CN108315468B - Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof - Google Patents
Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof Download PDFInfo
- Publication number
- CN108315468B CN108315468B CN201810305129.1A CN201810305129A CN108315468B CN 108315468 B CN108315468 B CN 108315468B CN 201810305129 A CN201810305129 A CN 201810305129A CN 108315468 B CN108315468 B CN 108315468B
- Authority
- CN
- China
- Prior art keywords
- enteromorpha
- sample
- primer
- sequence
- floating
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of algae molecular markers, and particularly relates to a mitochondrial molecular marker primer of an enteromorpha population and application thereof. The application of the primer in identifying the high-resolution mitochondrial molecular marker (rps2-trnL intergenic region sequence) of the enteromorpha prolifera population. The method can detect the algae sample and the microcosmic propagule sample, is more reliable and practical than the conventional method, and further has important significance for the research of green tide disasters, the germplasm identification of enteromorpha, the phylogeny and the systematic geographic research, and the protection and the utilization of enteromorpha resources.
Description
Technical Field
The invention belongs to the technical field of algae molecular markers, and particularly relates to a mitochondrial molecular marker primer of an enteromorpha population and application thereof.
Background
Enteromorpha (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B) and (C)Ulva prolifera) Belongs to the genus Ulva of the family Ulvaceae, is a common green alga in intertidal zones, and is widely distributed in China. In recent years, the explosive proliferation of enteromorpha prolifera causes green tide disasters due to large-area floating in yellow sea in China, causes serious negative effects on coastal economy and ecology, and arouses wide attention of society, media, governments and related scholars. The current research shows that the floating enteromorpha is different from the close-shore and adventitious enteromorpha and is a single and unique floating ecotype, so that the relation between the floating enteromorpha and the adventitious enteromorpha is the key for tracing the green tide enteromorpha and is an important basis for guiding the enteromorpha disaster prevention and control. Morphologically, enteromorpha of different populations are on microscopic propagule scaleThe sections are not different, and in the development and maturation period, the floating enteromorpha prolifera is usually yellow green, hollow and tubular, and has more branches, while the stationary enteromorpha prolifera has various forms according to different environments. On the molecular level, the resolution of molecular markers ITS, rbcL and 18S commonly used in the existing ulva research cannot effectively distinguish enteromorpha populations, and the 5S spacer molecular marker for sub-species analysis is often unstable in operation due to the existence of multiple copies in a genome, so that different enteromorpha populations cannot be accurately distinguished. Therefore, the development of the reliable molecular marker with high resolution is beneficial to prevention and control of enteromorpha disasters.
Disclosure of Invention
The invention aims to provide a mitochondrial molecular marker primer of an enteromorpha population and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a mitochondrial molecular marker primer of an enteromorpha prolifera population is as follows:
the sequence of the upstream primer is as follows: 5'AAAATCAAAATCTAGTAAACCAGGC3' (SEQ ID 1)
The sequence of the downstream primer is as follows: 5'GCTAGCGATTCTTAACGCGATTGGG 3' (SEQ ID 2).
The primer is applied to identifying high-resolution mitochondrial molecular marker (rps2-trnL intergenic region sequence) of enteromorpha prolifera population.
A high-resolution mitochondrial molecular marking method for identifying enteromorpha prolifera population,
1) performing PCR amplification by using DNA of a sample to be detected as a template and the primer pair for identifying the enteromorpha population as a primer;
2) and (3) carrying out electrophoresis detection on the product obtained by amplification, and further sequencing and analyzing a sample with a band (a mitochondrial rps2-trnL intergenic region) of about 500bp in the electrophoresis detection, so as to identify the enteromorpha population with high resolution.
The primer pair for identifying the enteromorpha prolifera population is an upstream primer sequence: 5'AAAATCAAAATCTAGTAAACCAGGC3' (SEQ ID 1);
the sequence of the downstream primer is as follows: 5'GCTAGCGATTCTTAACGCGATTGGG 3' (SEQ ID 2).
