CN108823327B - Camphor tree whole genome SSR molecular marker and preparation method and application thereof - Google Patents
Camphor tree whole genome SSR molecular marker and preparation method and application thereof Download PDFInfo
- Publication number
- CN108823327B CN108823327B CN201810474571.7A CN201810474571A CN108823327B CN 108823327 B CN108823327 B CN 108823327B CN 201810474571 A CN201810474571 A CN 201810474571A CN 108823327 B CN108823327 B CN 108823327B
- Authority
- CN
- China
- Prior art keywords
- camphor tree
- dna
- primer
- molecular marker
- whole genome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a camphor tree whole genome SSR molecular marker and a preparation method and application thereof. The camphor tree microsatellite molecular marker developed by the invention can be applied to population genetic diversity detection and genetic relationship analysis of ancient camphor trees, provides a new batch of microsatellite molecular markers for Lauraceae plants, and also provides a new tool for researches on population genetic differentiation and structure of Lauraceae plants, population genetic diversity level, gene flow and mating system among populations, species evolution, molecular assisted breeding and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of forestry molecular biology, particularly relates to a camphor tree molecular marker technology, and more particularly relates to a camphor tree whole genome SSR molecular marker, a preparation method thereof, and application thereof in genetic diversity analysis and genetic relationship identification of ancient camphor trees.
Background art:
microsatellites (Microsatelites) are also known as Short Tandem Repeats (STRs), Simple Repeat sequences (SSRs), and Simple Sequence Length Polymorphisms (SSLPs). It refers to a DNA sequence which is repeated in tandem for many times by taking 1-6 nucleotides as a unit, and the DNA sequence is commonly present in genomes of eukaryotes and prokaryotes and even viral genomes. The SSR molecular marker has the following characteristics: the repeat unit and the repeat number are highly variable, and the polymorphism information capacity is high; co-dominant, inherited following the mendelian rule; the flanking sequences are relatively conserved; choosing neutral, etc.
Based on the characteristics, the microsatellite molecular marker is widely applied to the research fields of fingerprint identification, genetic map construction, population genetic structure analysis, comparative genome, evolution research and the like of species. According to the different sequence properties of established SSR markers, SSR markers can be divided into genome SSR (gSSR) and expressed sequence tag SSR (EST-SSR). Compared with EST-SSR molecular markers, the genome SSR has the following advantages: (1) EST-SSR is derived from an expressed gene sequence, has incomplete information and lacks important genomic information such as a genome regulatory sequence, an intron sequence and the like; (2) compared with SSR molecular markers derived from genomes, the EST-SSR molecular markers have low polymorphism; (3) EST-SSR is affected by EST sequencing errors, development software and parameters. With the rapid development of high-throughput sequencing technology and the unique advantages of genome SSR markers, gSSR molecular markers can be widely developed and applied.
Cinnamomum camphora (L.) Presl is widely distributed in southern provinces of China, including Henan, Taiwan, Fujian, Jiangxi, Guangdong, Guangxi, Hubei, Hunan, Sichuan, Chongqing, Yunnan, Guizhou, Zhejiang, etc. The camphor tree has wide application, is a multipurpose tree species integrating material use, medicine use, spice use, oil use, chemical industry, appreciation, ecological environment and ecological culture construction, and has extremely important development and utilization values. According to the difference of the main chemical components of the leaf essential oil, the camphor trees can be divided into 5 chemical types such as cinnamomum camphora (mainly containing linalool), cinnamomum camphora (mainly containing camphor), cinnamomum camphora (mainly containing eucalyptus oil), cinnamomum camphora (mainly containing nerolidol), cinnamomum camphora (mainly containing d-borneol) and the like, and the cinnamomum camphora and the like have different development values. At present, chemical types of camphor trees are mostly identified by adopting a fragrance-smelling classification method, and certain randomness and subjectivity are provided, so that the obtained reliable SSR molecular markers of camphor tree genome can be used for identifying different chemical types of camphor trees, and have important significance for seedling stage directional selection.
The camphor tree is a species lacking in genetic research and genetic background information. Currently, the number of markers that can be effectively utilized by camphor trees is very limited. Further research on camphor tree genomes is carried out from depth and breadth, and new DNA molecular markers need to be developed. The existing research proves that the mass development of SSR and SNP markers can be economically and rapidly carried out by using a bioinformatics means based on an EST database. However, compared with other species such as rice, corn, grape, etc., camphor tree EST data recorded in databases of the National Center for Biotechnology Information (NCBI) is quite lacking, so that the requirement of camphor tree EST-SSR marker development is difficult to meet, and sufficient information cannot be provided for camphor tree DNA molecular marker development and research. Meanwhile, the development of molecular biology research of camphor trees is also greatly limited.
The invention content is as follows:
the invention aims to provide a whole genome SSR molecular marker development method suitable for camphor trees based on a camphor tree whole genome sequence which is automatically determined, develop gSSR molecular markers of a batch of camphor trees, and apply to genetic diversity detection and genetic relationship analysis of ancient camphor trees.
