CN114480668B - Microsatellite marker for fine tail plateau loach fish, amplification primer and application thereof - Google Patents
Microsatellite marker for fine tail plateau loach fish, amplification primer and application thereof Download PDFInfo
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Abstract
The invention discloses a microsatellite marker for a fine tail plateau loach, and an amplification primer and application thereof. The invention provides microsatellite markers of 22 fine tail loaches and corresponding amplification primer pairs, provides analysis data for genetic analysis of fine tail loach populations in different flow domains, provides a basis for further analysis of differences in later-stage combination morphological characters, and is applied to the fields of research on the genetic diversity of fine tail loach populations, genetic structure analysis, species resource protection and the like.
Description
Technical Field
The invention belongs to the technical field of molecular biology DNA (deoxyribonucleic acid) markers, and particularly relates to a microsatellite marker for fine tail plateau loach fishes, an amplification primer and application thereof.
Background
Fine tail loaches (Triplophysa stenura (Herzenstein)) belong to the genus loach (triprophysa) of the superfamily (cobiodea) of the order Cypriniformes (Cypriniformes) of the order Cyprinidus, are widely distributed in the dry streams of Jinshajiang, langjiang, guangjiang, yangtze, yarutibujiang and the like and the tributary water systems thereof, and are dominant species in a production area. In recent years, research on the fish of the genus loach is mainly focused on the content of morphological differences among species, homonymies of species and the like, molecular research is mainly performed on the classification status of the fish of the genus by adopting mitochondrial marking and combining morphology, and little research is performed on genetic diversity and genetic structure of specific species, and marking based on nuclear gene development is almost absent. Morphological research results show that the characteristics of the fine tail loaches in different drainage basins such as body spots, intestinal shapes, thickness of tail handles, positions of dorsal fins and ventral fins and the like are different to a certain extent, so that the research on genetic diversity and genetic structure of the fine tail loaches can provide analysis data for genetic analysis of the fine tail loaches in different drainage basins, provide a basis for further analysis of the differences in the aspect of combining morphological characteristics in later stages, and can provide analysis data for systematic development of the fish of the genus of the high-altitude loaches.
Microsatellite marker (microsatellite), also known as short tandem repeat (short tandem repeats, STR) or simple repeat (simple sequence repeats, SSR), refers to a DNA fragment formed by repeating a core sequence in multiple tandem with a few nucleotides (1-6 bp) as a basic unit. Microsatellite loci refer to loci that contain microsatellite markers on the genome, the microsatellite loci are abundant and evenly distributed on the genome, and the development of microsatellite loci refers to the process of finding microsatellite markers on the genome. The length polymorphism of each site is caused by the different repetition times and the incomplete repetition degree of the microsatellite marker core sequence. The flanking sequences connected with the core sequence have high conservation, specific primers can be designed at two ends of the flanking sequences, microsatellite sequences at corresponding sites can be amplified by a PCR technology, and the microsatellite marker length can be displayed by electrophoresis analysis, so that the length polymorphism of the microsatellite marker can be obtained. The microsatellite marking technology can be applied to the aspects of constructing genetic linkage maps, evaluating genetic diversity, drawing phylogenetic trees and the like. Therefore, the development and screening of the polymorphic microsatellite markers of the fine tail loaches are of great significance in the research of genetic diversity, genetic structure analysis, genetic resource protection and the biological and geographic research of the fish of the genus loaches.
Disclosure of Invention
The invention aims to provide a microsatellite marker of fine-tail high-altitude loach and amplification primers and application thereof, namely, microsatellite markers of 22 fine-tail high-altitude loaches and corresponding amplification primer pairs are provided, analysis data are provided for genetic analysis of fine-tail high-altitude loach populations in different flow domains, a basis is provided for further analysis of differences in later-stage combined morphological characters, and an effective tool is provided for research on genetic diversity of fine-tail high-altitude loach populations and species resource protection.
In order to solve the technical problems, the invention provides the following technical scheme:
providing a microsatellite marker for the tail plateau loach, wherein the nucleotide sequences of the microsatellite markers are respectively shown in SEQ ID NO: 1-22.
