of forestry science. Respectively collecting about 30g of fresh tender leaves, immediately placing into liquid nitrogen for quick freezing, and storing in a refrigerator at-80 deg.C in dark for later use. The bamboo cypress seeds are collected from mature cones of about 25-year-old bamboo cypress trees, and the seeds with plump seeds are selected by airing and stored in a laboratory at 4 ℃. Placing the bamboo cypress seeds in a hole tray with moist nutrient soil, and culturing in a greenhouse at 25 ℃ for more than 1 month to obtain the bamboo cypress seedlings.

Example 1:

I. extracting and sequencing of bamboo cypress chloroplast DNA

The leaves of the young bamboo cypress seedlings adopt a high-throughput sequencing method combining chloroplast DNA separation and Illumina Hiseq. Chloroplast Isolation was accomplished using the SIGMA plant Chloroplast Isolation Kit (chloreplast Isolation Kit, USA), and the Isolation procedure was modified appropriately based on the instructions, as follows:

separation of chloroplast: the amount extracted in this experiment was to isolate chloroplasts from 30g of bamboo cypress leaves:

(1) Diluting 5 × CIB (chloroplatinic isolation buffer) to 1 × CIB with 4-fold deionized water (about 135mL of 1 × CIB is required for 30g of leaves);

(2) Preparing 0.1% (w/v) BSA (bovine serum albumin) solution with 1 × CIB (135mL of 135mg BSA in 1 × CIB);

(3) Folding the filter screen into a funnel shape, and placing the funnel into a funnel with a proper size;

(4) Washing 30g of bamboo leaves subjected to dark treatment for 12h with deionized water, draining, cutting into pieces of about 2mm with scissors, and placing in a clean beaker;

(5) To the treated leaves, 120mL of 1 XCIB buffer mixed with BSA (4 mL/g leaves) was added;

(6) Adjusting to 2-3 strokes by using a homogenizer to grind and crudely extract the blades, filtering residues by using a filter screen to collect a solution (forcibly squeezing the filter screen to collect filtrate to the maximum extent), and subpackaging the filtrate into 4 precooled 50mL centrifuge tubes, wherein each tube is not more than 35mL;

(7) Centrifuge at 200Xg for 3min to remove unwanted cell debris and transfer the resulting supernatant to a fresh pre-cooled 50mL centrifuge tube;

(8) Centrifuging at 1000Xg for 7min to obtain green granular chloroplast precipitate

(9) The supernatant was discarded, the vessel wall was flicked by hand, 1mL of 1 XCIB buffer with BSA was added to each vessel and gently flicked with a tip cut off at about 5mm tip to resuspend the particles and place all chloroplast suspensions in 1 centrifuge tube

(II) purifying chloroplasts: intact chloroplasts were isolated from chloroplast suspensions using 40%/80% Percoll density gradient centrifugation:

(1) 2.5mL of an 80% Percoll solution was prepared by mixing 2mL of Percoll solution with 0.5mL of 1 XCIB buffer mixed with BSA and placed in a 14mL centrifuge tube;

(2) Mixing 2mL of Percoll solution with 3mL of 1 × CIB buffer mixed with BSA to prepare 5mL of 40% Percoll solution, carefully placing it on top of 80% Percoll solution;

(3) The chloroplast suspension was gently pipetted on top of the Percoll solution and centrifuged at 3200Xg for 15min. Intact chloroplasts will be present between 40% and 80% percoll solution;

(4) Gently aspirate intact chloroplasts (with tip-clipped tips) and prepare them into suspension with 3 volumes of 1 × CIB buffer without BSA;

(5) Centrifugation at 1700Xg for 1min, discarding the supernatant, resuspending the chloroplast granules in 0.5mL of 1 XCIB buffer without BSA, and storing at 4 ℃ in the dark for further use.

Detecting the integrity of the separated chloroplast by using a microscope, observing whether the chloroplast has other substance fragments and is broken, and extracting DNA of the separated chloroplast.

Chloroplast DNA was extracted using DNeasy Plant Mini Kit (Qiangen, CA, USA), and the concentration and purity of chloroplast DNA was determined. The quality of the obtained cpDNA is detected by a NanoDrop2000c ultraviolet spectrophotometer, and then sequencing work is completed by Beijing Nuo He biogenic science and technology GmbH.

