CN109897905A - A kind of and the associated SNP marker of lefteye flounder Malpigmentation and its application - Google Patents
A kind of and the associated SNP marker of lefteye flounder Malpigmentation and its application Download PDFInfo
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- CN109897905A CN109897905A CN201910343278.1A CN201910343278A CN109897905A CN 109897905 A CN109897905 A CN 109897905A CN 201910343278 A CN201910343278 A CN 201910343278A CN 109897905 A CN109897905 A CN 109897905A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention belongs to aquaculture fish assistant breeding technical fields, more particularly to a kind of and the associated SNP marker of lefteye flounder Malpigmentation and its application, SNP marker is as follows: adc-2677-C/A, positioned at the position 2677bp of gene base sequence as shown in SEQ ID NO:1, base A is mutated into C, threonine becomes histidine, normal lefteye flounder genotype is 68.75%AA, 31.25%AC and 0%CC, melanism lefteye flounder genotype is 0%AA, 37.5%AC and 62.5%CC, and albefaction lefteye flounder genotype is 14.29%AA, 64.29%AC and 21.42%CC.The present invention screens SNP site relevant to Malpigmentation from gene angle, and this site is used in lefteye flounder breeding work, solves the problems, such as lefteye flounder Malpigmentation from hereditary angle essence.
Description
Technical field
The invention belongs to aquaculture fish assistant breeding technical fields, are related to SNP marker technology, and in particular to one
Kind and the associated SNP marker of lefteye flounder Malpigmentation and its application.
Background technique
The flatfish such as lefteye flounder are as good economic fish in a large amount of large-scale cultivations of northern China.But often than
In mesh in-one fish artificial hatch group it can be found that the phenomenon that individual Malpigmentation, ratio is also relatively high, this has become China and is
The significant problem faced to world's artificial breeding lefteye flounder, is also a problem to be solved.
Flatfish Malpigmentation can be divided into two kinds of main classes of albefaction (albinism) and melanism (hypermelanism)
Type, they have been referred respectively to, and there is hickie i.e. chromatophore in a side or pigment is reduced；Melanism refers to that blackspot occurs in anophthalmia side
I.e. chromatophore increases.Because of albefaction or the difference of melanized region, make the type of flatfish class Malpigmentation that diversification be presented.Its
In, the albefaction of flatfish class can be divided into true albefaction (albino) and false albefaction (pseudo-albino) again.True albefaction is because of junket
Propylhomoserin enzyme gene mutate caused by melanin cannot synthesize caused by, the body surface of true albefaction individual is entirely white, eyes
The aobvious red because melanin granule is lacked；False albefaction is that have a side chromatophore development and break up affected as a result, false
The more or less reserve part fission color of albefaction individual body.There are many research in relation to Malpigmentation in recent years, such as the cultivation of lefteye flounder
Environment, such as intensity of illumination, water velocity, the depth of water, water temperature, cultivation density etc., there are also trophic factors to the shadow of lefteye flounder Malpigmentation
It rings, these researchs have certain effect for improving lefteye flounder Malpigmentation phenomenon, but few researchs are from hereditary angle
Solve the problems, such as Malpigmentation.
Summary of the invention
The purpose of the present invention is to provide a kind of and associated SNP marker of lefteye flounder Malpigmentation, SNP, that is, mononucleotide
Polymorphism passes through with many characters such as speed of growth, disease resistance and resistance close relation of biology from inherent cause
SNP site relevant to lefteye flounder body colour carries out educating preceding Molecular screening to breeding lefteye flounder, and it is different that lefteye flounder offspring body colour is reduced from germplasm
Normal incidence solves the problems, such as lefteye flounder Malpigmentation in the prior art.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
It is of the invention with the associated SNP marker of lefteye flounder Malpigmentation, specific as follows: adc-2677-C/A is located at base
Because of the position 2677bp of adenylate cyclase type 6-like base sequence as shown in SEQ ID NO:1, base A is prominent
Become C, threonine becomes histidine, and normal lefteye flounder genotype is 68.75%AA, 31.25%AC, 0%CC, melanism lefteye flounder gene
Type is 0%AA, 37.5%AC, 62.5%CC, and albefaction lefteye flounder genotype is 14.29%AA, 64.29%AC, 21.42%CC.
