CN110343773A - SV108 molecular labeling and its detection technique in a kind of structure variation TRO gene for identifying fragrant pig variety - Google Patents
SV108 molecular labeling and its detection technique in a kind of structure variation TRO gene for identifying fragrant pig variety Download PDFInfo
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Abstract
The invention discloses a kind of for identifying the structure variation SV108 of fragrant pig variety, it is characterised in that: SV108 is located at the chrx:47770108-47770216 that pig refers to genome Sscrofa 11.1, and SV108 missing gene is set to D, and the gene not lacked normally is set to I.Using detection technique of the present invention, the SV108 genotype of test sample, it was found that fragrant pig includes three kinds of genotype both DD type, DI type and II type, based on DD type, Deng being 97.1% for gene frequency D, for the black pig of Large White and Guizhou Province north without homozygous deletion type (DD) and heterozygous (DI), only normal type (II), waiting as gene frequency D is 0%.Therefore can application structure variation SV108 as molecular markers for identification perfume (or spice) pig variety.
Description
Technical field
The present invention relates to Animal molecular biology detection fields, it particularly relates to a kind of for identifying fragrant pig variety
SV108 molecular labeling and its detection technique in structure variation TRO gene.
Background technique
Fragrant pig multiplies and lives in closed forest for a long time, gradually forms inbreeding and does not degenerate, and gene height is overlapped small-sized
Pig kind, producing region scattered settlement seedling, strong, precious jade, Shui Deng ethnic group, plough less, have inconvenient traffic, crops are with glutinous paddy and japonica rice
Based on, the masses to live here do not plant the habit of vegetables, and wild feed also compares shortage, only grow edible wild herbs, water plant etc., pig
Cultivation predominantly from numerous self-fertile, raise competence is lower, 1~2, several villages boar, due to breeding too early, using excessive so that
Hypogenetic boar, using once, eliminate within 2 years, the boar newly selected comes from same group again, and inbreeding forms small for a long time
The adaptation environment of type pig existence heredity.As the improvement of people's living standards, some consumers have to the quality of pork it is higher
Requirement.The outer ternary of " fast large-scale ", which is not able to satisfy, pursues high-quality meat consumer demand, this gives many Native Pigs in China
Kind brings crisis.Using China's local pig breed characteristic with good meat quality by hybridizing production with external pig kind, and cause consumer without
Method correctly judges whether it is high meat or purebred pig kind, and it is also possible to purebred local pig breed is caused to move towards rare.
Insertion/deletion, repetition, inversion, transposition and the DNA of DNA fragmentation present on genomic level greater than 1Kb are copied
Shellfish number variation is referred to as structure variation.Currently, having detected that structure relevant to phenotypic variation in livestock and poultry genomics research
Variation.Structure variation site TRO-E12-sv108 is located at the 12nd exon of TRO gene, in chrX:47770108-47770216
Place missing 108bp.Through crowd surveillance, structure variation TRO-E12-sv108 detects three kinds of genotype, D equipotential base in fragrant pig
Because accounting for 97.1%, the black pig in Guizhou Province north and Large White are II genotype.The exons 12 of fragrant pig TRO gene high frequency lacks, very
It is possible that influencing the function of TRO albumen, low with fragrant litter size of pig there may be certain contact.
Nutrient TRO gene (Trophinin) is located on X chromosome, encodes memebrane protein, mediates trophocyte and uterus
The aminoterminal of adherency between intimal epithelium cell, TRO albumen is located at cytoplasm.Nutrient may be combined by homodimer
In cell, the adherency of apical cell when mediating mankind's embryo implantation, the cell adherence that nutrient mediates, which can activate, nourishes outer embryo
The invasion of confluent monolayer cells, however the influence adhered to parent side is unclear (Suzuki N, et al.1999).It is reported that TRO
The albumen of gene coding participates in cellular signal transduction during embryo implantation, may be during mammal early stage placentation
It plays an important role, also participates in the formation (Saburi S, et al.2001) of cancer.
Summary of the invention
The technical problem to be solved by the present invention is SV108 in a kind of structure variation TRO gene for identifying fragrant pig variety
Molecular labeling and its detection technique.
