CN105936937B - A kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei and its screening and application - Google Patents
A kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei and its screening and application Download PDFInfo
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Abstract
The present invention relates to a kind of relevant SNP marker of low to litopenaeus vannamei dissolved oxygen tolerance and its screening and application, belong to that aquatic livestock is hereditary and molecular marker assisted selection breeding technical field；The present invention is cloned from sod gene obtains a SNP marker, the SNP marker is that R of the nucleotide sequence shown in SEQ ID NO.1 from 5 ' ends in the 118th bit base is C or T, the label and the resistance to low dissolved oxygen character of litopenaeus vannamei are significant related, can be applied to the molecular marker assisted selection breeding of the degeneration-resistant character of the low dissolved oxygen of litopenaeus vannamei；Seedling selection can be carried out to vannamei boone breeding material according to practical breeding demand using technical solution of the present invention, effectively improve the efficiency and accuracy of breeding, the genetic level of litopenaeus vannamei reproductive population is improved, so as to accurately and efficiently select the strong litopenaeus vannamei kind of resistance；The method of the present invention is practical, applicability is wide.
Description
Technical field
The present invention relates to a kind of relevant SNP marker of low to litopenaeus vannamei dissolved oxygen tolerance and its screening and application, categories
In aquatic livestock heredity and molecular marker assisted selection breeding technical field.
Background technique
Litopenaeus vannamei (Litopenaeus vannamei) it is also referred to as Penaeus Vannmei, it is subordinate to Arthropoda, Crustachia,
Decapoda, Penaeidae, shore Penaeus, for wide warm eurysalinity torrid zone shrimps.Litopenaeus vannamei is because of energy fast, disease-resistant with the speed of growth
Power is strong, resistance is strong, adapts to the good characteristics such as high-density breeding, it has also become one of most important prawn culturing kind in China.Closely
The high-density breeding mode of Nian Lai, litopenaeus vannamei are widely promoted, but during prawn high-density breeding, due to supporting
Grow water body by the variation of seasonal temperature and salinity, the respiratory metabolism of organism in water, organic matter decomposition rot etc. factors influenced,
It has arrived cultivation later period breeding water body and anaerobic condition has easily occurred, to influence the growth of prawn, ingest and cast off a skin, prawn has been caused to exempt from
There is slow growth, to phenomena such as sensibility of bacterium and virus improves, the death rate is high in epidemic disease power, premunition, eupepsy decline.
It therefore, is to solve the effective way of problem above using the litopenaeus vannamei new varieties that Biotechnology in Genetic Breeding cultivates resistance to low dissolved oxygen.
Single nucleotide polymorphism (Single nucleotide polymorphisms, SNP) refers to certain in genome sequence
The variation of base on one position can occur wherein the form to make a variation includes conversion, transversion, insertion, missing etc. in any of DNA
On position, and occurrence frequency of any allele in population is higher than 1% on any position.SNP is as third generation molecule mark
Note, because its with enormous amount, widely distributed, codominance, it is highly stable, be easy to automation size analysis, can show other skills
The undetectable hiding polymorphism of art, may many merits relevant to gene function, obtained in molecular mark
It is widely applied.The effect for understanding functional gene is not only facilitated to the research of animal functional gene SNPs, but also can get
Functional gene molecular genetic marker associated with economic characters.In the research of litopenaeus vannamei molecular mark,
Many domestic and foreign scholars carry out the screening of SNP site using the SNP marker technology functional gene important to litopenaeus vannamei
And correlation analysis, the mononucleotide that such as existing scholar carries out litopenaeus vannamei AMY gene using SNP marker technology
Polymorphism research, screening goes out 9 SNP sites altogether in litopenaeus vannamei AMY gene；There is scholar to have studied litopenaeus vannamei group again
Protease CTSL gene polynorphisms and associated with growth traits are knitted, discovery 2 is significant SNP relevant to growth traits
Point；There are also the correlations that scholar has studied litopenaeus vannamei Hsp70 gene SNP s and ntiviral characteristic, as a result, it has been found that Hsp70 base
Because disease resistance of the different genotype to litopenaeus vannamei has a significant impact.The above research focuses mostly on raw in analysis litopenaeus vannamei
The polymorphism of the related genes such as long, disease-resistant, but there are no and the resistance to low dissolved oxygen trait related gene SNP marker of litopenaeus vannamei
Relevant report.