II. Extraction and detection of total DNA of bamboo cypress and long-leaf bamboo cypress

Fresh leaves of the bamboo cypress and the bamboo cypress are stored in a refrigerator at-80 ℃ through quick freezing by liquid nitrogen before use, and the total DNA of the bamboo cypress is extracted by utilizing a plant genome DNA extraction kit (Jiang Union organism, beijing). Extracting DNA according to the kit operation instruction, detecting the quality and concentration of a DNA sample by using NanoDrop2000c (ThermoFisher Scientific), detecting a DNA band by using 2% agarose gel electrophoresis, and observing the band definition to judge whether dispersion exists or not. The DNA template was diluted to 50 ng/. Mu.L and stored at-20 ℃ until use.

III, bamboo cypress and long-leaf bamboo cypress chloroplast genome amplification primers and PCR amplification

The chloroplast plant genome sequence of the podocarpus plant published by Chaw et al is used for carrying out bamboo cypress chloroplast genome amplification primer design and chloroplast genome sequence amplification, and the average fragment of the product is about 3.7kb. Specific primer sequences are shown in table 1:

TABLE 1 amplification primers of chloroplast genomes of Bambusa vulgaris and Bambusa vulgaris

The above primers were synthesized by Nanjing Kingsler Biotech, inc., and product amplification was performed using PrimeSTAR Max DNA Polymerase (Takara, dalian).

PCR reaction (50. Mu.L): mu.L of DNA (50 ng/. Mu.L), 1. Mu.L of F-Primer (10. Mu.M), 1. Mu.L of R-Primer (10. Mu.M), 25. Mu.L of Primer STAR Max Premix (2X), 21. Mu.L of ddH ₂ O。

PCR amplification procedure: 3min at 98 ℃; 10s at 98 ℃, 5s at 55 ℃, 5s/kb at 72 ℃ and 32 cycles; 4min at 72 ℃;4 ℃ forever.

IV, sequencing of chloroplast genome PCR products of bamboo cypress and annotation of genome

The band definition and the band size of the PCR products of chloroplast genomes of the bamboo cypress and the bamboo cypress are detected through 1% agarose gel electrophoresis, and the DNA product purification and sequencing process are finished by Nanjing Jinsley Biotech Co., ltd (the experiment carries out Sanger sequencing through an ABI 3730 sequencer). The published chloroplast genome (NC _ 023805) of the Pacific Arhat pine (Podocarpus lambertii) is taken as a reference genome, sequence assembly is carried out by using software CAP3, and splicing is completed after artificial confirmation. The chloroplast genomes of bamboo cypresses and bamboo cypresses longleaf were annotated with Dual Organillar GenoMe annotor (DOGMA) (default parameters) and the positions of the start and stop codons were determined manually, with the tRNAs binding software tRNAscan-SE v2.0 (http:// lowelab. Ucsc. Edu/tRNAscan-SE /) being confirmed manually. The complete map of the Genome of the bamboo cypress and the bamboo cypress with long leaves is drawn by the software Organella Genome DRAW v1 (http:// ogdraw. Mpimp-golm. Mpg. De /).

Results and analysis:

sequencing a DNA library constructed by the bamboo and cypress genome by using a high-throughput sequencing platform Illumina HiSeq 2500, and obtaining a complete circular chloroplast genome sequence through sequence splicing. Meanwhile, the result of verification by using a PCR clonal amplification method and a Sanger sequencing method is consistent with the result of high-throughput sequencing, the length of the result is 133724bp, and a 133777bp chloroplast genome sequence of the sabina longifolia (shown in figure 1) is obtained, the lengths of the chloroplast genome sequence and the chloroplast genome sequence are larger than those of other 2 published species of the Podocarpus (Podocarpus lambertii) and the Podocarpus takara (Podocarpus totara) respectively (the size of the chloroplast genome is 133734bp (NC _ 023805) and the size of the chloroplast genome sequence and the chloroplast genome sequence are 133259bp (NC _ 020361.1)), and the length difference of the chloroplast genome sequence and the chloroplast genome sequence is mainly existed in intergenic regions. The GC contents of the complete sequences of the bamboo cypress and bamboo juniper longleaf cpDNA were both 37.3%, and the GC content of the protein Coding Sequence (CDS) was 38.2%, and it can be seen that the GC contents of the bamboo cypress and bamboo juniper longleaf were significantly less than the AT content.

