CN104351096A - Paramisgurnus dabryanus selective breeding method - Google Patents
Paramisgurnus dabryanus selective breeding method Download PDFInfo
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- 238000009394 selective breeding Methods 0.000 title abstract description 3
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a paramisgurnus dabryanus selective breeding method. Concretely, the method comprises the following steps that (1) 12 microsatellite molecular markers relevant to the growth properties are provided; (2) a parent group 1 is selected for electronic marking and DNA (deoxyribonucleic acid) extraction; (3) the molecular markers are adopted for carrying out genotype screening on the parent group 1, and in addition, a parent group 2 is selected; (4) the molecular markers are adopted for carrying out genotype screening on the parent group 2, and parent groups 3 and 4 with different homozygous genotypes are respectively built; (5) the parent groups 3 and 4 are used as breeding groups to be respectively subjected to artificial reproduction to obtain parent groups 5 and 6; (6) hybridization is carried out between the parent groups 5 and 6, and in addition, scaled reproduction is carried out. Compared with a conventional breeding method, the method has the characteristics of high speed, stability and reliability, the practicability is high, the cost is low, the accuracy is high, the influence due to factors such as environment is avoided, the relying on the phenotypic character authentication can be avoided, the breeding years are shortened, the breeding efficiency is improved, and great application values are realized.
Description
Technical field
The invention belongs to fish breeding field, be specifically related to a kind of Misgurnus auguillicaudatus fine-variety breeding method.
Background technology
Misgurnus auguillicaudatus (
paramisgurnus dabryanus) belong to Cobitidae, colored loach subfamily, secondary loach genus, be a kind of small-sized fresh water benthic fishes being distributed in south east asia, domestic, be distributed in the ground such as Sichuan, Zhejiang, Taiwan, Liaoning, Heilungkiang more.The nearly cylindrical shape of body, head is shorter.Mouth is the next, and the shape of a hoof, there are a small gap in lower lip central authorities.Nostril is near eye, and stingless now, gill opening is little.Head is without squama, and body squama comparatively loach is large.Palpus 5 is right, and wherein kiss palpus 2 is right, and bicker palpus 1 is right, and chin palpus 2 is right, respectively must be all long, stretches and can reach gill cover trailing edge after bicker palpus.Eye is covered by epithelium.Caudal peduncle place skin pleat rib is flourishing, is connected with tail fin.Caudal peduncle is long about equal with height.Tail fin is circular.The nearly anal fin starting point of anus.Body back and side first half taupe, outside of belly white.Side has many irregular black brown spots.Dorsal fin, tail fin tool black point, other each fin canescence.Misgurnus auguillicaudatus delicious meat, nutritious, be rich in calcium, vitamin A, vitamin D and multiple proteins, amino acid and mineral matter, have the good reputation of " in water ginseng ", there is higher edible, medicinal and economic worth, develop into the cultivation object that China is important, one of main aquatic products of Ye Shi China's export Korea S.
China has become whole world Misgurnus auguillicaudatus and has cultivated largest country, and Jiangsu Province's Misgurnus auguillicaudatus cultivation scale has occupied again more than 70% of the whole nation.But the fingerling at present for Misgurnus auguillicaudatus cultivation is mainly fished for from natural water area, the difficult quality guarantee of wild seed, growth performance and culture efficiency also extremely unstable.As everyone knows, planting matter is the material base of aquaculture, and in China's aquatic products industry, from fishing for, to be main industry should not be underestimated to the effect played based on the transition process of the industry of cultivation breeding.Tradition prevalent variety cultivation technology is divided into two classes substantially: genetic manipulation and selection operation, and Genetic Manipulative Technology is the most successfully crossbreeding technology, selects operating technology then to mainly contain family and unexpected mass incident.Selection cross faces to be introduced bad proterties and to still need the problem that many generations backcross; Traditional breeding wants to build up target variety, at least needs could be bred as improved seeds (inheritance stability, in the basically identical colony having some of biology, morphology and economic characters) through too much generation (4-5 generation) artificial selection.Tradition selects the drawback of operation be the explanation of genotype enrichment and control inbred method and be still in the experience stage, only indirect utilization modern genetics is theoretical, concept obfuscation, equivalence relation is lacked between operation and theory, so that breeding process occurs that improved seeds heterozygosity is low, deleterious gene shows by isozygotying, and heterozygosity and polymorphism decline, and have the hidden danger that integral economic trait declines.