The invention also provides a primer pair for amplifying the polymorphic microsatellite markers of the tail-biting-fish, which is:
the upstream and downstream sequences are SEQ ID NOs: 23 (Tst 001-F), SEQ ID NO:24 (Tst 001-R) primer pair for amplifying a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 (Tst 001);
the upstream and downstream sequences are SEQ ID NOs: 25 (Tst 002-F), SEQ ID NO:26 (Tst 002-R) primer set for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:2 (Tst 002);
the upstream and downstream sequences are SEQ ID NOs: 27 (Tst 003-F), SEQ ID NO:28 (Tst 003-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:3 (Tst 003);
the upstream and downstream sequences are SEQ ID NOs: 29 (Tst 005-F), SEQ ID NO:30 (Tst 005-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:4 (Tst 005);
the upstream and downstream sequences are SEQ ID NOs: 31 (Tst 007-F), SEQ ID NO:32 (Tst 007-R) a primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:5 (Tst 007);
the upstream and downstream sequences are SEQ ID NOs: 33 (Tst 011-F), SEQ ID NO:34 (Tst 011-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:6 (Tst 011);
the upstream and downstream sequences are SEQ ID NOs: 35 (Tst 012-F), SEQ ID NO:36 (Tst 012-R) primer pair for amplifying a nucleic acid sequence of SEQ ID NO: microsatellite marker of 7 (Tst 012);
the upstream and downstream sequences are SEQ ID NOs: 37 (Tst 015-F), SEQ ID NO:38 (Tst 015-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:8 (Tst 015);
the upstream and downstream sequences are SEQ ID NOs: 39 (Tst 018-F), SEQ ID NO:40 (Tst 018-R) primer pair for amplifying a nucleic acid sequence of SEQ ID NO:9 (Tst 018);
the upstream and downstream sequences are SEQ ID NOs: 41 (Tst 029-F), SEQ ID NO:42 (Tst 029-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:10 (Tst 029);
the upstream and downstream sequences are SEQ ID NOs: 43 (Tst 036-F), SEQ ID NO:44 (Tst 036-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:11 (Tst 036);
the upstream and downstream sequences are SEQ ID NOs: 45 (Tst 038-F), SEQ ID NO:46 (Tst 038-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:12 (Tst 038);
the upstream and downstream sequences are SEQ ID NOs: 47 (Tst 041-F), SEQ ID NO:48 (Tst 041-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:13 (Tst 041);
the upstream and downstream sequences are SEQ ID NOs: 49 (Tst 050-F), SEQ ID NO:50 A primer pair of (Tst 050-R) for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:14 Microsatellite markers of (Tst 050);
the upstream and downstream sequences are SEQ ID NOs: 51 (Tst 051-F), SEQ ID NO:52 (Tst 051-R) primer pair for amplifying a nucleic acid sequence of SEQ ID NO:15 Microsatellite markers of (Tst 051);
the upstream and downstream sequences are SEQ ID NOs: 53 (Tst 053-F), SEQ ID NO:54 (Tst 053-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:16 (Tst 053) microsatellite marker;
the upstream and downstream sequences are SEQ ID NOs: 55 (Tst 059-F), SEQ ID NO:56 (Tst 059-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:17 Microsatellite marker of (Tst 059).
The upstream and downstream sequences are SEQ ID NOs: 57 (Tst 062-F), SEQ ID NO:58 (Tst 062-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:18 Microsatellite markers of (Tst 062);
the upstream and downstream sequences are SEQ ID NOs: 59 (Tst 068-F), SEQ ID NO:60 (Tst 068-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:19 (Tst 068);
the upstream and downstream sequences are SEQ ID NOs: 61 (Tst 077-F), SEQ ID NO:62 (Tst 077-R) primer pair for amplifying a nucleic acid sequence of SEQ ID NO:20 (Tst 077);
the upstream and downstream sequences are SEQ ID NOs: 63 (Tst 085-F), SEQ ID NO:64 (Tst 085-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:21 (Tst 085).
The upstream and downstream sequences are SEQ ID NOs: 65 (Tst 088-F), SEQ ID NO:66 (Tst 088-R) primer pair for amplifying a nucleic acid having the nucleotide sequence of SEQ ID NO:22 (Tst 088).
The invention provides an application of the microsatellite marker of the fine tail loach fish or the primer pair for amplifying the microsatellite marker in detecting the genetic diversity of the fine tail loach fish population.
According to the scheme, the application comprises the following steps:
(1) Extracting genomic DNA of the fine tail plateau loach;
(2) Microsatellite PCR amplification: the sequence of SEQ ID NO: performing PCR amplification by taking the fine tail plateau loach genome DNA obtained in the step (1) as a template after performing fluorescent marking on the primer pair of 23-66 respectively to obtain a microsatellite amplification product;
(3) Detecting amplified products by a sequencer: storing the amplification product obtained in the step (2) in a dark place, and carrying out capillary electrophoresis and STR analysis;
(4) Analysis of genetic diversity: and determining the genotype according to the molecular weight of each individual microsatellite amplification product of the fine tail highland loach fish, and calculating the genetic diversity parameter.
Preferably, the step (1) adopts a magnetic bead method genome DNA extraction kit (manufacturer, nanoMagBio) to extract genome DNA of fin tissues of the fine tail plateau loach;
preferably, the fluorescent label in step (2) is a FAM label.
Preferably, in step (3) capillary electrophoresis and STR analysis are performed using an ABI 3730XL sequencer.
Preferably, the genetic diversity parameter is calculated in step (4) using Cervus 3.0.
The invention provides application of a primer pair for microsatellite marking or amplifying the microsatellite marking in detecting the genetic diversity level of a fine tail plateau loach population, analyzing a genetic structure, protecting species resources and the like.
The beneficial effects of the invention are as follows:
1. according to the invention, 22 microsatellite markers are selected from the genomic DNA of the fine-tail plateau loach, and corresponding amplification primer pairs are obtained, so that analysis data are provided for genetic analysis of fine-tail plateau loach populations in different flow domains, a foundation is provided for further analysis of differences in later-stage combined morphological characters, and the method is applied to the fields of research on genetic diversity of fine-tail plateau loach populations, genetic structure analysis, species resource protection and the like.
