CN110041422A - Micropterus salmoides grows relevant SNP site and its application - Google Patents
Micropterus salmoides grows relevant SNP site and its application Download PDFInfo
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Abstract
Relevant SNP site and its application are grown the invention discloses Micropterus salmoides, and the relevant SNP site of the growth is located at HSC70-1 gene order the 821st, 2804 bit bases.Present invention discover that in the position C+821G of HSC70-1, there are allele Cs and allele G, form three kinds of genotype CC, GC and GG, and in the position A+2804T, there are allele A and allele T, form three kinds of frequency of genotypes AA, AT and TT.When the 821st and 2804 base positions genotype are that the individual growth traits of CC and TT is substantially better than the individual of other genotype.The largemouth black bass parent of CC and TT genotype will be retained in production using the phenomenon, remove the individual of other genotype, the speed of growth can be quickly obtained fastly and the Micropterus salmoides kind of inheritance stability, i.e., SNP site of the present invention can be applied to the screening of fast-growth largemouth black bass parent.
Description
Technical field
The invention belongs to field of molecular biotechnology, and in particular to the associated SNP site of Micropterus salmoides growth traits and its
Using.
Background technique
Micropterus salmoides (Micropterus salmoides L.), also known as largemouth bass, originate in North America, adaptable
By force, the advantages that growth is fast, disease is few, low temperature resistant, growth cycle is short and delicious flavour, be important cultured freshwater fish it
One.It introduces a fine variety from Taiwan to Guangdong Province within 1983, whole nation most area has cultivation now.But introduce a fine variety more than 30 years
Come, do not focus on parent due to production unit and reserve seed for planting the operating instruction that must be abided by, also periodically could not supplement and introduce parent from source area
This, causes the genetic diversity for cultivating Micropterus salmoides to reduce, so that production performance be made also to reduce, is mainly manifested under the speed of growth
Drop, bait transformation efficiency are low, premunition also declines to a great extent.Wherein, the speed of growth is related to the height of Micropterus salmoides yield, cultivation
The size of benefit.The trend that Micropterus salmoides growth is degenerated directly restricts the development of Culture Techniques of Micropterws Salmoides industry.Therefore, big mouth is improved
The work of the sea bass speed of growth is extremely important.
Selecting high-quality parent population is one of the main method for improving the cultured fishes speed of growth, and its purpose is to change cultivation
The genetic structure of group makes offspring obtain the genetic characteristics of the faster speed of growth, better disease resistance.In production,
Conventional method is that the fish that picking individual is larger, healthy and strong is used as the parent that reserves seed for planting, i.e., determines whether parent population reserves seed for planting according to phenotype.
This method is convenient, fast, simple, but be affected by human factors it is very big, in addition Micropterus salmoides belong to it is carnivorous based on fish,
Grazing eclipse is strong, and bait deficiency is to kill and devour mutually, causes its growth great disparity larger.So as it can be seen that only judging that big mouth is black from phenotype
Ideal effect is often not achieved in perch parent mass.With the development of molecular biology and science of heredity, a variety of something lost have been emerged
Passing label, such as AFLP, RAPD, SSR, SNP label, wherein SNP marker is suitable for high throughput automated analysis because widely distributed,
Inheritance stability has become genetic marker preferred in genetic breeding research.If these genetic markers can be related to the production traits
Be linked togather, can be realized and carry out selection and use from DNA level, overcome conventional method be affected by human factors it is big unfavorable
Factor, improves the accuracy of selection, and may be implemented in and identify the individual with merit in early days, filters out excellent
Standby parent accelerates breeding process so as to shorten breeding cycle.By finding molecule mark relevant to Micropterus salmoides fast-growth
Note improves the screening efficiency of fast long parent.
Summary of the invention
One of the objects of the present invention is to provide a kind of SNP sites relevant to Micropterus salmoides growth traits.
It is another object of the present invention to provide a kind of methods for screening fast-growth largemouth black bass parent.
