CN104152444A - SNP (single-nucleotide polymorphism) marker related to Crassostrea gigas glycogen content character and application thereof - Google Patents
SNP (single-nucleotide polymorphism) marker related to Crassostrea gigas glycogen content character and application thereof Download PDFInfo
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Abstract
The invention relates to an SNP (single-nucleotide polymorphism) marker related to Crassostrea gigas glycogen content character and application thereof. The site of the SNP marker is named TY202 and positioned on the 3rd exon of the glycogen debranching enzyme gene, and the genotype is Y. The TY202 is positioned on the 7022nd base at the 5' end of the glycogen debranching enzyme gene. The 3rd exon is positioned on the 6446-7043 base pair at the 5' end of the glycogen debranching enzyme gene. The glycogen content of the individual with the SNP marker site genotype of T/T or T/C is higher than that of the individual with the genotype of C/C. The method for detecting the individual genotype can be utilized to predict the glycogen content of the individual. When being used in breeding, the SNP marker can be used for selecting the target individual and for molecular marker assisted selective breeding. The SNP marker avoids using concentrated acid or concentrated alkali in the glycogen detection process, thereby being safer and more convenient to operate.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, specifically a kind of SNP mark relevant with long oyster glycogen content proterties.
Background technology
Long oyster, belongs to the lamellibranchiata (or Bivalvia) in Mollusca, and meat fertilizer is tender, nutritious, glycogen is a kind of important flavor nutrition material in oyster, can directly be absorbed by body, thereby alleviate pancreas burden, therefore very effective to the control of diabetes.
Glycogen debranching factor is a kind of enzyme in oyster glycogen degradation process, and its effect is mainly hydrolyzing alpha-1, and 6 glycosidic links are removed the branch of glycogen, make side chain glycogen become straight chain glycogen.People's glycogen debrancher deficiency can cause glycogen storage disease one 111 type diseases.The DNA total length of at present existing glycogen debranching factor, has carried out Mutation Screening and has found mutational site, many places it both at home and abroad, but not found generally acknowledged mutantional hotspot.
SNP is single nucleotide polymorphism (single nucleotide polymorphism), mainly refers in genomic level the polymorphism by the caused DNA sequence dna of variation of single core thuja acid.SNP distributes wide, and density is high, has the genetic stability of height.Compared with the molecule marker of other kinds, SNP mark can react the sequence information in biological genome better, can study in more detail in genome associated between sequence polymorphism and phenotypic character, effectively instructs the work of gene identification and location.
Aquatic animal breeding research starting is late, mainly concentrates on the research to growth traits, and method adopts traditional selection mostly, hybridization, and the cycle is long, takes effect slow.Quality trait research to long oyster is also rare.In recent years, along with genomics research obtains a series of breakthroughs, molecular marker assisted selection equimolecular breeding technique is day by day ripe, has greatly improved genetic breeding level.Utilize molecular marker assisted selection can reduce blindness, shortening the breeding cycle, improve the efficiency of breeding.Association analysis is the qualification that grew up the in recent years molecule marker associated with objective trait and the popular method of gene.Association analysis, claim again linkage disequilibrium mapping (LD mapping) or associated mapping (Association Mapping), be a kind of taking linkage disequilibrium as basis, identify the analytical procedure of a certain colony internal object proterties and genetic marker or candidate gene relation.Association analysis can detect all restructuring and the variance event of special group, has stronger precision.
Summary of the invention
The object of this invention is to provide the SNP mark that a kind of and long oyster glycogen content is relevant, for the molecular marker assisted selection of long oyster provides reference.
Technical scheme of the present invention is the SNP mark that a kind of and long oyster glycogen content is relevant for achieving the above object, and it is positioned on the 3rd exon of glycogen debranching factor gene, and genotype is Y.Obtain SNP site TY202, be positioned on the 3rd exon at 5 ' end 6446-7043 base pair place of glycogen debranching factor gene.Described TY202 is positioned at 5 ' end the 7022nd base place of glycogen debranching factor gene.