The technical scheme is that it is a kind of for identifying the structure variation SV108 of fragrant pig, it is located at pig and refers to genome
Three kinds of genotype of the position chrX:47770108-47770216 of Sscrofa11.1, structure variation SV108 are respectively designated as
DD, DI and II type, wherein II is insert type genotype, DD is the genotype of homozygous deletion, the genotype that DI is heterozygous deletion.
For the primer pair of the relevant SV108 label of Testing and appraisal perfume (or spice) pig variety, the primer pair are as follows: forward primer
SV108F:GGTACAGAAGAAAGACCCCAAGGA, reverse primer SV108R:GAAACTAGAACTGGTGCTGGCTG.Expand piece
The base sequence of section is respectively SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.
The present invention also provides detection method of the structure variation SV108 in different pig variety groups.
It is described that detection method includes the following steps:
(1) genomic DNA of fragrant pig is extracted；
(2) using above-mentioned fragrant pig genomic DNA as template, using the primer SV108F and SV108R, PCR amplification is carried out,
Detect the genotype of pig genome SV108；
(3) according to above-mentioned testing result, if three kinds of genotype of purpose segment all occur, and based on pure and mild deletion form DD,
Then pig to be measured is fragrant pig；If this segment only exists homozygous II type, test individual is the black pig of Large White or Guizhou Province north.
Amplification system used in PCR is carried out in step (2) by taking 20 μ L as an example:
| 2×Taq PCR Master Mix | 10.00μL |
| F-primer(0.10μg/μL) | 0.40μL |
| R-primer(0.10μg/μL) | 0.40μL |
| gDNA | 1.00μL |
| ddH2O | 8.20μL |
| Total Volume | 20.00μL |
PCR reaction condition are as follows:
The present invention further provides the structure variations in the detection technique for identifying fragrant pig.
The described application the following steps are included:
(1) distribution situation of the structure variation in different pig variety groups is detected using regular-PCR technology；
(2) frequency (i.e. homozygous deletion type DD, heterozygous DI, insertion of 3 kinds of genotype in group in each pig variety are calculated
The percentage of the type II sample number difference total laboratory sample number of Zhan) and allele frequency, analyze the structure variation in fragrant pig and
Difference size between other pig varieties.
It (3) whether is the fragrant pig variety of deletion Genotype auxiliary mirror according to structure variation SV108.
It in an embodiment of the present invention, is candidate with the site structure variation SV108 (chrX:47770108-47770216)
Structure variation distribution situation of the site in group is detected by round pcr in site；If test individual SV108 section with
All missings (DD), then pig kind to be measured is fragrant pig, if test individual SV108 section (II) genotype occurrence rate is 100%,
Pig kind to be measured is the black pig of Large White or Guizhou Province north.The detection in the structure variation site, to identify that fragrant pig is provided fundamental basis.
Beneficial effects of the present invention:
(1) fragrant pig structure variation provided by the invention is not by factors such as the age of fragrant pig, gender and feeding environment conditions
Limitation.
(2) method for the fragrant pig structure variation SV108 of detection that the present invention establishes is accurate and reliable, easy to operate.
(3) detection technique for the structure variation SV108 that the present invention is mentioned can be the molecular marking supplementary breeding of the fragrant pig of identification
Technology establishes theory and technology basis.
Detailed description of the invention
Fig. 1 is that agarose gel electrophoresis detects SV108 genotype results, M:DL2000 DNA in the embodiment of the present invention 1
Marker；1:DD type；2:DI type；3:II type；
Fig. 2 is the gene frequency of 3 pig variety SV108 in the embodiment of the present invention 2.
Specific embodiment
The present invention will be further explained for following instance, but does not limit the scope of the invention, if implementing without specializing
The technical method and conventional laboratory conditions that technical method used in example and condition are well known to the skilled person, or press
The method and condition of reagent manufacturers instruction suggestion is closed in photograph.
Embodiment 1 is detection method of the invention, and steps are as follows:
1, extracting genome DNA
The present invention uses blood/cell/tissue extracting genome DNA purchased from TIANGEN Biotech (Beijing) Co., Ltd.
Kit, for extracting genomic DNA from blood or tissue, the specific method is as follows:
(1) experiment sample pre-processes
A. blood sample
It takes 300 μ L fresh or the blood of -80 DEG C of preservations is added in the sterile EP tube of 1.5mL, less than 300 μ L GA buffers
Supply volume.
