CN104152444B - A kind of SNP marker related to long oyster glycogen content character and its application - Google Patents
A kind of SNP marker related to long oyster glycogen content character and its application Download PDFInfo
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Abstract
The present invention relates to a kind of SNP marker related to long oyster glycogen content character and its application.SNP marker site is named as TY202, and on the 3rd extron of glycogen debranching enzyme gene, genotype is Y.The TY202 is located at 5 ' the 7022nd bases of end of glycogen debranching enzyme gene.3rd extron is located at 5 ' 6,446 7043 base-pairs of end of glycogen debranching enzyme gene.SNP marker loci gene type is T/T or T/C individual glycogen content is higher than the individual that genotype is C/C.The method of present invention detection idiotype can predict the height of individual glycogen content, selection target individual be can be used in breeding, for molecular marker assisted selection breeding.The use of concentrated acid concentrated base in glycogen detection process is avoided, makes operation safer and simpler.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding field, specifically a kind of and long oyster glycogen content
The related SNP marker of character.
Background technology
Lamellibranchiata (or Bivalvia) in long oyster, category Mollusca, meat fertilizer is tender, and nutritious, glycogen is oyster
A kind of middle important flavor nutrition material, can directly be absorbed by organisms, so that mitigate pancreas burden, therefore to the preventing and treating of diabetes
It is largely effective.
Glycogen debranching enzyme is a kind of enzyme in oyster glycogen degradation process, and it, which is acted on, is mainly hydrolyzing alpha -1,6 glycosidic bond, moves
The branch of glycogen is gone to, side chain glycogen is changed into straight chain glycogen.People's glycogen debrancher deficiency can cause the type disease of glycogen storage disease 1
Disease.The DNA total lengths of glycogen debranching enzyme are had at present, and Mutation Screening has been carried out to it both at home and abroad and many places mutational site is found, but
Generally acknowledged mutantional hotspot is not found.
SNP is SNP (single nucleotide polymorphism), is primarily referred to as in genome
As the polymorphism of the DNA sequence dna caused by the variation of single nucleotide acid in level.SNP distributions are wide, and density is high, the something lost with height
Pass stability.Compared with other kinds of molecular labeling, SNP marker can preferably react the sequence letter in biological genome
Breath, associating between sequence polymorphism and phenotypic character in genome can be studied in more detail, identified for genes is effectively instructed
With the work of positioning.
In aquatic livestock breeding research starting evening, the research to growth traits is focused primarily upon, method is mostly using traditional
Selection, hybridization, the cycle is long, slowly effect.Quality trait research to long oyster is also rare.In recent years, as genomics is ground
Study carefully a series of breakthroughs of acquirement, molecular marker assisted selection equimolecular breeding technique is increasingly ripe, substantially increases genetic breeding water
It is flat.Blindness can be reduced using molecular marker assisted selection, shortens the breeding time limit, the efficiency of breeding is improved.Association analysis is
Molecular labeling and the popular method of gene that the identification grown up in recent years is associated with objective trait.Association analysis, also known as connects
The uneven mapping (LD mapping) of lock or association mapping (Association Mapping), be it is a kind of using linkage disequilibrium as
Basis, identifies objective trait and genetic marker or the analysis method of candidate gene relation in a certain colony.Association analysis can be examined
All restructuring and the variance event of special group are surveyed, with stronger precision.
The content of the invention
It is an object of the invention to provide a kind of SNP marker related to long oyster glycogen content, for the molecule mark of long oyster
Remember that assisted Selection provides reference.
To achieve the above object the technical scheme is that a kind of SNP marker related to long oyster glycogen content, it
On the 3rd extron of glycogen debranching enzyme gene, genotype is Y.SNP site TY202 is obtained, positioned at glycogen debranching enzyme base
On the 3rd extron at 5 ' end 6446-7043 base-pairs of cause.The TY202 is located at 5 ' ends the of glycogen debranching enzyme gene
At 7022 bases.
Its specific primer sequence is：F：5’-TAGTAAAGACAGGCAGCA-3’；R：5’-
TCATTTGTAGACAGGGAG-3’；Primer expanding 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, is higher than genotype in the loci gene type for T/T or T/C individual glycogen content
For C/C individual.
