CN106167825A - Microsatellite marker that a kind of yellow catfish growing characteristic is relevant and detection thereof and application - Google Patents

Abstract

The invention discloses the relevant microsatellite marker of a kind of yellow catfish growing characteristic and detection thereof and application；In Pelteobagrus fulvidraco GH sequence, i.e. find 4 microsatellite locus, by screening special primer amplification microsatellite locus, analyze the relatedness of itself and growth traits, find the preponderant genotype favourable to growth traits.Result shows wherein to have three sites and growth traits to have notable or pole significant correlation, and site GA has with body weight, body length, height, body thickness and significantly associates, and has with total length, caudal peduncle height and significantly associates, and wherein CD is preponderant genotype；Site TCTT is high with long, caudal peduncle to be had and significantly associates, and wherein BD is preponderant genotype；Site AC and body be thick, long have significantly associate, wherein DD is preponderant genotype, 4 microsatellite locus utilizing primer amplification of the present invention to obtain all have higher genetic diversity, and have significantly or extremely significant relatedness with growth traits, the present invention puts into practice tool for molecular breeding and is of great significance.

Description

Microsatellite marker that a kind of yellow catfish growing characteristic is relevant and detection thereof and application

Technical field

The present invention relates to molecular marker Biotechnology in Genetic Breeding field, more particularly, to a kind of yellow catfish growing characteristic phase The microsatellite marker closed and detection and application.

Background technology

Pelteobagrus fulvidraco is the small-sized economic freshwater Fish that China extensively cultivates；Because of its fine and tender taste, nutritious, dressed fish is high Very popular, Pelteobagrus fulvidraco breeding parent is mainly the wild population fishing for or cultivating number generation, not through artificially breeding, Its speed of growth, resistance against diseases and culture performance are more weak, and the two kinds of selection-breeding work therefore carrying out Pelteobagrus fulvidraco have the meaning of reality.

The most existing many scholars have carried out substantial amounts of research in terms of Pelteobagrus fulvidraco genetic breeding, and achieve many one-tenth Really.Along with the development of biotechnology, genetics-breeding in fish technology has had increasing method, and molecular marker is primarily referred to as can Reflection is individual biological or the genetic fragment of some diversity in genome between colony, has that polymorphism is high, widely distributed, codominance Etc. a series of advantages, at aspects such as genetics-breeding in fish and genetic linkage maps structure, Relationship iden-tification, research of fruit germplasm resources Have a wide range of applications.

Microsatellite marker (microsatellite), be also called STR (short tandem repeats, STRs) or simple repeated sequence (SSRs), it is the simple repeated sequence being uniformly distributed in eukaryotic gene group, by 2～6 The tandem repeat of nucleotide is constituted, due to the number of repetition of recurring unit between individuality in variability and quantity is rich Rich.Each microsatellite DNA is made up of core sequence and flanking sequence, and its core sequence is tandem sequence repeats arrangement, flanking DNA Sequence is positioned at the two ends of core sequence, for conservative special single-copy sequence, microsatellite can be made to be positioned chromosome specifically normal The specific part in chromatin district.

Microsatellite marker is the second filial generation molecular marker based on PCR, and up to now, micro-satellite labeling technique is important Economic fish is widely used, becomes research population genetic diversity, population genetic variations, genetic map construction, merit One important technology of energy gene mapping and QTL analysis etc..Molecular marker is utilized to cultivate the high-yield variety that growth traits is excellent It is one of important channel of increasing economic efficiency, it is thus achieved that the gene relevant to yellow catfish growing characteristic and gene loci can assist height Produce the selection-breeding of Pelteobagrus fulvidraco, there is presently no the report of the gene relevant to yellow catfish growing characteristic and gene loci.

Summary of the invention

The technical problem to be solved is the drawbacks described above overcoming prior art to exist, it is provided that a kind of and Pelteobagrus fulvidraco The microsatellite marker that growth characteristics are relevant.

Second object of the present invention is to provide the primer for the above-mentioned microsatellite marker of specific detection.

Third object of the present invention is to provide the application of above-mentioned microsatellite marker.

