CN106544410A - A kind of and megalobrama amblycephala disease resistance trait related SNP molecular labeling and its application - Google Patents
A kind of and megalobrama amblycephala disease resistance trait related SNP molecular labeling and its application Download PDFInfo
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- CN106544410A CN106544410A CN201610608136.XA CN201610608136A CN106544410A CN 106544410 A CN106544410 A CN 106544410A CN 201610608136 A CN201610608136 A CN 201610608136A CN 106544410 A CN106544410 A CN 106544410A
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- megalobrama amblycephala
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Abstract
The invention belongs to fish molecular marker screening technical field, and in particular to a kind of and megalobrama amblycephala disease resistance trait related SNP molecular labeling and its application.Described molecular labeling is obtained by cloning in megalobrama amblycephala NLRC3 like genes, in NLRC3 like gene extrons, the nucleotide sequence such as SEQ ID NO of the molecular labeling：Shown in 1, there is an allelic mutation (C/T) at 100 bit bases of the sequence, it is valine by alanine mutation that the mutation causes amino acid.Association analysis has been carried out to megalobrama amblycephala disease resistance trait using the molecular labeling of present invention screening.The molecular labeling of present invention screening can be applied in megalobrama amblycephala disease resistance trait assisted Selection.
Description
Technical field
The invention belongs to fish molecular labeling preparation field, and in particular to a kind of to divide with megalobrama amblycephala disease resistance trait related SNP
Son mark and its application, described molecular labeling can be applied in megalobrama amblycephala disease resistance trait assisted Selection.
Background technology
Megalobrama amblycephala (Megalobrama amblycephala) is under the jurisdiction of Cypriniformes Cypriniformes, Cyprinidae
Cyprinidae, catfish subfamily Culterinae, triangular bream belong to Megalobrama, are commonly called as blunt snout bream, are distributed across the middle and lower reach of Yangtze River big-and-middle
Type lake relatively large economic fish (Chen Yiyu, 1998).Have that feeding habits are wide, growth is fast, disease is few, easily fish for due to which, it is numerous
The advantages of simplicity, survival rate are high, meat tenderness is delicious is educated, it is deep to be welcome by production and consumption person.But, with cultivation intensive degree
Raising, caused by inbreeding, economic characters are degenerated, deterioration of abuse and breeding environment of fish medicine etc., and megalobrama amblycephala was cultivated
Disease in journey is also increasingly severe.Common disease has ich, trichodinasis, anchor head pulicosis, bacterial septicemia, thin
Bacterium property fin rot etc. (Wang Weimin, 2009), wherein, caused by Aeromonas hydrophila (Aeromonas hydrophila) infection
Bacterial septicemia, can cause each position hyperemia of fish body whole body, ulcer, intestinal bleeding, liver to be turned white, hydroabdomen, fester
Symptom, this disease eventually result in megalobrama amblycephala mortality once falling ill it is difficult to be controlled with medicine and treated, and affect
The development of megalobrama amblycephala aquaculture.
Fish are nospecific immunity and specific immunity the vertebrate deposited, but, compared with mammality, which is special
Property immune system is flourishing not enough, imperfection, make resisting to rely primarily on nospecific immunity and play when pathogenic microorganism is invaded
With.Nospecific immunity be by germline gene encode pattern recognition receptors (Pattern recognition receptor,
PRR) identification invades pathogen-associated molecular pattern (the Pathogen-associated molecular of internal exogenous molecules
Patterns, PAMP), so as to start inherent immunity reaction, direct, quick immune response is provided for body, PRR is whole solid
Have immune response research hinge (Sha et al, 2009).PAMP is some structures of pathogen, such as the cell membrane of bacterium into
Divide the peculiar DNA or RNA structures of lipopolysaccharides, peptide glycan, cilium, flagellin and bacterium, the non-parcel double-strand and single stranded RNA of virus
Deng, PRR is activated the expression of gene in downstream passages by combining with these ligand specificities, for body provide directly, it is quick
Immune response, infection early stage play a significant role (Ao Jingqun and CHEN XINHUA, 2012).NOD sample acceptor (NOD like
Receptors, NLRs) it is the important cytoplasmic region PRR of a class, during the regulation and control and opposing pathogen invasion of immune response
Play an important role (Biswas and Kobayashi, 2013).Much study and show, the exception of some molecules in NLRs
The generation of various diseases can be caused, the row such as mutation of NOD2 genes is closely related with the individual neurological susceptibility to Crohn ' s diseases
(Ogura et al 2001), the SNP mutation of NALP3 genes it is also related to many diseases (Pontillo et al, 2010).Mesh
Research in terms of the correlation analysis of front NLRs gene pleiomorphisms and disease focuses mostly on the person, to the research of aquatic livestock compared with
It is few.