Its specific primer sequence is: F:5 '-TAGTAAAGACAGGCAGCA-3 '; R:5 '-TCATTTGTAGACAGGGAG-3 '; Primer amplification fragment length is 75bp, and amplification of nucleotide acid sequence is shown in SEQ ID NO.1.
Genotype is T/T, T/C or C/C, the individuality that the individual glycogen content that is T/T or T/C at this loci gene type is C/C higher than genotype.
In wild population, detect each individual genotype and phenotypic data, predict that by association analysis the proterties degree of correlation that draws TY202 and long oyster glycogen content reaches conspicuous level (P<0.05) by genotype and phenotypic data, associated with the proterties of long oyster glycogen content, can be used for the marker assisted selection breeding of long oyster.
The detection method of the SNP mark relevant to long oyster glycogen content proterties, its screening step is:
A) gather multiple individualities in the wild long oyster colony of Jiangnan, Qingdao, it dissected, point tissue (closed shell flesh, gill tissue etc.) sampling, with after liquid nitrogen flash freezer in-80 DEG C of preservations, as experiment material;
B) measure the glycogen content in each individual closed shell flesh, obtain phenotypic data;
C) use phenol-chloroform extraction process to extract total DNA of each individual gill;
D) selected long oyster glycogen metabolism related gene, as candidate gene, is transcribed the SNP site design primer of group data prediction according to long oyster, after screening primer, utilizes HRM method to detect idiotype for each SNP;
E) utilize GAPIT software to carry out association analysis, predict the SNP site relevant to long oyster glycogen content proterties.
Advantage of the present invention:
The present invention can predict the height of individual glycogen content by the Id method of detection, can be used for select target individuality, for molecular marker assisted selection breeding in breeding.Avoid the use of concentrated acid concentrated base in glycogen testing process, made operation safer and simpler.
Brief description of the drawings:
Fig. 1 is primer screening result.Wherein, swimming lane 5 is Marker, and Marker used is Marker1.Swimming lane 10-13 is SNP site TY202 primer screening result.
Fig. 2 is SNP site TY202 HRM the result.
Fig. 3 is SNP site TY202 colony somatotype result.
Embodiment
The invention will be further elaborated for following examples, and embodiment only, for the present invention is described, the invention is not restricted to this.Experimental article that the present invention adopts is all from commercial.
The information of sequence table (1) SEQ ID NO.1
Sequence signature length: 75bp
Type: nucleic acid
Chain: two strands
Topological framework: linear
Molecule type: DNA
Source: long oyster
Sequence description:
TAGTAAAGACAGGCAGCATGCTGAYGGTGAAGGGTACTTTTTGGTGGACCCCATCCTCTCCCTGTCTACAAATGA
Embodiment 1
The screening in the SNP site relevant to glycogen content
A) collection of sample: gather multiple individualities in the wild long oyster colony of Jiangnan, Qingdao, totally 144, it to be dissected, point tissue (closed shell flesh, gill tissue etc.) sampling, with saving backup in-80 DEG C after liquid nitrogen flash freezer.
B) glycogen content detects: build up the muscle glycogen of Bioengineering Research Institute and liver starch with Nanjing and measure test kit and detect the glycogen relative content in each individual closed shell flesh, the operation steps of detection method in to specifications carried out.
1) sampling: get after the rinsing of frozen muscle samples physiological saline, filter paper blots, and (sample weight≤100mg) weighs.
2) hydrolysis: by sample weight (mg): alkali lye volume in test kit (μ l)=1:3, add together in test tube, boiling water bath boils 20min, flowing water is cooling, obtains glycogen hydrolyzed solution.
3) prepare glycogen and detect liquid: glycogen hydrolyzed solution adding distil water dilution is obtained to glycogen and detect liquid, the amount of adding distil water is (μ l): sample weight (mg) * 16.
Operation table is as shown in table 1.