2. And designing an applicability primer according to flanking regions at two ends of a microsatellite repeated sequence, and amplifying to obtain an amplified product with higher polymorphism and better stability.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, and many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention.
For the implementation methods in which specific conditions are not noted in the examples, it is generally possible to operate under conventional conditions, such as those described in the molecular cloning implementation guidelines written by J.Sam Brooks (Sambrook) et al, or according to the conditions recommended by the manufacturer.
Example 1
1. Search of sequences containing microsatellite repeat units
Searching a microsatellite repeated unit sequence from the sequence of the constructed fine tail plateau loach fish gene library by using software SSRHINTER 1.3; the parameters are set to find sequences containing more than 5 repeats of two bases, three bases and four bases. 96 sequences containing microsatellite repeating units are screened out, and primers are designed from the sequences to detect polymorphism.
2. Design of microsatellite primers:
from the gene sequences containing the microsatellite repeating units, a sequence conforming to the Primer design was selected and Primer Premier 5.0 was used for Primer design. The main parameters are set as follows: the primer length is 17-25bp,20bp is the optimal length, the PCR product fragment length range is 100-350bp, and the optimal annealing temperature is 55-65 ℃. The GC content is generally between 40% and 60%, and secondary structures are avoided as much as possible.
3. Polymorphism detection is carried out on amplification products of the designed primers:
(1) Extraction of genomic DNA:
extracting genomic DNA of the fin tissues of the 50 tail fine tail plateau loach bodies by using a magnetic bead method genomic DNA extraction kit (manufacturer, nanoMagBio);
(2) Microsatellite PCR amplification:
amplification in two steps using FAM fluorescent linker primers, wherein the M13 sequence 5'-3': TGTAAAACGACGGCCAGT, amplification system and procedure were as follows:
the first step: adapter primer amplification
(1) The amplification system is as follows:
| reagent(s) | Volume (mul) |
| 2X Taq PCR Master Mix (manufacturer, geneTech) | 5.0 |
| Stencil (genome DNA) | 1.0 |
| Upstream primer of adapter (concentration 10 pmol/. Mu.l) | 0.3 |
| Downstream primer (concentration 10 pmol/. Mu.l) | 0.3 |
| ddH 2 O | 3.4 |
| Total volume of | 10.0 |
(2) The amplification procedure was:
and a second step of: fluorescent primer amplification
(1) The amplification system is as follows:
| reagent(s) | Volume (mul) |
| 2X Taq PCR Master Mix (manufacturer, geneTech) | 5.0 |
| First step PCR product | 1.0 |
| M13 fluorescent primer (concentration 10 pmol/. Mu.l) | 0.3 |
| Downstream primer (concentration 10 pmol/. Mu.l) | 0.3 |
| ddH 2 O | 3.4 |
| Total volume of | 10.0 |
(2) The amplification procedure was:
(3) Detecting amplified products by a sequencer:
the amplified products were stored in the dark and capillary electrophoresis and STR analysis were performed on an ABI 3730XL sequencer to determine the allele sizes of the microtitech fish microsatellite markers on different individuals.
(4) Analysis of genetic diversity:
determining the genotype according to the allele size of each microsatellite amplification product, and calculating genetic diversity parameters by adopting Cervus 3.0 so as to screen microsatellite primers with polymorphism and corresponding microsatellite markers.
Through diversity detection, 22 microsatellite markers with genetic polymorphism are screened out, and the nucleotide sequences of the microsatellite markers are respectively shown in SEQ ID NO:1-22, and the information of the corresponding amplification primers is shown in Table 1.
TABLE 1 microsatellite markers for fine tail loach and corresponding primers
The 22 microsatellite markers were analyzed for genetic diversity in 50 samples of the fine tail plateau loach fish, and the results are shown in Table 2. The results in table 2 show that: the number of alleles (N) per microsatellite marker varies from 4 to 13, the average number of alleles is 6.3, the observed Heterozygosity (HO) ranges from 0.24 to 0.82, the average value is 0.593, the desired Heterozygosity (HE) ranges from 0.248 to 0.812, the average value is 0.629, the polymorphic information content PIC ranges from 0.224 to 0.779, and the average value is 0.577. Therefore, the microsatellite markers and the designed primers screened by the invention have higher genetic polymorphism.
TABLE 2 relevant information about fragment Length and polymorphism of 22 Fine tail high loach microsatellite markers
The microsatellite marker and the amplification primer thereof can also be used in the research fields of genetic structure analysis, species resource protection and the like of fine tail plateau loach fishes.