The above-mentioned screening technique with the associated SNP marker of lefteye flounder Malpigmentation, comprising the following steps:
(1) normal skin of normal lefteye flounder, the normal pale skin in lefteye flounder anophthalmia side of anophthalmia lateral body color anomaly are selected respectively
And the anophthalmia side casting skin of anophthalmia lateral body color anomaly carries out gene bidirectional sequencing, then carries out snp analysis to sequencing data, tentatively
Detect the site SNP, analysis obtains the access that conspicuousness is enriched in difference expression gene；
(2) snp analysis is carried out in access relevant to body colour in step (1), filtered out non-synonymous prominent positioned at code area
The site SNP of change；
(3) parting is carried out in the SNP site that step (2) obtains to lefteye flounder；
(4) screening and the associated SNP site of lefteye flounder Malpigmentation.
Further, in step (1), the analysis method for the access that conspicuousness is enriched in the difference expression gene includes gene
Annotation, differential genes expression analysis, differential gene GO enrichment analysis and differential gene KEGG enrichment analysis.
Further, in step (3), the primer such as SEQ ID NO:2 and SEQ ID NO:3 of the SNP site parting use
It is shown.
Further, the above-mentioned screening technique with the associated SNP marker of lefteye flounder Malpigmentation, specifically:
(I) acquire the normal skin of normal lefteye flounder, anophthalmia lateral body color anomaly the normal pale skin in lefteye flounder anophthalmia side and
The anophthalmia side casting skin of anophthalmia lateral body color anomaly extracts RNA, and quality inspection, and the sample rna of extraction carries out transcript profile bidirectional sequencing,
Snp analysis is carried out to sequencing data using bioinformatics means；
(II) (Arachidonic acid metabolism, Thyroid in 6 accesses relevant to body colour
hormone synthesis、 Phototransduction、Melanogenesi、Retinol metabolism、PPAR
Signaling pathway) snp analysis is carried out, there are the genes of SNP site 59 in 6 accesses, passes through the website NCBI
On Blast software screening method go out to be located at the SNP site of code area nonsynonymous mutation, 22 finally obtained in 12 genes are located at
The SNP site of code area carries out SNP parting, method are as follows: acquired under same growing environment respectively just to this 22 SNP sites
16 tail of normal lefteye flounder, 16 tail of melanism lefteye flounder sample, 16 tail of albefaction lefteye flounder sample, extract DNA, draw for the design of each SNP site
Object carries out PCR as shown in SEQ ID NO:2 and SEQ ID NO:3, by template of the DNA of extraction, with direct sequencing pair
SNP site carries out parting, and corresponding position in peak figure is sequenced and only has the peak C, then the idiotype is CC, bimodal if C and A, then
The idiotype is C/A, if the only peak A, which is AA；
(III) to 16 normal lefteye flounder samples of acquisition, 16 melanism lefteye flounder samples, 16 albefaction lefteye flounder samples it is each
The genotype in the site SNP is counted, with SPSS18.0 software to normal lefteye flounder sample and albefaction lefteye flounder sample, normal lefteye flounder
The genotype statistical result of sample and melanism lefteye flounder sample carries out one-way analysis of variance (P < 0.05: significant difference；P<
0.01: extremely significant difference), retain the SNP site with significant difference, obtain a SNP site in normal lefteye flounder and melanism
Genotype distribution in lefteye flounder sample has extremely significant difference, that is, is located at gene adenylate cyclase type 6-like
Such as the position the 2677bp adc-2677-C/A of SEQ ID NO:1 sequence.
Above-mentioned the answering in terms of the breeding for reducing lefteye flounder Malpigmentation rate with the associated SNP marker of lefteye flounder Malpigmentation
With.