The application on aquatic products genetic breeding along with molecular biology and genomics, different kinds of molecules mark (as SSR/SNP etc.) occurs and application efficiently solves the problems referred to above.But, not yet start the systematic genetic seed selection work of Misgurnus auguillicaudatus both at home and abroad at present, particularly molecular marking supplementary breeding, the shortage of high-quality seed seriously constrains the development of Misgurnus auguillicaudatus aquaculture, therefore urgently develops a kind of fine-variety breeding method of Misgurnus auguillicaudatus.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of Misgurnus auguillicaudatus fine-variety breeding method, thus can reliable, the growth performance of quick breeding mass and the stable Misgurnus auguillicaudatus improved seeds of culture efficiency, technique is applicable to the fine-variety breeding that fairly large seed multiplication farm or national Seed multiplication base carry out Misgurnus auguillicaudatus.
To achieve these goals, the present invention is achieved through the following technical solutions:
A kind of Misgurnus auguillicaudatus fine-variety breeding method, it comprises the following steps:
1) provide the microsatellite molecular marker that 12 are relevant to growth traits, its each self-corresponding primer is as follows:
The primer of microsatellite molecular marker Pda48: L:5 '-TGAGGCACTTTGTTTACGCTC-3 ';
R：5’-GCTCATGGGACACAAGATGG-3’；
The primer of microsatellite molecular marker Pda66: L:5 '-TACCCTGTGCTAAGGAGGC-3 ';
R：5’-AGAGGGAACGGCCAACAG-3’；
The primer of microsatellite molecular marker Pda174: L:5 '-GTCGGACCTGAAGAGGCAC-3 ';
R：5’-TGCCGTCTGTCATCCATGC-3’；
The primer of microsatellite molecular marker Pda242: L:5 '-TGCCAGCAATTGACATAAAGGG-3 ';
R：5’-GATTTCTCAAGCCCAGCGG-3’；
The primer of microsatellite molecular marker Pda247: L:5 '-ATAGTGCCCGTTTCCTGCC-3 ';
R:5 '-TGGTGAAAGAGTGAAACAATTACC-3 '; The primer of microsatellite molecular marker Pda260: L:5 '-GCTCTGCTCCGAACATTCC-3 ';
R：5’-CTTTCCGTCGCACCTTTCC-3’；
The primer of microsatellite molecular marker Pda288: L:5 '-AGGTGTGGTTTCAGGTTGC-3 ';
R：5’-ACAGAGGAAACCGGTGAGG-3’；
The primer of microsatellite molecular marker Pda301: L:5 '-CCTGTAGTTGACCCATTTCGC-3 ';
R：5’-AGAAGGGTGACTTGTCCAAAC-3’；
The primer of microsatellite molecular marker Pda310: L:5 '-GGGTCTCCACAGTGACGC-3 ';
R：5’-CGCTGTCTGAGTGATCCCG-3’；
The primer of microsatellite molecular marker Pda315: L:5 '-ACATCCTGCCTGAGGTTCC-3 ';
R：5’-TCTTAATGATGACGGCAGAGC-3’；
The primer of microsatellite molecular marker Pda316: L:5 '-TGAGTGGCACGCTCCAAG-3 ';
R：5’-CGAGGCCAAACTAACTGCG-3’；
The primer of microsatellite molecular marker Pda371: L:5 '-CTGGTTTCAACGGTGGACG-3 ';
R：5’-GTGCGTGTCCCTGAACTTG-3’；
2) select build, body colour normal, the anosis Misgurnus auguillicaudatus without wound is individual as alternative parental population 1, carries out electronic marker, and extract DNA to all individualities in alternative parental population 1;
3) adopt Pda66, Pda174, Pda242, Pda247, Pda260, Pda288, Pda310, Pda316, Pda371 to carry out genotype screening to alternative parental population 1, choosing genotype is the corresponding Pda66 of AA(), the corresponding Pda174 of FF(), the corresponding Pda242 of EE(), the corresponding Pda247 of NN(), the corresponding Pda260 of CC(), the corresponding Pda288 of CC or DD(), the corresponding Pda310 of CC(), the corresponding Pda316 of BB or AA(), the corresponding Pda371 of AA or BB() individuality as alternative parental population 2;
4) Pda48, Pda301, Pda315 is adopted to carry out genotype screening to alternative parental population 2, obtain genotype and be respectively the corresponding Pda48 of AA or FF(), the corresponding Pda301 of AA or BB(), the corresponding Pda315 of BB or CC() individuality, the individuality that same gene site has homologous genes type is cultivated separately, sets up alternative parental population 3 and alternative parental population 4 respectively;