Sequence listing
<110> institute of Water engineering and ecology of national academy of sciences of Water conservancy
<120> microsatellite marker for fine tail highland loach and amplification primer and application thereof
<160> 66
<170> PatentIn version 3.5
<210> 1
<211> 278
<212> DNA
<213> microsatellite molecular marker Tst001 of fine tail plateau loach fish
<400> 1
CCGAAACTCTGCAGCAGCATCTTCTCTCTCGCCGTTGCCCTGGTGACGGTTCATTCTCACTCTTTATTCTTGCCATTATAGCATAATTCAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATACTGTATTTAAGACACTTCTCACGTCATTTAAAAAACGTTTTTTGGTGTCAAAGTGTCAGTGGACACATTTCATGTTAGAGTTTATCTGTGTAATACGTACGCGTCACTGATGTAGATGTTGGGCCAGACCTCATTACAGGCCCCTGAGGGTCG
<210> 2
<211> 278
<212> DNA
<213> microsatellite molecular marker Tst002 of fine tail plateau loach fish
<400> 2
TCTAAGTACCTCTTAGTATCACATAACAAGTGGGAGTCTTCCAAAGGAGGGAGTGTATGTGGAAGGGAGGGTGGAAATAAGACGTTTTGGACCAGAATGCCAGGCAAATAGAGCCTAAACTGAATGCTGAGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAGAATAACCCAACTTTCATGCTTCACTAACCAACGTCTGCTCCTGCCAAAATACAAACCAAGACCAGTTATTTAGCCAGTATAAAAGAAAGCTAAAATACTGATGGTGATTCTAG
<210> 3
<211>278
<212> DNA
<213> microsatellite molecular marker Tst003 of fine tail plateau loach fish
<400> 3
AATGAGAAACGACACCATTTTGATGCAATTCATTTCTGTCTTCAGCTTTTAGGAGGCGTACAACATGGCCTGAAAATTGATTAAATGTCTCCCTTGAGAGAGCGAGCGAGCGAGAGAGAGAGAGAGAGAGAGAGTCTAAACTTACCTGTTGTTGATTGTTGTGCCAAAAATCCAACGTCATTTGGTGCCTCTGAAGATTAAAGACATTTAAAAAAGATTAGACATGTCATATTTTATGCCTAATATATGTGCCTTAATGTTTGATTCTCTTCAGCCTG
<210> 4
<211> 276
<212> DNA
<213> microsatellite molecular marker Tst005 of fine tail highland loach fish
<400> 4
CAGGAACATTACCCTCCCCCACGTCTGGACTGAAAATCGGAGGCTTATTGTGGGTTGGTGTGTAGGTGTGCGTAGGTGTCTCTGAGGGAAGAGTTTGTGAGTAAGTGTGTGTGTGTTTGGAGGTCAAGGACGGTCTCATATGCCCTGCCTCTGGCTGCTCTTGCGAGTACCCTTGTGGCGTTTATTTAAAGAGCTCATCAGCCTTCGCACACGCACGCACGCACACACACACACACACAAAAACTCTATACCCAAAGTGTCCTGCCTCCCAGTCTG
<210> 5
<211> 275
<212> DNA
<213> microsatellite molecular marker Tst007 of fine tail plateau loach fish
<400> 5
TGAATCTGCTTTCATCTTTACGCTTCATTCTGCCTTCAGTACGCTTCTGTTTCACACACACACACACACACACACACACACACACACACAGTCGCGTCGGTCGCTCAGTGGGTTGACAAACAGCTTTCAGTACTTTCTGTAATTTCTGCTGTTCTGTTGAAATAACTATATCAAGCTTTCTTCAAATGAATGTTTCAAATAATAATCTATTTCTCATTAAAATGATATATTTAATAAGAAGAGCCATGCTGGTTTGTACCACGCGTGTGTTAGAG
<210> 6
<211> 274
<212> DNA
<213> microsatellite molecular marker Tst011 of fine tail plateau loach fish
<400> 6
ACTCAGCTACCTCACAGTCTGAGCTCCGTTATGATGGCGCTGGCGTCTGAGGCCTGCGCTGTTTCACTCTTCGTCATCACCTGGTGAGACTGAGCACCTGTCGTTGTGTGTGTCTGTGTGCGTCTGCCAGTCAGTCAGTCTGTCTGTCTCTTTATGTGTGTGTGTGTGTGTGTGTGTCTGTCTGTATGTACTTGTACATCTATCTTCATGAGGACCAGTGTGAGTGTTAGACCAGCAGAGTGAGGACTAAGAGAGTAAAGTGAGCACATTTTGT
<210> 7
<211> 274
<212> DNA
<213> microsatellite molecular marker Tst012 of fine tail plateau loach fish
<400> 7
TTCCAGCCGTACTCGCTGGTGGCGTAGTCGTATGGAAACGTGTGGTGGTAATTATGGAAGCCCTCGCCTGAGAAAGGCAGCAAAAGTTGGCATTGTGAAAATACATTCACGACAACATTTCATTTTGGGGGTTTTGTGTAAGCATGTTCACAAGCTCGTAAGCGTGAGGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCGACGCACCTATAGCGCTGAAGGTGACGAATCGGTTCTCTCTGGGATTGATGGTGCTGTCATACGG
<210> 8
<211> 272
<212> DNA
<213> microsatellite molecular marker Tst015 of fine tail plateau loach fish
<400> 8
CACATGTGCACATATAGACACATATAGAGCCAATAAAAGTGACTTCACTCCCCCTGCTGGTGATGATGAGAATATCTTCACTTTATATCACAAAAAAATATATAAAACATTAATGAGAGATAAAACATAAATCAGTTAATGGAGACACACAGCTTTCAAGCTCAGACACCTCATTCAATATTCTATTCAACCATTTAATGAGAGGAATGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGATAAGAGACCTGCAGTGGACGATGACAGGA