Each individual genotype and phenotypic data are detected in wild population, by genotype with phenotypic data by associating
Analysis prediction show that TY202 and the character degree of correlation of long oyster glycogen content reach the level of signifiance (P<0.05), with long oyster sugar
The trait associations of former content, the marker assisted selection breeding available for long oyster.
The detection method of the SNP marker related to long oyster glycogen content character, it, which screens step, is：
A) collection Qingdao Jiangnan wild long oyster colony in multiple individuals, it is dissected, packet knit (closed shell flesh,
Gill tissue etc.) sampling, with liquid nitrogen flash freezer after -80 DEG C of preservations, as experiment material；
B) glycogen content in each individual closed shell flesh is determined, phenotypic data is obtained；
C) STb gene of each individual gill is extracted with phenol-chloroform extraction process；
D) long oyster glycogen metabolism related gene is selected as candidate gene, according to long oyster transcript profile data prediction
SNP site is designed after primer, screening primer using HRM methods for each SNP detection idiotypes；
E) analysis is associated using GAPIT softwares, predicts the SNP site related to long oyster glycogen content character.
Advantages of the present invention：
The method of present invention detection idiotype can predict the height of individual glycogen content, can be used for selecting in breeding
Target individual is selected, for molecular marker assisted selection breeding.The use of concentrated acid concentrated base in glycogen detection process is avoided, makes operation
It is 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 results.
Fig. 2 is SNP site TY202 HRM the results.
Fig. 3 is SNP site TY202 colonies genotyping result.
Embodiment
The invention will be further elaborated for following examples, and embodiment is merely to illustrate the present invention, and the present invention is not limited
In this.It is purchased in market that the present invention uses experimental article to be all from.
Sequence table (1) SEQ ID NO.1 information
Sequence signature length：75bp
Type：Nucleic acid
Chain：Double-strand
Topological structure：It is linear
Molecule type：DNA
Source：Long oyster
Sequence description：
TAGTAAAGACAGGCAGCATGCTGAYGGTGAAGGGTACTTTTTGGTGGACCCCATCCTCTCCCTGTCTAC
AAATGA
Embodiment 1
The screening of the SNP site related to glycogen content
A) collection of sample：Multiple individuals in the wild long oyster colony of Qingdao Jiangnan are gathered, totally 144, it are carried out
(closed shell flesh, gill tissue etc.) sampling is knitted in dissection, packet, is saved backup with liquid nitrogen flash freezer after -80 DEG C.
B) glycogen content is detected：The muscle glycogen and hepatic glycogen that Bioengineering Research Institute is built up with Nanjing determine kit detection
Glycogen relative amount in each individual closed shell flesh, detection method to specifications in operating procedure carry out.
1) sample：Take after the muscle samples frozen rinse with physiological saline, filter paper is blotted, weigh (sample weight≤
100mg)。
2) hydrolyze：By sample weight (mg)：Alkali lye volume (μ l)=1 in kit:3, add together in test tube, boiling water bath
20min is boiled, flowing water cooling obtains glycogenolysis liquid.
3) glycogen detection liquid is prepared：Distilled water diluting is added to obtain glycogen detection liquid, plus distilled water glycogenolysis liquid
Measure as (μ l)：Sample weight (mg) * 16.
Operate table as shown in table 1.
Table 1
| Blank tube | Standard pipe | Determine pipe | |
| Distilled water (ml) | 1.0 | 0.9 |
| 0.01mg/ml standards (ml) | 1.0 | ||
| Glycogen detection liquid (ml) | 0.1 | ||
| Nitrite ion (ml) | 2 | 2 | 2 |
Standard liquid and nitrite ion use in kit and carry solution.
Mix and 5min is boiled in rearmounted boiling water, cool down after under 620nm wavelength, 1cm optical paths, returned to zero with blank tube, survey each pipe
OD values.
Calculation formula is as follows：
C) DNA is extracted：The STb gene of each individual gill is extracted with phenol-chloroform extraction process.
1) 1.5 ml centrifuge tubes are taken, 700 μ l DNA Extraction buffers (100 mM Tris-HCl, 5 mM EDTA) are added,
3-10mg oyster gill tissue is taken, is shredded with scissors.Vessel used must use flame sterilization and ddH between two individuals2O is rushed
Wash to prevent the cross pollution between individual.35 μ lSDS are added, are mixed.2 μ l Proteinase Ks are added, are mixed.