It is an object of the invention to be achieved by the following technical programs:

A kind of primer of the microsatellite relevant to yellow catfish growing characteristic for amplification, the nucleotide sequence of primer such as SEQ ID Shown in NO:4～9, wherein, the repetition motif of the microsatellite fragment of primer SEQ ID NO:4 and SEQ ID NO:5 amplification is (GA)_n1, the span of n1 is 17～35, described microsatellite fragment and the body weight of Pelteobagrus fulvidraco, total length, body length, height, body be thick, Caudal peduncle height is correlated with；The repetition motif of the microsatellite fragment of primer SEQ ID NO:6 and SEQ ID NO:7 amplification is (TCTT)_n2, n2 Span be 6～14, described microsatellite fragment and Pelteobagrus fulvidraco long, caudal peduncle is high relevant；Primer SEQ ID NO:8 and The repetition motif of the microsatellite fragment of SEQ ID NO:9 amplification is (AC)_n3, the span of n3 is 25～35, described microsatellite Fragment and the body of Pelteobagrus fulvidraco be thick, long relevant.

Preferably, the solution temperature of the microsatellite fragment of primer SEQ ID NO:4 and SEQ ID NO:5 amplification is 56 DEG C, Clip size is 311bp；The solution temperature of the microsatellite fragment of primer SEQ ID NO:6 and SEQ ID NO:7 amplification is 61 DEG C, Clip size is 272bp, and the solution temperature of the microsatellite fragment of primer SEQ ID NO:8 and SEQ ID NO:9 amplification is 56 DEG C, Clip size is 220bp.

Applicant is found by research, and by above-mentioned micro-satellite primers amplified sample, what energy was special amplifies microsatellite marker (or being called microsatellite locus), these microsatellite markers are closely related with the growth form of Pelteobagrus fulvidraco, therefore can be used for yellow forehead The molecular breeding of fish.

The present invention also provides for the test kit containing described micro-satellite primers.

According to embodiments of the present invention, can be by detecting above-mentioned microsatellite marker, it is possible to effectively determine the complete of Pelteobagrus fulvidraco Long character, therefore, the present invention also provides for described micro-satellite primers or the application in Pelteobagrus fulvidraco genetic breeding of the described test kit.

The present invention also provides for a kind of method detecting Pelteobagrus fulvidraco total length character, comprises the following steps:

S1. sample DNA is extracted；

S2. utilize the primer amplification sample DNA of SEQ ID NO:4～9 described in claim 1 or 2, analyze micro-in sample DNA defending The type of asterisk note and genotype, determine the total length character of Pelteobagrus fulvidraco.

The present invention also provides for the method for the excellent Pelteobagrus fulvidraco of a kind of selection-breeding, comprises the following steps:

S1. sample DNA is extracted；

S2. utilize the primer amplification sample DNA of SEQ ID NO:4～9 described in claim 1 or 2, analyze micro-in sample DNA defending The type of asterisk note and genotype；

S3. result judges: if containing repeating motif (GA) in sample DNA_n1, then Select gene type be the sample of CD be excellent yellow forehead Fish；If the motif containing restructuring (TCTT) in sample DNA_n2, then Select gene type be the sample of BD be excellent Pelteobagrus fulvidraco；If sample DNA In containing repeat motif (AC)_n3, then Select gene type be the sample of DD be excellent Pelteobagrus fulvidraco.

Compared with prior art, the method have the advantages that

The present invention obtains Pelteobagrus fulvidraco GH full length gene sequence first, by SSRHunter software find in GH sequence 4 micro- Satellite site, analyzes the relatedness of Pelteobagrus fulvidraco microsatellite locus and growth traits first, through variance by SAS software GLM model Analytical control is the site of significant difference, utilizes Duncan method that the genotype in significant difference site is carried out multiple ratio Relatively, the preponderant genotype favourable to growth traits is found.Assay is: wherein have three sites and growth traits have notable or Pole significant correlation, site GA has with body weight, body length, height, body thickness and significantly associates, and has with total length, caudal peduncle height and significantly associates, its Middle CD is preponderant genotype；Site TCTT is high with long, caudal peduncle to be had and significantly associates, and wherein BD is preponderant genotype；Site AC with Body is thick, long have notable association (P ＜ 0.05), and wherein DD is preponderant genotype, the microsatellite locus of this test and growth traits The probability having notable relatedness is bigger, and this is likely to be due to newfound 4 microsatellites and is positioned on GH gene, and its polymorphism is to yellow forehead The growth promoter of fish has directly impact.4 microsatellite locus that the study find that all have higher genetic diversity, and Site GA, TCTT, AC have significantly with growth traits or extremely significant relatedness, and the present invention has for molecular breeding practice Highly important meaning.