<210> 9
<211> 271
<212> DNA
<213> microsatellite molecular marker Tst018 for fine tail plateau loach fish
<400> 9
ACAGACCGACACACAGCAGTCATGCATATACACTGTATGTACAAATATCCTTATGCACGTATACAACTACACATATTTACAGTATAATTAGGTGTATTGTATGTACATCATTTGCATATTACAGTGTGTGTAAATGTGCATGCACACACAAACAAACACACACACACACACACACACACACACACTGTAAGATATATATGCAATGTTCATATTGTAAGGTGTGTATGTGACACGTTTCTCCGTCTCTTATCGCAGCGCCCAGCGCCGCTCC
<210> 10
<211> 265
<212> DNA
<213> microsatellite molecular marker Tst029 of fine tail plateau loach fish
<400> 10
TCGTTTTAATGAAATCATGTCTACATGTATATTTATAGTATATATAACGTATCGTTTAACGGTTTGGCCGTCGATGTGTTGTGTGTAAACTAAGTCAATAGGAATAAATAAAAGCGATATTGTTAAAACTAACTAACTAACTAACTAACTAACTAACTAACTAACTAACTAACTAACTGGGCAAGTTCAATTGAAATATGTCGCTTCCTGCCACGACGTACCTTTAAATGAATGAGAGTTGCTCAGGTAACCTGAGACCCAGGCG
<210> 11
<211> 262
<212> DNA
<213> microsatellite molecular marker Tst036 of fine tail plateau loach fish
<400> 11
TAGAAGCCAAACCTGTACCAGTAAAAAACACAAAGCAATTCAGTACCCACTTTGTTGAATCTATATATGATACGAAAGATAAAATGATAGGGGGTGTTATGTGAGCGTAGGATATGAAACGTGAATTCATAAAAGTTTGACAGACATCACAGTGTGTGTTTGACAGCATGAGGTCGGTCATGGCTCACAGTGCTGAAACACATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAACTCTTTCATATCGTCCTGAGGTCAAAT
<210> 12
<211> 253
<212> DNA
<213> microsatellite molecular marker Tst038 for fine tail plateau loach fish
<400> 12
AAAATTGAATTTGTAATTAATAATAATTCCTAAGATTGATTAAACTTGAATTTAAAATGCTTGTATTTCAGCTGGCTAAAGGGTCCAAAATGGATATATACAAAACTGAAAGGGAAGTCTTTTGCTGCATCACCCCTCATGTCTCTCTTGCGGCCTCTCTCTCTCTCTCTTTGTGCAGGAGCTAATAAGAAAAGGCATTCCTCATCATTTTCGAGCAATCGTTTGGCAGCTGTTATGCAACGCCACCGAAATG
<210> 13
<211> 258
<212> DNA
<213> microsatellite molecular marker Tst041 of fine tail plateau loach fish
<400> 13
TGGGAGGAAGACCACCCACCGCTCGACCATCAGTAAACGTTGTGTGTAATCTACACGAAGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCCCCACGGAATAAGACTCCAGTCTGGGCAAATTAGACGTAGGGAGGGCAGTGAGACAGAGGCAGGCGGCCACTGATGGCTCCATCCCAATAATGATTGCACAGGCACAGCCTCACATCCGCAGAGATAGACCTGCAATCTATTTCCCTTCACCTCACTGC
<210> 14
<211> 251
<212> DNA
<213> microsatellite molecular marker Tst050 of fine tail plateau loach fish
<400> 14
CTGTCACTTTTGCTTAGGGGCTGAATTACGGGTGGAAAGAGGCTCTCGTCTACGCCCAGGCCCGCTGCCAAATTGGCTGAGGATTCAGACGCTGCTATATCCCTCTTTTATTTAATTGAAGTGCTAATGAACGAGGAGACGGAGATTGCCAGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATGAGCACACACAAAAAGAAGAAAAAAAACAGAACGGCGAGAGCTTTGAGAGAGATAAAGGCTTAAA
<210> 15
<211> 250
<212> DNA
<213> microsatellite molecular marker Tst051 for fine tail plateau loach fish
<400> 15
TGTTCTTCTGTGATGGCTTTATACACCTGTGATAAACCCAGACCTGGTTTAGATGGTGGGTTACATGAGGCTTGCCATGTGTGGACATTCAGCAGCATTGAAGGTTTATTATGTCATGACGTGTGTGTGTGTGTGTGTGTGTGTGTCAGGTGCTTGCCCCACACGTGCTGACTACAGAGGGCTGATGGTGGAACGCAGCGGTCTGGCTGAAACCCTTGTGATGTCACTGCCCCTCATGGAAAACCAGCTG
<210> 16
<211> 247
<212> DNA
<213> microsatellite molecular marker Tst053 of fine tail plateau loach fish
<400> 16
TGTCAATTTCAAGTAAATTCCCACTACAAAGCTCTTTCTTCCATTTATTAGCATACGGCCGTCATGGTTTGTGTCGAGACAGGTCTGCCGTGTTGTGGCACAGCTGGGCGATTCTGAAGGGCTCCTGACACCTTCTGTCTGTTGTGTTGCCTGGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTATTACACCCGAGCAAATCTTGAGATTAAAAGGGGAAAATGTTCCTCCCACTTCCAAAGGT
<210> 17
<211> 242
<212> DNA
<213> microsatellite molecular marker Tst059 of fine tail plateau loach fish
<400> 17