The technical solution used in the present invention is:
The amino acid sequence of Micropterus salmoides HSC70-1 is as shown in SEQ ID NO:1.
The gene order of Micropterus salmoides HSC70-1 described above is nucleotide sequence shown in SEQ ID NO:2, the
821 S are base C or G, and the 2804th W is base A or T.
Micropterus salmoides HSC70-1 gene order described above is judging the application in Micropterus salmoides growth speed.
The relevant SNP site of the Micropterus salmoides speed of growth is located at shown in HSC70-1 gene order SEQ ID NO:2
821st bit base, the 2804th bit base.
SNP site described above is judging the application in Micropterus salmoides growth speed.
Application of the SNP site described above in screening fast-growth Micropterus salmoides.
A method of screening fast-growth Micropterus salmoides detects the 821st bit base of Micropterus salmoides HSC70-1 gene order
Whether the SNP site at place is CC homozygote, if so, being the Micropterus salmoides of fast-growth；Or detection HSC70-1 gene sequence
Arrange whether the SNP site at the 2804th bit base is TT homozygote, if so, being the Micropterus salmoides of fast-growth.
Further, method described above, includes the following steps:
1) Micropterus salmoides DNA is extracted；
2) to extract obtained DNA as template, PCR detects the 821st, 2804 alkali of Micropterus salmoides HSC70-1 gene order
Whether the SNP site of Ji Chu is respectively CC, TT homozygote.
Further, the concrete operations of PCR detection are to be utilized respectively primer P1F and P1R, P2F and P2R to big mouth
Sea bass DNA carries out primary PCR amplification, obtains first PCR product,
P1F:5'-TGAAGCCTACCTCGGAAAAGT-3'(SEQ ID NO.3),
P1R:5'-AGCATCCTTAGTGGCCTGGCGC-3'(SEQ ID NO.4),
P2F:5'-TCCACCAGCTTATCAGACTGT-3'(SEQ ID NO.5),
P2R:5'-GGTCAACCCTCCAAGTAACTT-3'(SEQ ID NO.6)；
First PCR product is subjected to extension amplification with primer P1 and P2 respectively again, by products therefrom through sequencing analysis, is determined
Whether the SNP site at Micropterus salmoides HSC70-1 gene order the 821st, 2804 bit bases is respectively CC, TT homozygote；
P1:5'-TTACTTAGCATAGCTCTGGACA-3'(SEQ ID NO.7),
P2:5'-CTGTAGGGGGTAACTGAAGGGT-3'(SEQ ID NO.8).
Further, the reaction system of primary PCR amplification are as follows:
Primary PCR amplification response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C extend
30s, 24 circulations；72 DEG C of extension 3min.
Further, extend the reaction system of amplification are as follows:
Extend amplified reaction program are as follows: 96 DEG C of initial denaturation 1min；96 DEG C of denaturation 10s, 52 DEG C of annealing 5s, 60 DEG C of extension 30s,
30 circulations.
The beneficial effects of the present invention are:
(1) present invention will retain the largemouth black bass parent of CC and TT genotype in production, remove the individual of other genotype,
It can be quickly obtained that the speed of growth is fast and the kind of the Micropterus salmoides of inheritance stability.This method has compared with traditional method
Purpose is strong, the direct advantage of function and effect, and it is easy to operate, detection quickly, testing cost it is low, convenient for being widely used to promote.
(2) present invention greatly reduces the blindness of largemouth black bass parent screening, can be quickly obtained the speed of growth it is fast and
The Micropterus salmoides individual of inheritance stability.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The acquisition of the SNP marker relevant to Micropterus salmoides growth traits of embodiment 1
The application is the study found that Micropterus salmoides heat shock protein HSC70-1 (heat shock cognate protein 70)
Amino acid sequence as shown in SEQ ID NO:1, Micropterus salmoides HSC70-1 coding sequence overall length be 3382bp (SEQ
ID NO:2), it is made of 8 exons and 7 intrones.In Micropterus salmoides heat shock protein HSC70-1 gene order (SEQ
ID NO:2) the 821st and 2804 respectively find a SNP site, find the 821st by Snapshot method parting
There are allele Cs and allele G for base positions, form three kinds of genotype CC, GC and GG；It is deposited in the 2804th bit base position
In allele A and allele T, three kinds of frequency of genotypes AA, AT and TT are formed.