The primers of the camphor tree whole genome SSR molecular markers are respectively as follows:
cc-19: f: 5'-ATTTGCCTCGTGTTCCATTC-3' R: 5'-TGGAATTTCAGATCCCCAAA-3', respectively; (sequence derived from SEQ ID NO. 1)
Cc-25: f: 5'-GCGCCATTTGTTTTCTTCAT-3' R: 5'-AATCACTAGGGTCGGAAGGG-3', respectively; (sequence derived from SEQ ID NO. 2)
Cc-49: f: 5'-GCATCTCCCTACCAAATCCA-3' R: 5'-TTGCTCATTTTGAAGCATCG-3', respectively; (sequence derived from SEQ ID NO. 3)
Cc-55: f: 5'-CAGCCATTCAGAAGGGAAAG-3' R: 5'-CAACTTCTTCTATGGGGGCA-3', respectively; (sequence derived from SEQ ID NO. 4)
Cc-60: f: 5'-CCGAACGTCAACTCAAACAA-3' R: 5'-TTTGATGGGTTCATTGGTGA-3', respectively; (sequence derived from SEQ ID NO. 5)
Cc-76: f: 5'-TGGAATGCAAAGAAGGAACC-3' R: 5'-CTCTGGTCCCCTGATTTCTG-3', respectively; (sequence derived from SEQ ID NO. 6)
Cc-80: f: 5'-TCTCTCTCATGGTCAAATTGTTG-3' R: 5'-AGGTCCCCAAGGTTCCTAGA-3', respectively; (sequence derived from SEQ ID NO. 7)
Cc-81: f: 5'-CCATTTCTCAATAGGAATATTGATTGT-3' R: 5'-AGCCCATACCTTTTCATTTCA-3', respectively; (sequence derived from SEQ ID NO. 8)
Cc-83: f: 5'-CACGGTCCCCAATCTCTAAA-3' R: 5'-TCAAATTTGGGTTGGACCAT-3', respectively; (sequence derived from SEQ ID NO. 9)
Cc-85: f: 5'-GGAACGTCCGGCTATGTAAA-3' R: 5'-AAAGTGGCAAACAAAACCCT-3', respectively; (sequence derived from SEQ ID NO. 10)
Cc-96: f: 5'-CACGGTACTGACCAGGGTTC-3' R: 5'-GGCCCAGTTGTTCCACATTA-3', respectively; (sequence derived from SEQ ID NO. 11)
Cc-100: f: 5'-AGAGATCGAAAGGGCGATG-3' R: 5'-CGCTCCCTACAGAACCCAT-3', respectively; (sequence derived from SEQ ID NO. 12)
Cc-114: f: 5'-TGATGAGGATGGGGTCATTT-3' R: 5'-TGCCATGTTTTGGAGGTAAA-3', respectively; (sequence derived from SEQ ID NO. 13)
Cc-131: f: 5'-AGAATAGGGAAAGGGGCAGA-3' R: 5'-CCCTTCCTTGATCAAACCCT-3', respectively; (sequence derived from SEQ ID NO. 14)
Cc-133: f: 5'-ATGAGCGTCATCCAATGTCA-3' R: 5'-GCACATACCCAGTTCCGATT-3', respectively; (sequence derived from SEQ ID NO. 15)
Cc-134: f: 5'-GGGTCGGGGTAAATTAGGTC-3' R: 5'-GACCGGTTTGCTAAGGTCAA-3', respectively; (sequence derived from SEQ ID NO. 16)
Cc-143: f: 5'-CTATCCACTTCCCTGCCGTA-3' R: 5'-CCAGAATCTTCGAGAAAGCG-3', respectively; (sequence derived from SEQ ID NO. 17)
Cc-185: f: 5'-TGCTCTAATTCACCCCTTGC-3' R: 5'-AGGCTCCACACTAAAACCCA-3', respectively; (sequence derived from SEQ ID NO. 18)
Cc-188: f: 5'-ACCATCCAGAGTTTGATCGG-3' R: 5'-GAGGGTTCTCCTTCGGATTC-3', respectively; (sequence derived from SEQ ID NO. 19)
Cc-191: f: 5'-GTTCTCCTTCTGATCCACGG-3' R: 5'-GACCCATTATGCGTTGAACC-3' are provided. (sequence derived from SEQ ID NO. 20)
The annealing temperatures at both ends of the primers of SSR molecular markers, which are respectively numbered Cc-19, Cc-25, Cc-49, Cc-55, Cc-60, Cc-76, Cc-80, Cc-81, Cc-83, Cc-85, Cc-96, Cc-100, Cc-114, Cc-131, Cc-133, Cc-134, Cc-143, Cc-185, Cc-188 and Cc-191, are respectively Cc-19: 57 ℃; cc-25: at 58 ℃; cc-49: 56 ℃; cc-55: at 58 ℃; cc-60: 55 ℃; cc-76: at 58 ℃; cc-80: 59 ℃; cc-81: 55 ℃; cc-83: 55 ℃; cc-85: 55 ℃; cc-96: at 58 ℃; cc-100: 59 ℃; cc-114: at 54 ℃; cc-131: at 58 ℃; cc-133: 56 ℃; cc-134: at 58 ℃; cc-143: at 58 ℃; cc-185: at 58 ℃; cc-188: at 58 ℃; cc-191: at 58 ℃.
The second purpose of the invention is to provide the application of the primer of the camphor tree whole genome SSR molecular marker in the detection of the genetic diversity of the cinnamomum camphora population and/or the clustering analysis of the cinnamomum camphora genetic relationship.
The specific application method is as follows:
(1) extracting genome DNA of a camphor tree sample to be detected;
(2) taking the extracted DNA sample to be detected as a template, and respectively carrying out PCR amplification on each pair of primers in the primers of the camphor tree whole genome SSR molecular marker to obtain PCR amplification products;
(3) typing the PCR amplification product.
And (4) performing genetic diversity analysis and genetic relationship analysis on the camphor trees according to the typing statistical result.
The PCR amplification system comprises the following steps: 10 XBuffer 1. mu.l, 25mmol/L MgCl2Mu.l, 0.6. mu.l of 10mmol/L dNTP, 0.1. mu.l of 5U/. mu.l DNA polymerase, 50ng of DNA template, 0.5. mu.l of each of primers F and R marked by 25mmol/L SSR molecules, and fixing the volume to 10. mu.l by double distilled water.
The PCR amplification comprises the following amplification reaction procedures: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30sec, renaturation at annealing temperature of primer pair of each camphor tree whole genome SSR molecular marker for 30sec, extension at 72 ℃ for 30sec, and 32 cycles; and finally, extending for 5min at 70 ℃, wherein the annealing temperatures at two ends of the primers of the SSR molecular markers, which are respectively numbered Cc-19, Cc-25, Cc-49, Cc-55, Cc-60, Cc-76, Cc-80, Cc-81, Cc-83, Cc-85, Cc-96, Cc-100, Cc-114, Cc-131, Cc-133, Cc-134, Cc-143, Cc-185, Cc-188 and Cc-191, are respectively Cc-19: 57 ℃; cc-25: at 58 ℃; cc-49: 56 ℃; cc-55: at 58 ℃; cc-60: 55 ℃; cc-76: at 58 ℃; cc-80: 59 ℃; cc-81: 55 ℃; cc-83: 55 ℃; cc-85: 55 ℃; cc-96: at 58 ℃; cc-100: 59 ℃; cc-114: at 54 ℃; cc-131: at 58 ℃; cc-133: 56 ℃; cc-134: at 58 ℃; cc-143: at 58 ℃; cc-185: at 58 ℃; cc-188: at 58 ℃; cc-191: at 58 ℃.