The PCR system is as follows:
the PCR conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, and extension at 72 ℃ for 40s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
The high-resolution mitochondrial molecular marking method for identifying the enteromorpha prolifera population comprises the steps of carrying out base sequence global comparison on the amplification product and an enteromorpha prolifera population standard reference sequence (SEQ ID 3-10), intercepting front and back homologies of a comparison matrix, carrying out adjacency method phylogenetic analysis by using phylogenetic software, and further realizing high-resolution identification of the enteromorpha prolifera population according to a result of a phylogenetic tree.
The sequence of the sample to be detected and the floating ecological enteromorpha are on the same phylogenetic branch, so that the sample has the same or similar genotype, namely the sample to be detected is the floating ecological enteromorpha; and if the sequence of the sample to be detected and the fixed-growth type enteromorpha are on the same phylogenetic branch, the sample has the same or similar genotype with the known fixed-growth type enteromorpha, namely the sample to be detected is the fixed-growth type enteromorpha.
The invention has the advantages that:
according to the invention, specific primers are utilized to amplify specific fragments with extremely high specificity among enteromorpha populations, and rapid identification of the populations can be completed only by carrying out PCR amplification and one-time conventional sequencing reaction; the primer disclosed by the invention has extremely high resolution and strong stability, and can be used for quickly, conveniently and effectively identifying the enteromorpha populations.
According to the invention, by means of a bioinformatics method, on the basis of analyzing and comparing sequence fragments of an enteromorpha mitochondrial rps2-trnL intergenic region, a molecular marking method for identifying enteromorpha population mitochondria is developed, the problem that the enteromorpha population cannot be identified according to external forms and traditional molecular means is solved, automation and standardization can be realized in the sample identification process, rapid and effective identification can be carried out by using organic fragments and microscopic propagule samples, and an application system which is easy to use can be established and formed in a short time.
The method can detect the algae sample and the microcosmic propagule sample, is more reliable and practical than the conventional method, and further has important significance for the research of green tide disasters, the germplasm identification of enteromorpha, the phylogeny and the systematic geographic research, and the protection and the utilization of enteromorpha resources.
Drawings
FIG. 1 is an agarose gel electrophoresis result of rps2-trnL fragment amplified by PCR of Enteromorpha prolifera of floating type and Enteromorpha prolifera of fixed type provided in the embodiment of the present invention, wherein Nos. 1-6 represent samples of Enteromorpha prolifera of floating type, Nos. 7-12 represent samples of Enteromorpha prolifera of fixed type, M represents a molecular weight standard, and 8 bands from top to bottom represent 2500bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively.
FIG. 2 is a phylogenetic diagram of rps2-trnL based sequences according to an embodiment of the present invention. In the figure, a solid circle represents a floating enteromorpha individual, and a hollow circle represents a fixed enteromorpha individual.
FIG. 3 is a phylogenetic diagram of rps2-trnL based sequences according to an embodiment of the present invention. In the figure, a solid circle represents a floating enteromorpha individual, a hollow circle represents a stationary enteromorpha individual, and a solid triangle represents a detection sample.
Detailed Description
The invention is further explained below with reference to the figures and examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1) Obtaining of enteromorpha DNA standard reference sequence rps2-trnL intergenic region
Analyzing the high mutation region of the enteromorpha mitochondrial genome: .
Mitochondrial genome sequences (GeneGenbank Access No. KT428794.1 and KT 428794.1) of two different enteromorpha strains are obtained from the national center for Biotechnology information (NCBI, http:// www. ncbi. nlm. nih. gov /), genome comparison is carried out by utilizing a Clustal W module in MEGA 7.0 software, a section with high sequence height between the two strains is firstly found according to the comparison result of the mitochondrial genome, a section belonging to a gene spacer is further screened according to genome annotation information, and finally a gene with a candidate section being rps2-trnL is determined.