Table 1
| ? | Blank tube | Standard pipe | Measure pipe |
| Distilled water (ml) | 1.0 | ? | 0.9 |
| 0.01mg/ml standard (ml) | ? | 1.0 | ? |
| Glycogen detects liquid (ml) | ? | ? | 0.1 |
| Nitrite ion (ml) | 2 | 2 | 2 |
Standardized solution and nitrite ion all adopt and in test kit, carry solution.
Mix in rearmounted boiling water and boil 5min, cooling after in 620nm wavelength, under 1cm optical path, with blank tube zeroing, survey each pipe OD value.
Calculation formula is as follows:
C) DNA extraction: the total DNA that extracts each individual gill with phenol-chloroform extraction process.
1) get 1.5 ml centrifuge tubes, add 700 μ l DNA extraction damping fluids (100 mM Tris-HCl, 5 mM EDTA), the oyster gill tissue of getting 3-10mg, shreds with scissors.Vessel used must be used flame sterilization and ddH between two individualities
2o rinses in case the crossed contamination between individuality.Add 35 μ lSDS, mix.Add 2 μ l Proteinase Ks, mix.
2) centrifuge tube is hatched in 65 DEG C of metal baths, after 1.5 hours, every pipe is added 2 μ l Proteinase Ks, shakes gently during this time more than centrifuge tube organizes and continue to hatch half an hour after complete digestion total incubation time at least 3 hours.
3) sample after hatching is cooled to room temperature, adds isopyknic PCI, fully mix, centrifugal 10 min of 13000rpm.Repeat above-mentioned steps once.PCI is phenol: chloroform: the mixing solutions of primary isoamyl alcohol (25:24:1).
4) by step 3) supernatant liquor use again chloroform: primary isoamyl alcohol (24:1) extracting 1 time, centrifugal 5 min of 13000 rpm.
5) by step 4) supernatant liquor move into equal-volume-20 DEG C Virahols, put upside down fully and mix back and forth, place 20min for-20 DEG C.
6) by step 5) mixed solution at 4 DEG C, centrifugal 5 min of 13000 rpm.Carefully pour out all liquid, note the white precipitate particle moving bottom not allowing or pour out.
7), with 2 pouring liquids of-20 DEG C of 75% washing with alcohol, opening is placed centrifuge tube and is dried ethanol,
Become colorless when transparent and add 50-100 μ l (specifically seeing the amount of DNA) ddH2O to carry out dissolving DNA until white precipitate.Be placed in-20 DEG C for subsequent use.
D) design of primers: transcribe the SNP site that group data prediction goes out according to long oyster, utilize Primerpremier 5 softwares to carry out design of primers, amplified production size is controlled at 50-100bp, and this is tested final primer sequence and is: F:5 '-TAGTAAAGACAGGCAGCA-3 '; R:5 '-
TCATTTGTAGACAGGGAG-3’。Primer amplification fragment length is 75bp, and amplification of nucleotide acid sequence is shown in SEQ ID NO.1.
E) primer screening: random 4 individual DNA of picking carry out pcr amplification as template from experimental population, PCR reaction adopts 10 μ l systems, and with 15 μ l mineral oil sealings, reaction conditions is as shown in table 2.
Table 2
Amplified production detects with 10% non-denaturing polyacrylamide gel, electrophoresis 20min under voltage 200V.Observations (Fig. 1) under ultraviolet gel imaging instrument.Electrophoretic band is clear single and consistent in 4 templates, shows that this primer specificity is good and amplification efficiency is high, can be used for next step experiment; F) HRM method checking SNP site:
1) from experimental population random 8 individual DNA of picking as template, re-start pcr amplification with the primer filtering out, amplified reaction carries out in the 96 hole PCR Sptting plates with shirt rim, and PCR reaction adopts 10 μ l systems, with 15 μ l mineral oil sealings, reaction conditions is as shown in table 2.
2) reaction adds mark and 1 μ l LC-green dyestuff in 1 μ l after finishing, and instantaneous centrifugal rear 95 DEG C of sex change 10min, are cooled to room temperature.