5) with alternative parental population 3 for reproductive population, carry out an artificial propagation generation and obtain standby parental population 5; With alternative parental population 4 for reproductive population, carry out an artificial propagation generation and obtain standby parental population 6;
6) carry out biological control according to the matching method of the female individuals in the male in the female individuals × parental population 6 in parental population 5 or the male × parental population 6 in parental population 5, Misgurnus auguillicaudatus breeding can be obtained, complete Breeding Process.
In actual production operation, after step 6) completes, can step 2 be repeated) to the operation of step 6), and monitor breeding line genetic background, prevent inbreeding.
Preferably, in such scheme, described in step 1), microsatellite molecular marker obtains according to laxative remedy:
A) Misgurnus auguillicaudatus of all ages and classes and sex is chosen, gather Various Tissues and extract RNA, equivalent pipettes and is mixed into total serum IgE, isolate mRNA, reverse transcription obtains cDNA library, reclaims object fragment after pcr amplification enrichment, adopts second generation high-flux sequence platform (Illumina Hiseq 2000 platform) to check order quantitatively, adopt sequence assembling software (Trinity software) to assemble sequenced fragments after order-checking, obtain transcript sequence;
B) microsatellite molecular marker is adopted to catch software (Msatcommander software) scanning step a) the middle transcript sequence obtained, filter out the microsatellite molecular marker that core sequence length is 10-360 bp, design of primers is carried out to the microsatellite molecular marker that flank sequence length is 50 more than bp;
C) according to the result of transcript profile GO functional annotation, choose the microsatellite molecular marker being categorized into process of growth entry, in addition other microsatellite molecular markers of random selecting, amount to synthesis micro-satellite primers 400 right;
D) collect the Misgurnus auguillicaudatus wild population of different geographical, observe and compare its shape difference, the colony choosing difference maximum hybridizes, and artificial induced spawning obtains family full-sibs colony;
E) microsatellite molecular marker in random employing step b) scans the individuality in the Misgurnus auguillicaudatus family full-sibs colony obtained in step d), correlation analysis between the growth traits of carrying out microsatellite molecular marker and family colony, filters out 12 microsatellite molecular markers relevant to growth traits.
In addition, the present invention also asks the primer protecting 12 microsatellite molecular markers used in above-mentioned Misgurnus auguillicaudatus fine-variety breeding method.
Compared with prior art, Misgurnus auguillicaudatus fine-variety breeding method of the present invention has following beneficial effect: molecular mark (marker-assistant selection, be called for short MAS) utilize molecular labeling to select breeding material from DNA level, thus quickening process obtains the offspring with object proterties.Compared with traditional breeding method, MAS method has rapidly, stablize, feature reliably, practical, cost is low, accuracy is high, not by the impact of the factors such as environment, the dependence to phenotype Characters Identification can be broken away from, early selecting from generation to generation, thus shortening the breeding cycle (just can select desirable high-quality population through 3 generations in theory) greatly, improve breeding efficiency, have using value.
Accompanying drawing explanation
Fig. 1 is the electrophoretogram of the RNA sample never gathered in each tissue in other Misgurnus auguillicaudatus body of the same sex.
Fig. 2 is transcript sequence staple diagram.
Fig. 3 is transcript profile GO functional annotation (bioprocess level) result schematic diagram.