The electrophoresis detection of the PCR amplification product is to detect the PCR amplification product by 6 percent modified polyacrylamide gel electrophoresis, and the product is silver-stained and developed.
The third purpose of the invention is to provide a preparation method of SSR molecular marker primers developed based on camphor tree genome, which is characterized by comprising the following steps:
(1) obtaining the complete genome sequence of camphor tree
Extracting camphor tree genome DNA by a CTAB method, establishing a library of the extracted DNA sample, and performing high-throughput sequencing to obtain a camphor tree complete genome database;
(2) screening of camphor tree whole genome microsatellite DNA sequence
Searching microsatellite loci in a camphor tree full-genome database by using a microsatellite analysis program, and screening the obtained microsatellite loci by using online microsatellite locus analysis software to finally obtain a camphor tree DNA sequence containing the microsatellite loci;
(3) designing a batch primer: aiming at the camphor tree DNA sequence containing the microsatellite loci, designing and amplifying DNA polymerase chain reaction primers containing the microsatellite loci by adopting batch primer design software, and carrying out comprehensive evaluation on the obtained SSR primers by using Oligo 7;
(4) and (3) primer e-PCR amplification detection:
performing simulated amplification on a batch designed primer in a camphor tree genome sequence by using e-PCR, outputting a primer which can theoretically amplify a predicted amplification product, detecting whether the designed primer can amplify the product and polymorphism of the product, obtaining a matched primer sequence in a camphor tree genome database, the length of the predicted amplification product and the position of an initial point, performing simulated amplification in NCBIPrimer-BLAST, screening according to the obtained simulated amplification product, and rejecting a repeated primer to finally obtain a specific primer;
(5) primer polymorphism detection
And (2) performing DNA polymerase chain reaction amplification on the selected specific primers by taking camphor tree genome DNA as a template, performing electrophoretic separation and silver staining detection on amplification products, and selecting the primers which meet the requirements and are the camphor tree genome SSR molecular markers.
The invention has the beneficial effects that:
1. compared with other development methods of camphor trees, the method has the advantages of comprehensiveness, completeness, accuracy and reliability, and has important practical value for developing molecular markers of lauraceae plants.
2. The camphor tree genetic background information is lack, the EST sequence data is limited, the requirement of camphor tree EST-SSR marker development is difficult to meet, the number of markers which can be effectively utilized is very limited, a new batch of camphor tree SSR molecular markers are developed, the problem of camphor tree gSSR lack is solved, and sufficient information and available gSSR molecular markers are provided for the development and research of DNA molecular markers of other camphor plants.
3. The camphor tree microsatellite molecular marker developed by the invention can be applied to population genetic diversity detection and genetic relationship analysis of ancient camphor trees, provides a new batch of microsatellite molecular markers for Lauraceae plants, and also provides a new tool for researches on population genetic differentiation and structure of Lauraceae plants, population genetic diversity level, gene flow and mating system among populations, species evolution, molecular assisted breeding and the like.
Drawings
FIG. 1 shows the primary screening results of all primers for DNA polymerase chain reaction amplification;
FIG. 2 shows the result of DNA polymerase chain reaction amplification of primer Cc-19 in Guzhang;
FIG. 3 shows the result of DNA polymerase chain reaction amplification of primer Cc-25 in Guzhang;
FIG. 4 shows the result of DNA polymerase chain reaction amplification of primer Cc-49 in Guzhang;
FIG. 5 shows the result of DNA polymerase chain reaction amplification of primer Cc-55 in Guzhang;
FIG. 6 shows the result of DNA polymerase chain reaction amplification of primer Cc-60 in Guzhang;
FIG. 7 shows the result of DNA polymerase chain reaction amplification of primer Cc-76 in Guzhang;
FIG. 8 shows the result of DNA polymerase chain reaction amplification of primer Cc-80 in Guzhang;
FIG. 9 shows the result of DNA polymerase chain reaction amplification of primer Cc-81 in Gucamphor;
FIG. 10 shows the result of DNA polymerase chain reaction amplification of primer Cc-83 in Guzhang;
FIG. 11 shows the result of DNA polymerase chain reaction amplification of primer Cc-85 in Guzhang;
FIG. 12 shows the result of DNA polymerase chain reaction amplification of primer Cc-96 in Guzhang;
FIG. 13 shows the result of DNA polymerase chain reaction amplification of primer Cc-100 in Guzhang;
FIG. 14 shows the result of DNA polymerase chain reaction amplification of primer Cc-114 in Guzhao;
FIG. 15 shows the result of DNA polymerase chain reaction amplification of primer Cc-131 in Guzhao;
FIG. 16 shows the result of DNA polymerase chain reaction amplification of primer Cc-133 in Guzhang;
FIG. 17 shows the result of DNA polymerase chain reaction amplification of primer Cc-134 in Guzhang;
FIG. 18 shows the result of DNA polymerase chain reaction amplification of primer Cc-143 in Guzhang;
FIG. 19 shows the result of DNA polymerase chain reaction amplification of primer Cc-185 in Guzhang;
FIG. 20 shows the result of DNA polymerase chain reaction amplification of primer Cc-188 in Guzhang;
FIG. 21 shows the result of DNA polymerase chain reaction amplification of primer Cc-191 in Cinnamomum camphora;
FIG. 22 is a result of clustering of the UPGM of the Cinnamomum camphora;
FIG. 23 is a diagram of cluster analysis of GUZHANG by the software UPGM method of NTSYS-pcversion 2.11.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
a preparation method of an SSR molecular marker primer developed based on camphor tree genome comprises the following steps:
1. camphor tree DNA extraction
Extracting genome DNA of camphor tree leaves by adopting a CTAB method, detecting the DNA concentration and purity of the camphor tree leaves by using a kit and a spectrophotometer after extraction, detecting the integrity of a camphor tree leaf genome DNA sample by using an agarose gel electrophoresis DNA sample, and detecting whether other components exist.