Mononucleotide polymorphic (Single Nucleotide Polymorphisms, SNP) refer in genomic level by
The polymorphism of DNA sequence dna caused by the variation of single nucleotide acid.Due to its have quantity it is many, widely distributed, suitable for quick, rule
Modelling examination.Simultaneously because gene frequency is readily estimated with the advantages of being easy to Genotyping, current SNP is had become after micro-
Third generation genetic marker after satellite.Many phenotypic differences, all may be relevant with SNP to neurological susceptibility of medicine or disease etc..
SNP polymorphisms are widely used in economic animal breeding process.Such as build high density genetic linkage mapses, objective trait with
The association analysis of molecular labeling, linkage disequilibrium and haplotyping, marker assisted selection breeding, population genetic variations and system
The aspects such as developmental analysis, cultivar identification all show good application prospect.Again due to high-resolution melting curve technology (high
Resolution melting, HRM) be at home and abroad rose in recent years it is a kind of for be mutated scanning and Genotyping most
New genetics analysis method, has the advantages that high flux, low cost, accuracy is high and is widely used (Wittwer et al
2003)。
Have not yet to see about the SNP molecule related to megalobrama amblycephala disease resistance trait is screened from NLRC3-like genes
Mark and its report applied.
The content of the invention
This research purpose is the defect for overcoming prior art, and screening obtains a kind of and megalobrama amblycephala disease resistance trait related SNP
Molecular labeling and its application, from NLRC3-like genescreens obtain with the disease-resistant correlated traits of megalobrama amblycephala especially with a head
The related SNP marker of the anti-infected by Aeromonas hydrophila proterties of triangular bream, the cultivation for megalobrama amblycephala disease-resistant variety provide useful something lost
Pass mark.
Technical scheme is as described below：
The present invention extracts genomic DNA by infecting megalobrama amblycephala with Aeromonas hydrophila, is expanded using design of primers, PCR
Sequencing, SNP site screening obtain SNP site, then SNP site are analyzed by high-resolution melting curve method, are utilized
The genotype frequency of SPSS software counterworks group and susceptible group carries out significance difference analysis, finds disease-resistant related locus.
1 related SNP site 100C/T disease-resistant to megalobrama amblycephala is found in NLRC3-like genes.Analyzed by Chi-square Test and disease-resistant
Association analysis finds, the genotype frequency and gene frequency in the site difference extremely significantly (P in susceptible group and resistance group<
0.01)。
Applicant obtains megalobrama amblycephala gene involved in immunity NLRC3-like gene cDNA sequences by gene clone technology,
Confirm 1 related gene NLRC3-like polymorphism SNP marker disease-resistant to megalobrama amblycephala first, and using HRM methods entering
Molecular labeling described in row flux distribution, obtains the SNP marker on a megalobrama amblycephala NLRC3-like gene extron, its
Nucleotide sequence such as SEQ ID NO：1 (base at 100 bit bases of the sequence is base T after mutation)) shown in, in the sequence
There is an allelic mutation (C/T) at 100 bit bases of row.
Specifically, applicant screens a kind of and megalobrama amblycephala disease resistance trait related SNP molecular labeling, and its nucleotide sequence is such as
Shown in lower：
CACGGGAAGAGACAACACGCTACATTAATAAGAAAATAAAAGAAGGCCACTTCCCAGAAAAACATGAGACCCTTTTG
CGGTGCATTGATGAACTTAACGR(C/T)
ACACTGACGTGTTGGTTAAAAGAGTCTCATGATGAACTTCTGAGAGTGATGCTATTGCATTGTTTCTTGCCAAAAGA
GAAGTGGTAGAAATATAATCTACCATCAACAATCTTG
R at 100 bit bases of above-mentioned sequence is the mutation (mutation be located at gene extron subdivision) of C or T, the mutation
Amino acid is caused to be valine by alanine mutation.