Designing a rps2-trnL sequence primer pair:
using the obtained upstream and downstream sequences of the intergenic region of rps2-trnL as templates, the sequence information of primers was designed as follows:
the sequence of the upstream primer is as follows: 5'AAAATCAAAATCTAGTAAACCAGGC3' (SEQ ID 1)
The sequence of the downstream primer is as follows: 5'GCTAGCGATTCTTAACGCGATTGGG 3' (SEQ ID 2)
Primer amplification and result detection:
the amplification primers are respectively applied to PCR amplification and identification of known different enteromorpha populations (floating enteromorpha and fixed enteromorpha) samples:
taking the obtained upstream and downstream primers as a primer pair, and taking the DNA of the enteromorpha population sample as a template to amplify;
the PCR system is as follows:
the conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, and extension at 72 ℃ for 40s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
The amplification products were detected by 1.5-fold agarose gel electrophoresis. As a result, the DNA fragments amplified using the pair of primers were about 500bp in length, respectively, and the rps2-trnL fragments were efficiently amplified from the samples, respectively (FIG. 1).
2) Determination of DNA Standard reference sequences
The PCR amplification product obtained by the gel electrophoresis was cut, and sent to a sequencing company (Qingdao Optimaea Co.) for DNA sequencing by Sanger method, using the amplification primer (SEQ ID 1) as a sequencing primer. The sequencing result shows that the DNA fragments (fragments of various enteromorpha populations rps2-trnL intergenic region DNA) of the floating enteromorpha and the fixed enteromorpha can be effectively amplified by using the known different enteromorpha population samples as templates and using the primers of the embodiment as standard reference sequences.
The DNA standard reference sequence of the floating enteromorpha obtained by sequencing has the following nucleotide sequence (SEQ ID 3)
GAGGGTCAATCCGACCGTTGGGGTATCTCTGGTATAGCAACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGTATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAATAAGCCAAACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGTTCAAAGATAACAAAAAAATAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAAATTCGCTA
The DNA standard reference sequence 1 of the sedentary enteromorpha is shown as the following nucleotide sequence (SEQ ID 4)
TGTTTAATCCCGACCGTTGGGGTATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGTTAACTCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCAATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCCATATTTAATGCCTATT
The DNA standard reference sequence 2 of the sedentary enteromorpha prolifera has a nucleotide sequence shown as (SEQ ID 5) TGTTTAAATCCCGAACCGTTGGGGTATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGTTAACTCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCA
The DNA standard reference sequence 3 of the sedentary enteromorpha is shown in the specification, and the nucleotide sequence is as follows (SEQ ID 6) GTCAAAAAATCCGACCGTTTGGGGTAATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCACGA
The DNA standard reference sequence 4 of the sedentary enteromorpha is shown in the specification, and the nucleotide sequence is as follows (SEQ ID 7) AATCCCGAACCGTTGGGGTATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGATCCGCTAGCA
The DNA standard reference sequence 5 of the sedentary enteromorpha prolifera has a nucleotide sequence shown as (SEQ ID 8) TTTAAATTCCCGACCGTTGGGGTAATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGTTAACTCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCCGCTTTTGTTTTGCTT
The DNA standard reference sequence 6 of the sedentary enteromorpha is shown as the following nucleotide sequence (SEQ ID 9)
AATCCCGACCGTTGGGGTAATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGTTAACTCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCAATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAAAAACCCCTAGCAAGAATGG
The DNA standard reference sequence 2 of the sedentary enteromorpha is shown as the following nucleotide sequence (SEQ ID 10)
TTTAATAAATCCCGACCGTTTGGGGTAATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCAAGA
3) Phylogenetic analysis
The base sequences obtained by sequencing are subjected to global base sequence comparison, phylogenetic analysis is carried out by using an adjacency method module in MEGA 7.0 software, and the results of a phylogenetic tree show that: all floating ecotype enteromorpha and sedentary enteromorpha were gathered on independent phylogenetic branches, respectively (see fig. 2). The branch length of the floating ecological enteromorpha is 0, which indicates that the sequences of the samples are completely consistent, and the detected floating enteromorpha has completely consistent genotypes; the branch length of the fixed-growth type enteromorpha branch line is not 0, which indicates that the sequences of the samples are not completely consistent, and the determined biological type enteromorpha has different genotypes.