B. tissue sample
The tissue sample saved in -80 DEG C of refrigerators is taken out, clip 50mg is thoroughly shredded, and is transferred to sterile 1.5mL EP
The GA buffer of 200 μ L, abundant suspended tissue's particle is added in Guan Zhong.4 μ L RNaseA (100mg/mL) are added into centrifuge tube
Solution sufficiently vibrates 15sec with vortex oscillator, mixes well convenient for enzyme and solution, pollutes for RNA in removal sample, by sample
Product placement stands 5min at room temperature, and centrifuge tube is carried out brief centrifugation.
(2) 20 μ L Proteinase K are added into centrifuge tube, mixing fullys shake.
A. when blood sample genome extracts, step 4 is directly carried out.
B. when ear tissue sample gene group is extracted, 56 DEG C of water-baths dissolve tissue pieces in sample thoroughly, brief centrifugation.
(3) 200 μ L GB buffers are added into centrifuge tube again, vortex oscillator mixes well, and EP pipe is placed in 70 DEG C
In water-bath, water bath with thermostatic control 10min, until solution becomes limpid, brief centrifugation in EP pipe.
(4) 200 μ L dehydrated alcohols are added into EP pipe again, it is reverse to mix solution, at this time in EP pipe it is possible that
EP pipe is carried out of short duration centrifugation by white flock precipitate.
(5) solution that above-mentioned steps obtain and flocculent deposit are transferred completely into adsorption column CB3 that (adsorption column is put into collection
In pipe), 12000rmp is centrifuged 1min, outwells waste liquid.Adsorption column CB3 is placed back in collecting pipe.
(6) the buffer GD that dehydrated alcohol is added in 500 μ L in advance is added into adsorption column CB3, stands 2min, 12000rmp
It is centrifuged 1min, waste liquid is outwelled, adsorption column CB3 is placed back in collecting pipe.
(7) the rinsing liquid PW that dehydrated alcohol is added in 600 μ L is added into adsorption column CB3, stands 2min, 12000rmp centrifugation
1min outwells waste liquid, and adsorption column CB3 is placed back in collecting pipe.
(8) repetitive operation step (8) is primary.
(9) the adsorption column CB3 and collecting pipe for obtaining previous step, 12000rmp are centrifuged 2min, waste liquid are outwelled, by adsorption column
CB3 lid is opened, and is placed on clean blank sheet of paper and is placed at room temperature for several minutes, and remaining rinsing liquid in adsorbent material, Yi Mianying are removed
Ring subsequent experimental.
(10) the adsorption column CB3 thoroughly dried is transferred in a 1.5mL sterile EP tube, is added dropwise to adsorbed film center is hanging
The elution buffer TE of 200 μ L is stored at room temperature 3min, and 12000rmp is centrifuged 2min, and genomic DNA is in 1.5mL centrifuge tube.
The solution being collected by centrifugation can be added to again in adsorption column CB3,12000rpm is centrifuged 2min, collects DNA again
Solution, to improve the yield of genomic DNA.Obtaining genomic DNA can be directly used for testing or being placed in next step -20 DEG C of preservations.
2. the amplification of target sequence and reference sequences
According to ncbi database log in the region primers, the primer pair are as follows: forward primer SV108F:
GGTACAGAAGAAAGACCCCAAGGA, reverse primer SV108R:GAAACTAGAACTGGTGCTGGCTG.Target fragment difference
For 592bp and 485bp.In terms of 20 μ L:
| 10×PCR Mix | 10.00μL |
| Up-primer(0.10μg/μL) | 0.40μL |
| Down-primer(0.10μg/μL) | 0.40μL |
| gDNA | 1.00μL |
| ddH2O | 8.20μL |
| Total Volume | 20.00μL |
PCR reaction condition are as follows:
The primer that this kit is included meets PCR amplification requirement, expanding effect stabilization, high specificity, PCR amplification result
Can in accurate response genome target sequence structure variation situation.
3, the genotyping of structure variation SV108
After PCR amplification, 5 μ L PCR products is taken to detect through 0.7% agarose electrophoresis, 0.5 μ g/mL ethidium bromide dye
Color, gel imaging system photograph record strip band.