The gene annotation of chloroplast genomes of bamboo cypress and bamboo cypress with DOGMA software is carried out, and the two genes are annotated to 120 genes, wherein 118 is a single copy gene and comprises 82 coding protein genes, 32 tRNA genes and 4 rRNA genes. In addition, there are two double-copy genes, trnD-GUC and trnN-GUU, which are presumed to be related to the inverted repeat structure of chloroplast (Table 2). Of the 120 annotated genes, 15 genes contained introns, with the rps12 and ycf3 genes containing two introns, and 7 genes encoding proteins (petB, petD, atpF, ndhA, ndhB, rpl2 and rpl 16) and 6 tRNA genes (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA and trnV-UAC) containing one intron.

The genetic contents of the bamboos and bamboos longipes are highly similar to the published chloroplast genomes of the wood species of the podocariaceae family, and in contrast to other conifer species, the bamboos and bamboos longipes also lack the rps16 gene, consistent with the previously reported conclusion that the rps16 gene is lost in the podocariaceae and araucaceae (araucaceae).

TABLE 2 chloroplast genome gene composition of annotated-based bamboo cypress and bamboo cypress

Note: ¹ is a gene comprising an intron; ² is a gene repeatedly existing on the genome

V, chloroplast genome system evolution

7 angiosperms are taken as an exogeneous group, 21 chloroplast whole genome sequences of the plants are selected to construct a phylogenetic tree, and besides 2 bamboo cypress plants, other two published chloroplast genome sequences of the Podocarpaceae are downloaded in GeneBank. Sequence alignment is carried out by using MAFFT v7 (http:// MAFFT. Cbrc. Jp/alignment/server) software, and an Akaike Information Criterion (AIC) value is taken as a standard, and JModeltest2 software is used for screening out an optimal nucleotide substitution Model (Model: GTR + G) and corresponding parameters. Bayesian analysis was performed using MrBayes v.3.2.6 software. Adopting 1000000 iteration mode of sampling 1 time every 1000 times, discarding 25% of aged samples in burn-in stage, and when the average standard deviation is reduced to below 0.01, using the rest samples to construct evolutionary tree. And finally, putting the obtained operation result into the FigTree to check the result.

Results and analysis:

the chloroplast genome can provide valuable phylogenetic analysis between plant genera and species due to structural conservation. 21 species were selected for phylogenetic analyses, of which 7 angiosperms were used as the exopopulation, including Chinese unlined long-gown (Liriodenron chinensis), liriodenron tulipera (Liriodenron tulipera) and Chinese forest (dryys grandiensis), tobacco (Nicotiana tabacum), arabidopsis thaliana (Arabidopsis thaliana), populus eupatoria (Populus eupatoria), and Populus trichocarpa (Populus trichocarpa). In addition, in addition to 4 species of mataires, gymnosperms such as araucaceae (araucaceae), pinaceae (Pinaceae), taxodiaceae (Taxodiaceae), and ginkgo biloba (Ginkgoaceae) were selected for phylogenetic tree construction. A phylogenetic tree is constructed by utilizing a Bayes method on the basis of a chloroplast whole genome sequence, and the result has high support rate which is almost 100 (figure 2). In the phylogenetic tree, the bamboo cypress and the long-leaf bamboo cypress are gathered into one branch, and the two branches are gathered with the south Arhat pine (Podocarpus lambertii) and the peach Tuohan pine (Podocarpus totara) of the genus Podocarpus; the evolutionary tree shows that podocarpus family is more closely related to the species of the araucaceae family and slightly more distant from the plants of the pinaceae and the cedaceae families. According to the result of the evolutionary tree, the bamboo cypress and the long-leaf bamboo cypress are presumed to belong to the natural group under the genus of bamboo cypress.

Sequence listing

<110> Nanjing university of forestry

<120> bamboo cypress and long-leaf bamboo cypress chloroplast genome PCR (polymerase chain reaction) amplification primer and application thereof

<130> 100

<160> 58

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> cp1F(Artificial)

<400> 1

ccttgactgt caactacgga ttg 23

<210> 2

<211> 23

<212> DNA

<213> cp1R(Artificial)

<400> 2

actacgactg atcctgaaag cga 23

<210> 3

<211> 20

<212> DNA

<213> cp2F(Artificial)

<400> 3

tgaacccgct gactaaacct 20

<210> 4

<211> 23

<212> DNA

<213> cp2R(Artificial)

<400> 4

ctcacattgg gacattacga gta 23

<210> 5

<211> 23

<212> DNA

<213> cp3F(Artificial)

<400> 5

tataataaga tcgggacgct cct 23

<210> 6

<211> 21

<212> DNA

<213> cp3R(Artificial)