2) centrifuge tube is incubated in 65 DEG C of metal baths, often pipe adds 2 μ l Proteinase Ks after 1.5 hours, during which gently shakes
Continue to be incubated more than half an hour after centrifuging tubing digestion completely, total incubation time at least 3 hours.
3) sample after incubation is cooled to room temperature, adds isometric PCI, fully mix, 13000rpm centrifugations 10
min.Repeat the above steps once.PCI is phenol：Chloroform：Isoamyl alcohol (25:24:1) mixed solution.
4) by step 3) supernatant use chloroform again：Isoamyl alcohol (24:1) extract 1 time, 13000 rpm centrifuge 5 min.
5) by step 4) supernatant move into isometric -20 DEG C of isopropanols, overturn fully mix back and forth, -20 DEG C of placements
20min。
6) by step 5) mixed liquor at 4 DEG C, 13000 rpm centrifuge 5 min.All liq carefully is poured out, is careful not to
The white precipitate particle of bottom is allowed to move or pour out.
7) 2 times are washed with -20 DEG C of 75% ethanol and pours out liquid, opening places centrifuge tube and dries ethanol,
50-100 μ l (amount for specifically seeing DNA) ddH2O is added when white precipitate becomes colorless transparent and carrys out dissolving DNA.Put
It is standby in -20 DEG C.
D) design of primers：The SNP site gone out according to long oyster transcript profile data prediction, it is soft using Primerpremier 5
Part carries out design of primers, and amplified production size is controlled in 50-100bp, and the final primer sequence of this experiment is：F：5’-
TAGTAAAGACAGGCAGCA-3’；R：5’-
TCATTTGTAGACAGGGAG-3’.Primer expanding fragment length is 75bp, and amplification of nucleotide acid sequence is SEQ ID
Shown in NO.1.
E) primer screening：Random 4 individual DNA of picking enter performing PCR amplification as template from experimental population, and PCR is anti-
10 μ l systems should be used, with 15 μ l mineral oil seals, reaction condition is as shown in table 2.
Table 2
Amplified production detected with 10% non-denaturing polyacrylamide gel, the electrophoresis 20min under voltage 200V.It is purple
Result (Fig. 1) is observed under outer gel imager.Electrophoretic band is clear single and consistent in 4 templates, shows the primer specific
Property good and amplification efficiency it is high, available for next step experiment；F) HRM methods checking SNP site：
1) random 8 individual DNA of picking, as template, PCR are re-started with the primer filtered out from experimental population
Amplification, amplified reaction is carried out in the 96 hole PCR reaction plates with shirt rim, and PCR reactions use 10 μ l systems, with 15 μ l mineral oil sealings
Mouthful, reaction condition is as shown in table 2.
2) reaction adds after 1 μ l internal standards and 1 μ l LC-green dyestuffs, brief centrifugation 95 DEG C of denaturation 10min after terminating, cold
But to room temperature.
The stable short nucleotide sequence of annealing temperature is inside designated as, long 50bp is mixed in equal volume by high temperature internal standard and low temperature internal standard
Conjunction is formed.
3) take out 96 hole PCR reaction plates be put into the machines of LightScanner 96 carry out HRM detections, collect 55 DEG C -95 DEG C it
Between fluorescence signal, operation is finished carries out interpretation of result (Fig. 2) according to melting curve, and the neat site of melting curve is verifies
SNP site.
G) HRM methods detection idiotype：Enter performing PCR with 144 individuals in the qualified primer pair experimental population filtered out
Amplification, draws each individual melting curve (Fig. 3).Concrete operation method is ibid.
H) association analysis：Phenotypic data and genotype data are imported into GAPIT softwares, runs software is associated analysis.
The result of full-length genome prediction is obtained, being predicted the outcome according to full-length genome, it is related to long oyster glycogen content character to filter out
SNP site TY202 (P=0.008), in SEQ ID NO.1 from 5 ' latter ends the 25th nucleotides.
The glycogen content of each individual of statistics draws the average value of 144 individual glycogen contents, is T/T's in genotype
Individual glycogen content average out to 5.48mg/g, genotype is T/C individual glycogen content average out to 5.59mg/g.Genotype is C/
C individual glycogen content average out to 4.12mg/g.So, it is higher than in the loci gene type for T/T or T/C individual glycogen content
Genotype is C/C individual.