Inside be designated as the short nucleotide sequence that annealing temperature is stable, long 50bp, mixes by marking equal-volume in mark and low temperature in high temperature.
3) taking out 96 hole PCR Sptting plates puts into LightScanner 96 machines and carries out HRM detection, collect the fluorescent signal between 55 DEG C-95 DEG C, move and completely carry out interpretation of result (Fig. 2) according to melting curve, the neat site of melting curve is the SNP site verifying.
G) HRM method detects idiotype: use 144 individualities in the qualified primer pair experimental population filtering out to carry out pcr amplification, draw the melting curve (Fig. 3) of each individuality.Concrete operation method is the same.
H) association analysis: phenotypic data and genotype data are imported to GAPIT software, and operating software carries out association analysis.The result that obtains the prediction of full genome, predicts the outcome and filters out the SNP site TY202 (P=0.008) relevant to long oyster glycogen content proterties according to full genome, in SEQ ID NO.1 from 5 ' latter end the 25th Nucleotide.
The glycogen content of adding up each individuality draws the mean value of 144 individual glycogen contents, the individual glycogen content average out to 5.48mg/g that is T/T in genotype, the individual glycogen content average out to 5.59mg/g that genotype is T/C.Genotype is the individual glycogen content average out to 4.12mg/g of C/C.So, the individuality that the individual glycogen content that is T/T or T/C at this loci gene type is C/C higher than genotype.
Embodiment 2
The application of the SNP mark that a kind of and long oyster glycogen content is relevant
A) collection of sample: gather totally 96 of the wild long oyster colonies of Qingdao Shen Tanggou, it is dissected, get the gill and closed shell flesh, with saving backup in-80 DEG C after liquid nitrogen flash freezer.
B) extraction of DNA: extracting method with in embodiment mono-c).
C) SNP site TY202 genotype detection:
1) taking 96 wild long oyster DNA of refreshing soup ditch as template, carry out pcr amplification with the Auele Specific Primer of TY202, amplified reaction carries out in the 96 hole PCR Sptting plates with shirt rim, and PCR reaction adopts 10 μ l systems, with 15 μ l mineral oil sealings, reaction conditions is as shown in table 2.
2) reaction adds mark (interior mark is the same) and 1 μ l LC-green dyestuff in 1 μ l after finishing, and instantaneous centrifugal rear 95 DEG C of sex change 10min, are cooled to room temperature.
3) take out 96 hole PCR Sptting plates and put into LightScanner 96 machines and carry out HRM detection, collect the fluorescent signal between 55 DEG C-95 DEG C, move and completely carry out interpretation of result according to melting curve, add up the genotype of each individuality.
After detection, the genotype that only has the 40th group is C/C, and other are genotype is that T/T or genotype are T/C, predicts judgement according to the conclusion of embodiment 1, and the glycogen content of the 40th group of individuality is respectively organized content lower than other, then carries out the detection of glycogen content.
D) detection of glycogen content: detection method with in embodiment mono-d).
The individual glycogen content average out to 6.11mg/g that table 3 data presentation is T/T in SNP site TY202 genotype, the individual glycogen content average out to 6.76mg/g that genotype is T/C, the individual glycogen content that genotype is C/C is 2.50mg/g.Above result proves, genotype is the individuality that the individual glycogen average content of T/T or T/C is C/C higher than genotype.
After testing, our predicting the outcome is correct, and the glycogen content of the 40th group of individuality is lower than other each group content.
In sum, SNP site TY202 is associated with glycogen content, by detecting the genotype of SNP site TY202, can dope the height of individual glycogen content.