Applicant devises a kind of detection and megalobrama amblycephala disease resistance trait related SNP molecular labeling and detection NLRC3-like
The primer pair of Genotyping, its DNA sequence dna are as follows：
Forward primer F：CACGGGAAGAGACAACACGC,
Reverse primer R：CAAGATTGTTGATGGTAGATTAT.
SNP site of the present invention is obtained using conventional HRM methods screening.
The SNP marker of present invention system screening can be used to detect that megalobrama amblycephala immune character related gene NLRC3-like is more
State property, can effective assisted Selection disease resistance trait associated genotype.
Description of the drawings
Sequence table SEQ ID NO：1 is the nucleosides with megalobrama amblycephala disease resistance trait related SNP molecular labeling of present invention screening
Acid sequence (length 214bp), mutational site (C/T) is at 100 bit bases of the sequence.
Sequence table SEQ ID NO：2 is the upstream primer sequence for expanding megalobrama amblycephala NLRC3-like gene 100C/T sites.
Sequence table SEQ ID NO：3 is the downstream primer sequence for expanding megalobrama amblycephala NLRC3-like gene 100C/T sites.
Sequence table SEQ ID NO：4 is the megalobrama amblycephala NLRC3-like gene global cDNA sequences of present invention clone.Sequence
Total length is 2863bp, the sequence of the 2364-2577 positions of the sequence be the present invention with megalobrama amblycephala disease resistance trait related SNP molecule mark
Note.
Fig. 1：Megalobrama amblycephala DNA extracts result electrophoretogram.Description of reference numerals:Swimming lane M:Marker；A sections (swimming lane 1-
11):Disease-resistant individuality；B sections (swimming lane 1-11).
Fig. 2：The sequencing peak of megalobrama amblycephala NLRC3-like gene 100C/T sites different genotype in embodiments of the invention
Figure.
Fig. 3：The HRM knots of megalobrama amblycephala NLRC3-like gene 100C/T sites different genotype in embodiments of the invention
Really.Description of reference numerals：In Fig. 3, two kinds of colors represent only two kinds genotype.
Fig. 4：Be the present invention screening with the sequence directly perceived with megalobrama amblycephala disease resistance trait related SNP molecular labeling.In the sequence
100 generations, one allelic mutation of row, is represented with R (C/T).
Specific embodiment
Embodiment 1
1. subjects (test material)
Megalobrama amblycephala used by experiment picks up from Hubei hundred and holds aquatic products breeding Co., Ltd, and megalobrama amblycephala used is an age megalobrama amblycephala,
Totally 1500 tail.First temporarily support in bucket to after stablizing after salvaging from pond, the manual-induced sense of Aeromonas hydrophila is injected to which
Dye, the injection concentration of Aeromonas hydrophila is 1 × 107Cfu/mL, water temperature are maintained at 27-29 DEG C.Gather respectively dead at first in 12h
The fish (susceptible group) for dying and the fin ray sample of the fish (resistance group) still survived after 5d, preserve in being placed on absolute ethyl alcohol, take back
Laboratory is preserved at being placed in -20 DEG C, used as the sample for extracting genomic DNA.
2. test method
2.1 extract megalobrama amblycephala genomic DNA using phenol-chloroform method, comprise the following steps that described：
(1) fin ray for taking 0.2g megalobrama amblycephalas is put in 2mL centrifuge tubes, adds 600 μ L cell pyrolysis liquids (commercially available), with disappearing
Fin ray is shredded by the scissors of poison, adds 6 μ L Proteinase Ks (20mg/ μ L), 60 DEG C of water-baths to digest to clarification (2-4h).
(2) with equal-volume (600 μ L) phenol：Chloroform：Isoamyl alcohol (volume ratio 25：24：1) extract 2 times, 4500r/min centrifugations
20min, takes supernatant into 1.5mL centrifuge tubes.
(3) absolute ethyl alcohol of 2 times of volume precoolings is added to be precipitated, -20 DEG C of standing 30min or so.
(4) 4 DEG C, 12500r/min centrifugation 20min abandon supernatant.
(5) washed with 70% ethanol and precipitated 2 times, 4 DEG C, 12500r/min is centrifuged 10min.