Example 2:
the species type of enteromorpha samples (sample 1) collected from the eastern Shandong Dashan mountain and the Qingdao Kangquan Bay (sample2) could not be confirmed. The method of the invention is used for comparative identification:
the method comprises the following specific steps:
1) extracting DNA of a sample to be detected by using a conventional method for standby;
2) and (3) PCR amplification:
using the sample obtained in step 1) as a template, and performing PCR amplification by using the primers obtained in the above example, wherein the PCR conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, and extension at 72 ℃ for 40s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
The PCR system is as follows:
3) the products amplified above were detected by 1.5-fold agarose gel electrophoresis. As a result, the DNA fragments amplified using the primers (SEQ ID 1 and SEQ ID 2) were about 500bp in length, respectively, and rps2-trnL fragments of the sample were efficiently amplified, respectively.
4) Sequencing the PCR amplification product:
determining the nucleotide sequence of rps2-trnL fragment of sample1 as follows: SEQ ID 11
GAGGGTCAATCCGACCGTTGGGGTATCTCTGGTATAGCAACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGTATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAATAAGCCAAACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCCTTTGCGGGTTCAAAGATAACAAAAAAATAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAAATTCGCTA
Determining the nucleotide sequence of rps2-trnL fragment of sample2 as follows: SEQ ID 12
GTCAAAAAATCCGACCGTTTGGGGTAATCTCTGGTATAGCGACAAGCCAACTAGGTCGTCGTAGCTGCAATCATTATAGTTTAAACAATCTTGTAAGTTATCCCATCCTAGGGAATCCATCGAGTAGTTTTTTTATTTATAGCCTTATTGGGATCTTTATAAAAACACTTCGTTCAAGCTAATTATACGTATTAATAATAATATTTATAATTAATTATACACTACCTTTCAAAGAAGCCAAACCTTTCAAAGAAGCCAAAGGTGGCTTTCTCGCTTTCTCGCTTTCTCCTTTGCGGGGGCAAGGGTTACAATATCTTAATTTTATTTGCAATATGGGTCCTTTAGAGGGCTTAATTAATCGTTCGATTTGCTACACTATAAAAGTTAAGTTGGTAAAACTAAAATATCATATTATTACAAATGAGCCGAGCAAACCCAATCGCGTTAAGAATCGCTAGCACGA
5) Base sequence alignment and phylogenetic analysis:
the detected rps2-trnL fragment was globally aligned with DNA standard reference sequence SEQ ID 3-10 using the Clustal W module in MEGA 7.0 software. And then, carrying out adjacent phylogenetic analysis by using a phylogenetic module in MEGA 7.0 software, and according to the result of a phylogenetic tree: the sequence of sample1 is integrated with the floating ecological enteromorpha, which indicates that the sample belongs to the floating ecological enteromorpha; the sequence of sample2 is integrated with the above enteromorpha taperta, which indicates that the sample belongs to the enteromorpha taperta (fig. 3).