TAGAATGACACTCACTGTAAATCTAGTTTAGTGTGGGTTATTAAATAGCCTTCATCAATCAGGGTCAAGGTGTTGTGAGTGTGCGCCCTTTCAGCTGCCTGTGAAAACTGAGCCTGAAGTGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATCTCAAAAGGAAGGCGGTAAACTCTCACCTTTGACCCCTTTTCGTCTTTCTTTGGAGGGGCGAGAGGTTTCTTGAACAAGTCATCTGCA
<210> 18
<211> 238
<212> DNA
<213> microsatellite molecular marker Tst062 of fine tail highland loach fish
<400> 18
AGACTGAGGAGAGTTTGAGGAACAGAGTGAGAGGAGGAACTCACGGGCGGTGCTTTTTGAGACAGTTCTCTGTTGAGTCTCTCTGTGAAGCCCTGCAGGAGTGTGTTCCCTGCAGTCACCATCACACTGCCATACAGACACTGAACACACACACACACACACACACACACACACACACACTGATGTTTGAGAACACACTGTTCTCCTTCAGTTTCTTACACACACAGATGCTTTCTTA
<210> 19
<211> 227
<212> DNA
<213> microsatellite molecular marker Tst068 of fine tail plateau loach fish
<400> 19
TGACAAGTACTAAAGAAAAAACTGTCGACACCACACAACAATCTAGCAGAAAGAGCGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGGTTGTAGACAGCAGACAGATGGAGTTGTCACAACTGTCTTTCCTCTGCGTGTCTGTTTATCAGTTTCTCTCGCCTGATTTTCATCCACTTGTACTGAAATCACTCCATGTTCATTCTGACTAAA
<210> 20
<211> 275
<212> DNA
<213> microsatellite molecular marker Tst077 of fine tail plateau loach fish
<400> 20
ATTATCTCATCGCTCTGTGTGTGTGTTCTAGAGGGAGAGATGTTTGTGTTATAATATTACGACACAATGGGATGTGAGGGATTGTTCAGAGCCCACCTGCTATGAGCATTGAATGAAGGAAACCCCAAAATAATATCTGTGTGTGTGTGTGTGTGTGTGTTAGGGGGTTATCCTGGTGGGGATTTGTGAAGCTTGTTTTGTGTGGTAAGGCCTGTAGATTAAAGTGACAAACAGTTGTTCTGAACAATAGTGAGGCGGATGTCCTGCTGCTGCTG
<210> 21
<211> 276
<212> DNA
<213> microsatellite molecular marker Tst085 of fine tail plateau loach fish
<400> 21
GCGTGACAGCCCTGTGTTTATTCATGAGGGTTTGTGTTCTTGTGGAATGTTACAGACCCATCGGACCTCACGGCAGCATCAGACAGATGTGTGTGTGTGTGTGTGTGTGTGCGGTCGAGGTCTCTGTGTGTTTTTTACGTCTCACGCAGATATCGAGAAGCGTCTCTATATCCAGTAAAACTTTATTGACTGAAAAACATTTCTTTGTTTTTGTTCAAACTCATTTTGTGTTGCTTCACAGTTTACTGCTTGAACTTTATACGGCACGAGGAATGT
<210> 22
<211> 258
<212> DNA
<213> microsatellite molecular marker Tst088 of fine tail plateau loach fish
<400> 22
CCTCTGTACTCCTGCTGGGCAGAAACAGGTGCAATACTTCTCCCATGGCTGTCAGAGCTGGAGAATTTGTTACACTATAAAAATCTGTGAGGAAACCAACCATTAGTGCCCTTTATTCCTCTCTTGCTCTCTCTGACCTGTCCCTCCCTCCCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCTACCCTAGGTGATATTTGAAACCATATTAATCAAAAGATTCATCTACTTTGTAGCCTACTGTGGACAGTGAGCGCC
<210> 23
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst001-F
<400> 23
AGCAGCATCTTCTCTCTCGC
<210> 24
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst001-R
<400> 24
AGGGGCCTGTAATGAGGTCT
<210> 25
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst002-F
<400> 25
GGAAGGGAGGGTGGAAATAA
<210> 26
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst002-R
<400> 26
TGTATTTTGGCAGGAGCAGA
<210> 27
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst003-F
<400> 27
TCTTCAGCTTTTAGGAGGCG
<210> 28
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst003-R
<400> 28
AATGACGTTGGATTTTTGGC
<210> 29
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst005-F
<400> 29
CCCACGTCTGGACTGAAAAT
<210> 30
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst005-R
<400> 30
GAGGCAGGACACTTTGGGTA
<210> 31
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst007-F
<400> 31
TACGCTTCATTCTGCCTTCA
<210> 32
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst007-R
<400> 32
ACAAACCAGCATGGCTCTTC
<210> 33
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst011-F
<400> 33
CTGAGCACCTGTCGTTGTGT
<210> 34
<211> 22
<212> DNA
<213> microsatellite primer sequence Tst011-R
<400> 34
TCCTCACTCTGCTGGTCTAACA
<210> 35
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst012-F
<400> 35
TCATTTTGGGGGTTTTGTGT
<210> 36
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst012-R
<400> 36
AACCGATTCGTCACCTTCAG
<210> 37
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst015-F
<400> 37
CCTGCTGGTGATGATGAGAA
<210> 38
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst015-R
<400> 38
ATCGTCCACTGCAGGTCTCT
<210> 39
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst018-F
<400> 39
GACCGACACACAGCAGTCAT
<210> 40
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst018-R
<400> 40
GCTGCGATAAGAGACGGAGA
<210> 41
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst029-F
<400> 41
CCGTCGATGTGTTGTGTGTA
<210> 42
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst029-R
<400> 42
GTGGCAGGAAGCGACATATT
<210> 43
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst036-F
<400> 43
GGGGGTGTTATGTGAGCGTA
<210> 44
<211> 22
<212> DNA
<213> microsatellite primer sequence Tst036-R
<400> 44
TTGACCTCAGGACGATATGAAA
<210> 45
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst038-F
<400> 45
TGGCTAAAGGGTCCAAAATG
<210> 46
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst038-R
<400> 46
TTGCATAACAGCTGCCAAAC
<210> 47
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst041-F
<400> 47
CCGCTCGACCATCAGTAAAC
<210> 48
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst041-R
<400> 48
CCCTACGTCTAATTTGCCCA
<210> 49
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst050-F
<400> 49
GCTAATGAACGAGGAGACGG
<210> 50
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst050-R
<400> 50
TCTCTCTCAAAGCTCTCGCC
<210> 51
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst051-F
<400> 51
CTTGCCATGTGTGGACATTC
<210> 52
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst051-R
<400> 52
ACATCACAAGGGTTTCAGCC
<210> 53
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst053-F
<400> 53
GGCTCCTGACACCTTCTGTC
<210> 54
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst053-R
<400> 54
CTTTGGAAGTGGGAGGAACA
<210> 55
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst059-F
<400> 55
TCAGGGTCAAGGTGTTGTGA
<210> 56
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst059-R
<400> 56
GACGAAAAGGGGTCAAAGGT
<210> 57
<211> 20
<212> DNA
<213> microsatellite primer sequences Tst062-F
<400> 57
GTCTCTCTGTGAAGCCCTGC
<210> 58
<211> 24
<212> DNA
<213> microsatellite primer sequence Tst062-R
<400> 58
AAGAAACTGAAGGAGAACAGTGTG
<210> 59
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst068-F
<400> 59
CTGTCGACACCACACAACAA
<210> 60
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst068-R
<400> 60
AAGTGGATGAAAATCAGGCG
<210> 61
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst077-F
<400> 61
CCACCTGCTATGAGCATTGA
<210> 62
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst077-R
<400> 62
CAGGACATCCGCCTCACTAT
<210> 63
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst085-F
<400> 63
TTACAGACCCATCGGACCTC
<210> 64
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst085-R
<400> 64
ACGCTTCTCGATATCTGCGT
<210> 65
<211> 20
<212> DNA
<213> microsatellite primer sequence Tst088-F
<400> 65
TACTCCTGCTGGGCAGAAAC
<210> 66
<211> 23
<212> DNA
<213> microsatellite primer sequence Tst088-R
<400> 66
TCACTGTCCACAGTAGGCTACAA
Claims (5)
1. The microsatellite marker group for the fine tail plateau loach fishes is characterized by comprising the following components in sequence as shown in SEQ ID NO: 1-22.
2. A primer pair set for amplifying polymorphic microsatellite markers of the fine tail highland loach fish of claim 1, wherein the primer pair set is:
the upstream and downstream sequences are SEQ ID NOs: 23. SEQ ID NO:24 for amplifying a nucleic acid sequence as set forth in SEQ ID NO:1, a microsatellite marker shown in figure 1;
the upstream and downstream sequences are SEQ ID NOs: 25. SEQ ID NO:26 for amplifying a nucleic acid sequence of SEQ ID NO:2, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 27. SEQ ID NO:28 for amplifying a nucleic acid sequence of SEQ ID NO:3, microsatellite marking;
the upstream and downstream sequences are SEQ ID NOs: 29. SEQ ID NO:30 for amplifying a nucleic acid sequence of SEQ ID NO:4 microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 31. SEQ ID NO:32 for amplifying a nucleic acid sequence of SEQ ID NO:5, microsatellite marking;
the upstream and downstream sequences are SEQ ID NOs: 33. SEQ ID NO:34 for amplifying a nucleic acid sequence of SEQ ID NO:6 microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 35. SEQ ID NO:36 for amplifying a nucleic acid sequence of SEQ ID NO:7 microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 37. SEQ ID NO:38 for amplifying a nucleic acid sequence of SEQ ID NO:8 microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 39. SEQ ID NO:40 for amplifying a nucleic acid sequence of SEQ ID NO: 9;
the upstream and downstream sequences are SEQ ID NOs: 41. SEQ ID NO:42 for amplifying a nucleic acid sequence of SEQ ID NO:10, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 43. SEQ ID NO:44 for amplifying a nucleic acid sequence having the nucleotide sequence of SEQ ID NO:11, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 45. SEQ ID NO:46 for amplifying a nucleic acid sequence of SEQ ID NO: 12;
the upstream and downstream sequences are SEQ ID NOs: 47. SEQ ID NO:48 for amplifying a nucleic acid sequence of SEQ ID NO:13, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 49. SEQ ID NO:50 for amplifying a nucleic acid sequence of SEQ ID NO:14, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 51. SEQ ID NO:52 for amplifying a nucleic acid sequence of SEQ ID NO:15, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 53. SEQ ID NO:54 for amplifying a nucleic acid sequence of SEQ ID NO:16, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 55. SEQ ID NO:56 for amplifying a nucleic acid sequence of SEQ ID NO:17, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 57. SEQ ID NO:58 for amplifying a nucleic acid sequence of SEQ ID NO:18, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 59. SEQ ID NO:60 for amplifying a nucleic acid sequence having the nucleotide sequence of SEQ ID NO:19, a microsatellite marker;
the upstream and downstream sequences are SEQ ID NOs: 61. SEQ ID NO:62 for amplifying a nucleic acid sequence of SEQ ID NO:20, microsatellite markers;
the upstream and downstream sequences are SEQ ID NOs: 63. SEQ ID NO:64 for amplifying a nucleic acid sequence having the nucleotide sequence of SEQ ID NO:21, microsatellite markers; and
the upstream and downstream sequences are SEQ ID NOs: 65. SEQ ID NO:66 for amplifying a nucleic acid sequence of SEQ ID NO: 22.
3. Use of a microsatellite marker set of a fine-tailed high-altitude loach fish according to claim 1 or a primer pair set for amplifying the microsatellite marker according to claim 2 for detecting genetic diversity of a fine-tailed high-altitude loach fish population.
4. The application according to claim 3, characterized in that it comprises the steps of:
(1) Extracting genomic DNA of the fine tail plateau loach;
(2) Microsatellite PCR amplification: the sequence of SEQ ID NO: performing PCR amplification by taking the fine tail plateau loach genome DNA obtained in the step (1) as a template after performing fluorescent marking on the primer pair of 23-66 respectively to obtain a microsatellite amplification product;
(3) Detecting amplified products by a sequencer: storing the amplification product obtained in the step (2) in a dark place, and carrying out capillary electrophoresis and STR analysis;
(4) Analysis of genetic diversity: and determining the genotype according to the molecular weight of each individual microsatellite amplification product of the fine tail highland loach fish, and calculating the genetic diversity parameter.
5. Use of the microsatellite marker set of the fine tail loach fish of claim 1 or the primer pair set for amplifying the microsatellite marker of claim 2 for detecting the genetic diversity level of the fine tail loach population, analyzing the genetic structure and protecting species resources.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017215055A1 (en) * | 2016-06-14 | 2017-12-21 | 中国科学院南海海洋研究所 | Litopenaeus vannamei osmotic-pressure adjustment-related functional gene est-ssr label and specific primer thereof and method for detection |
| CN108559782A (en) * | 2018-04-26 | 2018-09-21 | 水利部中国科学院水工程生态研究所 | Short body pair loach microsatellite locus and its primer and application |
| CN113373241A (en) * | 2021-06-29 | 2021-09-10 | 水利部中国科学院水工程生态研究所 | Microsatellite marker of fishes in loach, and amplification primer and application thereof |
| CN113528677A (en) * | 2021-08-11 | 2021-10-22 | 华中农业大学 | Leaf-specific notopterygium plateau loach microsatellite molecular marker, and primer and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017215055A1 (en) * | 2016-06-14 | 2017-12-21 | 中国科学院南海海洋研究所 | Litopenaeus vannamei osmotic-pressure adjustment-related functional gene est-ssr label and specific primer thereof and method for detection |
| CN108559782A (en) * | 2018-04-26 | 2018-09-21 | 水利部中国科学院水工程生态研究所 | Short body pair loach microsatellite locus and its primer and application |
| CN113373241A (en) * | 2021-06-29 | 2021-09-10 | 水利部中国科学院水工程生态研究所 | Microsatellite marker of fishes in loach, and amplification primer and application thereof |
| CN113528677A (en) * | 2021-08-11 | 2021-10-22 | 华中农业大学 | Leaf-specific notopterygium plateau loach microsatellite molecular marker, and primer and application thereof |
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