This experiment for the random population that the sample number of association analysis is 430 tails be with a batch breeding, with pool cultivated, and adopt
Sample time consistency, therefore the difference of time, environment and Artificial feeding conditions is not considered when establishing model.Micropterus salmoides HSC70-
Different genotype frequency distribution in random population is shown in Table 1 and 2 respectively at 1 gene the 821st, 2804.Wherein, the 821st
SNP site be the homozygotic genotype frequency of CC it is lower be 7.21%, the 2804th SNP site is the homozygotic gene of TT
Type frequency is lower, is also 7.21%.
The 821st site different genotype frequency distribution in random population of 1 Micropterus salmoides HSC70-1 gene of table
The 2804th site different genotype frequency distribution in random population of 2 Micropterus salmoides HSC70-1 gene of table
The association analysis of SNP and character
The Multiple range test of growth traits between different genotype is individual at Micropterus salmoides HSC70-1 gene the 821st, 2804
As a result it is shown in Table 3 and table 4 respectively.5 growth traits (weight, overall length, the long, body of 821st CC genotype individuals measurement
It is high, caudal peduncle is long) mean phenotypic value be above the mean value of CG type and GG type individual, wherein CC genotype individuals respectively with CG and
There are significant difference (P < 0.05) on weight and overall length for GG genotype individuals.2804th TT genotype individuals measurement
The mean phenotypic values of 5 growth traits (weight, overall length, long, body is high, caudal peduncle is long) be above AT type and AA type individual
Mean value, wherein there are significant difference (P < on weight and overall length with AT and AA genotype individuals respectively for TT genotype individuals
0.05)。
Above-mentioned association analysis the result shows that, the genotype that the SNP site of Micropterus salmoides HSC70-1 gene of the present invention is constituted
Weight and overall length character are had a significant impact (P < 0.05), the growth traits for the individual that genotype is CC and TT is substantially better than it
The individual of his genotype.
The Multiple range test of growth traits between the 821st site different genotype individual of 3 HSC70-1 gene of table
The Multiple range test of growth traits between the 2804th site different genotype individual of 4 HSC70-1 gene of table
From the data in table 1~4 it is found that the 821st and 2804 of HSC70-1 gene order (SEQ ID NO:2)
SNP site and Micropterus salmoides growth traits are closely related.By detection Micropterus salmoides HSC70-1 gene order (SEQ ID NO:
2) whether the SNP site at the 821st is CC homozygote, or whether the SNP site at detection the 2804th is TT homozygosis
Body, the largemouth black bass parent of fast-growth required for can quickly and accurately filtering out.
A kind of method for screening fast-growth Micropterus salmoides of embodiment 2
Fast-growth largemouth black bass parent is screened using above-mentioned SNP site, is included the following steps:
(1) primer sequence:
Two pairs of primers are devised according to Micropterus salmoides heat shock protein HSC70-1 gene order and carry out PCR amplification, are designed and are closed
At primer it is as follows:
P1F:5'-TGAAGCCTACCTCGGAAAAGT-3'(SEQ ID NO.3),
P1R:5'-AGCATCCTTAGTGGCCTGGCGC-3'(SEQ ID NO.4),
P2F:5'-TCCACCAGCTTATCAGACTGT-3'(SEQ ID NO.5),
P2R:5'-GGTCAACCCTCCAAGTAACTT-3'(SEQ ID NO.6)；
(size is by primer 2 DNA bands of expected amplification, i.e. SEQ ID NO:9 (size 176bp) and SEQ ID NO:10
222bp)。
(2) sample DNA submits (alkaline lysis):
1, clip Micropterus salmoides fin ray 10-20mg to be detected is fitted into clean EP pipe；
2, the 50mmol/L NaOH solution of 180 μ L is added, water-bath 20min (room temperature), during which concussion is for several times；
3, the 1mol/L Tris-HCL solution (PH=8.0) of 20 μ L, sufficient vortex concussion is added；
4, sample cell being put into centrifuge 12000rpm centrifugation 10min, Aspirate supernatant obtains Micropterus salmoides genomic DNA,
It is spare.