Further, above-mentioned SNP marker for reducing lefteye flounder Malpigmentation rate breeding specific steps are as follows: parent is done to choosing
This lefteye flounder clip part fin ray extracts tissue DNA, carries out PCR amplification, PCR product sequencing, sieve by template of the DNA of extraction
Selecting the lefteye flounder that SNP site adc-2677-C/A is AA homozygosis is male parent and maternal progress biological engineering, establishes SNP site adc-
2677-C/A is the lefteye flounder family of AA homozygosis, then thereafter on behalf of the lower seed of Malpigmentation rate.
Compared with prior art, the beneficial effects of the present invention are:
Compared to existing improvement lefteye flounder Malpigmentation technology, the present invention screens and Malpigmentation from gene angle
Relevant SNP site, and this site is used in lefteye flounder breeding work, it is expected to the solution lefteye flounder from hereditary angle essence
Malpigmentation problem.
Specific embodiment
The present invention is further described below in conjunction with specific embodiment, following embodiment be it is descriptive, be not
Limited, this does not limit the scope of protection of the present invention.
With the screening technique of the associated SNP marker of lefteye flounder Malpigmentation, specifically:
(1) acquire the normal skin of normal lefteye flounder, the normal pale skin in lefteye flounder anophthalmia side of anophthalmia lateral body color anomaly and
The anophthalmia side casting skin of anophthalmia lateral body color anomaly extracts RNA, and quality inspection.
(2) sample rna extracted carries out transcript profile bidirectional sequencing, is carried out using bioinformatics means to sequencing data
Snp analysis.
(3) (Arachidonic acid metabolism, Thyroid in 6 accesses relevant to body colour
hormone synthesis、 Phototransduction、Melanogenesi、Retinol metabolism、PPAR
Signaling pathway) snp analysis is carried out, there are the genes of SNP site 59 in 6 accesses, passes through the website NCBI
On Blast software screening method go out to be located at the SNP site of code area nonsynonymous mutation, 22 finally obtained in 12 genes are located at
The SNP site of code area carries out parting to this 22 SNP sites.
(4) SNP parting: 16 tail of normal lefteye flounder, 16 tail of melanism lefteye flounder sample, albefaction under same growing environment are acquired respectively
16 tail of lefteye flounder sample extracts DNA, for each SNP site design primer such as SEQ ID NO:2 and SEQ ID NO:3 institute
Show, carry out PCR by template of the DNA of extraction, parting is carried out to SNP site with direct sequencing.
(5) parting rule: corresponding position only has the peak C in sequencing peak figure, then the idiotype is CC, bis- if C and A
Peak, then the idiotype is C/A, if the only peak A, which is AA.
(6) to 16 normal lefteye flounder samples of acquisition, 16 melanism lefteye flounder samples, 16 albefaction lefteye flounder samples it is each
The genotype of SNP site is counted, with SPSS18.0 software to normal lefteye flounder sample and albefaction lefteye flounder sample, normal lefteye flounder sample
The genotype statistical result of this and melanism lefteye flounder sample carries out one-way analysis of variance (P < 0.05: significant difference；P < 0.01:
Extremely significant difference), retain the SNP site with significant difference, finally obtains a SNP site in normal lefteye flounder and melanism tooth
Genotype distribution in flounder sample has extremely significant difference (see Table 1 for details for result, table 2), that is, is located at gene adenylate
The position the 2677bp adc-2677-C/A of cyclase type 6-like such as SEQ ID NO:1 sequence.
Genotype distribution statistics result between the different type sample of the site table 1:adc-2677-C/A
Table 2: the conspicuousness X compared between sample2Distribution
| Normal lefteye flounder | Melanism lefteye flounder | |
| Normal lefteye flounder | - | 21.09** |
| Albefaction lefteye flounder | 10.29** | 6.26* |
Note: X2 0.05=5.991, X2 0.01=9.21
The significance test result X of normal lefteye flounder and melanism lefteye flounder site adc-2677-C/A parting2=21.09 > 9.21,
P < 0.01 shows that genotype distribution has extremely significant sex differernce between normal lefteye flounder sample and melanism lefteye flounder sample on this site,
This SNP site is related to lefteye flounder melanism.The significance test knot of normal lefteye flounder and albefaction lefteye flounder site adc-2677-C/A parting
Fruit X2=10.29>9.21>5.991, P<0.01 shows on this site gene between normal lefteye flounder sample and albefaction lefteye flounder sample
Type distribution has extremely significant difference, this SNP site is related to lefteye flounder albefaction.SNP site adc-2677-C/A and lefteye flounder albefaction are black
Change has correlation.