Accompanying drawing explanation

Fig. 1 is common amplification PCR result, and wherein, Fig. 1 (1) is site AT, GA primer electrophoresis result；Fig. 1 (2) is site TCTT, AC primer electrophoresis result；The most left band is MarkerDL2000, from top to bottom stripe size respectively 2000bp, 1000bp, 750bp、500bp、250bp、100bp。

Fig. 2 is microsatellite molecular marker PCR primer genotyping result on ABI3730xl；Wherein, Fig. 1 (1) is AT site Typing；1(2) it is GA site typing；1(3) it is TCTT site typing；1(4) it is AC Locus Analysis in Shoots.

Detailed description of the invention

Further illustrate present disclosure below in conjunction with Figure of description and specific embodiment, but should not be construed as right The restriction of the present invention.In the case of present invention spirit and essence, the amendment that the inventive method, step, condition are made Or replace, belong to the scope of the present invention.Unless otherwise noted, experimental technique used in embodiment is people in the art Conventional method known to Yuan and technology, reagent or material are and are obtained by commercial sources.

The present invention uses the method for homologous clone to obtain Pelteobagrus fulvidraco GH total length (sequence is as shown in SEQ ID NO:1), altogether 3117bp, 5 exons (starting to be called exons 1, exon 2, exon 3, exon 4 and exon 5 from 5 ' ends), 4 introns (starting to be called introne 1, intron 2, introne 3, intron 4 from 5 ' ends), 5 ' UTR are 165bp, 3 ' UTR is 485bp.

The screening of embodiment 1 microsatellite locus and typing

Utilize the microsatellite locus in SSRHunter software search Pelteobagrus fulvidraco GH full length gene sequence, determine microsatellite locus class The information such as type, position and length.Use Primer Premier 5.0 software about upstream and downstream 150bp of SSRs site Design upstream and downstream primer.Primer information is shown in Table 1.

In order to realize the semi-automatic detection of the fluorescence of microsatellite polymorphism, to general primer 5 ' or 3 ' ends are needed to carry out glimmering The labelling of light modification group.The PCR primer that fluorescent primer amplification obtains is carried out on 96 hole capillary tube 3730xl DNA analysis instrument Electrophoresis detection, fluorophor can send coloured light under the effect of laser, and system collects the fluorescence letter of different colours automatically Breath, the final data message obtaining PCR primer.

The primer in table 1 is utilized to carry out regular-PCR amplified reaction, the reaction condition optimal to grope primer, reaction system For table 2.

PCR response procedures: 94 DEG C of 5 min of denaturation；(degeneration 94 DEG C of 30 s, Tm draw according to different in 38 circulations Thing is different, and anneal 51～56 DEG C of 30s, extends 72 DEG C of 25s)；72 DEG C of 10 min of rear extension, 4 DEG C of forever. Product carries out 2% agargel electrophoresis detection, and electrophoresis uses gel imaging system take pictures and preserve after terminating；Whole process lucifuge Operation, wraps the PCR primer masking foil that can amplify purpose band and send Hua Da genome company to carry out typing detection.

3 kinds of fluorescent decoration groups of this experimental selection, AT and AC site selects FAM(green, green), GA site be HEX (yellow, yellow), TCTT site are TAMRA(yellow-orange, crocus).