Fig. 4 is the part electrophoretograms of 12 microsatellite molecular markers in Misgurnus auguillicaudatus colony, wherein each swimming lane represents a Misgurnus auguillicaudatus individuality, M representation DNA molecular weight standard, band is followed successively by 1000bp from top to bottom, 700bp, 500bp, 400bp, 300bp, 200bp and 100bp, the banding pattern (genotype) of 12 molecular labelings Different Individual in colony has notable difference as seen from the figure.
Embodiment
Unless stated to the contrary, following use term in the specification and in the claims has following implication.
" breeding " refers to compared with the infraspecific most cultured population used in aquaculture, has the kind of one or more economic characters of comparative advantages, and the economic characters of wherein cultured fishes are generally growth property, dressing percentage, resistance etc.
" fine-variety breeding " refers to that carrying out the generation by the one or more excellent economic characters of direct or indirect screening technique to basic population selects with the process obtaining breeding.
" microsatellite molecular marker (Microsatellite Molecular Marker; MMM) " refers to a kind of genetic marker, also known as Short tandem repeatSTR (Short Tandem Repeats, or simple repeated sequence (Simple Sequence Repeats STR), SSR), be with the DNA sequence dna of the sequence tandem sequence repeats of 1-6 base, length generally arrives a hundreds of base tens.
" high-flux sequence platform " comprises the second generation sequencing technologies of three kinds of main flows, be respectively Roche/454 Manganic pyrophosphate complex initiation, Illumina/Solexa polymerase synthesis order-checking and ABI/SOLiD ligase sequencing technologies, it is large that the total prominent features of three kinds of sequencing technologies is that single runs output series data volume, so be commonly referred to as again " high throughput sequencing technologies ".
" transcript profile GO functional annotation " is the annotation of gene function and sorting technique that carry out transcript profile data based on gene ontology (Gene Ontology, GO), and it comprises bioprocess, molecular function and cell components three branches, belongs to bioinformatics category.
" genotype " refers to genetic material (gene) form of expression determining species specific trait, and such as AA genotype represents red pattern proterties, and BB genotype represents white pattern proterties, and AB genotype represents pink colour pattern proterties.
" growth traits " refers to and grows relevant proterties, and as body length, height etc., wherein " total length " refers to the length of fish kiss end to tail fin end; " body is long " refers to that fish kiss end arrives the length of tail fin base portion or end vertebra; " height " refers to the maximum height of fish body; " body weight " refers to the quality of fish body; " ghost weight " refers to the quality of the rear fish body of the group of gilling.
" genotype and growth traits correlation analysis " refers to and utilizes statistical method analyzing gene type and growth traits (as body is long) to have non-correlation and correlation significance degree.
" family colony " refers to the test colony of drawing for genetic map, comprises family full-sibs (deriving from identical parental colony) and family half sibs (with the different mother of father or uterine colony).
" alternative parental population " refers to for selecting the artificial propagation sexual maturity colony of female, male parent population.
" standby parental population " refers in the future for selecting artificial propagation colony that is female, male parent, individuality not necessarily sexual maturity in colony.
" electronic marker " is also called electronic tag, RF tag, responder, data medium, refers to utilize to be different from conventional physical mark, based on electronic chip, the method for different animal subject can be marked by the label of injector to inject.
Preferred embodiment: the fine-variety breeding of Misgurnus auguillicaudatus.