Embodiment 1
The present embodiment is intended to verify whether screened SNP site has generally in normal lefteye flounder and Malpigmentation lefteye flounder
Property feature.Specific implementation method are as follows:
The acquisition of different type lefteye flounder sample: the normal lefteye flounder sample of 50 tails, 50 tail melanism lefteye flounder samples, 50 tail albefaction lefteye flounders
Sample, extracts sample DNA, and the primer provided using claim 4 carries out PCR amplification, product as template using the sample DNA extracted
Sequencing, analyzes sequencing result, the genotype in the site statistical sample adc-2677-C/A, with SPSS18.0 software to normal
The genotype statistical result of lefteye flounder sample and albefaction lefteye flounder sample, normal lefteye flounder sample and melanism lefteye flounder sample carries out single factor test side
Difference analyses (P < 0.05: significant difference；P < 0.01: extremely significant difference), see Table 3 for details and table 4 for statistical result.
Genotype distribution statistics result between the different type sample of the site table 3:cAMP-976-G/A
Table 4: the conspicuousness X2 distribution compared between sample
| Normal lefteye flounder | Melanism lefteye flounder | |
| Normal lefteye flounder | - | 53.61** |
| Albefaction lefteye flounder | 23.93** | 5.04 |
Note: X20.05=5.991, X20.01=9.21
The significance test result X of normal lefteye flounder and melanism lefteye flounder site adc-2677-C/A parting2=53.61 > 9.21,
P < 0.01 shows that genotype distribution has extremely significant sex differernce between normal lefteye flounder sample and melanism lefteye flounder sample on this site,
This SNP site is related to lefteye flounder melanism；The significance test knot of normal lefteye flounder and albefaction lefteye flounder site adc-2677-C/A parting
Fruit X2=23.93>9.21>5.991, P<0.01 shows on this site gene between normal lefteye flounder sample and albefaction lefteye flounder sample
Type distribution has extremely significant difference, this SNP site is related to lefteye flounder albefaction.Illustrate that the SNP site screened and lefteye flounder melanism are white
It is all related to change phenomenon, and conclusion is stablized, and is the associated SNP marker of lefteye flounder Malpigmentation.
Embodiment 2
The present embodiment applies to the SNP site screened in breeding work, to improve lefteye flounder Malpigmentation phenomenon.Body colour
The preponderant genotype of normal lefteye flounder SNP site adc-2677-C/A homozygous, this site of melanism lefteye flounder preponderant genotype for AA
For CC is homozygous, preponderant genotype of this site of albefaction lefteye flounder is AC heterozygous, the breeding method of the normal lefteye flounder of breeding body colour is
Screening SNP site adc-2677-C/A is that the lefteye flounder of AA homozygosis is male parent and maternal progress biological engineering.
(1) selection body colour is normal, and the good lefteye flounder of upgrowth situation is as parent.
(2) the lefteye flounder clip part fin ray that parent is to choosing, extracts tissue DNA, carries out PCR by template of the DNA of extraction
Amplification filters out the lefteye flounder that SNP site adc-2677-C/A is AA homozygosis and is used as breeding parent.
(3) lefteye flounder that screening is walked more than is male parent and maternal progress biological engineering, and establishing SNP site adc-2677-C/A is
The lefteye flounder family of AA homozygosis, then offspring has lower Malpigmentation incidence.
(4) the SNP site adc-2677-C/A established is the lefteye flounder family a and common lefteye flounder family b of AA homozygosis same
Artificial propagation, hatching, seed rearing are carried out in environment, rearing conditions, illumination, water temperature, the depth of water, density etc. are all consistent.Training
It educates by 120 days, then selects 100 tail lefteye flounders from family a and family b group at random, whether the body colour for counting every fish is normal,
Statistical result is shown in Table 5.