<400> 6

actggaacat tcacggaaca a 21

<210> 7

<211> 23

<212> DNA

<213> cp4F(Artificial)

<400> 7

gcttctgctt gttccgtgaa tgt 23

<210> 8

<211> 21

<212> DNA

<213> cp4R(Artificial)

<400> 8

tggagagtta gccgcgccta c 21

<210> 9

<211> 19

<212> DNA

<213> cp5F(Artificial)

<400> 9

agagcgcctg accagttag 19

<210> 10

<211> 23

<212> DNA

<213> cp5R(Artificial)

<400> 10

ctgccatgaa acagcttatt aga 23

<210> 11

<211> 22

<212> DNA

<213> cp6F(Artificial)

<400> 11

gtagcatgcc atatttcgat ca 22

<210> 12

<211> 23

<212> DNA

<213> cp6R(Artificial)

<400> 12

tatctattta cgcaacgctt ggt 23

<210> 13

<211> 23

<212> DNA

<213> cp7F(Artificial)

<400> 13

ctttcatctt ctccaggtaa tag 23

<210> 14

<211> 21

<212> DNA

<213> cp7R(Artificial)

<400> 14

cggactgtaa attcgttgtg c 21

<210> 15

<211> 18

<212> DNA

<213> cp8F(Artificial)

<400> 15

gccatagtct gtagcaag 18

<210> 16

<211> 23

<212> DNA

<213> cp8R(Artificial)

<400> 16

ccagtaggag aacgcataag ata 23

<210> 17

<211> 23

<212> DNA

<213> cp9F(Artificial)

<400> 17

gttcaacaat acggcttatc cta 23

<210> 18

<211> 22

<212> DNA

<213> cp9R(Artificial)

<400> 18

gcaaatcttg ggtttcgctt tc 22

<210> 19

<211> 19

<212> DNA

<213> cp10F(Artificial)

<400> 19

aatcctccta gccgttatc 19

<210> 20

<211> 23

<212> DNA

<213> cp10R(Artificial)

<400> 20

atccgtcgtg caaattctat cat 23

<210> 21

<211> 23

<212> DNA

<213> cp11F(Artificial)

<400> 21

gatctaggca tagtcaatta gag 23

<210> 22

<211> 23

<212> DNA

<213> cp11R(Artificial)

<400> 22

ttacatgaat ttaccgaacc tcc 23

<210> 23

<211> 18

<212> DNA

<213> cp12F(Artificial)

<400> 23

ccaaaggtct atgagcta 18

<210> 24

<211> 23

<212> DNA

<213> cp12R(Artificial)

<400> 24

attattgtct atcttggcga atc 23

<210> 25

<211> 18

<212> DNA

<213> cp13F(Artificial)

<400> 25

tcacaaccct ttttcgta 18

<210> 26

<211> 23

<212> DNA

<213> cp13R(Artificial)

<400> 26

gaacctcctc ttcatcaact tcc 23

<210> 27

<211> 23

<212> DNA

<213> cp14F(Artificial)

<400> 27

agttttaatg atatgcgaga gtg 23

<210> 28

<211> 23

<212> DNA

<213> cp14R(Artificial)

<400> 28

gaccgggaac aggacctatt aca 23

<210> 29

<211> 23

<212> DNA

<213> cp15F(Artificial)

<400> 29

aaatacttat tacaaccggg tga 23

<210> 30

<211> 23

<212> DNA

<213> cp15R(Artificial)

<400> 30

atccgcacta tccagggtac aac 23

<210> 31

<211> 18

<212> DNA

<213> cp16F(Artificial)

<400> 31

ccggttcact gatagaga 18

<210> 32

<211> 23

<212> DNA

<213> cp16R(Artificial)

<400> 32

tcataagtgc ttgcccatag gaa 23

<210> 33

<211> 23

<212> DNA

<213> cp17F(Artificial)

<400> 33

gtcgacgtaa gtaggaatag ttg 23

<210> 34

<211> 19

<212> DNA

<213> cp17R(Artificial)

<400> 34

aggacatccg ctcgtttca 19

<210> 35

<211> 23

<212> DNA

<213> cp18F(Artificial)

<400> 35

ttcgagcaag ttttaataat acc 23

<210> 36

<211> 18

<212> DNA

<213> cp18R(Artificial)

<400> 36

ggcacggcgc tagaactc 18

<210> 37

<211> 18

<212> DNA

<213> cp19F(Artificial)