2. Obtaining the complete genome sequence of camphor tree
Sending the extracted genome DNA sample with excellent quality to a sequencing company, constructing a library, then carrying out high-throughput sequencing analysis, filtering and selecting to remove low-quality data, and obtaining the first camphor tree whole genome database. The camphor tree whole genome sequence and the genome annotation file are both from the research center of camphor tree engineering technology of the national forestry bureau. The camphor tree whole genome sequencing is completed by utilizing an Illumina HiSeq2000 sequencing platform of Shenzhen Huada Gen Limited company, the total size of the assembled genome is 727Mb, and the sequence data is stored in a FASTA format and is used for searching SSR loci.
3. Screening of camphor tree whole genome microsatellite DNA sequence
And (3) screening the Microsatellite loci of the processed sequence by using a Microsatellite analysis program (MISA) compiled by Perl language and online Microsatellite locus analysis software (Microstellite reports Finder), and obtaining the camphor tree DNA sequence containing the Microsatellite loci.
4. Designing a batch primer: aiming at the camphor tree DNA sequence containing the microsatellite loci, a DNA polymerase chain reaction Primer (SSR Primer) containing the microsatellite loci is designed and amplified by adopting batch Primer design software Primer 3.0, and the obtained SSR Primer is comprehensively evaluated by using Oligo 7. The principle of primer design is as follows: 1) the number of the primers is controlled to be 18-24, and the optimal primer length is 20 bp; 2) the annealing temperature range is 50-60 ℃, and the annealing temperature difference of the two primers is less than 4 ℃; 3) the GC content range is 40-60%, and the optimal GC content is 50%; 4) the size of the amplified DNA fragment is within 400 bases. When one DNA sequence contains two microsatellite loci, and the distance between the two microsatellite loci is more than 100 basic groups, respectively designing primers for the two microsatellite loci; when two microsatellite loci are within 100 bases apart in the same sequence, primers are designed without placing the two loci within the amplification range of a pair of primers, thereby obtaining batch-designed primers.
5. And (3) primer e-PCR amplification detection: and (3) performing simulated amplification on the primers designed in batches in the camphor tree genome sequence by using e-PCR, and outputting the primers which can theoretically amplify expected amplification products so as to detect whether the designed primers can amplify the products and polymorphism of the products. The matched primer sequences, the expected amplification product length and the position of the starting point in the camphor tree genome database can be obtained. Meanwhile, mock amplification was performed at NCBI Primer-BLAST with parameters set to default values. And screening according to the obtained simulated amplification product, and rejecting repeated primers to obtain the selected specific primer.
6. Final acquisition of microsatellite loci
Selecting 5 cinnamomum camphora samples, extracting leaf genome DNA, constructing a mixed DNA pool, performing DNA polymerase chain reaction amplification on the selected specific primers, and primarily screening to obtain SSR primers with clear bands and good repeatability; and (3) adopting an ancient camphor tree population to re-screen the primers obtained by the primary screening to finally obtain 20 pairs of specific primers with stability and good polymorphism, namely primers of the camphor tree genome SSR molecular markers.
The specific sequences of 20 pairs of primers of the camphor tree genome SSR molecular markers are as follows:
Cc-19：F：5'-ATTTGCCTCGTGTTCCATTC-3'R：5'-TGGAATTTCAGATCCCCAAA-3'；
Cc-25：F：5'-GCGCCATTTGTTTTCTTCAT-3'R：5'-AATCACTAGGGTCGGAAGGG-3'；
Cc-49：F：5'-GCATCTCCCTACCAAATCCA-3'R：5'-TTGCTCATTTTGAAGCATCG-3'；
Cc-55：F：5'-CAGCCATTCAGAAGGGAAAG-3'R：5'-CAACTTCTTCTATGGGGGCA-3'；
Cc-60：F：5'-CCGAACGTCAACTCAAACAA-3'R：5'-TTTGATGGGTTCATTGGTGA-3'；
Cc-76：F：5'-TGGAATGCAAAGAAGGAACC-3'R：5'-CTCTGGTCCCCTGATTTCTG-3'；
Cc-80：F：5'-TCTCTCTCATGGTCAAATTGTTG-3'R：5'-AGGTCCCCAAGGTTCCTAGA-3'；
Cc-81：F：5'-CCATTTCTCAATAGGAATATTGATTGT-3'R：5'-AGCCCATACCTTTTCATTTCA-3'；
Cc-83：F：5'-CACGGTCCCCAATCTCTAAA-3'R：5'-TCAAATTTGGGTTGGACCAT-3'；
Cc-85：F：5'-GGAACGTCCGGCTATGTAAA-3'R：5'-AAAGTGGCAAACAAAACCCT-3'；
Cc-96：F：5'-CACGGTACTGACCAGGGTTC-3'R：5'-GGCCCAGTTGTTCCACATTA-3'；
Cc-100：F：5'-AGAGATCGAAAGGGCGATG-3'R：5'-CGCTCCCTACAGAACCCAT-3'；
Cc-114：F：5'-TGATGAGGATGGGGTCATTT-3'R：5'-TGCCATGTTTTGGAGGTAAA-3'；
Cc-131：F：5'-AGAATAGGGAAAGGGGCAGA-3'R：5'-CCCTTCCTTGATCAAACCCT-3'；
Cc-133：F：5'-ATGAGCGTCATCCAATGTCA-3'R：5'-GCACATACCCAGTTCCGATT-3'；
Cc-134：F：5'-GGGTCGGGGTAAATTAGGTC-3'R：5'-GACCGGTTTGCTAAGGTCAA-3'；
Cc-143：F：5'-CTATCCACTTCCCTGCCGTA-3'R：5'-CCAGAATCTTCGAGAAAGCG-3'；
Cc-185：F：5'-TGCTCTAATTCACCCCTTGC-3'R：5'-AGGCTCCACACTAAAACCCA-3'；
Cc-188：F：5'-ACCATCCAGAGTTTGATCGG-3'R：5'-GAGGGTTCTCCTTCGGATTC-3'；
Cc-191：F：5'-GTTCTCCTTCTGATCCACGG-3'R：5'-GACCCATTATGCGTTGAACC-3'。
secondly, the application of primers of the camphor tree whole genome SSR molecular markers in the detection of the genetic diversity of the cinnamomum camphora population and/or the clustering analysis of the genetic relationship of the cinnamomum camphora, which comprises the following steps:
(1) DNA polymerase chain reaction amplification