Table 3
| Individual numbering | Glycogen content | Genotype | Individual numbering | Glycogen content | Genotype | Individual numbering | Glycogen content | Genotype |
| 1 | 8.43 | TT | 33 | 2.19 | TC | 65 | 5.89 | TT |
| 2 | 5.98 | TT | 34 | 3.93 | TT | 66 | 8.35 | TC |
| 3 | 7.84 | TC | 35 | 6.36 | TT | 67 | 5.53 | TT |
| 4 | 4.50 | TT | 36 | 5.23 | TT | 68 | 6.42 | TT |
| 5 | 6.58 | TT | 37 | 4.58 | TT | 69 | 8.99 | TC |
| 6 | 2.85 | TT | 38 | 5.55 | TT | 70 | 9.71 | TT |
| 7 | 7.13 | TT | 39 | 5.46 | TC | 71 | 8.05 | TC |
| 8 | 8.44 | TT | 40 | 2.50 | CC | 72 | 6.75 | TT |
| 9 | 9.17 | TT | 41 | 4.31 | TT | 73 | 5.34 | TC |
| 10 | 6.53 | TT | 42 | 6.40 | TT | 74 | 4.50 | TT |
| 11 | 3.45 | TT | 43 | 8.04 | TT | 75 | 5.66 | TT |
| 12 | 6.08 | TT | 44 | 4.95 | TT | 76 | 4.43 | TT |
| 13 | 6.29 | TT | 45 | 5.53 | TT | 77 | 9.15 | TT |
| 14 | 6.85 | TT | 46 | 7.80 | TT | 78 | 6.79 | TT |
| 15 | 4.25 | TT | 47 | 7.63 | TC | 79 | 8.06 | TT |
| 16 | 8.19 | TT | 48 | 6.38 | TT | 80 | 5.08 | TT |
| 17 | 3.44 | TT | 49 | 6.19 | TT | 81 | 5.70 | TT |
| 18 | 3.47 | TT | 50 | 5.79 | TT | 82 | 7.62 | TT |
| 19 | 5.58 | TT | 51 | 6.90 | TT | 83 | 6.59 | TT |
| 20 | 5.99 | TT | 52 | 8.10 | TT | 84 | 5.89 | TT |
| 21 | 7.07 | TC | 53 | 7.55 | TT | 85 | 3.42 | TC |
| 22 | 7.84 | TT | 54 | 3.48 | TT | 86 | 10.20 | TT |
| 23 | 5.20 | TT | 55 | 4.50 | TT | 87 | 6.01 | TT |
| 24 | 4.00 | TC | 56 | 3.95 | TT | 88 | 9.12 | TT |
| 25 | 6.15 | TT | 57 | 4.23 | TT | 89 | 7.80 | TT |
| 26 | 4.05 | TT | 58 | 5.17 | TT | 90 | 8.66 | TC |
| 27 | 5.17 | TC | 59 | 5.91 | TT | 91 | 4.85 | TT |
| 28 | 5.25 | TT | 60 | 3.90 | TT | 92 | 5.31 | TT |
| 29 | 7.15 | TT | 61 | 6.91 | TT | 93 | 5.75 | TT |
| 30 | 2.19 | TT | 62 | 5.76 | TT | 94 | 5.65 | TT |
| 31 | 7.37 | TT | 63 | 10.11 | TC | 95 | 4.78 | TT |
| 32 | 5.37 | TT | 64 | 9.79 | TT | 96 | 6.20 | TT |
Note: glycogen content unit is mg/g.
Claims (5)
1. a SNP mark relevant to long oyster glycogen content proterties, is characterized in that, described SNP marker site called after TY202 is positioned on the 3rd exon of glycogen debranching factor gene, and genotype is Y.
2. the SNP mark relevant with long oyster glycogen content according to claim 1, is characterized in that: described TY202 is positioned at 5 ' end the 7022nd base place of glycogen debranching factor gene.
3. the SNP mark relevant with long oyster glycogen content according to claim 1, is characterized in that: described genotype is T/T, T/C or C/C.
4. the SNP mark relevant with long oyster glycogen content according to claim 1, is characterized in that: the specific primer sequence in described SNP site is: F:5 '-TAGTAAAGACAGGCAGCA-3 '; R:5 '-TCATTTGTAGACAGGGAG-3 '.
5. a SNP relevant with long oyster glycogen content according to claim 1 is marked at the application in glycogen content detection.
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