<400> 37

aaccgtctga acaactta 18

<210> 38

<211> 23

<212> DNA

<213> cp19R(Artificial)

<400> 38

atcaaagaaa tagtcggcaa ctt 23

<210> 39

<211> 19

<212> DNA

<213> cp20F(Artificial)

<400> 39

ggaaaaacgt actcgtaga 19

<210> 40

<211> 23

<212> DNA

<213> cp20R(Artificial)

<400> 40

aatctcatac tcttccgctg ttg 23

<210> 41

<211> 19

<212> DNA

<213> cp21F(Artificial)

<400> 41

agaaatgaaa ccaccgatt 19

<210> 42

<211> 23

<212> DNA

<213> cp21R(Artificial)

<400> 42

tgcggattca taaactgttc gat 23

<210> 43

<211> 18

<212> DNA

<213> cp22F(Artificial)

<400> 43

cgccacattg ttctactt 18

<210> 44

<211> 23

<212> DNA

<213> cp22R(Artificial)

<400> 44

gtatccggca accaggtatg taa 23

<210> 45

<211> 23

<212> DNA

<213> cp23F(Artificial)

<400> 45

atttagggtt ctttatcgct tat 23

<210> 46

<211> 21

<212> DNA

<213> cp23R(Artificial)

<400> 46

tccatagcat cgggtaacca t 21

<210> 47

<211> 23

<212> DNA

<213> cp24F(Artificial)

<400> 47

atttcggact cttattagga atc 23

<210> 48

<211> 19

<212> DNA

<213> cp24R(Artificial)

<400> 48

gcttatggac ccgaacctg 19

<210> 49

<211> 22

<212> DNA

<213> cp25F(Artificial)

<400> 49

ataaccatct ttcggctaac tt 22

<210> 50

<211> 23

<212> DNA

<213> cp25R(Artificial)

<400> 50

ggacaggcgg tggaaactac taa 23

<210> 51

<211> 18

<212> DNA

<213> cp26F(Artificial)

<400> 51

ggtgttcttt ccgatctc 18

<210> 52

<211> 23

<212> DNA

<213> cp26R(Artificial)

<400> 52

aaaaggctac tctgtttcgt tcc 23

<210> 53

<211> 23

<212> DNA

<213> cp27F(Artificial)

<400> 53

caataaatta gaataataga gcc 23

<210> 54

<211> 22

<212> DNA

<213> cp27R(Artificial)

<400> 54

cgggacttct ttacttacga ta 22

<210> 55

<211> 18

<212> DNA

<213> cp28F(Artificial)

<400> 55

ttcgcaaaga cctaacat 18

<210> 56

<211> 23

<212> DNA

<213> cp28R(Artificial)

<400> 56

aaatatcaga atcccgtagc tcc 23

<210> 57

<211> 22

<212> DNA

<213> cp29F(Artificial)

<400> 57

tgatatgggt aaaaagacct aa 22

<210> 58

<211> 23

<212> DNA

<213> cp29R(Artificial)

<400> 58

ttctaaccat gaccgcaatt cta 23

Claims

1. The application of the PCR amplification primers of chloroplast genomes of bamboo cypress and long-leaf bamboo cypress in the PCR amplification of the chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress is characterized by comprising the following steps of:

2) Constructing a PCR amplification system by using the PCR amplification primers of chloroplast genomes of the bamboo cypress and the long-leaf bamboo cypress, and performing PCR amplification on the DNA extracted in the step 1);

the PCR amplification primers of the chloroplast genomes of the bamboo cypresses and the bamboo cypresses comprise 29 pairs of primers, and the nucleotide sequences of the 29 pairs of primers are as follows:

the cp1 forward primer is shown as SEQ ID NO. 1; the cp1 reverse primer is shown as SEQ ID NO. 2;

2. The use according to claim 1, wherein the PCR amplification system is: 100ng DNA, 10. Mu.M forward Primer, 10. Mu.M reverse Primer, 25. Mu.L Primer STAR Max Premix (2X), ddH ₂ And O is supplemented to 50 mu L.

3. The use of claim 2, wherein the procedure of PCR amplification is: 3min at 98 ℃; 10s at 98 ℃, 5s at 55 ℃, 5s/kb at 72 ℃ and 32 cycles; 4min at 72 ℃;4 ℃ forever.

4. Use of the primer set forth in claim 1 for studying phylogeny, parental analysis, variety identification or phylogeny of Bambusa vulgaris or Bambusa longissima.