And (2) performing DNA polymerase chain reaction amplification on the 20 selected primers of the camphor tree genome SSR molecular markers by using optimized reaction conditions and amplification programs, wherein the amplification system and conditions are as follows: 10 XBuffer 1. mu.l, 25mmol/L MgCl2Mu.l, 0.6. mu.l of 10mmol/L dNTP, 0.1. mu.l of 5U/. mu.l DNA polymerase, 50ng of DNA template, 0.5. mu.l of each of 25mmol/L primers F and R, and a volume of 10. mu.l by double distilled water. The polymerase chain reaction procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30sec, renaturation at annealing temperature of primer pair of each camphor tree whole genome SSR molecular marker for 30sec, extension at 72 ℃ for 30sec, and 32 cycles; finally, extending for 5min at 70 ℃, wherein the annealing temperature is as follows: the annealing temperatures at both ends of the primers of SSR molecular markers, which are respectively numbered Cc-19, Cc-25, Cc-49, Cc-55, Cc-60, Cc-76, Cc-80, Cc-81, Cc-83, Cc-85, Cc-96, Cc-100, Cc-114, Cc-131, Cc-133, Cc-134, Cc-143, Cc-185, Cc-188 and Cc-191, are respectively Cc-19: 57 ℃; cc-25: at 58 ℃; cc-49: 56 ℃; cc-55: at 58 ℃; cc-60: 55 ℃; cc-76: at 58 ℃; cc-80: 59 ℃; cc-81: 55 ℃; cc-83: 55 ℃; cc-85: 55 ℃; cc-96: at 58 ℃; cc-100: 59 ℃; cc-114: at 54 ℃; cc-131: at 58 ℃; cc-133: 56 ℃; cc-134: at 58 ℃; cc-143: at 58 ℃; cc-185: at 58 ℃; cc-188: at 58 ℃; cc-191: at 58 ℃. Thereby obtaining an amplification product. The sequences, annealing temperatures, repeating motifs, etc. of the primers are detailed in Table 1.
TABLE 120 SSR primer information
(2) Electrophoretic separation and silver staining detection
The amplification product was subjected to silver staining detection after adding a denaturing buffer (containing 98% formamide, 10mmol/L EDTA, pH8.0, 0.25% bromophenol blue and 0.2% xylene cyanide) to the amplification product, applying 8% (by mass) of a denatured polyacrylamide gel (thickness of about 1.0 mm) and 1 XTBE (electrophoresis buffer) to the amplification product, and performing electrophoresis at a constant pressure of 120V for 90 minutes. The silver staining method mainly comprises the following steps: after electrophoresis, placing the glass plate adhered with the gel into a plastic basin for silver staining; adding fixing solution (containing 10% anhydrous ethanol and 0.5% acetic acid), fixing by slightly shaking on shaking table for 15 min, and rinsing with deionized water for 2 times (each time for 2 min); placing the mixture into 0.1% silver nitrate staining solution, slightly shaking the mixture on a shaking table for 10 minutes for staining, and then rinsing the mixture for 2 times with deionized water, wherein each time lasts for 5 seconds; placing the gel in color development solution (containing 1.5% sodium hydroxide and 0.014% sodium tetraborate), and slightly shaking on a shaking table until the bands are clear and the number of bands is not increased any more; adding a stationary liquid, stopping the color reaction, and rinsing with distilled water for several minutes; the gel and water beads on the glass plate were removed. The film was observed under a white light and photographed with a digital camera. The results are shown in FIG. 1.
(3) Application of microsatellite molecular marker in genetic diversity detection of cinnamomum camphora population
Because the original natural population of the camphor tree exists less and most of the existing artificial cultivation population and individuals, the effectiveness and polymorphism of the camphor tree genome SSR molecular marker primer are detected by selecting an ancient camphor tree sample with the age of more than 100 years. The method comprises the steps of collecting cinnamomum camphora samples from three counties of Anyi, Anfu and Ruijin of Jiangxi province, wherein the samples are shown in Table 2, extracting cinnamomum camphora genome DNA by a CTAB method, taking the extracted DNA sample to be detected as a template, and applying the obtained SSR molecular marker primers of cinnamomum camphora genome to genetic diversity detection of cinnamomum camphora groups through DNA polymerase chain reaction amplification, electrophoretic separation and silver staining detection. The 42 DNA polymorphic sites of the Cinnamomum camphora were characterized by analyzing the genotype data and gene frequency using Popgene 1.32(Yeh and Boyle, 1997), calculating the observed and expected heterozygosity of the Cinnamomum camphora, and performing Hardy-Weinberg (Hardy-Weinberg equibrium, HWE) and linkage disequilibrium tests. As can be seen from fig. 2-21 and table 3, the primers for 20 pairs of camphor tree genome SSR molecular markers show polymorphism in 42 tested cinnamomum camphora individuals, and thus, the primers for 20 pairs of camphor tree genome SSR molecular markers can be used for analyzing genetic diversity and genetic structure of cinnamomum camphora, and are an effective and reliable molecular marker.
TABLE 2 Experimental collection of Cinnamomum camphora and its sources
TABLE 3 genetic diversity of Cinnamomum camphora
(4) Application of microsatellite molecular marker in clustering analysis of genetic relationship of cinnamomum camphora
Collecting ancient camphor tree samples from Anyi, Anfu and Ruijin counties in Jiangxi province, wherein the samples are shown in Table 2, extracting camphor tree genome DNA by a CTAB method, and performing PCR amplification on primers of SSR molecular markers of camphor tree genomes by using the extracted DNA sample to be detected as a template and developed 20 to obtain PCR amplification products (the specific steps are shown in steps (1) - (2)); detecting PCR amplification products by adopting 6% modified polyacrylamide gel electrophoresis, carrying out silver staining and developing, and counting electrophoresis results; the genetic relationship of the three groups is analyzed by Popgene 1.32(Yeh and Boyle, 1997) (see the attached figure 22), and meanwhile, the clustering analysis is carried out on the cinnamomum camphora by the software UPGM method of NTSYS-pcversion2.11 (see the attached figure 23), as can be seen from the attached figures 22 and 23, the genetic relationship of the groups in Anyi, Anfu and Ruijin is more similar to that of the groups in Anfu and Ruijin, and is consistent with the research result of the gene flow trend in the current research on the lineage geography of the cinnamomum camphora. Therefore, the primer of the 20 pairs of camphor tree genome SSR molecular markers can be used for analyzing the genetic diversity and the genetic relationship analysis of camphor trees, and is an effective and reliable molecular marker.