Sequence listing
<110> oceanographic institute of Chinese academy of sciences
<120> mitochondrial molecular marker primer of enteromorpha prolifera population and application thereof
<140> 2018103051291
<141> 2018-04-08
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaatcaaaa tctagtaaac caggc 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gctagcgatt cttaacgcga ttggg 25
<210> 3
<211> 465
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gagggtcaat ccgaccgttg gggtatctct ggtatagcaa caagccaact aggtcgtcgt 60
agctgcaatc attatagttt aaacaatctt gtaagttatc ccatcctagg gaatccatcg 120
agtagttttt ttatttatag ccttattggt atctttataa aaacacttcg ttcaagctaa 180
ttatacgtat taataataat atttataatt aattatacac tacctttcaa ataagccaaa 240
cctttcaaag aagccaaacc tttcaaagaa gccaaaggtg gctttctcgc tttctccttt 300
gcgggttcaa agataacaaa aaaataattt tatttgcaat atgggtcctt tagagggctt 360
aattaatcgt tcgatttgct acactataaa agttaagttg gtaaaactaa aatatcatat 420
tattacaaat gagccgagca aacccaatcg cgttaaaatt cgcta 465
<210> 4
<211> 471
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgtttaatcc cgaccgttgg ggtatctctg gtatagcgac aagccaacta ggtcgtcgta 60
gctgcaatca ttatagttta aacaatcttg taagttatcc catcctaggg aatccatcga 120
gtagtttttt tatttatagc cttattggga tctttataaa aacacttcgt tcaagctaat 180
tatacgtatt aataataata tttataatta attatacact acctttcaaa gaagccaaac 240
ctttcaaaga agttaactcc aaaggtggct ttctcgcttt ctcctttgcg ggggcaaggg 300
ttacaatatc ttaattttat ttgcaatatg ggtcctttag agggcttaat taatcgttca 360
atttgctaca ctataaaagt taagttggta aaactaaaat atcatattat tacaaatgag 420
ccgagcaaac ccaatcgcgt taagaatcgc tagccatatt taatgcctat t 471
<210> 5
<211> 457
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgtttaaatc ccgaaccgtt ggggtatctc tggtatagcg acaagccaac taggtcgtcg 60
tagctgcaat cattatagtt taaacaatct tgtaagttat cccatcctag ggaatccatc 120
gagtagtttt tttatttata gccttattgg gatctttata aaaacacttc gttcaagcta 180
attatacgta ttaataataa tatttataat taattataca ctacctttca aagaagccaa 240
acctttcaaa gaagttaact ccaaaggtgg ctttctcgct ttctcctttg cgggggcaag 300
ggttacaata tcttaatttt atttgcaata tgggtccttt agagggctta attaatcgtt 360
cgatttgcta cactataaaa gttaagttgg taaaactaaa atatcatatt attacaaatg 420
agccgagcaa acccaatcgc gttaagaatc gctagca 457
<210> 6
<211> 463
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtcaaaaaat ccgaccgttt ggggtaatct ctggtatagc gacaagccaa ctaggtcgtc 60
gtagctgcaa tcattatagt ttaaacaatc ttgtaagtta tcccatccta gggaatccat 120
cgagtagttt ttttatttat agccttattg ggatctttat aaaaacactt cgttcaagct 180
aattatacgt attaataata atatttataa ttaattatac actacctttc aaagaagcca 240
aacctttcaa agaagccaaa ggtggctttc tcgctttctc gctttctcct ttgcgggggc 300
aagggttaca atatcttaat tttatttgca atatgggtcc tttagagggc ttaattaatc 360
gttcgatttg ctacactata aaagttaagt tggtaaaact aaaatatcat attattacaa 420
atgagccgag caaacccaat cgcgttaaga atcgctagca cga 463
<210> 7
<211> 461
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aatcccgaac cgttggggta tctctggtat agcgacaagc caactaggtc gtcgtagctg 60
caatcattat agtttaaaca atcttgtaag ttatcccatc ctagggaatc catcgagtag 120
tttttttatt tatagcctta ttgggatctt tataaaaaca cttcgttcaa gctaattata 180
cgtattaata ataatattta taattaatta tacactacct ttcaaagaag ccaaaccttt 240
caaagaagcc aaaggtggct ttctcgcttt ctcgctttct cgctttctcc tttgcggggg 300
caagggttac aatatcttaa ttttatttgc aatatgggtc ctttagaggg cttaattaat 360
cgttcgattt gctacactat aaaagttaag ttggtaaaac taaaatatca tattattaca 420