5: two family lefteye flounder body colour statistics of table
| Normal lefteye flounder (%) | Melanism lefteye flounder (%) | Albefaction lefteye flounder (%) | |
| Family a | 62 | 35 | 3 |
| Family b | 33 | 56 | 11 |
The normal lefteye flounder ratio of body colour is 62% in family a, and melanism lefteye flounder ratio is 35%, and albefaction lefteye flounder ratio is
3%, the normal lefteye flounder ratio of body colour is 33% in family b, and melanism lefteye flounder ratio is 56%, and albefaction lefteye flounder ratio is 11%,
Family a is compared with family b, and normal lefteye flounder ratio increases 29%, and melanism lefteye flounder ratio reduces 21%, albefaction lefteye flounder ratio
Reduce 9%.Show the SNP site adc-2677-C/A screened to apply in lefteye flounder breeding, lefteye flounder can be effectively reduced and supported
Malpigmentation phenomenon during growing.
Sequence table
<110>Shanghai Ocean University
<120>a kind of and the associated SNP marker of lefteye flounder Malpigmentation and its application
<141> 2019-04-26
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gtgttacagt tactcctgta acgaggatat ctaatgaaga acaggtggtc tttagaggcg 120
ctaggcggag tctagggctg acacgctgaa ccaagggtgg ctcaaatgca gacacacctc 180
tgatgcctct ggccagtttg agctctatgg tgtgtgctcg ctgggataaa ctagtggcct 240
ggccaaagag caaagtgttg ctacacacag taaagcccac acaggatgtc gtgggccaga 300
ggcctctttg taggcagagt ggacgatcgg aagacagcat ggggagagcg caacggcaaa 360
aagaggaagg acagctcatc cctgtgcaac ccgcgctaca tgagctgctt gagggaccct 420
gaggacctgg agccctctaa tgtggcccct cagcattacc actgtgcagc gggcaccagc 480
gggggcaact ttggcttgcg ctgtggctgg caggaggacc actatgggaa aaggatgggt 540
ggtggtgggg atctgggtct aaaggcggtg aaccttggct tcgaagatgg cgacctgaag 600
ggaaggaaag aggaggaatc aggggaatta gctgtgggca gctgcaacac gatgactgct 660
gcagactgct ggatgcggtt tgtgtgggtt ttccaatcca agaagtttca gtccaccaag 720
ctggagcgcc tgtaccagcg ctacttcttc aggctcaacc agagctccct gaccatgctg 780
atgggggtgc tggtggtggt gtgtggggtc atgcttgcct tccactgcat ccacacgacc 840
cctgatgatg cctatgtctc agtactctgt gtggctatgg cgctcttctt ggccatgatg 900
gtggtgtgca accgcaatgg cttccatcag gactacatgt ggattgtgag ctatctggtg 960
atcggggtgc tgattgtggt gcaggttttt ggggtgctga agatgtatcc ccgcagtgcc 1020
tctgagggca tctggtggac agtcttcttc atctacatta tctacacact gctgccggtc 1080
agaatgagag ccgctgtggt cagtggagca gtgctgtcca ccatacatgt aatcaccgcc 1140
tggcagctca accaagagga caagtacatt ttcaaacagg tcagtgccaa tgtgctgatc 1200
ttcctgtgca ccaacatgat tgggatctgt acccactacc ccgcggaggt ctcccagaga 1260
caagccttcc aggagacgag aggatacatc caggctagac tgcacctgca gagggaaaac 1320
cagcagcagg agcgcctact gctgtcagta ctgccaaggc acgttgccat ggagatgaag 1380
gctgatatca atgccaagaa ggaagatatg atgttccaca agatctacat tcaaaaacat 1440
gacaatgtta gcatactgtt tgcagacatc gagggcttca ccagcctggc atctcagtgc 1500
acggcccagg agctggtcat gacgctgaac gagctgttcg cccgcttcga caagctggcc 1560
tcggagaacc actgcctgcg cattaaaatc ctcggggact gttactactg tgtgtcaggg 1620
ctgcctgagc ccagggctga ccacgcccac tgctgtgtgg agatgggagt agacatgatc 1680
gaagccatct cgctggtgag agaggtgaca ggcgttaatg tgaacatgcg ggtgggcatc 1740
cacagcggtc gcgttcactg cggggtgctg ggcctgcgca agtggcaatt tgatgtctgg 1800