Sequence listing
<110> scientific college for forestry in Jiangxi province
<120> camphor tree whole genome SSR molecular marker and preparation method and application thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 296
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgttctcgga tacgaacgat tgcatcaaac acttctgcga gctatcgagt tcgaaaagga 60
aaggattaag gtttctcaaa gttaaaccca aagatgtttt tttttttttt tttttttttt 120
tttttttttt tttttttgga agatccctaa cggactcatc aaagagaaga gatgccccat 180
tccccgacac gaacgattgc atcaaacatt tctatgagct atcgagttcg aagaggaaaa 240
gattaaggct tctcaaagtt gaacccaaag atgagaaatc tgaaaaaaaa aatttg 296
<210> 2
<211> 154
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcgccatttg ttttcttcat atctttttta ccactagtat ttcctccttg aaatataata 60
catgttacca ttccaaaaat aaataaataa ataaataaat aaatagtgca ttaggtgagt 120
agcagtactc ctgccccttc cgaccctagt gatt 154
<210> 3
<211> 214
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcatctccct accaaatcca tccaatatct aggcatcatt gatcatatct gatacaatct 60
caaaatgcat gttcatggat atgatatgag gttaacaatt ttttctgtaa ttgagcaaga 120
actttagagc ctcataaaaa aaaaaacccg attagaccat gtgaggtaaa atccgtgtca 180
tatcccatga tgcccgatgc ttcaaaatga gcaa 214
<210> 4
<211> 261
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cagccattca gaagggaaag atagagggag ggatagttta agaactaata tatatatata 60
tatatgcaaa gtgaaaagca atgtacatgc agcagctctc aaaaataagt ttccacaagt 120
gggaattcct tcgtgaattc gttttagaat tcaaaagtga aaaagatctc gacttaaata 180
tttctgggca agacaagtgg atcacatttg gtcggatggt caaataaatc accgaaccac 240
gtgcccccat agaagaagtt g 261
<210> 5
<211> 142
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccgaacgtca actcaaacaa ttctgcgagc tattgtccta aaagggaaag aactaaagtt 60
tctcaaattt gattccaaag gtgagaaatc tgattatata tatatatata ttttttgaaa 120
gatcaccaat gaacccatca aa 142
<210> 6
<211> 157
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tggaatgcaa agaaggaacc taatttacaa catattgaaa aaagaaaaaa aaaaagaact 60
ccaactctgt gacaatagaa gactgtaatt cctgtaaaca aagataacaa actgatattt 120
tgttttagag cgagcaccag aaatcagggg accagag 157
<210> 7
<211> 234
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tctctctcat ggtcaaattg ttgactctag gaaccttggg gaccttccag ctctctctct 60
ctctctctct catggtcaaa ttgttgactc taggaacctt ggggaccttc cagctctctc 120
tctctctctc tctcatggtc aaattgttga ctctaggaac cttggggacc ttccagctct 180
ctctctctct ctctctcatg gtcaaattgt tgactctagg aaccttgggg acct 234
<210> 8
<211> 162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccatttctca ataggaatat tgattgtgga aaaaaaaaag tactgaaata aaaaagtatg 60
ggctatttac aaataataga accagagata gttacccatt tctcaatagg aatattgttt 120
gtggaaaaaa aaaaaaagta ctgaaatgaa aaggtatggg ct 162
<210> 9
<211> 162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cacggtcccc aatctctaaa attgtccaca cgacaattaa atctaattca gcctaaagaa 60
taattagttg aaaaaaattt aaaaaaatag aatgattagt tgaaaaaaaa acagagagag 120
agagagagat tagcctcatc acatggtcca acccaaattt ga 162
<210> 10
<211> 280
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggaacgtccg gctatgtaaa agactaagga gaagcattct ctactttaaa atttatctct 60
atatattaaa aaatttgcat ctcactttct ctctctacat ttaaaacaca ctctctctct 120
ctcagaaatc tgagaagcat ttattcaatt tatctcgaag atcatcacgt cagtaagctg 180
tagacgactc tctcaatttc tatttgcttc tttttcttct tcttctcctc ttcattcgtc 240
tctttcgatt ctctattatt agggttttgt ttgccacttt 280
<210> 11
<211> 184
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cacggtactg accagggttc gattccctgg atgcgcattc ttttttggat attttttgtt 60
ttttttttgt tgctagaatt caagtaatgt ggaacaactg acccctcaaa tgtatcacat 120
gtgcattttt ttttttggat cttgtttttt gctaagaatt caagtaatgt ggaacaactg 180
ggcc 184
<210> 12
<211> 131
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
agagatcgaa agggcgatgg aaatgggttc tgcagggagc gatcaaaagg gagagagaga 60
gagagagaga gagagcagag agattgagtg gagagatcga gcggcgatgg aaatgggttc 120
tgtagggagc g 131
<210> 13
<211> 190
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tgatgaggat ggggtcattt tcctccaaaa catggcaaaa gattgaactt aaaagctgcc 60
atccatcatt ggcccatatg atgattgatt ggtcgtagct aaaaaatgac tccatttcat 120
aatcccaacc attaaagttg atggattgta tgatgatgat gatgggttca tttacctcca 180
aaacatggca 190
<210> 14
<211> 122
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agaataggga aaggggcaga aacaaagagg aaagagagac tagccaaaat agtgggatgg 60
agagagagag agagagggtg ttttttttct gttcagagaa caagggtttg atcaaggaag 120
gg 122