Embodiment 2
A kind of application of the SNP marker related to long oyster glycogen content
A) collection of sample：Gather Qingdao Shen Tanggou wild long oyster colony totally 96, it is dissected, take the gill and
Closed shell flesh, is saved backup with liquid nitrogen flash freezer after -80 DEG C.
B) DNA extraction：C in extracting method be the same as Example one).
C) SNP site TY202 genotype detections：
1) using 96 of refreshing soup ditch wild long oyster DNA as template, enter performing PCR with TY202 specific primer and expand, expand
Increase reaction to carry out in the 96 hole PCR reaction plates with shirt rim, PCR reactions use 10 μ l systems, with 15 μ l mineral oil seals, reaction
Condition is as shown in table 2.
2) reaction adds 95 DEG C of changes after 1 μ l internal standards (internal standard is ibid) and 1 μ l LC-green dyestuffs, brief centrifugation after terminating
Property 10min, is cooled to room temperature.
3) take out 96 hole PCR reaction plates be put into the machines of LightScanner 96 carry out HRM detections, collect 55 DEG C -95 DEG C it
Between fluorescence signal, operation is finished carries out interpretation of result, the genotype of each individual of statistics according to melting curve.
After detection, only the 40th group of genotype is C/C, and other are that genotype is T/T or genotype is T/C, according to
The conclusion of embodiment 1 is predicted judgement, and the glycogen content of the 40th group of individual is less than other each group contents, then carries out glycogen content
Detection.
D) detection of glycogen content：D in detection method be the same as Example one).
The data display of table 3, in the individual glycogen content average out to 6.11mg/g that SNP site TY202 genotype is T/T, base
Because of the individual glycogen content average out to 6.76mg/g that type is T/C, the individual glycogen content that genotype is C/C is 2.50mg/g.With
Upper result proves that genotype is T/T or T/C individual glycogen average content is higher than the individual that genotype is C/C.
After testing, we predict the outcome correct, and the glycogen content of the 40th group of individual is less than other each group contents.
In summary, SNP site TY202 is associated with glycogen content, by detecting SNP site TY202 genotype,
The height of the glycogen content of individual can be predicted.
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 (1)
1. a kind of SNP marker specific primer related to long oyster glycogen content to being detected in glycogen content in application, institute
State specific primer is to sequence：F：5’-TAGTAAAGACAGGCAGCA-3’；R：5’-TCATTTGTAGACAGGGAG-3’；
The SNP marker is located on the 3rd extron of glycogen debranching enzyme gene, and genotype is Y.
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| CN105506162B (en) * | 2016-01-31 | 2020-05-05 | 中国海洋大学 | SNP (single nucleotide polymorphism) marker related to rapid growth of crassostrea gigas as well as identification method and application thereof |
| CN107841561B (en) * | 2016-09-18 | 2021-04-16 | 中国海洋大学 | SNP (Single nucleotide polymorphism) marker related to oyster shell color and screening method |
| CN107475413B (en) * | 2017-09-20 | 2021-05-28 | 中国科学院海洋研究所 | Method for screening crassostrea gigas parent shellfish with high content of unsaturated fatty acid C20:3 omega 6 |
| CN107354234B (en) * | 2017-09-20 | 2020-12-25 | 中国科学院海洋研究所 | Method for screening parent oysters with high glycogen content and related primer pair thereof |
| CN107475414B (en) * | 2017-09-20 | 2021-06-08 | 中国科学院海洋研究所 | Method for screening parent oysters with high glycogen content |
| CN107475415A (en) * | 2017-09-20 | 2017-12-15 | 中国科学院海洋研究所 | A kind of SNP primer pairs of the method for screening the high long oyster parent shellfish of glycogen content and its correlation |
| CN109355400B (en) * | 2018-11-30 | 2022-02-22 | 中国科学院海洋研究所 | Method for identifying genes related to content of glycogen of crassostrea gigas and SNP (Single nucleotide polymorphism) marker and screening high glycogen individuals |
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| CN103740702B (en) * | 2014-01-07 | 2015-11-18 | 中国科学院海洋研究所 | A kind of SNP marker relevant to bay scallop heat tolerance and authentication method thereof and potential application |
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