1, the structure of Misgurnus auguillicaudatus transcript profile database:
Select the parent of Misgurnus auguillicaudatus family colony for experiment material, male loach, female loach are respectively from Dongting Lake and Hongchehu Lake.After anaesthetizing with the gavaculine ethyl ester mesylate (MS-222) of 100 mg/L, gather the Various Tissues (ovum of the muscle of female, milter, bone, fin ray, brain, spleen, liver pancreas and raun) of male and female loach, by specification (D311A reagent, precious biotechnology (China) Co., Ltd) requirement be placed in RNA conserving liquid, frozen for subsequent use.Concrete operations are as follows:
Adopt TRIzol
?reagent (100ml, life science (China) company) extract its electrophoretogram of RNA(of above-mentioned various tissue as shown in Figure 1, in figure, each swimming lane comprises two to three bands, illustrate that RNA quality is better, meet order-checking to require), the qualified rear equivalent of quality testing pipettes various RNA and is finally mixed into 5 μ g total serum IgE, the mRNA of polyA tail is gone out to have with the Beads enrichment with oligo (dT), ion method interrupts mRNA, then obtain a chain cDNA with 6 base random primed reverse transcription, add dNTPs, RNaseH and DNA polymerase i synthesizes two chain cDNA.Connect " A " base by T4 ligase end-filling after obtaining double-strand cDNA, finally connect index joint.The kit more than building library is Truseq
tMrNA sample prep Kit.Pcr amplification enrichment is carried out to the library obtained, then reclaims object fragment with the agarose gel of 2%.Carry out quantitatively with TBS380 to library, check order on Hiseq2000 checks order platform.After order-checking after de-redundancy and low quality segment are filtered, residue 39,751, article 380, sequence fragment, Misgurnus auguillicaudatus two muscle transcription group sequence samples amount to 105,308 in addition, 078 fragment (10.47Gb), adopt Trinity software combination sequenced fragments, obtain 71 after assembling, 887 transcript profile sequences, all long 1464.58 bp, total length 105,284,263 bp, all long 1464.58 bp of sequence, the sequence being wherein greater than 1000bp has 34,603(48.1%) bar, the batch being suitable for microsatellite molecular marker excavates (concrete data are see Fig. 2).
2, batch exploitation Misgurnus auguillicaudatus microsatellite molecular marker:
Msatcommander software is adopted to carry out SSR screening to all transcript profile sequences, parameter designing is that core sequence is greater than 12 bp's, find that SSR site has 15106, carry out design of primers with all microsatellite molecular markers of Primer 3.0 software to flanking sequence >50bp, concrete design of primers parameter refers to table 1.
3, synthesis comprises the primer of the microsatellite molecular marker relevant with process of growth:
According to transcript profile GO functional annotation result (as shown in Figure 3), choose the microsatellite molecular marker being categorized into process of growth entry (asterisk mark) in bioprocess classification, in addition other microsatellite molecular markers of random selecting, amount to synthesis micro-satellite primers 400 to (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), purification process is ULTRAPAGE.
4, Misgurnus auguillicaudatus family full-sibs colony is built:
Collect Taihu Lake, Hongchehu Lake, Dongting Lake, the wild Misgurnus auguillicaudatus colony in the lakes such as Chaohu, find that Dongting Lake and Hongchehu Lake colony are at morphology, genetic structure aspect difference is maximum, therefore using Dongting Lake one tail milter and Hongchehu Lake one tail raun as parent, artificial propagation obtains family full-sibs colony (AB2011-F6), oxytocic hormone is raun 2 microgram LRH-A2 and 1 milligram of DOM mixing, milter reduces by half, after injecting pin of hastening parturition, water purification is hatched, put fortune windmillpalm sheath-fibre and make fish nest, hatching in 2-3 days to be fed cultivation 1 year after emerging, measure total length, body is long, height, weight, fish body heavily etc. is placed in absolute ethyl alcohol after index and saves backup by ghost.
5, the correlation analysis of Misgurnus auguillicaudatus microsatellite molecular marker and growth traits:
The 117 tail individualities of random use 177 EST-SSRs molecular labelings to Misgurnus auguillicaudatus family full-sibs colony (AB2011-F6) carry out genomic DNA detection, screen 58 polymorphic micro-satellite molecular labelings altogether.Microsatellite molecular marker and family colony (AB2011-F6) are carried out marking-growth traits correlation analysis with the regression analysis-linear model of SPSS software (Version 17.0), its result is as shown in following table 2 and Fig. 4.
As shown in Table 2, the microsatellite molecular marker relevant to Misgurnus auguillicaudatus primary growth 12 can be screened by above-mentioned correlation analysis, wherein have 12 genotype of isozygotying relevant to growth traits in 9 marks, the genotype being heterozygosis in other 3 marks is relevant to growth traits.