Summary of the invention
The object of the present invention is to provide a kind of relevant SNP marker of low to litopenaeus vannamei dissolved oxygen tolerance, the molecule marks
Note can be applied to the molecular marker assisted selection breeding of the degeneration-resistant character of the low dissolved oxygen of litopenaeus vannamei, and it is resistance to low molten to carry out litopenaeus vannamei
The marker assisted selection and application of oxygen character.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei, clones from sod gene and obtains a SNP
Molecular labeling, the SNP are C or T labeled as R of the nucleotide sequence shown in SEQ ID NO.1 from 5 ' ends in the 118th bit base；
Nucleotide sequence shown in SEQ ID NO.1 is as follows:
CCTAGCTTCTCAGCTATCAGTTCATGGAGGCGGCCACTTGAACCACACCATCTTCTGGACCAACATGGC
TCCCGATGCTGGTGGCGAGCCGCAAGGAGCTGTTGCACAAGCCATTGA
R[C/T]GAGAGCTTTGGATCATTCCAGTCCTTCAAGGTGGGTTTCTTGTGATGGTCA。
Second object of the present invention is to provide the primer pair for expanding above-mentioned SOD genetic fragment, can be used as detection and be somebody's turn to do
One of composition of kit of SNP marker, the PCR for the segment where SNP marker of the present invention are expanded, and by sequencing, are sentenced
Break the genotype in the litopenaeus vannamei to be measured SNP marker site.Primer pair of the present invention is specifically primer SOD-FP and primer
SOD-RP is with litopenaeus vannamei superoxide dismutase sod gene (GenBank Accession NO.DQ298206.1) sequence
It is classified as template, using obtained by 3.0 software design of Primier Premier.The primer SOD-FP is shown in SEQ ID NO.2
Sequence, primer SOD-RP are sequence shown in SEQ ID NO.3:
(1) SEQ ID NO.2:AGGCAGCCAATGACGTAAGCG；
(2) SEQ ID NO.3:GACCATCACAAGAAACCCACC.
It is another object of the present invention to provide a kind of relevant SNP markers of low to litopenaeus vannamei dissolved oxygen tolerance
Screening technique includes the following steps:
(1) using the genomic DNA of litopenaeus vannamei individual dead and survival after the experiment of 96h low dissolved oxygen stress as mould
Plate, using above-mentioned primer pair, i.e. primer SOD-FP and primer SOD-RP carry out PCR amplification, obtain amplified fragments, amplified fragments
Length be 169bp；
(2) amplified fragments that step (1) obtains are sequenced, obtain sod gene partial sequence and sequencing peak figure, it will
Sequence results carry out BLACT comparison, filter out base mutation site i.e. SNP site；
(3) Genotyping analysis is carried out using peak figure of the chromas software to sequencing, if occurring at SNP site bimodal
It is heterozygous genotypes, it is unimodal, it is homozygous genotype；With the Chi-square Test of SPSS19.0 software to the low dissolved oxygen of litopenaeus vannamei
Tolerance and genotype carry out correlation analysis.
The extracting method of step (1) described genomic DNA is not particularly limited, and can use any of genome
DNA extraction method or kit carry out；The condition for carrying out PCR amplification to genomic DNA is also not particularly limited, PCR amplification item
Part can be in optimized selection by those skilled in the art.
Since direct Sequencing is a kind of accuracy highest, strong flexibility, the detection technique that flux is big, detection cycle is short.It should
Method only need to design pair of primers in the two sides of SNP site, and the product expanded can directly detect SNP by sequencing
Site.Therefore, present invention preferably employs the detections that the method for direct Sequencing carries out SNP marker.
It can be applied to the choosing of the resistance to low dissolved oxygen of litopenaeus vannamei by the SNP site that step described in above-mentioned technical proposal obtains
Process is educated, specifically during the selection and use of litopenaeus vannamei, SNP site is carried out to litopenaeus vannamei breeding candidate population
Parting preferentially selects SNP site for TT genotype or TC gene in conjunction with the parting information of other and degeneration-resistant character related locus
The individual of type as resistance to low dissolved oxygen litopenaeus vannamei breeding parent or carry out large-scale cultivation, avoid selecting SNP site for
The litopenaeus vannamei individual of CC genotype is as parent or carries out breeding scale.
The beneficial effects of the present invention are:
(1) it is found through statistics, genotype is the low of the litopenaeus vannamei of TT or TC at the site of SNP marker of the present invention
Dissolved oxygen tolerance is significantly higher than the litopenaeus vannamei that genotype is homozygosis CC, in turn, by the above-mentioned SNP for detecting litopenaeus vannamei
Site can effectively determine the tolerance performance of its low dissolved oxygen；The resistance to low dissolved oxygen of SNP marker and litopenaeus vannamei of the invention
Shape is closely related, can be effective for the molecular mark of litopenaeus vannamei.