(3) PCR system that primer expands for the first time:
The reaction system and amplification condition of primary PCR amplification are as follows:
(4) PCR amplification program that primer expands for the first time:
(5) Single base extension is carried out to the PCR product of purifying
The use of Snapshot method with the first PCR product that obtains is template, makes one base of primer extend in polymorphic site
It terminates, is detected on sequenator, whether be CC and TT according to the base of the color polypeptide site at peak.
Extension system are as follows:
Extension primer sequence are as follows: P1:5'-TTACTTAGCATAGCTCTGGACA-3'(SEQ ID NO.7) and P2:5'-
CTGTAGGGGGTAACTGAAGGGT-3'(SEQ ID NO.8)。
Extension condition are as follows:
(6) on sequenator, the color of extension products size and peak is detected, determines the HSC70-1 of parent to be measured
Whether the SNP site at the 821st bit base of gene order is CC homozygote, if so, being the Micropterus salmoides of fast-growth；Or
Whether the SNP site detected at the 2804th bit base of HSC70-1 gene order is TT homozygote, if so, for fast-growth
Micropterus salmoides.
This detection method can operate completion within 10 hours, and can detect simultaneously to multiple samples, energy
Fast and accurately testing result is provided for the Micropterus salmoides good variety selection next to be carried out and identification.We pass through to excellent
The identification of gesture allele, is the assessment on DNA level to Micropterus salmoides germplasm quality, and purpose is stronger.It is examined with this method
Cost needed for surveying a sample is about 3 yuan, and cost is relatively low, is suitble to promote the use of.
In conclusion applicants have found that, in the position C+821G of Micropterus salmoides heat shock protein HSC70-1 gene order
A SNP is found respectively with the position A+2804T, and by the discovery of Snapshot method parting, in the position C+821G, there are equipotential bases
Because of C and allele G, three kinds of genotype CC, GC and GG are formed, there are allele A and allele in the position A+2804T
T forms three kinds of frequency of genotypes AA, AT and TT.Detection random population it was found that, two SNP sites to weight and complete
Long character has a significant impact, in the growth traits for the individual that 821 base positions and 2804 base positions genotype are CC and TT
It is substantially better than the individual of other genotype.Therefore, it according to Micropterus salmoides heat shock protein HSC70-1 gene order design primer, builds
The parent that effectively identification has good development character and inheritance stability is stood.CC and TT base will be retained in production using this method
Because of the largemouth black bass parent of type, remove the individual of other genotype, can be quickly obtained that the speed of growth is fast and inheritance stability it is big
Mouth sea bass kind.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
<120>Micropterus salmoides grows relevant SNP site and its application
<130>
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 650
<212> PRT
<213>artificial sequence
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Met Ser Lys Gly Pro Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser
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Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Ser Glu
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Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn Pro Thr
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Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Arg Phe Asp Asp
65 70 75 80