Result shows: 4 microsatellites detected on GH gene, and site is distributed in introne 1, introne 3,3 ' UTR, all Belonging to simple SSR, core sequence is respectively (AT) n, (GA) n1, (TCTT) n2, (AC) n3.Be respectively designated as AT, GA, TCTT, AC.Before fluorescent primer synthesizes, first carry out PCR amplification to check the quality of primer amplification, then resynthesis with general primer Fluorescent primer, and at 5 ' end mark fluorescent groups of upstream (or downstream) primer, the most each SSRs site energy Enough find the respective value of multiple annealing temperature and prove that these 4 SSRs sites can be amplified.

The genotype utilizing microsatellite PCR product application 96-capillary tube 3730xl DNA analysis instrument to carry out SSRs judges, tool Body step is:

(1) diluting the pcr amplification product of SSR: take one piece of 96 hole PCR plate, every hole adds 10ul ddH₂O, then every hole adds sample Product 1ul(multitube mixing is also so operation).Dilution factor on-fixed, depending on the concentration of each SSR amplified production.Slightly centrifugal mixed Even.

(2) preparation internal standard ROX500 mixed liquor: take a 1.5ml centrifuge tube, adds and changes 1020ul Hi-Di into Formamide and 60ul ROX500, vortex oscillation mixing (the amount of reagent ratio of each reaction is Hi-Di Formamide: ROX500=8.5ul:0.5ul).

(3) separately taking one piece of 96 new hole PCR plate, every hole adds diluted sample mixed liquor 1ul, and the most every hole is separately added into 9ul ROX500 mixed liquor, slightly centrifugal mixing.

(4) denaturing samples, 95 DEG C 4 minutes, 4 DEG C 1 minute.

(5) upper sequenator 3730xl electrophoresis.

(6) application software GeneMapper ID v3.1 software is to the repeatability of electrophoresis, the analysis result of fragment and base Because genotyping result checks, finally export result, such as Fig. 2.

Wherein the small peak on main peak side is shadow peak, the most then be referred to as shadow band/ghost band, be because The slip mispairing of Taq enzyme causes.STRs genotyping result is from top to bottom:

AT: fluorescent labeling is FAM, and molecular size range is 252 to be homozygote；

GA: fluorescent labeling is HEX, and molecular size range is 321,342 to be heterozygote；

TCTT: fluorescent labeling is TAMRA, and molecular size range is 286 to be homozygote；

AC: fluorescent labeling is FAM, and molecular size range is 216,230 to be heterozygote.

Embodiment 2 SSRs allele and distribution

The present embodiment experiment sample totally 160 tail, site AT detects that 3 allele, site TCTT and site AC detect 4 Allele, site GA detects 5 allele.Allele and the distribution in each site are shown in Table 3.

Embodiment 3 SSRs heredity growth characteristics are analyzed

In research, the Pelteobagrus fulvidraco Population genetics analysis of 4 SSR polymorphic sites is shown in Table 4.The average heterozygosity in 4 sites is 0.5661, site AT and TCTT are moderate polymorphic, and site GA and AC is that height is polymorphic, the polymorphism of these 4 microsatellites is described relatively High.

This research carries out typing at 96-capillary tube ABI3730xl to SSRs, utilizes the general linear model in SAS software (GLM), to microsatellite locus genotype and Pelteobagrus fulvidraco primary growth character (body weight, total length, body length, height, body thickness, long a, tail Handle length, caudal peduncle are high) it is associated analyzing, the Analysis of variance inspection site in significant difference, use Duncan method to carry out many Anharmonic ratio is relatively.Result shows: in the AT of site, the growth traits between each genotype is without significant difference (table 5).In the GA of site, genotype BE and CD body weight significantly overweights genotype AB (P < 0. 05)；Genotype BE, DD, EE total length are considerably longer than genotype AB (P < 0. , and genotype CD total length pole is considerably longer than genotype AB (P < 0. 01) 05)；Genotype BE, CD, EE body length are considerably longer than gene Type AB (P < 0. 05)；Genotype BE, CD height are significantly higher than genotype AB (P < 0. 05)；Genotype CD body thickness is significantly thicker than Genotype AB and BB (P < 0. 05)；Genotype BB, BC, BD, CD, DD, EE caudal peduncle height are significantly higher than genotype AB (P < 0. 05), And genotype BE caudal peduncle height pole is significantly higher than genotype AB (P < 0. 01) (table 6 and table 7).