<210> 15
<211> 122
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgagcgtca tccaatgtca tttgatgtgt gaagtgaggt aacttctctc tctctctcta 60
tatatatata tatatgtatg tatgagagat atttaaggtc ttaatcggaa ctgggtatgt 120
gc 122
<210> 16
<211> 157
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gggtcggggt aaattaggtc aggccaaaga ttttatatat atatatatat atatgaagcg 60
aaatggtttc aaattgaaat cactttcaat tcatttcaat tgattttaac taatccaatt 120
taccaattta atttagtttg accttagcaa accggtc 157
<210> 17
<211> 223
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctatccactt ccctgccgta ctaggtaaaa ttcaacctga gccgaccacc ttgtctcaat 60
tgcaagtggc ttaaccggcc cgtaaaggtg tttgtttcca tctcacaaga aaaagtagta 120
ataataataa taaaaagatt tccatcattc caaaatccag cccaaggaaa caaacgcgtg 180
gcactacttt tctctgcttt caacgctttc tcgaagattc tgg 223
<210> 18
<211> 204
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tgctctaatt caccccttgc attgatgggt ttgtatgagt caatgctgaa acctttttat 60
gggtttgttg gatgattgtg ttatgttcta attactggga tttctttctt tctttctttc 120
ctcctctgta tctaattttg aagattttct tgagaaatgt ttttgcagtt ttcccttttt 180
ttgatgggtt ttagtgtgga gcct 204
<210> 19
<211> 207
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
accatccaga gtttgatcgg aaagagggag aagacttatt ttaggaagca attgagaagt 60
gagaatttgt ttatttattt atttatttaa ctttcagtca ttgtttgggc catacaacta 120
ttccgcttga ttttcttgcg ttaatattgg gacccaggtg ggccaccaga tgactcatcc 180
aatccatgaa tccgaaggag aaccctc 207
<210> 20
<211> 259
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gttctccttc tgatccacgg atcgaatggg tcatcgggtg gcctacctgg gtttcagtat 60
caacgcaaga aactaaatag cccaaacaat gactaaaact taaaaaataa ataaataaat 120
aaataaataa caaataaaca aaaacaaatt ccctattctt aattgtttcc taaaacacgt 180
actctccctc tctctaatca gacgcaagat ggtgtaccaa atagggctca catcacatgg 240
gttcaacgca taatgggtc 259
Claims (6)
1. A primer group of a camphor tree whole genome SSR molecular marker is characterized in that the primer group of the camphor tree whole genome SSR molecular marker is as follows:
Cc-19：F：5'- ATTTGCCTCGTGTTCCATTC -3' R：5'- TGGAATTTCAGATCCCCAAA -3'；
Cc-25：F：5'-GCGCCATTTGTTTTCTTCAT-3' R：5'-AATCACTAGGGTCGGAAGGG-3'；
Cc-49：F：5'-GCATCTCCCTACCAAATCCA-3' R：5'-TTGCTCATTTTGAAGCATCG-3'；
Cc-55：F：5'-CAGCCATTCAGAAGGGAAAG-3' R：5'-CAACTTCTTCTATGGGGGCA-3'；
Cc-60：F：5'- CCGAACGTCAACTCAAACAA -3' R：5'- TTTGATGGGTTCATTGGTGA -3'；
Cc-76：F：5'-TGGAATGCAAAGAAGGAACC-3' R：5'-CTCTGGTCCCCTGATTTCTG-3'；
Cc-80：F：5'-TCTCTCTCATGGTCAAATTGTTG-3' R：5'-AGGTCCCCAAGGTTCCTAGA-3'；
Cc-81：F：5'-CCATTTCTCAATAGGAATATTGATTGT-3' R：5'-AGCCCATACCTTTTCATTTCA-3'；
Cc-83：F：5'-CACGGTCCCCAATCTCTAAA-3' R：5'-TCAAATTTGGGTTGGACCAT-3'；
Cc-85：F：5'-GGAACGTCCGGCTATGTAAA-3' R： 5'-AAAGTGGCAAACAAAACCCT-3'；
Cc-96：F：5'- CACGGTACTGACCAGGGTTC -3' R：5'- GGCCCAGTTGTTCCACATTA -3'；
Cc-100：F：5'- AGAGATCGAAAGGGCGATG-3' R：5'- CGCTCCCTACAGAACCCAT -3'；
Cc-114：F：5'- TGATGAGGATGGGGTCATTT -3' R：5'- TGCCATGTTTTGGAGGTAAA -3'；
Cc-131：F：5'-AGAATAGGGAAAGGGGCAGA-3' R：5'-CCCTTCCTTGATCAAACCCT-3'；
Cc-133：F：5'-ATGAGCGTCATCCAATGTCA-3' R：5'-GCACATACCCAGTTCCGATT-3'；
Cc-134：F：5'-GGGTCGGGGTAAATTAGGTC-3' R：5'-GACCGGTTTGCTAAGGTCAA-3'；
Cc-143：F：5'- CTATCCACTTCCCTGCCGTA -3' R：5'-CCAGAATCTTCGAGAAAGCG-3'；
Cc-185：F：5'-TGCTCTAATTCACCCCTTGC-3' R：5'-AGGCTCCACACTAAAACCCA-3'；
Cc-188：F：5'- ACCATCCAGAGTTTGATCGG -3' R：5'- GAGGGTTCTCCTTCGGATTC -3'；
Cc-191：F：5'-GTTCTCCTTCTGATCCACGG-3' R：5'-GACCCATTATGCGTTGAACC-3'。
2. the use of the primer set of the camphor tree whole genome SSR molecular marker according to claim 1 in the detection of genetic diversity of camphor tree population and/or the clustering analysis of camphor tree genetic relationship.
3. Use according to claim 2, characterized in that the steps are as follows:
(1) extracting genome DNA of a camphor tree sample to be detected;
(2) taking the extracted DNA sample to be detected as a template, and respectively carrying out PCR amplification on each pair of primers in the primer group of the camphor tree whole genome SSR molecular marker in claim 1 to obtain PCR amplification products;
(3) typing the PCR amplification product.
4. The use according to claim 3, wherein the PCR amplification system comprises: 10 XBuffer 1. mu.l, 25mmol/L MgCl2Mu.l, 0.6. mu.l of 10mmol/L dNTP, 0.1. mu.l of 5U/. mu.l DNA polymerase, 50ng of DNA template, 0.5. mu.l of each of primers F and R marked by 25mmol/L SSR molecules, and fixing the volume to 10. mu.l by double distilled water.