6, the foundation of parental population and scale artificial culture:
The individuality that build is normal in the Misgurnus auguillicaudatus colony of Hongchehu Lake, body colour is normal, anosis nothing is hindered is as alternative parental population 1.Electronic marker is carried out to tail fish every in alternative parental population 1, and clip tail fin, preserve with 95% alcohol, be used for extracting DNA.Carry out genotype screening with Pda66, Pda174, Pda242, Pda247, Pda260, Pda288, Pda310, Pda316, Pda371 with to alternative parental population 1, choosing genotype is Pda66(AA), Pda 174(FF), Pda242(EE), Pda247(NN), Pda260(CC), Pda288(CC or DD), Pda310(CC), Pda316(BB or AA), Pda371(AA or BB) individuality as alternative parental population 2.With Pda48, Pda315, Pda301, parental line selection is carried out to alternative parental population 2, obtain genotype be respectively Pda48(AA or FF), Pda301(AA or BB), Pda315(BB or CC) individuality, the individuality of same gene site homologous genes type is cultivated separately, sets up alternative parental population 3 and alternative parental population 4 respectively.Carry out the expansion of parental population, with parental population 3 for reproductive population, carry out artificial propagation and obtain high-quality standby parent 5 in enormous quantities; With parental population 4 for reproductive population, carry out artificial propagation and obtain high-quality standby parent 6 in enormous quantities.Carry out seed rearing, adult fish culture and parent culture subsequently, set up parental population 5 or parental population 6.Carry out biological control by the matching method of the female individuals of the male of the female individuals × parental population 6 of parental population 5 or the male × parental population 6 of parental population 5, obtain large quantities of high-quality seed, carry out popularization cultivation.
7, seed selection interpretation of result:
Since 2008, Misgurnus auguillicaudatus large-scale artificial breeding work has been carried out in University Of Suzhou's related item group and relevant enterprise cooperation, and meanwhile, project team successively establishes Misgurnus auguillicaudatus mixing breeding line and family.In March, 2013 starts to carry out molecular marking supplementary breeding work, artificial propagation in June, 2013, and survey in October, 2013 and produce, seed selection group average weight is 7.14 ± 2.14, control group body weight 4.41 ± 1.75, and breeding line has significant growth performance.
Claims (4)
1. a Misgurnus auguillicaudatus fine-variety breeding method, it comprises the following steps:
1) provide 12 microsatellite molecular markers, its each self-corresponding primer is as follows:
The primer of microsatellite molecular marker Pda48: L:5 '-TGAGGCACTTTGTTTACGCTC-3 ';
R：5’-GCTCATGGGACACAAGATGG-3’；
The primer of microsatellite molecular marker Pda66: L:5 '-TACCCTGTGCTAAGGAGGC-3 ';
R：5’-AGAGGGAACGGCCAACAG-3’；
The primer of microsatellite molecular marker Pda174: L:5 '-GTCGGACCTGAAGAGGCAC-3 ';
R：5’-TGCCGTCTGTCATCCATGC-3’；
The primer of microsatellite molecular marker Pda242: L:5 '-TGCCAGCAATTGACATAAAGGG-3 ';
R：5’-GATTTCTCAAGCCCAGCGG-3’；
The primer of microsatellite molecular marker Pda247: L:5 '-ATAGTGCCCGTTTCCTGCC-3 ';
R:5 '-TGGTGAAAGAGTGAAACAATTACC-3 '; The primer of microsatellite molecular marker Pda260: L:5 '-GCTCTGCTCCGAACATTCC-3 ';
R：5’-CTTTCCGTCGCACCTTTCC-3’；
The primer of microsatellite molecular marker Pda288: L:5 '-AGGTGTGGTTTCAGGTTGC-3 ';
R：5’-ACAGAGGAAACCGGTGAGG-3’；
The primer of microsatellite molecular marker Pda301: L:5 '-CCTGTAGTTGACCCATTTCGC-3 ';
R：5’-AGAAGGGTGACTTGTCCAAAC-3’；
The primer of microsatellite molecular marker Pda310: L:5 '-GGGTCTCCACAGTGACGC-3 ';
R：5’-CGCTGTCTGAGTGATCCCG-3’；
The primer of microsatellite molecular marker Pda315: L:5 '-ACATCCTGCCTGAGGTTCC-3 ';
R：5’-TCTTAATGATGACGGCAGAGC-3’；
The primer of microsatellite molecular marker Pda316: L:5 '-TGAGTGGCACGCTCCAAG-3 ';
R：5’-CGAGGCCAAACTAACTGCG-3’；
The primer of microsatellite molecular marker Pda371: L:5 '-CTGGTTTCAACGGTGGACG-3 ';