In the TCTT of site, genotype CC is long is considerably longer than genotype BB (P < 0. 05)；Genotype BD caudal peduncle height is significantly high In genotype AB (P < 0. 05) (table 8 and table 9).In the AC of site, genotype CC and DD body thickness are significantly thicker than frequency of genotypes AA (P < 0. 05)；Genotype BB, CC, CD, DD long considerably longer than genotype BD (P < 0. 05) (table 10 and 11).

Note: show significant difference (P < 0. 05) with a line difference lowercase alphabet, represent difference with a line difference capitalization Extremely notable (P < 0. 01).

Pelteobagrus fulvidraco GH gene find altogether 4, SSRs site, research show site AT and growth traits onrelevant；Site GA has with body weight, body length, height, body thickness and significantly associates (P ＜ 0.05), has with total length, caudal peduncle height and significantly associates (P ＜ 0.01), having eight kinds of genotype, wherein CD is preponderant genotype；Site TCTT is high with long, caudal peduncle to be had and significantly associates (P ＜ 0.05), having 7 kinds of genotype, wherein BD is preponderant genotype；Site AC and body be thick, long have significantly associate (P ＜ 0.05), Having 6 kinds of genotype, wherein DD is preponderant genotype.

Comparative example 1

When designing micro-satellite primers, it is two right that the every pair of primer all devises, and naming method is: site-F1/R1, micro-defends for totally four Championship point AT, GA, TCTT, AC.Redness is labeled as finally expanding PCR primer, and carries out fluorescent decoration.

When inventor is according to 4 microsatellite design primers, devising a lot of primer pair, primer given below all can not be special Specific amplification microsatellite locus, can not be used for gene type.

SEQ ID NO:10(AT-F1): 5'-GCTCTGTTACCTGATGAA-3'

SEQ ID NO:11(AT-R1): 5'-TGAGTATTGAAGCACTTT-3'

SEQ ID NO:12(GA-F2): 5'-GAGGGAAACCTGAGGAAGA-3'

SEQ ID NO:13(GA-R2): 5'-GGTGAATGGAGGAGAATAA-3'

SEQ ID NO:14(TCTT-F2): 5'-GATCCCTGGATTCCAACT-3'

SEQ ID NO:15(TCTT-R2): 5'-AGCACCATCGTGAAACTG-3'

SEQ ID NO:16(AC-F1): 5'-AAAATCCACCACTAGACC-3'

SEQ ID NO:17(AC-R1): 5'-ACTCACCAACACCACAGA-3'.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>a kind of yellow catfish growing characteristic is relevant microsatellite marker and detection thereof and application

<130>

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 3117

<212> DNA

<213>GH gene order

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tgatgttatt gtggaaggtg tgaggtgtct catgtcgatg cattaagcat gtagagatca 60