(2) Seedling selection can be carried out to vannamei boone breeding material according to practical breeding demand using technical solution of the present invention,
The efficiency and accuracy of breeding are effectively improved, the genetic level of litopenaeus vannamei reproductive population is improved, so as to accurate, efficient
Ground selects the strong litopenaeus vannamei kind of resistance；
(3) method is practical, extracting genome DNA, sequencing approach do not have particular requirement, and applicability is wide.
Detailed description of the invention
The 118th site haplotype of sod gene is TT peak value figure in Fig. 1 embodiment；
The 118th site haplotype of sod gene is TC peak value figure in Fig. 2 embodiment；
The 118th site haplotype of sod gene is CC peak value figure in Fig. 3 embodiment.
Specific embodiment
The present invention is described in further details below by example, these examples are only used to illustrate the present invention, and unlimited
The scope of the present invention processed.
Embodiment 1
The present embodiment by the partial sequence to litopenaeus vannamei superoxide dismutase sod gene carry out sequencing and
BLAST is compared, and screening goes out 1 SNP site, is located at the 118th bit base of litopenaeus vannamei sod gene, and C/T mutation has occurred.
The SNP marker can detect its loci polymorphism, and site of analysis base by direct sequencing in litopenaeus vannamei group
Because of the correlation of type frequency low dissolved oxygen tolerance with litopenaeus vannamei.
The correlation analysis of SNP site 118C > T and low dissolved oxygen tolerance in litopenaeus vannamei sod gene, according to following step
It is rapid to carry out:
(A) laboratory sample is grouped；
(B) litopenaeus vannamei extracting genome DNA: this implementation selection is using conventional phenol chloroform method；
(C) litopenaeus vannamei sod gene SNP site screening, site 118C > T parting；
(D) correlation analysis of 118C > T genotype and low dissolved oxygen tolerance trait；
(E) site 118C > T assists the breeding of the low dissolved oxygen tolerant varieties of litopenaeus vannamei.
Concrete operations are as follows:
(A) laboratory sample is grouped
It is tested by low dissolved oxygen stress and is grouped 64 tail litopenaeus vannamei: will be dead after the test of 96h low dissolved oxygen stress
It dies prawn and is included into low dissolved oxygen stress sensitive group, the prawn of survival is included into low dissolved oxygen stress tolerance group, and wherein stress sensitive group is all
Shore prawn number of receiving is 31 tails, and the litopenaeus vannamei number of stress tolerance group is 33 tails.
(B) litopenaeus vannamei DNA is extracted
Litopenaeus vannamei muscle 30-40mg is taken, 400 μ L 1 × TE lysates are added and shred, sequentially add 200 μ L's 10%
SDS, 8 μ L Proteinase Ks (20mg/mL), 37 DEG C of cracking 30min, then as 55 DEG C of digestion to clear, about 1-2h；Toward clarification
Lysate in isometric phenol/chloroform/isoamyl alcohol (25:24:1) solution is added, reverse to mix well, 12000rpm/min centrifugation
20min；Supernatant carefully is pipetted into new centrifuge tube, adds isometric chloroform/isoamyl alcohol (24:1), is overturned sufficiently mixed
It is even, it is centrifuged 15min, is extracted 2 to 3 times；Supernatant carefully is pipetted into new centrifuge tube, and the anhydrous second of 2 times of volumes pre-cooling is added
Alcohol, it is reverse to mix well, it is put into -20 DEG C of refrigerators and stands 30min；12000rpm/min is centrifuged 15min, abandons supernatant, is added 70%
Ethanol washing 2-3 times is centrifuged 2min every time, and room temperature is dried, and 150 μ L1 × TE Buffer are added and save.With 1% Ago-Gel
Test sample, UV spectrophotometer measuring concentration and purity.
(C) screening of litopenaeus vannamei sod gene SNP site and site 118C > T parting.
Litopenaeus vannamei superoxide dismutase sod gene (GenBank Accession is obtained in the website NCBI
NO.DQ298206.1) sequence, by 3.0 software design of Primier Premier obtain specific primer to SOD-FP and
SOD-RP.With group dead after experiment in low dissolved oxygen stress 96 hours and surviving populations litopenaeus vannamei (64 tail) genomic DNA
For template, expanded under the conditions of PCR using above-mentioned primer.PCR reaction system is 25 μ L:DNA template 40ng, 10 × PCR
Buffer(Mg2+ Plus) 2.5 μ L, dNTP s, 2 μ L, 2 μ L of upstream primer, 2 μ L of downstream primer, Taq enzyme (5U/ μ L) 0.2 μ
L, mending distilled water to total volume is 25 μ L.Pcr amplification reaction program is as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30S, and 58 DEG C
Anneal 35s, 72 DEG C of extension 1min, carries out 35 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservations altogether.PCR product is directly sent
In Shanghai, raw work is sequenced.