Thr Val Val Gln Ser Asp Met Lys His Trp Pro Phe Asn Val Ile Asn
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Asp Asn Thr Arg Pro Lys Val Gln Val Glu Tyr Lys Gly Glu Thr Lys
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Ser Phe Tyr Pro Glu Glu Val Ser Ser Met Val Leu Thr Lys Met Lys
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Glu Ile Ala Glu Ala Tyr Leu Gly Lys Thr Val Asn Asn Ala Val Ile
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Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp
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Ala Gly Thr Ile Ser Gly Leu Asn Val Leu Arg Ile Ile Asn Glu Pro
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Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Val Gly Ser Glu
180 185 190
Arg Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser
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Ile Leu Thr Ile Glu Asp Gly Ile Phe Glu Val Lys Ser Thr Ala Gly
210 215 220
Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Val Asn His
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Phe Ile Ala Glu Phe Lys Arg Lys Tyr Lys Lys Asp Ile Ser Asp Asn
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Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg
260 265 270
Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp Ser Leu Tyr
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Glu Gly Val Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu
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Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Asp Pro Val Glu Lys Ser
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Leu Arg Asp Ala Lys Met Asp Lys Gly Gln Ile His Asp Ile Val Leu
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Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys Leu Leu Gln Asp
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Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala
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Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala
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Asp Asp Val Gln Arg Asp Lys Val Ser Ala Lys Asn Gly Leu Glu Ser
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atgtctaagg gaccagcagt tggtatcgat cttgggacca cctactcctg tgttggtgtg 60
ttccagcatg gcaaagttga aatcatcgcc aatgaccaag gcaacaggac cacacccagc 120
tacgtggcct tcacagacag tgagaggctg atcggagatg cagccaagaa tcaggttgcc 180
atgaacccca ccaacacagt ctttggtaag tattaaacgg ggaaattgtg gtcttgtgtc 240
actcttgcct gcgtaaaatc aaacatttta tatatagtaa atagtaaaac gccttaaagt 300
ccaatactaa ctgcttataa agagactggc acctctgtcg gtggtaaaac agtaaagtct 360
tatctatgaa ctcgaggcaa gaatggaaca agactgaaat cttaagtggc cgtattaaac 420
aacttcctca ctgtgatgac gattttcctg ccattcgtag tttccaccag aggttgaaag 480
accaaagatg gcccaaatcc ttccttggat gtctgagtga tattcttaaa ggaagcccag 540
agcatattat attttttttc cattaaaccc taaactgtct tttgcagatg ccaaacgact 600
gattggccgc aggtttgacg acacagttgt gcagtcagat atgaagcact ggccatttaa 660
tgtcatcaat gataacaccc gccccaaggt tcaagttgaa tacaaaggcg agacaaagtc 720
cttctaccca gaggaggtct catctatggt gctgacaaag atgaaggaga ttgctgaagc 780
ctacctcgga aaagtatgtt acttagcata gctctggaca saactccaaa tataaaatta 840
tatgaagtga tgctaatttt tttcttattc ccttcaagac tgtcaacaat gctgtaatta 900
cggtacccgc ctacttcaac gactcccagc gccaggccac taaggatgct ggcacaatct 960
ctggcctcaa tgtcctgcgt atcatcaatg aaccaaccgc tgctgccatt gcctatgggt 1020
tggacaagaa ggtaaataaa actagaaaac ttccaattac acttgcatga tggccaatgt 1080
accatcttga attgaggtaa taaaacataa ctgagtagat tttaaatgtt actaacttaa 1140
tttcaggttg ggtctgaaag gaacgttctc atctttgatc ttggtggtgg caccttcgat 1200
gtttccatct tgaccatcga ggatggcatc tttgaggtca agtccactgc tggagatact 1260