tccaacgacg tgacgctcgc taaccaaatg gaagctggag gcaaggctgg gcgtatccac 1860
atcacaaaag ccacactgca gtatctgaac ggggactacg aagtggagcc gggatttgga 1920
gaagagagga atacttacct gaaggagaac gacattgaaa ccttcttggt gctgggctgc 1980
agccaaaaga ggaaagagga gaaagccatg atggccaaga tgcagagaac acgtgcaaat 2040
tctgctgagg gactgatgcc acgctgggtg cctgacagat ccttctccag acccaaggag 2100
cccaaagcct tcaggcagat gggcatcgac gaagcatcca gcaaagacaa ccgctgcact 2160
caggaggccc tgaaccccga ggatgaggtg gatgagtttc ttggacgggc catcgacgcg 2220
cgcagcattg accagctgcg gaaagatcac gtcaagaagt ttctgctcac ttttcagaca 2280
gctgacctcg agaaaaagta ctccaaaaag gttgatgatc gttttggagg atacgtggcc 2340
tgcacactcc ttgttttctg ctgcatctgt ttcattcaga tcatcatttt cccgaggaca 2400
acacttatgc taggtcttta catcagcatc ttcatcatcc ttaacaacat cctcttcatc 2460
tgtgccatct actcctgcat caaactcttc ccagttgcct tgcagactgt aaccaagaaa 2520
atagtccagt cacgtgcaaa cagcacactg gttggcgtct tcaccatcct cctcctcttc 2580
atctcctctt ttgctaatat gtttacctgc agcacagaga aactggagga ctgcgtggcc 2640
gacgagctga acacatcagt gactctctgc ctgctgcaca acctgaccaa gacagccgag 2700
gatggtttga ctctgtgctc cagccaggcc atgccctgcc atttccccga gtacttcagc 2760
tacagcgtgt tgctgaccct gctggcctgc tctgtgttcc tccacatcag cagcataggg 2820
aagctggctc tgatgctgct catccagctc actttcctcc tgctggtaga gtggcctcag 2880
gtggccctgt ttgacaacgc agacctgctg gtcattgcca acaacctgaa attatcaaat 2940
aatgattccc aacaacagcc tagtttcaat gaaactgcag aacagtgcct ggagagtgtg 3000
accaaggtat ccctaaaggt catgacccct gtaatcctga cagtttttgt tctggctctc 3060
tacttgcacg gccagcaggt ggagtccact gctcgcctgg acttcctctg gaagctgcag 3120
gccacagaag agaaggagga gatggaggag ctgcaagcat acaaccggcg cctgctgcac 3180
aacatcctac ccaaggacgt ggctgcccac tttcttgcga gggagcgtag gaatgacgag 3240
ttgtactacc agtcatgtga atgcgtcgct gtcatgtttg cctccattag caacttctct 3300
gagttttacg tggagctgga ggcaaacaac gagggagtgg aatgtctcag gctgctcaac 3360
gagatcatcg ctgattttga tgagatcatc agtgaggaga gattccgtca gctggagaaa 3420
atcaaaacca tcggctccac ctacatggca gcctccggcc tcaaccattc cacttatgat 3480
aaagaaggcc gctcacacat tctagctttg gctgactacg ccatgaggct tagagaacag 3540
atgaaataca tcaacgagca ctccttcaac aacttccaga tgaagatagg actgaacatg 3600
gggcctgtgg tggcaggggt gattggagcc aggaaacccc agtatgatat atgggggaac 3660
acagtcaacg tggctagccg catggacagc acaggagtac cagactacat ccaggtgacc 3720
acagacttgt accacgtgct ggtcaacaac ggataccagc tggactgccg aggtgtcgtc 3780
aaggtgaagg ggaaggggga gatgaccacc tacttcctca ccagcggtcc cccgggcagt 3840
taatgacaga gcggggctgc ccacagagtg actcccattg cactgacata acagagagca 3900
gcagagagac ttgagagttc agcccggcga cgagcaacgg agcgtggtgc gaacgcgcca 3960
gggacgggag atggccggca gagacgagcc aggcccttct cgcctattgt cacatcataa 4020
gatgcacaca cgtaggacgt tgttcacgtt tgaaggcccc cccaccccac cccaagctgc 4080
ttgagagata acgaagccca gttgccttag gacggaggtg gctatgcttg tgttgatgag 4140
caatagtatg agtatctcat ctggagtcaa tatttatttt catgaggatg gatttttttg 4200
tgataacctt gtacaaaaat gaaaatattt acatacaaaa aaagctatgt cacttaacag 4260
aaaatatgca aggacaatat gaaggatgaa aatgaccaaa gaacaaatgt ttgatatgaa 4320
acacacacac aaattattct gttcagaaaa tacagtaatt tattcaactg cagacagcaa 4380
gggccacggt aacgtcactg tctttgatgg ggacaaactt ttcttttttt agctgcggat 4440
tttttcatgg ggataacaca cacacacaca cac 4473