Sequence alignment is carried out to sequencing result using DNAMAN software, screens the SNP site of sod gene.Pass through Chromas
Software checks Sequencing chromatogram, and the genotype of each litopenaeus vannamei individual SNP site is judged by manually proofreading, and occurs bimodal
Then explanation is heterozygous, unimodal, is homozygous, and count low dissolved oxygen stress sensitive group and each genotype of tolerance group and equipotential base
The number of individuals of cause.Fig. 1 is that the 118th site haplotype of sod gene is TT peak value figure in the present embodiment；Fig. 2 is SOD in the present embodiment
The 118th site haplotype of gene is TC peak value figure；Fig. 3 is that the 118th site haplotype of sod gene is CC peak value in the present embodiment
Figure.
(D) correlation analysis of 118C > T genotype and resistance to low dissolved oxygen character.
According to sequencing result, statistics dead litopenaeus vannamei individual (31 tail) and survival after low dissolved oxygen stress is tested 96 hours
Group's litopenaeus vannamei is individual (33 tail), the case where totally 64 tail litopenaeus vannamei individual 118C > T loci gene type, calculates each group
In the polymorphic site genotype frequency and gene frequency, statistical result is shown in Table 1.
1 sod gene type of table and gene frequency
Utilize Chi-square Test method site of analysis 118C > T dissolved oxygen tolerance low with litopenaeus vannamei of SPSS19.0 software
Correlation, the results showed that in the 118C > T of site, the distribution of TT, TC and CC3 kind different genotype has with low dissolved oxygen tolerance
Significant relevance (X 2 =6.515, P=0.038), 2 kinds of allele Cs and T are also showed that be associated with low dissolved oxygen tolerance
Property (X 2 =6.874, P=0.009).The result shows that: the genotype polymorphism of sod gene site 118C > T point is to vannamei boone pair
The low dissolved oxygen tolerance of shrimp has extremely significant influence, and the resistance to low dissolved oxygen performance of TT and TC genotype individuals is better than CC genotype.Preferential choosing
The individual that site 118C > T is TT type or TC type is selected, is supported as the parent of resistance to low dissolved oxygen litopenaeus vannamei breeding or progress scale
It grows, avoids selecting the site 118C > T as far as possible being CC type as parent or progress breeding scale.
Sequence table
<110>Guangdong Ocean University
<120>a kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei and its screening and application
<160>3
<210>1
<211>168
<212>nucleotide
<213>unknown
<400>1
CCTAGCTTCT CAGCTATCAG TTCATGGAGG CGGCCACTTG AACCACACCA TCTTCTGGAC 60
CAACATGGCT CCCGATGCTG GTGGCGAGCC GCAAGGAGCT GTTGCACAAG CCATTGAGAG 120
AGCTTTGGAT CATTCCAGTC CTTCAAGGTG GGTTTCTTGT GATGGTCA 168
<210>2
<211>21
<212>nucleotide
<213>unknown
<400>2
AGGCAGCCAA TGACGTAAGC G
21
<210>3
<211>21
<212>nucleotide
<213>unknown
<400>3
GACCATCACA AGAAACCCAC C
21
Sequence table
<110>Guangdong Ocean University
<120>a kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei and its screening and application
<160>3
<210>1
<211>168
<212>nucleotide
<213>unknown
<400>1
CCTAGCTTCT CAGCTATCAG TTCATGGAGG CGGCCACTTG AACCACACCA TCTTCTGGAC 60
CAACATGGCT CCCGATGCTG GTGGCGAGCC GCAAGGAGCT GTTGCACAAG CCATTGAGAG 120
AGCTTTGGAT CATTCCAGTC CTTCAAGGTG GGTTTCTTGT GATGGTCA 168
<210>2
<211>21
<212>nucleotide
<213>unknown
<400>2
AGGCAGCCAA TGACGTAAGC G
21
<210>3
<211>21
<212>nucleotide
<213>unknown
<400>3
GACCATCACA AGAAACCCAC C
21
Claims (6)
1. a kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei, it is characterised in that: the SNP is labeled as SEQ ID
Nucleotide sequence shown in NO.1, wherein the R from 5 ' ends in the 118th bit base is C or T；
Nucleotide sequence shown in SEQ ID NO.1 is as follows:
CCTAGCTTCTCAGCTATCAGTTCATGGAGGCGGCCACTTGAACCACACCATCTTCTGGACCAACATGGCTCCC
GATGCTGGTGGCGAGCCGCAAGGAGCTGTTGCACAAGCCATTGA
RGAGAGCTTTGGATCATTCCAGTCCTTCAAGGTGGGTTTCTTGTGATGGTCA。
2. the relevant SNP marker of one kind according to claim 1 dissolved oxygen tolerance low to litopenaeus vannamei, feature exist
In: the litopenaeus vannamei sod gene, the primer pair of gene fragment amplification are primer SOD-FP and primer SOD-RP, can
Sentence as one of the composition for detecting the SNP marker kit for the PCR amplification of segment where SNP marker, and by sequencing
Break the genotype in the litopenaeus vannamei to be measured SNP marker site；
The primer SOD-FP is sequence shown in SEQ ID NO.2, and primer SOD-RP is sequence shown in SEQ ID NO.3:
SEQ ID NO.2:AGGCAGCCAATGACGTAAGCG；
SEQ ID NO.3:GACCATCACAAGAAACCCACC.