catcttggcg gtgaggattt cgacaaccgc atggtcaacc acttcattgc agagttcaag 1320
cgcaagtaca agaaagacat cagtgacaac aagagagctg tccgtcgcct gcgcaccgct 1380
tgcgagaggg caaagcgcac cctgtcttcc agcacccagg ccagcattga aatcgactct 1440
ctgtacgagg gagtcgactt ctatacctca atcaccaggg ctcgctttga ggagctcaac 1500
gctgacctct tccgtggcac cctggaccct gtggagaagt cgctccgtga tgccaagatg 1560
gataaagggc agattcacga catagtgttg gtcggtggct ccactcgtat ccccaagatc 1620
cagaagctgc tccaggattt cttcaatgga aaggagctca acaagagcat caatccagat 1680
gaagccgtgg cctatggagc tggtaagtaa accaaacatt tccatccaat tagagctcta 1740
gatataaatt tctactgaga agtatttttg ttgaattgtt aagagattaa catgaaaaat 1800
atccaccttc agtccctttc aaaatctaac cagccgccct tatttacagc tgtccaggct 1860
gccatcctgt ctggtgacaa gtctgagaat gtccaggact tgctgctttt ggacgtcacc 1920
cctctctccc tgggaattga gaccgctgga ggtgtcatga ctgtcctgat caaacgtaac 1980
accactattc ctaccaagca gacgcagacc ttcaccacct actctgacaa ccagcctggt 2040
gtgctcatcc aggtgaggac gctggttgat ggggttcagt tgtttgtgca attcataatt 2100
ctaacagaat taacataatt ataaattcat ttcggggatg ttggagagac tgaagtgaca 2160
ttgcatacgt ccaacaggtt tatgagggcg agcgtgccat gacaaaggac aacaacctgc 2220
tgggcaagtt tgagctgacg ggcatccccc ctgctcctcg tggtgttccc cagatagagg 2280
tgacgtttga tattgatgcc aatggaatcc ttaatgtctc tgctgtagac aagagcactg 2340
gcaaggagaa caagatcacc atcaccaatg acaagggtac aaaaaagtct gcttaaccca 2400
aataccgctg cagtgtccca acaacggatg attaaatggt aatcttgcct gttttatgcc 2460
aggtcgtctc agcaaggagg acattgaacg catggtccaa gaagctgaga agtacagggc 2520
tgaagacgac gtccagcgtg acaaggtgtc agccaaaaat ggcctggagt cgtatgcttt 2580
caacatgaag tcgaccatgg aagatgaaaa gcttgctggc aagatcagtg atgaagacaa 2640
gcagaagatc ttggacaagt gcaacgagat tattggctgg ctggacaaga accaggtggg 2700
aattgttggt tttggaacct gtgatccacc agcttatcag actgtctatt cctgtctgct 2760
aagcgaattt agagatatat actgtagggg gtaactgaag ggtwggggtc tatatataca 2820
aacaaatatg tttctgagaa ggagaaaatg aacgataact gaaaatacgg taataaacta 2880
atttaacaaa catagccttc tgatctcagt ataggaaaaa tgcttaagtt acttggaggg 2940
ttgaccccag agttttgatt tttttcaaca tgcaaatgta taatttacta ggaagtaagc 3000
aatgggtcta ggattttgca ggtctgctaa acgtcatcag agaatggtga ttttatgtgt 3060
agacactatc gttttgtcag gctaaattat cctcatctgt gtttcaacaa tagcagagtt 3120
taaactgaac tgagtattaa ctgtcaatta attccaattg agaatatatg atttgatttg 3180
tttgtactag actgcagaaa aagatgaata tgaacaccag caacaagaac tggagagggt 3240
gtgtaacccc atcatcacca agctgtacca gagtggtggt gatgtgacag gtgggatgtc 3300
cagtggaatg ccaggcggat tccctggggc tggtggtgct ccagccgctg gaggatcctc 3360
tggaccaact tgtggagggg ga 3382
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
tgaagcctac ctcggaaaag t 21
<210> 4
<211> 22
<212> DNA
<213>artificial sequence
<400> 4
agcatcctta gtggcctggc gc 22
<210> 5
<211> 21
<212> DNA
<213>artificial sequence
<400> 5
tccaccagct tatcagactg t 21
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
ggtcaaccct ccaagtaact t 21
<210> 7
<211> 22
<212> DNA
<213>artificial sequence
<400> 7
ttacttagca tagctctgga ca 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence
<400> 8
ctgtaggggg taactgaagg gt 22
<210> 9
<211> 176
<212> DNA
<213>artificial sequence
<400> 9
tgaagcctac ctcggaaaag tatgttactt agcatagctc tggacacaac tccaaatata 60
aaattatatg aagtgatgct aatttttttc ttattccctt caagactgtc aacaatgctg 120
taattacggt acccgcctac ttcaacgact cccagcgcca ggccactaag gatgct 176
<210> 10
<211> 222
<212> DNA
<213>artificial sequence
<400> 10
tccaccagct tatcagactg tctattcctg tctgctaagc gaatttagag atatatactg 60
tagggggtaa ctgaagggtt ggggtctata tatacaaaca aatatgtttc tgagaaggag 120
aaaatgaacg ataactgaaa atacggtaat aaactaattt aacaaacata gccttctgat 180
ctcagtatag gaaaaatgct taagttactt ggagggttga cc 222
Claims (10)
1. the amino acid sequence of Micropterus salmoides HSC70-1 is as shown in SEQ ID NO:1.