R：5’-GTGCGTGTCCCTGAACTTG-3’；
2) select build, body colour normal, the anosis Misgurnus auguillicaudatus without wound is individual as alternative parental population 1, carries out electronic marker, and extract DNA to all individualities in alternative parental population 1;
3) adopt Pda66, Pda174, Pda242, Pda247, Pda260, Pda288, Pda310, Pda316, Pda371 to carry out genotype screening to alternative parental population 1, choosing genotype is that the individuality of AA or BB that BB or AA, Pda371 that CC, Pda316 that CC or DD, Pda310 that CC, Pda288 that NN, Pda260 that EE, Pda247 that FF, Pda242 that AA, Pda174 that Pda66 is corresponding are corresponding are corresponding are corresponding are corresponding are corresponding are corresponding are corresponding are corresponding is as alternative parental population 2;
4) Pda48, Pda301, Pda315 is adopted to carry out genotype screening to alternative parental population 2, obtain the individuality that genotype is respectively AA or FF corresponding to Pda48, BB or CC that AA or BB, Pda315 that Pda301 is corresponding are corresponding, the individuality that same gene site has homologous genes type is cultivated separately, sets up alternative parental population 3 and alternative parental population 4 respectively;
5) with alternative parental population 3 for reproductive population, carry out an artificial propagation generation and obtain standby parental population 5; With alternative parental population 4 for reproductive population, carry out an artificial propagation generation and obtain standby parental population 6;
6) carry out biological control according to the matching method of the female individuals in the male in the female individuals × parental population 6 in parental population 5 or the male × parental population 6 in parental population 5, Misgurnus auguillicaudatus breeding can be obtained, complete Breeding Process.
2. Misgurnus auguillicaudatus fine-variety breeding method according to claim 1, is characterized in that, after step 6) completes, repeat step 2) to the operation of step 6).
3. Misgurnus auguillicaudatus fine-variety breeding method according to claim 1, it is characterized in that, described in step 1), microsatellite molecular marker obtains according to laxative remedy:
A) Misgurnus auguillicaudatus of all ages and classes and sex is chosen, gather Various Tissues and extract RNA, equivalent pipettes and is mixed into total serum IgE, isolate mRNA, reverse transcription obtains cDNA library, reclaims object fragment after pcr amplification enrichment, adopts Illumina Hiseq 2000 platform to check order quantitatively, adopt Trinity software combination sequenced fragments after order-checking, obtain transcript sequence;
B) adopt the transcript sequence obtained in Msatcommander software scans step a), filter out the microsatellite molecular marker that core sequence length is 10-360 bp, design of primers is carried out to the microsatellite molecular marker that flank sequence length is 50 more than bp;
C) according to the result of transcript profile GO functional annotation, choose the microsatellite molecular marker being categorized into process of growth entry, in addition other microsatellite molecular markers of random selecting, amount to synthesis micro-satellite primers 400 right;
D) collect the Misgurnus auguillicaudatus wild population of different geographical, observe and compare its shape difference, the colony choosing difference maximum hybridizes, and artificial induced spawning obtains family full-sibs colony;
E) microsatellite molecular marker in random employing step b) scans the individuality in the Misgurnus auguillicaudatus family full-sibs colony obtained in step d), correlation analysis between the growth traits of carrying out microsatellite molecular marker and family colony, filters out 12 microsatellite molecular markers relevant to growth traits.
4. the primer of 12 microsatellite molecular markers used in Misgurnus auguillicaudatus fine-variety breeding method according to any one of claim 1 to 3.
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