aatgagccga gcaaacccaa tcgcgttaag atccgctagc a 461
<210> 8
<211> 471
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tttaaattcc cgaccgttgg ggtaatctct ggtatagcga caagccaact aggtcgtcgt 60
agctgcaatc attatagttt aaacaatctt gtaagttatc ccatcctagg gaatccatcg 120
agtagttttt ttatttatag ccttattggg atctttataa aaacacttcg ttcaagctaa 180
ttatacgtat taataataat atttataatt aattatacac tacctttcaa agaagccaaa 240
cctttcaaag aagttaactc caaaggtggc tttctcgctt tctcctttgc gggggcaagg 300
gttacaatat cttaatttta tttgcaatat gggtccttta gagggcttaa ttaatcgttc 360
gatttgctac actataaaag ttaagttggt aaaactaaaa tatcatatta ttacaaatga 420
gccgagcaaa cccaatcgcg ttaagaatcg ctagccgctt ttgttttgct t 471
<210> 9
<211> 459
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aatcccgacc gttggggtaa tctctggtat agcgacaagc caactaggtc gtcgtagctg 60
caatcattat agtttaaaca atcttgtaag ttatcccatc ctagggaatc catcgagtag 120
tttttttatt tatagcctta ttgggatctt tataaaaaca cttcgttcaa gctaattata 180
cgtattaata ataatattta taattaatta tacactacct ttcaaagaag ccaaaccttt 240
caaagaagtt aactccaaag gtggctttct cgctttctcc tttgcggggg caagggttac 300
aatatcttaa ttttatttgc aatatgggtc ctttagaggg cttaattaat cgttcaattt 360
gctacactat aaaagttaag ttggtaaaac taaaatatca tattattaca aatgagccga 420
gcaaacccaa tcgcgttaaa aaacccctag caagaatgg 459
<210> 10
<211> 456
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tttaataaat cccgaccgtt tggggtaatc tctggtatag cgacaagcca actaggtcgt 60
cgtagctgca atcattatag tttaaacaat cttgtaagtt atcccatcct agggaatcca 120
tcgagtagtt tttttattta tagccttatt gggatcttta taaaaacact tcgttcaagc 180
taattatacg tattaataat aatatttata attaattata cactaccttt caaagaagcc 240
aaacctttca aagaagccaa aggtggcttt ctcgctttct cctttgcggg ggcaagggtt 300
acaatatctt aattttattt gcaatatggg tcctttagag ggcttaatta atcgttcgat 360
ttgctacact ataaaagtta agttggtaaa actaaaatat catattatta caaatgagcc 420
gagcaaaccc aatcgcgtta agaatcgcta gcaaga 456
<210> 11
<211> 465
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gagggtcaat ccgaccgttg gggtatctct ggtatagcaa caagccaact aggtcgtcgt 60
agctgcaatc attatagttt aaacaatctt gtaagttatc ccatcctagg gaatccatcg 120
agtagttttt ttatttatag ccttattggt atctttataa aaacacttcg ttcaagctaa 180
ttatacgtat taataataat atttataatt aattatacac tacctttcaa ataagccaaa 240
cctttcaaag aagccaaacc tttcaaagaa gccaaaggtg gctttctcgc tttctccttt 300
gcgggttcaa agataacaaa aaaataattt tatttgcaat atgggtcctt tagagggctt 360
aattaatcgt tcgatttgct acactataaa agttaagttg gtaaaactaa aatatcatat 420
tattacaaat gagccgagca aacccaatcg cgttaaaatt cgcta 465
<210> 12
<211> 463
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gtcaaaaaat ccgaccgttt ggggtaatct ctggtatagc gacaagccaa ctaggtcgtc 60
gtagctgcaa tcattatagt ttaaacaatc ttgtaagtta tcccatccta gggaatccat 120
cgagtagttt ttttatttat agccttattg ggatctttat aaaaacactt cgttcaagct 180
aattatacgt attaataata atatttataa ttaattatac actacctttc aaagaagcca 240
aacctttcaa agaagccaaa ggtggctttc tcgctttctc gctttctcct ttgcgggggc 300
aagggttaca atatcttaat tttatttgca atatgggtcc tttagagggc ttaattaatc 360
gttcgatttg ctacactata aaagttaagt tggtaaaact aaaatatcat attattacaa 420
atgagccgag caaacccaat cgcgttaaga atcgctagca cga 463
Claims (3)
1. A primer for identifying floating enteromorpha and sedentary enteromorpha is characterized in that: the primer is as follows:
the sequence of the upstream primer is as follows: 5'AAAATCAAAATCTAGTAAACCAGGC3' of the composition,
the sequence of the downstream primer is as follows: 5'GCTAGCGATTCTTAACGCGATTGGG 3'.