<210> 2
<211> 21
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
cctgcagcac agagaaactg g 21
<210> 3
<211> 21
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
acttcagcta cagcgtgttg c 21
Claims (6)
1. a kind of and associated SNP marker of lefteye flounder Malpigmentation, which is characterized in that the SNP marker are as follows:
Adc-2677-C/A, positioned at the position 2677bp of gene base sequence as shown in SEQ ID NO:1, base A is mutated into C,
Threonine becomes histidine, and normal lefteye flounder genotype is 68.75%AA, 31.25%AC and 0%CC, and melanism lefteye flounder genotype is
0%AA, 37.5%AC and 62.5%CC, albefaction lefteye flounder genotype are 14.29%AA, 64.29%AC and 21.42%CC.
2. described in claim 1 with the screening technique of the associated SNP marker of lefteye flounder Malpigmentation, which is characterized in that including
Following steps:
(1) select respectively the normal skin of normal lefteye flounder, the normal pale skin in lefteye flounder anophthalmia side of anophthalmia lateral body color anomaly and
The casting skin of anophthalmia lateral body color anomaly anophthalmia side carries out gene bidirectional sequencing, then carries out snp analysis, Preliminary detection to sequencing data
SNP site out, analysis obtain the access that conspicuousness is enriched in difference expression gene；
(2) it combines in step (1) access relevant to body colour known to 6 and carries out snp analysis, filter out positioned at code area
The SNP site of nonsynonymous mutation；
(3) parting is carried out in the SNP site that step (2) obtains to lefteye flounder；
(4) screening and the associated SNP site of lefteye flounder Malpigmentation.
3. the screening technique of SNP marker according to claim 2, which is characterized in that in step (1), the difference table
The analysis method for the access being enriched with up to conspicuousness in gene includes gene annotation, differential genes expression analysis, differential gene GO richness
Set analysis and differential gene KEGG enrichment analysis.
4. the screening technique of SNP marker according to claim 2, which is characterized in that in step (3), the SNP site
The primer that parting uses is as shown in SEQ ID NO:2 and SEQ ID NO:3.
5. described in claim 1 reducing the rate breeding of lefteye flounder Malpigmentation with the associated SNP marker of lefteye flounder Malpigmentation
On application.
6. the application of SNP marker according to claim 5, which is characterized in that the SNP marker is for reducing tooth
The method of flounder Malpigmentation rate breeding are as follows: do the lefteye flounder clip part fin ray of parent to choosing, tissue DNA is extracted, with the DNA of extraction
Carry out PCR amplification for template, PCR product sequencing, filter out the lefteye flounder that SNP site adc-2677-C/A is AA homozygosis be male parent with
Female parent carries out biological engineering, establishes the lefteye flounder family that SNP site adc-2677-C/A is AA homozygosis, then thereafter on behalf of Malpigmentation
The lower seed of rate.
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