ggtataaaaa ttggacctgc tagcaggagg gaaatcattg tctgaaagtc ggcgtctgat 120

ttttcagtga aaattcgaac caagtttctt cagagagatt ctaaaatggc tcgaggtaag 180

gctctgacct ggtgttacat tacgactgtg atgttacagt gaggaacttt tattgaygtt 240

tttgttttat aagattacta ttagattttc aaattacttt ctgtataaaa tccaccacta 300

gacctgtttt attttgtgcc taatttcaga attatccttg tagatgtatc ctcacagtac 360

acacacacac acacacacac acacacacac acacacacac acacacacac acacacacat 420

actgtatatg cattatataa tgtatgctac tctcctgctg ttcttcctct tccacagggt 480

tagtgttgct ctctgtggtg ttggtgagtt tgttctttag ccaaggcgcg acatttgaga 540

accagaggct cttcaacaac gccgtcattc gtgtgcaaca ccttcatcag ctggctgcca 600

agatgatgga tgactttgta agctgacttt acatcattta cagatgttta gtttactgaa 660

gtatgcttta gctaacatgg ttgctgtgct gcaaatataa aggaggaagc tctgttacct 720

gatgaacgca aacagctcag caagatcttc cccctgagtt tctgcaactc ggactccatc 780

gaagctccgg caggcaaaga cgagacccag aaaagctctg tgagtcacaa aactgcctca 840

agaattcatg cgattttaat atatatatat ttaaagatat ttgttattgt gataacgaat 900

ttggtgattt aatgatcatt agctgtgaca caaatgattt aaaggagcaa tatgtaatat 960

ataaaagtgc ttcaatactc ataaaaaatt gaatcatgtc aaagatgctt taaaatgttc 1020

aaaaaaatgt aataaagtag ttgttttact gtttttacag tttctttgat taaggcttct 1080

acttaattaa ttacccaata aaatatcccc cccccaatat ttaaatgttt cttaaaagaa 1140

ataactaaaa aacattactt tttctgaatt ttttaagagt agccctgcaa tgcaaggtat 1200

ctattttaaa gtttcagtta acatttttct ggcccagaaa caagctccac tgtttttttt 1260

tttttttgta aaaggcagct taagagtcaa acatgtctag ctagtagttg atgttttcat 1320

tgctctctta gcgtgatgaa tattgtgatt tacgtgctac agtctcatgc taaagatcgt 1380

ttataagcaa acatgagcat cggagagaag aatactgctt tgcatctctg tgaactttaa 1440

acgagtctgt tttttaacca ggtgctgaag ctactgcaca cttcctaccg tctgatcgag 1500

tcatgggagt tccccagcaa gaacctcggc aaccctaacc atatctctga gaagctggct 1560

gacctgaaga tgggcatcgg cgtgcttatc gaggtgagca cgagcgccac agatcagaca 1620

ccgagtcact gcaatgtgtg cagggagaac agaagagtta atagaatgtg ttagttaaca 1680

tgtatgagta tgtatttccc gtatggacct gtgtgcacaa aaatgatgtt ttgtacaact 1740

tggatgaaag tgtttttttt gtgtcattcc ccatccattt ctctataact aatacatgct 1800

ctggtgctct gggtggctca gaacttgcga tctaagtaca agacttagca gtacaagaaa 1860

cccgtcagtt cattcagcta tatgcggttt taaaacttaa aactaatcag aatagtagct 1920

atttatttac tgttggtaga atctgaccgt tcattcattc aatggaaaag ttggtaaggt 1980

aataataatg taacttttgt aactgtaagt ttgcagttct gatcaattga aataatttta 2040

atcattaaaa tgtaaatgtt tacaccatat gttgtagtct caggaggata aactcttcat 2100

accttccagc cctattaacg tccccggctc agacccggag ctctgctcct ttacacatga 2160

gtgaaattta aacagtaaca attattagta ttatttagat cattttctcc agtattgtag 2220

tttttatata gcgcaggttg gatttattta actcctacat gttaataaac acaataacca 2280

acaggcgtac ttaaggtggt cgttatttat gatcaattca tgtattgtta ttataaagtg 2340

tttcagttat gcagtatgat aaagtttagt gaaaattgtc tagttttatt gtagcaaggc 2400

ttataaagtc actctgctca ctccataggg atgtctggac ggacagacca gcatggacga 2460

gaacgacgct ctggctccgc cattcgagga tttctaccag accttgagcg agggaaacct 2520

gaggaagagc ttccgtctgc tgtcctgctt caagaaggac atgcacaagg tggagaccta 2580

tctaagcgtg gccaagtgca ggagatccct ggattccaac tgcaccctgt agaatgagag 2640

agagagagag agagagagag agagagagag attgattgat tgattgacta gtcacagtct 2700

gggatctctc caggcatgac tatcatcttt ctttctttct ttctttcctt ttgtttcttt 2760

gggtttaatt tctgtcttta tgtatttatt ctcctccatt cacctgttta acagatttct 2820

ccatctcagc aaccttaagg atgtaacata tgggaccagt ttcacgatgg tgctatgcaa 2880

attcagcttt atatcaaata acgaataaga aaatggatta aacgtaatta atttagaaat 2940

ggacgaggac gctcctggct aatgaatgta aaatattcag agggtatatt ttcctatcaa 3000

agtctatact cttctaattt taattatggc ctatttctgt gtgaagcttt gactgtctct 3060

gtgtgaaaat ctctctcatt gcttttttgt ggatttccaa taaacctaat ccctggg 3117

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<213> AT-F

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gtttctgcaa ctcggact 18

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<213> AT-R

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gcatctttga catgattc 18

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<213> GA-F

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ctaccagacc ttgagcga 18

<210> 5

<211> 18

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<213> GA-R

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ggtgaatgga ggagaata 18