2. The use of the primer of claim 1 for the identification of floating and sedentary enteromorpha.
3. A method for identifying floating enteromorpha and sedentary enteromorpha is characterized in that:
1) carrying out PCR amplification by using DNA of a sample to be detected as a template and the primer of claim 1;
2) carrying out electrophoresis detection on the product obtained by amplification, and further sequencing and analyzing a sample with a band of about 500bp in the electrophoresis detection to obtain a mitochondrial rps2-trnL intergenic fragment of the sample;
3) carrying out base sequence global comparison on the rps2-trnL intergene fragment and an enteromorpha population standard reference sequence SEQ ID 3-SEQ ID 9, intercepting front and back homologies of a comparison matrix, carrying out adjacent method phylogenetic analysis by using phylogenetic software, and further realizing high-resolution enteromorpha population identification according to the result of a phylogenetic tree;
4) if the sequence of the sample to be detected and the standard reference sequence SEQ ID 3 of the floating ecological enteromorpha are on the same phylogenetic branch, the sample has the same or similar genotype with the known floating ecological enteromorpha, namely the sample to be detected is the floating ecological enteromorpha; if the sequence of the sample to be detected and the standard reference sequence SEQ ID 4-SEQ ID 9 of the enteromorpha prolifera of the fixed type are on the same phylogenetic branch, the sample has the same or similar genotype with the known enteromorpha prolifera of the fixed type, namely the sample to be detected is the enteromorpha prolifera of the fixed type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810305129.1A CN108315468B (en) | 2018-04-08 | 2018-04-08 | Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810305129.1A CN108315468B (en) | 2018-04-08 | 2018-04-08 | Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108315468A CN108315468A (en) | 2018-07-24 |
| CN108315468B true CN108315468B (en) | 2021-07-27 |
Family
ID=62896097
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810305129.1A Active CN108315468B (en) | 2018-04-08 | 2018-04-08 | Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108315468B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278728B (en) * | 2021-07-15 | 2021-10-15 | 中国科学院海洋研究所 | Enteromorpha floating ecological chloroplast genome specific molecular marker and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102051417A (en) * | 2010-12-14 | 2011-05-11 | 宁波出入境检验检疫局检验检疫技术中心 | Detection kit and detection method for Enteromorpha compressa |
| KR101493955B1 (en) * | 2014-09-04 | 2015-02-24 | 대한민국 | Single Nucleotide Polymorphism Marker for Identification of Ulva Species |
| CN104561333A (en) * | 2015-01-20 | 2015-04-29 | 上海海洋大学 | Method for rapidly identifying green tide enteromorpha algae |
| CN106884055A (en) * | 2017-04-25 | 2017-06-23 | 上海海洋大学 | A kind of method that utilization gel electrophoresis differentiates green tide Enteromorpha algae |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942503B (en) * | 2009-12-28 | 2012-12-12 | 中国海洋大学 | Method for rapidly authenticating green tide algae enteromorpha |
| CN105695580A (en) * | 2016-03-10 | 2016-06-22 | 宁波检验检疫科学技术研究院 | LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection method for Ulva compressa |
-
2018
- 2018-04-08 CN CN201810305129.1A patent/CN108315468B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102051417A (en) * | 2010-12-14 | 2011-05-11 | 宁波出入境检验检疫局检验检疫技术中心 | Detection kit and detection method for Enteromorpha compressa |
| KR101493955B1 (en) * | 2014-09-04 | 2015-02-24 | 대한민국 | Single Nucleotide Polymorphism Marker for Identification of Ulva Species |
| CN104561333A (en) * | 2015-01-20 | 2015-04-29 | 上海海洋大学 | Method for rapidly identifying green tide enteromorpha algae |
| CN106884055A (en) * | 2017-04-25 | 2017-06-23 | 上海海洋大学 | A kind of method that utilization gel electrophoresis differentiates green tide Enteromorpha algae |