3. a kind of screening technique of the relevant SNP marker of low to litopenaeus vannamei dissolved oxygen tolerance according to claim 1,
It is characterized by comprising following steps:
(1) using the genomic DNA of litopenaeus vannamei individual dead and survival after 96 hours low dissolved oxygen stress are tested as template,
PCR amplification is carried out using primer pair SOD-FP and SOD-RP as claimed in claim 2, obtains amplified fragments；
(2) amplified fragments that step (1) obtains are sequenced, sod gene partial sequence and sequencing peak figure are obtained, by sequence
As a result BLACT comparison is carried out, base mutation site i.e. SNP site is filtered out；
(3) Genotyping analysis is carried out to the peak figure of sequencing using chromas software, if occur at SNP site it is bimodal if say
Bright is heterozygous, unimodal, is homozygote；With the Chi-square Test of SPSS19.0 software to the low dissolved oxygen tolerance of litopenaeus vannamei
Correlation between genotype is analyzed, and the significant relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei is obtained.
4. the screening technique of the relevant SNP marker of a kind of dissolved oxygen tolerance low to litopenaeus vannamei according to claim 3,
Be characterized in that: the length of step (1) described amplified fragments is 169bp.
5. the screening technique of the relevant SNP marker of a kind of dissolved oxygen tolerance low to litopenaeus vannamei according to claim 3,
Be characterized in that: the method that step (2) uses direct Sequencing to the sequencing of amplified fragments designs a pair in the two sides of SNP site and draws
Object, the product expanded can directly detect SNP site by sequencing.
6. a kind of application of the relevant SNP marker of low to litopenaeus vannamei dissolved oxygen tolerance according to claim 1, special
Sign is: being applied to the breeding of the low dissolved oxygen tolerance of litopenaeus vannamei using the SNP site that claim 3 the method obtains
Journey carries out SNP site point to litopenaeus vannamei breeding candidate population specifically during the selection and use of litopenaeus vannamei
Type selects SNP site for TT genotype or TC genotype in conjunction with the parting information with low dissolved oxygen tolerance trait related locus
Parent or progress breeding scale of the individual as resistance to low dissolved oxygen litopenaeus vannamei breeding, avoid selecting SNP site for CC gene
The litopenaeus vannamei individual of type is as parent or carries out breeding scale.
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| CN114752685B9 (en) * | 2022-03-02 | 2023-08-15 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei molecular marker, primer and application thereof |
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|---|---|---|---|---|
| CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
| CN102146460A (en) * | 2011-01-26 | 2011-08-10 | 中国科学院南海海洋研究所 | Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker |
| CN104789690A (en) * | 2015-05-14 | 2015-07-22 | 中国科学院海洋研究所 | DNA probe sequence for determining genetic sex of litopenaeus vannamei and acquiring method of DNA probe sequence |
| CN105200160A (en) * | 2015-11-12 | 2015-12-30 | 广东海洋大学 | SNP marker relevant to low dissolved oxygen tolerance of Litopenaeus vannamei as well as screening method and application of SNP marker |
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2016
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
| CN102146460A (en) * | 2011-01-26 | 2011-08-10 | 中国科学院南海海洋研究所 | Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker |
| CN104789690A (en) * | 2015-05-14 | 2015-07-22 | 中国科学院海洋研究所 | DNA probe sequence for determining genetic sex of litopenaeus vannamei and acquiring method of DNA probe sequence |
| CN105200160A (en) * | 2015-11-12 | 2015-12-30 | 广东海洋大学 | SNP marker relevant to low dissolved oxygen tolerance of Litopenaeus vannamei as well as screening method and application of SNP marker |
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