(6) precipitation is dried, adds 50-100 μ L ddH2O dissolution precipitations, stand overnight, and treat which is fully dissolved after -20
DEG C preserve.
2.2 obtain SNP site
Fish from megalobrama amblycephala transcript profile and take NKLC-like gene orders, design RACE primers clone the gene order, and with
Megalobrama amblycephala genomic library (result is not delivered) is compared, and obtains megalobrama amblycephala NKLC-like genome sequences, in susceptible group and resistance group
In respectively 5 sample DNAs of random selection as template.
, respectively 10 templates are entered with performing PCR amplification with 5.0 Software for Design PCR primers (being shown in Table 1) of Primer premier
(table 2), sending Wuhan to hold up Kechuang neoformation Science and Technology Ltd. amplified production carries out purifying sequencing.With DNAstar softwares to surveying
Sequence result is analyzed, and is analyzed by sequence alignment and peak figure, filters out candidate SNP locus.
Primer sequence of 1 present invention of table for SNPs amplifications
PCR response procedures：94 DEG C of denaturation 30s of circulating system are entered after 94 DEG C of denaturations 5min, anneal 30s, 72 DEG C of extensions
30s, 30 circulations, last 72 DEG C of extensions 5min.Electrophoresis, the imaging preservation in 1% Ago-Gel by product.
2.3 SNP partings and the correlation analysis with disease resistance
Parting, reactant are carried out to megalobrama amblycephala NLRC3-like gene SNP s sites using high-resolution melting curve method (HRM)
System's (high-resolution melting curve method) is shown in Table 3；Parting the primer is shown in Table 4；Statistical analysis is carried out to genotyping result with SPSS softwares.
3 high-resolution solubility curve law system of table
SNP parting programs：
Denaturation：95 DEG C, 5min
Amplification (45 circulations)：95 DEG C, 1min；60 DEG C, 10sec；72 DEG C, 15sec (collection fluorescent value)
High-resolution solubility curve：95 DEG C, 1min；40 DEG C, 1min；65 DEG C, 1sec；95 DEG C of continuous collection fluorescent values, 25
Secondary/DEG C, 0.02 DEG C/sec of rate of temperature change.
The primer sequence for genotyping of 4 present invention design of table
3 interpretations of result
3.1 use genotype frequency and allele of the SPSS softwares to SNP site in vulnerable groups and disease-resistance population
Frequency is counted, and obtains result as described in Table 5.
5 megalobrama amblycephala NLRC3-like gene nucleotide pleomorphism sites genotype of table is distributed
Find the genotype frequency and gene frequency in C/T sites at 100 bit bases easy by Chi-square Test analysis
Difference extremely significantly (P in sense group and resistance group<0.01), genotype is CC individual more disease-resistant for the individuality of CT than genotype.
Bibliography
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Claims (3)
1. a kind of with megalobrama amblycephala disease resistance trait related SNP molecular labeling, its nucleotide sequence is as follows：
CACGGGAAGAGACAACACGCTACATTAATAAGAAAATAAAAGAAGGCCACTTCCCAGAAAAACATGAGACCCT
TTTGCGGTGCATTGATGAACTTAACGR(C/T)
ACACTGACGTGTTGGTTAAAAGAGTCTCATGATGAACTTCTGAGAGTGATGCTATTGCATTGTTTCTTGCCAAAAGA
GAAGTGGTAGAAATATAATCTACCATCAACAATCTTG,
R at 100 bit bases of above-mentioned sequence is the mutation of C or T.
2. a kind of detection primer pair as claimed in claim 1 with megalobrama amblycephala disease resistance trait related SNP molecular labeling, its feature exist
In the sequence of the primer pair is as follows：
Forward primer F：CACGGGAAGAGACAACACGC,
Reverse primer R：CAAGATTGTTGATGGTAGATTAT.
3. the SNP marker related to megalobrama amblycephala disease resistance trait described in claim 1 is selected in megalobrama amblycephala disease resistance trait auxiliary
Application in selecting.
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| CN109385482A (en) * | 2018-11-19 | 2019-02-26 | 中国水产科学研究院北戴河中心实验站 | The relevant SNP marker of lefteye flounder fertility and its screening technique and application |
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