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<213> TCTT-F

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gatccctgga ttccaactg 19

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tagcaccatc gtgaaactg 19

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<213> AC-F

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aatccaccac tagacct 17

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<213> AC-R

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ctcaccaaca ccacaga 17

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<211> 18

<212> DNA

<213> AT-F1

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gctctgttac ctgatgaa 18

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tgagtattga agcacttt 18

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gagggaaacc tgaggaaga 19

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ggtgaatgga ggagaataa 19

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<213> TCTT-F2

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gatccctgga ttccaact 18

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agcaccatcg tgaaactg 18

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actcaccaac accacaga 18

Claims (6)

1. the primer being used for the amplification microsatellite relevant to yellow catfish growing characteristic, it is characterised in that the nucleotide of primer Sequence is as shown in SEQ ID NO:4～9, wherein, and the microsatellite fragment of primer SEQ ID NO:4 and SEQ ID NO:5 amplification Repeating motif is (GA)_n1, the span of n1 is 17～35, described microsatellite fragment and the body weight of Pelteobagrus fulvidraco, total length, body length, Height, body are thick, caudal peduncle height is correlated with；The repetition motif of the microsatellite fragment of primer SEQ ID NO:6 and SEQ ID NO:7 amplification is (TCTT)_n2, the span of n2 is 6～14, described microsatellite fragment and Pelteobagrus fulvidraco long, caudal peduncle is high relevant；Primer SEQ The repetition motif of the microsatellite fragment of ID NO:8 and SEQ ID NO:9 amplification is (AC)_n3, the span of n3 is 25～35, institute State microsatellite fragment and the body of Pelteobagrus fulvidraco thick, long relevant.

Micro-satellite primers for yellow catfish growing specificity analysis the most according to claim 1, it is characterised in that primer The solution temperature of the microsatellite fragment of SEQ ID NO:4 and SEQ ID NO:5 amplification is 56 DEG C, and clip size is 311bp；Primer The solution temperature of the microsatellite fragment of SEQ ID NO:6 and SEQ ID NO:7 amplification is 61 DEG C, and clip size is 272bp, primer The solution temperature of the microsatellite fragment of SEQ ID NO:8 and SEQ ID NO:9 amplification is 56 DEG C, and clip size is 220bp.

3. contain the test kit of micro-satellite primers described in claim 1 or 2.

4. test kit described in micro-satellite primers described in claim 1 or 2 or claim 3 in Pelteobagrus fulvidraco genetic breeding should With.

5. the method detecting Pelteobagrus fulvidraco total length character, it is characterised in that comprise the following steps:

S1. sample DNA is extracted；

S2. utilize the primer amplification sample DNA of SEQ ID NO:4～9 described in claim 1 or 2, analyze micro-in sample DNA defending The type of asterisk note and genotype, determine the total length character of Pelteobagrus fulvidraco.

6. the method for the excellent Pelteobagrus fulvidraco of selection-breeding, it is characterised in that comprise the following steps:

S1. sample DNA is extracted；

S2. utilize the primer amplification sample DNA of SEQ ID NO:4～9 described in claim 1 or 2, analyze micro-in sample DNA defending The type of asterisk note and genotype；

S3. result judges: if containing repeating motif (GA) in sample DNA_n1, then Select gene type be the sample of CD be excellent yellow forehead Fish；If the motif containing restructuring (TCTT) in sample DNA_n2, then Select gene type be the sample of BD be excellent Pelteobagrus fulvidraco；If sample DNA In containing repeat motif (AC)_n3, then Select gene type be the sample of DD be excellent Pelteobagrus fulvidraco.