2. the gene order of Micropterus salmoides HSC70-1 described in claim 1 is nucleotide sequence shown in SEQ ID NO:2,
821st S is base C or G, and the 2804th W is base A or T.
3. Micropterus salmoides HSC70-1 gene order described in claim 2 is judging the application in Micropterus salmoides growth speed.
4. the relevant SNP site of the Micropterus salmoides speed of growth, it is located at shown in HSC70-1 gene order SEQ ID NO:2 the
821 bit bases, the 2804th bit base.
5. SNP site described in claim 4 is judging the application in Micropterus salmoides growth speed.
6. application of the SNP site described in claim 4 in screening fast-growth Micropterus salmoides.
7. a kind of method for screening fast-growth Micropterus salmoides, which is characterized in that detection Micropterus salmoides HSC70-1 gene order
Whether the SNP site at the 821st bit base of SEQ ID NO:2 is CC homozygote, if so, the big mouth for fast-growth is black
Perch；Or/and whether the SNP site at the 2804th bit base of detection HSC70-1 gene order SEQ ID NO:2 is TT homozygosis
Body, if so, being the Micropterus salmoides of fast-growth.
8. the method according to the description of claim 7 is characterized in that including the following steps:
1) Micropterus salmoides DNA is extracted；
2) to extract obtained DNA as template, at PCR detection Micropterus salmoides HSC70-1 gene order the 821st, 2804 bit bases
SNP site whether be respectively CC, TT homozygote.
9. according to the method described in claim 8, it is characterized in that, the concrete operations of PCR detection are to be utilized respectively primer
P1F and P1R, P2F and P2R carry out primary PCR amplification to Micropterus salmoides DNA, obtain first PCR product,
P1F:5'-TGAAGCCTACCTCGGAAAAGT-3'(SEQ ID NO.3),
P1R:5'-AGCATCCTTAGTGGCCTGGCGC-3'(SEQ ID NO.4),
P2F:5'-TCCACCAGCTTATCAGACTGT-3'(SEQ ID NO.5),
P2R:5'-GGTCAACCCTCCAAGTAACTT-3'(SEQ ID NO.6)；
First PCR product is subjected to extension amplification with primer P1 and P2 respectively again, by products therefrom through sequencing analysis, determines big mouth
Whether the SNP site at sea bass HSC70-1 gene order the 821st, 2804 bit bases is respectively CC, TT homozygote；
P1:5'-TTACTTAGCATAGCTCTGGACA-3'(SEQ ID NO.7),
P2:5'-CTGTAGGGGGTAACTGAAGGGT-3'(SEQ ID NO.8).
10. according to the method described in claim 9, it is characterized in that, the reaction system of primary PCR amplification are as follows:
Primary PCR amplification response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 30s,
24 circulations；72 DEG C of extension 3min.
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| CN113637766A (en) * | 2021-07-30 | 2021-11-12 | 中国水产科学研究院珠江水产研究所 | InDel molecular marker related to genetic sex identification of micropterus salmoides and application thereof |
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