Non-Patent Citations (2)
| Title |
|---|
| "Genetic analyses of floating Ulva prolifera in the Yellow Sea suggest a unique ecotype";Jin Zhao等;《Estuarine-Coastal and Shelf Science》;20150514;第163卷;第96页摘要，第97页左栏第3段、右栏第3-4段，第98页图1、表1、右栏第3段-第99页左栏第2段、右栏第1段、表2、图2，第100页左栏第2-3段、图4，第101页左栏第3段 * |
| "浒苔的线粒体、叶绿体基因组研究及基于转录组数据的SSR分子标记开发";王灵珂;《中国优秀硕士学位论文全文数据库 基础科学辑》;20180215;第8页第1段，第14页表1-6，第15页第2段-第16页第2段，第17页表1-8，第19页第1段-第20页第3段、表1-9，第22页第2-3段，第25页第1-8段，第55页第1-2段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108315468A (en) | 2018-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tharachand et al. | Molecular markers in characterization of medicinal plants: an overview. | |
| CN111926100B (en) | Molecular marker of rice bacterial leaf blight resistance gene xa5 and application thereof | |
| CN104946640A (en) | Specificity labeling primer for tea-oil tree improved variety Chang Lin 18 and detection method | |
| CN116875736A (en) | SNP molecular marker of rice blast resistance gene pizz, primer set and application | |
| CN108315468B (en) | Mitochondria molecular marker primer of enteromorpha prolifera population and application thereof | |
| CN110846440A (en) | Complete primer pair for determining complete genome of passion flower virus in east Asia and application thereof | |
| CN107988408B (en) | Primer pair and DNA bar code for identifying enteromorpha kindred species and application and detection method thereof | |
| CN102181559B (en) | Specific primer system of EST (expressed sequence tag)-SSR (simple sequence repeat) molecular markers for Pleurotus ostreatus and application of specific primer system | |
| CN103667451A (en) | Quick molecular detection method of novel mycelium T4 injecting wheat stripe rust with AABBDDXX | |
| CN111996263A (en) | Asian gypsy moth detection method based on Taqman-MGB real-time fluorescence PCR technology, and primer and probe thereof | |
| Chen et al. | Developing a MtSNP-based genotyping system for genetic identification of forensically important flesh flies (Diptera: Sarcophagidae) | |
| Yang et al. | Mitochondrial DNA polymorphisms in Phytophthora infestans: New haplotypes are identified and re-defined by PCR | |
| CN104946641A (en) | Specificity labeling primer for camellia oleifera improved varieties changlin number three and twenty one and detection method | |
| CN111235303B (en) | Method for identifying cord-grass and spartina alterniflora | |
| CN113718050A (en) | Molecular characteristic SSR primer and method for identifying Chinese torreya polyploid varieties | |
| CN117821636B (en) | SNP molecular marker of rice blast resistance gene Pib, primer set and application | |
| CN112410459A (en) | KASP molecular marker for detecting rice blast resistance gene Pi25 and application thereof | |
| CN102443640B (en) | TRAP-SCAR252 (target region amplification polymorphism-sequence characterized amplified region 252) marker for identifying E group chromosomes of Agropyron elongatum and application thereof | |
| Shi et al. | A new insight into the SNP genotyping using high-resolution melting method after the correlation analysis of the SNPs with WSSV-resistant traits | |
| Cui et al. | An efficient method for developing polymorphic microsatellite markers from high-throughput transcriptome sequencing: a case study of hexaploid oil-tea camellia (Camellia oleifera) | |
| CN113481313B (en) | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties | |
| CN114058725B (en) | Primer and method for identifying variety of super-early-maturing yuhuan shaddock' early-corn denier | |
| CN113106164B (en) | SNP molecular marker for predicting red sage root flower color and application thereof | |
| CN118064428B (en) | MNP molecular marker combination and method for constructing rubber tree DNA fingerprint | |
| CN113444823B (en) | Primer group and method for identifying streptomyces albidoflavus W68 by using primer group |
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 |