5. The use of claim 3, wherein the PCR amplification is performed by the following amplification reaction procedures: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30sec, renaturation at annealing temperature of primer pair of each camphor tree whole genome SSR molecular marker for 30sec, extension at 72 ℃ for 30sec, and 32 cycles; finally, extension is carried out for 5min at 70 ℃, and the annealing temperature of the primer is as follows: cc-19: 57 ℃; cc-25: at 58 ℃; cc-49: 56 ℃; cc-55: at 58 ℃; cc-60: 55 ℃; cc-76: at 58 ℃; cc-80: 59 ℃; cc-81: 55 ℃; cc-83: 55 ℃; cc-85: 55 ℃; cc-96: at 58 ℃; cc-100: 59 ℃; cc-114: at 54 ℃; cc-131: at 58 ℃; cc-133: 56 ℃; cc-134: at 58 ℃; cc-143: at 58 ℃; cc-185: at 58 ℃; cc-188: at 58 ℃; cc-191: at 58 ℃.
6. The use of claim 3, wherein the typing of the PCR amplification product is performed by detecting the PCR amplification product by electrophoresis using 6% denaturing polyacrylamide gel electrophoresis, silver staining, and then typing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810474571.7A CN108823327B (en) | 2018-05-17 | 2018-05-17 | Camphor tree whole genome SSR molecular marker and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810474571.7A CN108823327B (en) | 2018-05-17 | 2018-05-17 | Camphor tree whole genome SSR molecular marker and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108823327A CN108823327A (en) | 2018-11-16 |
| CN108823327B true CN108823327B (en) | 2022-01-18 |
Family
ID=64148162
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810474571.7A Active CN108823327B (en) | 2018-05-17 | 2018-05-17 | Camphor tree whole genome SSR molecular marker and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108823327B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724760B (en) * | 2019-12-02 | 2021-03-19 | 江西省科学院生物资源研究所 | Polymorphic primer of cinnamomum camphora nuclear genome SSR molecular marker and application thereof |
| CN110760610B (en) * | 2019-12-02 | 2020-10-30 | 南京林业大学 | Polymorphic primer of camphor chloroplast SNP molecular marker and application thereof |
| CN116590448B (en) * | 2022-10-13 | 2024-08-27 | 江西省科学院生物资源研究所 | Application of SSR molecular marker of camphor tree |
| CN117887874A (en) * | 2023-05-15 | 2024-04-16 | 南昌工程学院 | G-SSR (G-simple sequence repeat) labeled primer combination and application thereof in construction of fingerprint spectrum of camphor tree |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017106274A1 (en) * | 2015-12-16 | 2017-06-22 | Syngenta Participations Ag | Genetic regions & genes associated with increased yield in plants |
| CN107190096A (en) * | 2017-07-25 | 2017-09-22 | 山东农业大学 | The general molecular labeled primer and its joint development methods and applications of trees |
-
2018
- 2018-05-17 CN CN201810474571.7A patent/CN108823327B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017106274A1 (en) * | 2015-12-16 | 2017-06-22 | Syngenta Participations Ag | Genetic regions & genes associated with increased yield in plants |
| CN107190096A (en) * | 2017-07-25 | 2017-09-22 | 山东农业大学 | The general molecular labeled primer and its joint development methods and applications of trees |
Non-Patent Citations (2)
| Title |
|---|
| DEVELOPMENT OF MICROSATELLITE MARKERS FOR CINNAMOMUM CAMPHORA (LAURACEAE);Y. KAMEYAMA等;《American Journal of Botany》;20121231;第e1页右栏第1段至第e3页左栏倒数第2段 * |
| Natural genetic differentiation and human-mediated gene flow:the spatiotemporal tendency observed in a long-lived;Yoshiaki Kameyama等;《Tree Genetics & Genomes》;20171231;第13卷(第38期);第1-12页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108823327A (en) | 2018-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108823327B (en) | Camphor tree whole genome SSR molecular marker and preparation method and application thereof | |
| CN108642208B (en) | General SSR molecular marker for cinnamomum and related plants and development method and application thereof | |
| CN105112518B (en) | A kind of HLA classifying method based on Pacbio RS II microarray dataset | |
| CN114686597A (en) | SNP molecular marker for sex identification of salangid and application thereof | |
| CN108060237B (en) | Forensic medicine composite detection kit based on 55Y chromosome SNP genetic markers | |
| CN108192893B (en) | Method for developing blumea balsamifera SSR primer based on transcriptome sequencing | |
| CN107988385B (en) | Method for detecting marker of PLAG1 gene Indel of beef cattle and special kit thereof | |
| CN109280696B (en) | Method for splitting mixed sample by SNP detection technology | |
| CN107354151B (en) | STR molecular marker developed based on sika whole genome and application thereof | |
| CN110964844B (en) | Primer, kit and method for qualitative determination of ginseng, poria cocos and bighead atractylodes rhizome powder | |
| KR102283596B1 (en) | InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof | |
| CN108642190B (en) | Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers | |
| CN112961932A (en) | Primer group combination for rapidly identifying purity of ginger variety, identification method and application | |
| CN112410441A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288479.1-95621 | |
| CN112430675A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288474.1-322717 | |
| Vajpayee et al. | Forensic DNA typing: inception, methodology, and technical advancements | |
| CN111647680A (en) | Method for rapidly identifying and tracing sedge variety at whole genome level based on second-generation high-throughput sequencing | |
| CN106591462B (en) | Primer, kit and identification method for identifying genetic relationship between Yangtze river finless porpoise individuals | |
| CN110616270A (en) | COI gene sequence-based molecular identification method of beta and beta | |
| CN114525362B (en) | Primer combination for identifying Apostichopus japonicus population of Anyuan No. 1 and application thereof | |
| CN114164282B (en) | Primer for identifying genetic sex of hynchus bifidus and PCR identification method | |
| CN114164283B (en) | PCR primer for sex identification of double-wishbone and identification method | |
| KR102380784B1 (en) | Molecular marker for discriminating genetic resources of Peucedanum japonicum and uses thereof | |
| Xiao et al. | Development of Genetic Novel SSR Markers by Transcriptome Sequencing in Ophicephalus argus Cantor | |
| CN114686595A (en) | SNP molecular marker for sex identification of salangid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |