CN107460236B - Mytilus edulis microsatellite loci and application thereof - Google Patents

Abstract

The invention relates to a common mussel microsatellite locus and application thereof, wherein 15 microsatellite loci have nucleic acid sequences of SEQ ID NO 1-15 and primer sequences of P1-P30; the invention screens 15 microsatellite loci from the genome DNA of the common mussel, and designs specific primers in flanking regions at two ends of a microsatellite repetitive sequence for amplification, so that the obtained product has high polymorphism and stability, and can be used in the fields of population genetics, genetic relationship analysis, molecular marker-assisted breeding and the like of the common mussel.

Description

Mytilus edulis microsatellite loci and application thereof

Technical Field

The invention belongs to the technical field of molecular biology DNA marking, and particularly relates to a common mussel microsatellite locus and application thereof.

Background

Mytilus edulis (Mytilus edulis) is commonly known as a rainbow, with a wedge-shaped shell, a sharp tip, a slightly straight ventral edge and an arc-shaped dorsal edge. The external surface is purple brown, the internal surface is purple black or black, and the pearl luster is provided. Live in shallow sea and the byssus is attached to the substrate such as rock reef. Widely distributed in coastal areas of Liaoning, Shandong, Zhejiang and other provinces in China. Meat is eaten and the dry product is called "mussel". In recent years, the wild resources of the common mussels are sharply reduced due to excessive exploitation of the wild resources of the common mussels and environmental pollution, and the genetic diversity is sharply reduced. In recent years, the work of protection and repair of proliferation and releasing and spawning sites of the artificial breeding is developed, the artificial breeding is successful, the current resource situation of the artificial breeding needs to be evaluated, and an evaluation mechanism of the effect of proliferation and releasing needs to be established to make a reasonable releasing plan, so that the artificial breeding has important significance for protecting and recovering ocean diversity.

Microsatellite DNA (Microsatelite DNA) is one of the genetic molecular markers that have rapidly developed in recent years. It is formed by repeating and connecting 2-6 basic group short nucleotides as basic units in series to form a sequence, one microsatellite can express dozens of to hundreds of bp, and length polymorphism of each site is caused by different repetition times and incomplete repetition degree. Because the sequences at both ends of each microsatellite are mostly relatively conservative single copy sequences, a pair of specific primers can be designed according to both ends of each microsatellite, the microsatellite sequences of corresponding sites are amplified by a PCR technology, and the polymorphism of individual microsatellites of different genotypes can be displayed by electrophoretic analysis. Microsatellites are widely distributed, have high density and rich polymorphism, follow the Mendel's law of separation, have the advantages of codominant inheritance, easy PCR amplification, extremely small quantity of required DNA, low requirement on DNA quality, good result repeatability and the like, and are widely applied to the fields of genetic diversity, genetic relationship analysis, linkage map construction, functional gene positioning, molecular Marker-assisted breeding (MAS) and the like. Particularly, for aquatic organisms, due to their living environments and the like, the use of microsatellites as markers allows families to which polyculture individuals belong to be distinguished without physical markers, and facilitates the development of breeding work and the provision of breeding efficiency. Therefore, the selection of the effective microsatellite marker has important significance for the protection and the reasonable development and utilization of the common mussels.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a common mussel microsatellite locus and a polymorphic primer aiming at the current situation of the prior art, to provide 30 common mussel microsatellite loci and corresponding primers, and to provide an effective tool for population genetics, family identification and molecular marker-assisted breeding technology of the common mussels.

The technical scheme adopted by the invention for solving the technical problems is as follows: 30 microsatellite loci are provided, and the nucleic acid sequences of the microsatellite loci are SEQ ID NO 1-15 respectively.

The invention also provides a primer pair of the 15 microsatellite loci, which has a sequence shown as SEQ ID NO:1, the upstream and downstream primers are respectively P1 and P2,

wherein P1: 5'-ACCTGTTTACGACAGGGTGC-3', respectively;

P2:5’-CACCCCTCTTTTGAGGGTAA-3’。

from the sequence of SEQ ID NO: 2, the upstream and downstream primers are respectively P3 and P4;

wherein P3: 5'-TTGGTAGAATTATCCCTTTTAATGA-3', respectively;

P4：5’-GGGAGGGTAATTTTGGGCTA-3’。

from the sequence of SEQ ID NO: 3, the upstream and downstream primers are respectively P5 and P6;

wherein P5: 5'-CGGCATTCCTTTACACTTCC-3';

P6：5’-GCGATAGTAGCATGTTGGAAA-3’。

from the sequence of SEQ ID NO: 4, the upstream and downstream primers of the primer pair designed on the microsatellite locus are respectively P7 and P8;

wherein P7: 22 is 5'-AGATGAGAGCAAAGTGGCGT-3';

p8: 23 is 5'-CAGCCATGTTTTCAGTCGAA-3'.

From the sequence of SEQ ID NO: 5, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P9 and P10 respectively;

wherein P9: 24 is 5'-TCAAAACATGCAAAGGAAAGAA-3';

p10: 25 is 5'-GTGGCAGTATACCAGAAAAGGT A-3'.

From the sequence of SEQ ID NO: 6, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P11 and P12 respectively;

wherein P11: 5'-TGCAACCATGTGTTCAGTTG-3', respectively;

P12：5’-GCCCTTGATGACAAAATGCT-3’。

from the sequence of SEQ ID NO: 7, the upstream and downstream primers of the primer pair designed on the microsatellite locus are respectively P13 and P14;

wherein P13: 5'-GATGTTCCAAATGAATGGAAGTT-3', respectively;

P14：5’-CAAACCTACAACCATAAACCACA-3’。

from the sequence of SEQ ID NO: 8, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P15 and P16 respectively;

wherein P15: 5'-ACCCGGTGTGACCTGTTAAA-3', respectively;

P16：5’-CAACAATTTTATTTGTAGTTTCCGA-3’。

from the sequence of SEQ ID NO: 9, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P17 and P18 respectively;

wherein P17: 5'-AACAGTCCCAGACAATCAGGA-3', respectively;

P18：5’-CATGCATGAGGGTTTGAAAT-3’。

from the sequence of SEQ ID NO: 10, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P19 and P20 respectively;

wherein P19: 5'-ACCCGACAGTTTGAAAATCG-3', respectively;

P20：5’-CAAGCTGCGAATGAATGAAA-3’。

from the sequence of SEQ ID NO: 11, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P21 and P22 respectively;

wherein P21: 5'-TGTTTTTCTCCCCAGGAGTG-3', respectively;

P22：5’-TGGTTTGGATGGAGGATTGT-3’。

from the sequence of SEQ ID NO: 12, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P23 and P24 respectively;

wherein P23: 5'-TTGTTGACAATGCTAGTTACCG-3', respectively;

P24：5’-GCTTTATTGCTTAAAGTCGTGGA-3’。

from the sequence of SEQ ID NO: 13, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P25 and P26 respectively;

wherein P25: 5'-GCTTTCAAACCCATTTTGGA-3', respectively;

P26：5’-TCCAATAGTTGTGGCCCTTT-3’。

from the sequence of SEQ ID NO: 14, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P27 and P28;

wherein P27: 5'-TTCCATTCTTTTCCTTTTTCCA-3', respectively;

P28：5’-GATTCCACATCTTAATACACAAACAA-3’。

from the sequence of SEQ ID NO: 15, the upstream and downstream primers are respectively P29 and P30;

wherein P29: 5'-TGTACATGAAAATTAGAAACCTAGCTG-3', respectively;

P30：5’-ATTGCTTTGCATATAGATGGATTC-3’。

the microsatellite polymorphism primer is used for detecting the genetic diversity of the Mytilus edulis population, and comprises the following steps:

1) designing and synthesizing a microsatellite PCR primer: screening out a segment containing a microsatellite sequence from the constructed library, and designing a primer for synthesis;

2) obtaining of genomic DNA: extracting genome DNA of the muscle tissue of the Mytilus edulis by adopting a bio-tech DNA kit;

3) amplification of microsatellite fragments: carrying out PCR by adopting a designed primer and taking the extracted Mytilus edulis genome DNA as a template to obtain an individual Mytilus edulis microsatellite amplification product;

4) and (3) detecting a PCR amplification product by electrophoresis: detecting the microsatellite amplification product by adopting modified polyacrylamide gel electrophoresis and silver staining methods;

5) and (3) genetic parameter evaluation: the genotype was determined based on the molecular weight of the microsatellite amplification product from each individual, and genetic diversity parameters were calculated using GENEPOP 4.0.10.

The invention screens 30 microsatellite loci from the genome DNA of the common mussel, and designs specific primers in flanking regions at two ends of a microsatellite repetitive sequence for amplification, so that the obtained product has high polymorphism and stability, and can be used in the fields of population genetics, genetic relationship analysis, molecular marker-assisted breeding and the like of the common mussel.

Drawings

FIG. 1 is a graph showing the relationship between the number of parents and the success rate of identification in the application of the microsatellite primer set of the present invention.

Detailed Description

The method of the present invention is described in detail below with reference to specific examples.

1. Finding sequences containing microsatellite loci:

from the sequences of the constructed Mytilus edulis Achellatus gene library using software SSRHUNTER1.3(http:// www.biosoftNet/dna/ssrfunter. htm) to perform a search for a microsatellite sequence; the parameters are set to find sequences containing two, three and four base repeats at 5 or more times. And screening out 500 sites containing microsatellite repeats in total, and designing primers from the sites to detect polymorphism.

2. Designing a microsatellite primer:

from the gene sequences containing the microsatellite, a sequence corresponding to the primer design was selected and primer design was performed using PrimerPremier5.0. The main parameters are set as follows: the length of the primers is 18-25p, 20 are the optimal length, the length range of the PCR product fragment is 100-350bp, and the optimal annealing temperature is 55-65 ℃. The GC content is generally between 40 and 60 percent, and the mismatch and hairpin structure are avoided as much as possible.

3. Detecting polymorphism of designed primer

1) Extraction of genomic DNA:

extracting genome DNA of 30 Mytilus edulis muscle tissues by adopting a bio-tech DNA kit;

2) amplification of microsatellite PCR:

amplifying the genome DNA of the Mytilus edulis by adopting a designed microsatellite primer sequence; 10 mu L of reaction system, including 7 mu L of 4 XPower Taq PCR Master Mix (BioTeke, Beijing, China), 1 mu L of each of forward and reverse primers, and about 100ng of genome DNA; the PCR procedure was 94 ℃ pre-denaturation for 3min, followed by 35 amplification cycles, each cycle consisting of 94 ℃ denaturation for 1min, annealing for 1min (for each primer annealing temperature see Table 1), 72 ℃ extension for 1min, and finally 72 ℃ extension for 5 min.

3) And (3) detecting an amplification product by electrophoresis:

detecting the amplified product by 8% modified polyacrylamide gel electrophoresis, performing 800W constant power electrophoresis for 1-1.5 hours, then performing spotting, and performing 1200V constant voltage electrophoresis for 4-5 hours. And (3) after electrophoresis, carrying out dyeing and developing treatment, soaking the rubber plate in 10% glacial acetic acid solution for 30min, washing the rubber plate for 3min by distilled water twice, dyeing the rubber plate for 30min by 0.2% silver nitrate solution, developing the rubber plate for 30 s by 3% sodium carbonate, terminating the reaction to show amplified product fragments, and detecting the allele position by using 10-bp DNA ladder (Invitrogen Inc.) as marker.

4) And (3) genetic parameter evaluation:

determining the genotype according to the molecular weight of each individual microsatellite amplification product, and calculating genetic diversity parameters by adopting GENEPOP 4.0.10 so as to screen the microsatellite primers with polymorphism and corresponding sites.

Through diversity detection, 25 microsatellite loci with genetic polymorphism are screened out in total, the nucleotide sequences of the microsatellite loci are respectively SEQ ID NO. 1-25, and the information of the designed polymorphic primers is shown in Table 1

Table 1: annealing temperature, fragment length, polymorphism, etc. of 15 microsatellite loci

Wherein Na represents the number of alleles, H_ORepresenting observation of heterozygosity, H_ERepresenting the desired heterozygosity.

The primers of the invention are used for amplifying the genome DNA of 30 mussel muscle tissues, and the genetic diversity analysis result shows that the number of alleles at each microsatellite locus is different from 4 to 17, and the average number of alleles is 7.3. Observed Heterozygosity (HO) ranges from 0.069 to 1, with expected Heterozygosity (HE) ranging from 0.102 to 0.891; thus proving that the microsatellite loci screened by the invention and the designed primer have genetic polymorphism.

5) And (3) identification effect analysis: the method comprises the steps of estimating the ability of the 15 microsatellite loci to carry out family identification in a population according to different allele frequencies of the 15 microsatellite loci of 30 Mytilus edulis individuals by using CERVUS 3.0 software, selecting parent pairs (2, 100 and 500) with different quantities, simulating the number of offspring as 10000, the parent detection rate as 100 percent, the locus detection rate as 100 percent, the typing error rate as 1 percent, the 95 percent confidence level and the unknown parent sex, and simulating the identification success rate of the 15 microsatellite loci.

6) The results show that: the higher the detection success rate with increasing number of polymorphic microsatellite loci (FIG. 1). Under the condition that the number of the offspring is 10000 and the genotype information of the parents is not clear, the identification success rate of 7-8 microsatellite locus numbers is about 80 percent, and the identification success rate of more than 9 microsatellite locus numbers can be 100 percent.

The results show that the 15 sites are available for inter-offspring identification below the 500 pairs parent. The primer designed by the invention can be used for successfully analyzing mass family materials of the Mytilus edulis and identifying any offspring to the family to which the Mytilus edulis belongs.

The microsatellite primer disclosed by the invention can also be used for the research in the fields of genetic diversity, releasing effect evaluation, molecular marker-assisted breeding and the like of the common mussels.

Sequence listing

<110> Ningbo university

<120> Mytilus edulis microsatellite loci and application thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 688

<212> DNA

<213> Mytilus edulis

<400> 1

atagtttaga caaggaacgt ttagtgatat atttgattct ttttccgaac accaccacca 60

atcttttcat ttggtaacct tatacctgtt tacgacaggg tgctccgaag tcaacggagg 120

aatttaccag cacaacaaca acaacaatac tgactgttga caattatcac tcacttattt 180

aaagtttatt ttagatttta gcaagtgtat atatattcac ggagtgtgta catacttaaa 240

gacttttatc catattaccc tcaaaagagg ggtgttccta atagaggaag ctcatacgaa 300

gaaaagaagg taaatcaata gaaagtagaa ttgcgaattt ttggattatt ctatttttag 360

aaaaaccaat acacataaaa gcactattta taaatgttgt ttcgtggtac ctaaataata 420

ggaaattgga tcgggaaaga aacgtgaaat tatcgcaatt agtgaagaaa ttgatcacta 480

cttcgaagtt ttataaatat ctacacagca gtgaacattt taaatttact agttaataaa 540

ttttatcaag ttaaaactga agaatatcct catctcatgg gcttttcgtg ctattaattt 600

tttgtgacat cacaatttta attgagaaag caagcatagt acatattcat gcatatataa 660

agtacgaaaa cattgatctg atcatatg 688

<210> 2

<211> 322

<212> DNA

<213> Mytilus edulis

<400> 2

atctgaaaaa cgaattaaga tgactaaaat aactataaag aacatttccg atgtaaaatt 60

tacaatccag agttagaatt tattggtaga attatccctt ttaatgataa aatcacaaaa 120

aaatgcaaaa ttaaccgata tatttgagat tcatatatat atatatatgg ctgttttcaa 180

acgttttatt ttgaagaaag ctttaaagta aaaacattaa acacaattat ttgcgaatag 240

aaaaaagaaa acaaacagtt gatttaagga aaatctattt atctatcatc gaaattagcc 300

caaaattacc ctccccccca ag 322

<210> 3

<211> 811

<212> DNA

<213> Mytilus edulis

<400> 3

taatgctctt cgaaatgaga ggggatacca acaatgtaat agaaaagtat tataattaca 60

tacttattta catcttttat tttacccacg tcaatagata ctatttcaca acattgaagt 120

ttcacttcgt tatgaaatat caaaatccaa ttaagtatcc atatgtttca ttagataagt 180

gattagataa cgttgttgga cggagtctga cggcattcct ttacacttcc tcacaggtat 240

aaatagacag gtaaacaaat catttcttca cgaatgaatt acttcaatat agatattcac 300

atatatatat atatatatat atttatttgt gtagattgtc aacatgagac cttatcacgt 360

tatctagggt aagtaaagct tatctatatt aactacacta tttaagtgca ctttaagttc 420

attttaatgt aataacaata tataatggaa acagtatgca aacttatttc caacatgcta 480

ctatcgcttt agcctgataa gaaaagtatc attcattcgc cgtgtaaatt aatcatatca 540

tcaacccgtc atgataaact ttttatataa gcgtaacgaa cggccctcga taaaagataa 600

ctatatttga ccaaatcata tctttagccc agctatcaat atcttttttt ctcaaataat 660

caaagtagtc tctactattc ttcgatttcc acggtagcag tcacaaaagc aggtccatac 720

gttgatccac attttatgaa gcttgatttg aatttgcatc aactattcaa atcagaccgt 780

attgaatata acgacagaga tgcacaatca t 811

<210> 4

<211> 412

<212> DNA

<213> Mytilus edulis

<400> 4

gcctttcttc cgctttcgtg ttgctgatat atagcaagat gagagcaaag tggcgtaaga 60

agcgtatgag gaggttgaag cgtaaaagga ggaagatgag ggcaaggtct aagtagacgt 120

tgccatcgtt tatttttaag actccaattg atgtgttcag ctatagacca aggtattgca 180

accatgtgtt cagttgagaa ctcctattga tgatgatgat gatgatgaaa acgatgatga 240

tgaattagcg tggtcacgtg atcaaagtct tagtttgacc aggtaccatg ctttcgactg 300

aaaacatggc tgttttaaca acaatatgtt gtcatggtca tccaacacga cttacaacaa 360

cagcaacaca aagcaacaac aacattttgt catcaagggc tcaacttttg gg 412

<210> 5

<211> 225

<212> DNA

<213> Mytilus edulis

<400> 5

ggtttcttta ttctttcttt ttatcttttt attttgcact aagattacac caagacagtc 60

aggttactgt attgaaagaa gaaaaataaa gagaaactta tttcaaaaca tgcaaaggaa 120

agaaataaaa ggataaatat atatagagag agagagacat acttattttc ttccaaatcc 180

tacagaaaaa acaagaatgt tatacctttt ctggtatact gccac 225

<210> 6

<211> 519

<212> DNA

<213> Mytilus edulis

<400> 6

tctaagtaga cgttgccatc gtttattttt aagactccaa ttgatgtgtt cagctataga 60

ccaaggtatt gcaaccatgt gttcagttga gaattcctat tgatgatgat gatgatgatg 120

aaaacgatga tgatgaatta gcgtggtcac gtgatcaaag tcttagtttg accaggtacc 180

atgctttcga ctgaaaacat ggctgtttta acaacaatat gttgtcatgg tcatccaaca 240

cgacttacaa caacagcaac acaaagcaac aacagcattt tgtcatcaag ggctcaactt 300

tgggaactat ctttgaagaa gaaagaggaa gaagaagaat tggtgtctac tgcgtggaag 360

tctccatggt gaccaggcta ggcatctgta tggtaaccac tgttgaggct ttggtggctc 420

ctctatgana acatattttg taatatctta aacttggcaa tgatccaatt tatccaaaga 480

catgaaattg catggattga attgttcagt tgaggtttt 519

<210> 7

<211> 299

<212> DNA

<213> Mytilus edulis

<400> 7

gatgttccaa atgaatggaa gttccataca tatttatata tgtgtgtgtg tgtgtgcact 60

tacataaggt ccatttcttt ctgatgcagc tcaaatatta tgattaagag atcttgtaga 120

acatgtattt taaaagaagt tctggtagtt gttaaatatg tatatctctc agtacttatt 180

aacagtaaat gtggtttatg gttgtaggtt tgtttatgag ctttaaattg ctgatttctt 240

tatatgttat caaagtatca gttttataca gatttataca agaaataaag tcttaacgc 299

<210> 8

<211> 200

<212> DNA

<213> Mytilus edulis

<400> 8

aatttaatta gtctattttt ctatgtttat cagaaatccc gcgttgtaca tacattgcct 60

ttgttaatca aaaaagttta ccacacccgg tgtgacctgt taaaagattt atactaatat 120

atatatattt atttatgtag aaaaatttgt tattttttta tacataaaat atcggaaact 180

acaaataaaa ttgttgttgg 200

<210> 9

<211> 563

<212> DNA

<213> Mytilus edulis

<400> 9

tgcgttctcc tggtcctgca ggagcacctc tcatcttggc accaagaatg caaaacatcc 60

ccacaagttc agccggcctg atcgggggtc ctccaccacc tcttgtttca ccaacagatg 120

ccagtggtac tgctagtctg atatataatc cttacagtga tttcccttat gggtattcac 180

cggcggcatc aatcattgaa tatcccagtc acttatacca gactgcagct ggtatgtttt 240

ctttcgtgcg ttaaatgttt cagtcatcag tcattccatt ttacacaaca tttaagaaca 300

gtcccaaact gacaatcaac agtcccagac aatcaggatt tattccaatc aaatcccatt 360

attattatga tttatatata tatatatatg tttaatttgt tttataatct acaattttaa 420

attatacctt aatatttata tgtacagtat ttctataaaa ttgttattac gttcataaat 480

tttttatttc aaaccctcat gcatgaaatt acagtgaaaa ttgctaggta tatttaattt 540

gactggcaaa aattagaggg aaa 563

<210> 10

<211> 809

<212> DNA

<213> Mytilus edulis

<400> 10

caacggtcta gtctatataa aagcatggat atataacatt agctgtgcac ttgagaggtg 60

taaatatgca tgtaaatatg ttttttaatt gtcaagtcac attcaagtca gaagttttat 120

ataaagtcca tgcgtattta cgtattttac ataagctatt gacattttgt ttaatcgact 180

atagctacat aaaacatata ttttttacat acaaggttat ataaaaagca atatttgtgg 240

caaatgatag agccttggtt gtcttaattt cttatcttga tcaattgcag ctatgattat 300

ggtattgaca taaatatgct gattgtttcc ttgaattttt catacgtttt tcgatttggg 360

aaccttttta tagtttaccc gacagtttga aaatcgaaca aaatgttttc aacgatacta 420

acaaacgaat gattaaatgt ctaatggaag atgataaaaa ctgaaagatt gttttattat 480

gaagacacaa agatgtagag tacaaggaag ctgacattat atatatatat atatataaca 540

attgtttttc attcattcgc agcttgtgtg ggcaaagagc atcgaagttg gatgtggtat 600

atatacttgt ccaccaaatg gtcagttccg agaaaagttt ctgtaccttt cctgttttta 660

caatccaccg taagttgttc aaaatttgtt ttttgtaaac aatggataaa gaaccacgaa 720

ggtgatgtat tgtatataaa tttcccaaag aaacactaag atagagcatt aactaaccgc 780

gctgtcattg gtgggctcaa accatctta 809

<210> 11

<211> 683

<212> DNA

<213> Mytilus edulis

<400> 11

gttatagatc tagtgcatgt ttaaatccca aggtgaaatc ttcttgtttt tctccccagg 60

agtgcagatc ttttttgtcg tagtatatat gcataataaa tatattttaa atctaagcag 120

gtaaattaaa catagggcat ttaaagttaa ctgcttgcta tttattaaaa caaaaatttg 180

aaaatatggc aaaactatat atatatatga ttgattgatg gtaaatagat ataagaatat 240

ttggtatgag tgccaatgag acaatcctcc atccaaacca ttataggtca aagtatgtcc 300

ttcaacactg agcctttaac agcaagcttt aaagggcccc aaaatgactg gtgtaaaaca 360

attaaaaatg gaaaccgatg gtctgatcta tattaaatac acttatgaac cacatcagcc 420

aatgacaacc aataaacatc aggttcctga ctaaggacag gtgcaaacaa atgcagcagg 480

gtttactgtt ttatgtgccc attttactgt agaaggaatg tttctaactt tttaagaaga 540

aacgtcttaa aatatctatg tgctgcagtg acacaaacac agtatttaaa cagaggggaa 600

agggagtatg gactatgtag ttgccattgg ggagtaactc tgtaaaccca gaaagatgtt 660

ggcaaaacaa agacaaaatc cct 683

<210> 12

<211> 402

<212> DNA

<213> Mytilus edulis

<400> 12

gagcaaccac tttgcaaaaa tcatcatcaa caatacctaa ttaattttta atatctgtat 60

tatggcatag aattgttgac aatgctagtt accgtatggt tccattaatg gtcatttatt 120

aataattaag acattaataa ttagttaacc acttttatga tcatagttct taaattcaat 180

tatggaagac aatataattg aaaacaaagg acatatggta tgtaaattcg cctattcaaa 240

atctaaaggc atatatatat atatgggtaa ataatgctct gtattgcttt tatgtccacg 300

actttaagca ataaagcatt ttagtgtact ctttgaaatg tttttcaaag agtatgaaac 360

agcaaactta agtgtcacga aataaacatt ttttagctct tg 402

<210> 13

<211> 649

<212> DNA

<213> Mytilus edulis

<400> 13

tttcatgatt gaattgtgta ttactagtaa tatatattgt aaattaatgt atgaatgtat 60

gaatgttgac tgtatgcatg tattttgttt gagggcctca atgaaaatta gaatatttct 120

aattgagtta ccctctttaa ataaagaatt tattattatt attattatta ttattatagg 180

atctggtggt atggaccctt cttttttctt attttttaaa acttttagta ttcaaataat 240

ctgtaatatc aaaagactta agtaaattaa tagtttatct aatggacttc tggttatatc 300

taattcattt tcactaattt gtttgaatta attttgttgt gctttcaaac ccattttgga 360

atcaggcatt tgtgttaaat ttttttaatg gtgaattctc tgtttaattt aaccaaggat 420

ttgtaatagt cttactcaca ggcgtgtcag aatttttgtt cttatatata ccttaatata 480

atataatata tatatatcga tttactaagg ggaaaaaaat tctcaagtgc atgtgacccc 540

ctaatccatc cccccctttt ttttttagca cattatgtac atttactaaa gggccacaac 600

tattggaaag gtcaattcaa tttgtcacaa tgatatacat gtaagaaac 649

<210> 14

<211> 250

<212> DNA

<213> Mytilus edulis

<400> 14

ttttcctttt tccatttttt tccagcatct ctctctctct ctctctgtct ttctgtcttt 60

atttagttat tgtttgtgta ttaagatgtg gaatcgatgt aagcacaccg acaacagaca 120

tgtctttctg tctttattta gttattgttt gtgtattaaa gatgtggaat cgatgtaagc 180

acaccgacaa tcagacaatg agacaaatgt gcccagccca aatgaaaatc ataccacatc 240

ttcttttttt 250

<210> 15

<211> 280

<212> DNA

<213> Mytilus edulis

<400> 15

tgtacatgaa aattagaaac ctagctgaac ccctcttccc ccccatctct ctctctctct 60

tttatttctt cttgttgaat ccatctatat gcaaagcaat acatagacaa aaaataaatt 120

ttatgatata tattatatac acacaagtct catcacaaaa aaaagggggg tgtttgttta 180

tcatggtaca tgtacgtcct tgtgtgtaga atattaaata gtacattata taaaaaaaag 240

atcaacatat gaatgtttct atctgtactt ctgttctttt 280

Claims (1)

1. A primer pair for amplifying microsatellite loci of Mytilus edulis, wherein the microsatellite loci comprise microsatellite loci with sequences of SEQ ID NO 1-15, the primer pair is designed from each microsatellite locus, and the sequence of each microsatellite locus is SEQ ID NO:1, the upstream and downstream primers are respectively P1 and P2;

wherein P1: 5'-ACCTGTTTACGACAGGGTGC-3', respectively;

P2:5’-CACCCCTCTTTTGAGGGTAA-3’；

from the sequence of SEQ ID NO: 2, the upstream and downstream primers are respectively P3 and P4;

wherein P3: 5'-TTGGTAGAATTATCCCTTTTAATGA-3', respectively;

P4：5’-GGGAGGGTAATTTTGGGCTA-3’；

from the sequence of SEQ ID NO: 3, the upstream and downstream primers are respectively P5 and P6;

wherein P5: 5'-CGGCATTCCTTTACACTTCC-3';

P6：5’-GCGATAGTAGCATGTTGGAAA-3’；

from the sequence of SEQ ID NO: 4, the upstream and downstream primers of the primer pair designed on the microsatellite locus are respectively P7 and P8;

wherein P7: 22 is 5'-AGATGAGAGCAAAGTGGCGT-3';

p8: 23 is 5'-CAGCCATGTTTTCAGTCGAA-3';

from the sequence of SEQ ID NO: 5, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P9 and P10 respectively;

wherein P9: 24 is 5'-TCAAAACATGCAAAGGAAAGAA-3';

p10: 25 is 5'-GTGGCAGTATACCAGAAAAGGT A-3';

from the sequence of SEQ ID NO: 6, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P11 and P12 respectively;

wherein P11: 5'-TGCAACCATGTGTTCAGTTG-3', respectively;

P12：5’-GCCCTTGATGACAAAATGCT-3’；

from the sequence of SEQ ID NO: 7, the upstream and downstream primers of the primer pair designed on the microsatellite locus are respectively P13 and P14;

wherein P13: 5'-GATGTTCCAAATGAATGGAAGTT-3', respectively;

P14：5’-CAAACCTACAACCATAAACCACA-3’；

from the sequence of SEQ ID NO: 8, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P15 and P16 respectively;

wherein P15: 5'-ACCCGGTGTGACCTGTTAAA-3', respectively;

P16：5’-CAACAATTTTATTTGTAGTTTCCGA-3’；

from the sequence of SEQ ID NO: 9, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P17 and P18 respectively;

wherein P17: 5'-AACAGTCCCAGACAATCAGGA-3', respectively;

P18：5’-CATGCATGAGGGTTTGAAAT-3’；

from the sequence of SEQ ID NO: 10, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P19 and P20 respectively;

wherein P19: 5'-ACCCGACAGTTTGAAAATCG-3', respectively;

P20：5’-CAAGCTGCGAATGAATGAAA-3’；

from the sequence of SEQ ID NO: 11, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P21 and P22 respectively;

wherein P21: 5'-TGTTTTTCTCCCCAGGAGTG-3', respectively;

P22：5’-TGGTTTGGATGGAGGATTGT-3’；

from the sequence of SEQ ID NO: 12, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P23 and P24 respectively;

wherein P23: 5'-TTGTTGACAATGCTAGTTACCG-3', respectively;

P24：5’-GCTTTATTGCTTAAAGTCGTGGA-3’；

from the sequence of SEQ ID NO: 13, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P25 and P26 respectively;

wherein P25: 5'-GCTTTCAAACCCATTTTGGA-3', respectively;

P26：5’-TCCAATAGTTGTGGCCCTTT-3’；

from the sequence of SEQ ID NO: 14, designing primer pairs on the microsatellite loci, wherein the upstream and downstream primers are P27 and P28;

wherein P27: 5'-TTCCATTCTTTTCCTTTTTCCA-3', respectively;

P28：5’-GATTCCACATCTTAATACACAAACAA-3’；

from the sequence of SEQ ID NO: 15, the upstream and downstream primers are respectively P29 and P30;

wherein P29: 5'-TGTACATGAAAATTAGAAACCTAGCTG-3', respectively;

P30：5’-ATTGCTTTGCATATAGATGGATTC-3’。

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