It is DD type by VDA genotypes if a sample only amplifies a kind of band of 485bp；When amplifying 485bp
It is DI type by VDA genotypes with two kinds of bands of 592bp；It is II by VDA genotypes if only amplified a kind of band of 592bp
Type (Fig. 1).The band that DD and II genotype is obtained distinguishes cloning and sequencing, and normal II genotype corresponds to the nucleotide of 592bp
Sequence, that is, SEQ ID No.1, nucleotide sequence, that is, SEQ ID No.2 of the DD genotype 485bp of missing, the 108bp sequence of missing
It is listed in SEQ ID No.3.
The application of 2 molecular labeling SV108 of embodiment
According to the method for embodiment 1, with pig genome area structure variation site (chrx:46186712-46187032)
For candidate locus, distribution situation of the genotype of the structure variation in 3 pig variety groups is detected using round pcr, 171
Fragrant pig ear tissue picks up from Xiang Zhu farm, Congjiang County, Guizhou Province and the Qingzhen City cultivation base Xiang Zhu, 31 Large White blood samples and 22
The black pig blood sample in Guizhou Province north.With the candidate region SV108, the structure variation SV108 in detection technique analysis group is established using the present invention
Distribution frequency, there are SV108 variations in fragrant pig groups as the result is shown.
3.8 3 pig variety TRO-E12-sv108 Genotypings of table
It can detect the genotype of structure variation SV108 in test individual genome using the detection technique that the present invention establishes,
It is not limited by factors such as the age of fragrant pig, gender, nutrition and environment, and easy to operate, as a result accurately.Although above
It, can be to it through the present invention is described in detail with a general description of the specific embodiments, but on the basis of the present invention
It makes some modifications or improvements, this is obvious to those skilled in the art.Therefore, without departing from spirit of that invention
On the basis of modifications or improvements, fall within the scope of the claimed invention.
Sequence table
SEQ ID No.1:
<110>Guizhou University
<120>SV108 molecular labeling and its detection technique in a kind of structure variation TRO gene for identifying fragrant pig variety.
<210> 1
<211> 592bp
<212> DNA
<213>fragrant pig SV108 section is normal
<400> 1
GGTACAGAAGAAAGACCCCAAGGATTGGGCTGCTCAGTACCGGGAGGCAGTAGAGATGGA 60
AGTCCAAGCTGCAGCTGTGGCTGTGGCTGAGGCTGAGGCCAGGGCTGAGGCAAGAGCCCA 120
AATGGGGATTGGAGAGGAAGCTGTGGCTGGGCCCTGGAATTGGGATGACATGGATATCGA 180
CTGCCTAACAAGGGAAGAGTTAGGCGATGATGCTCAGGCCTGGAGCAGATTTTCATTTGA 240
AATTGAGGCCAGAGACCAAGAAAATGCAAATGCCAGCACCAACATTGACTTCAGCAGAGG 300
AGCTAGCACCAGAGCTAACTTTAGCGATGGTGCTAGTATTTGTTTCAGTGGTGCACCCAG 360
CCCCAGTGATGGCTTTGGTGGCAGAGATGGCATTAGTTTTGGTAGCACATGCAGCACCAG 420
TGCCACCTTCAGCAGTACAGCCAGCATTAGCTTTGGTGGCACACACTACACTAGCGGCAG 480
CTTCAGCAGTGCAGCCAACATTTGCTTTGGTGGCATGCCCAACACTTGTTCTACTTTCAG 540
TGGTGGAGCCAGCATTAGCTTTGGTGGTGCAGCCAGCACCAGTTCTAGTTTC
SEQ ID No.2:
<210> 2
<211> 485bp
<212> DNA
<213>fragrant pig SV108 segment deletion
<400> 2
GGTACAGAAGAAAGACCCCAAGGATTGGGCTGCTCAGTACCGGGAGGCAGTAGAGATGGA 60
AGTCCAAGCTGCAGCTGTGGCTGTGGCTGAGGCTGAGGCCAGGGCTGAGGCAAGAGCCCA 120
AATGGGGATTGGAGAGGAAGCTGTGGCTGGGCCCTGGAATTGGGATGACATGGATATCGA 180
CTGCCTAACAAGGGAAGAGTTAGGCGATGATGCTCAGGCCTGGAGCAGATTTTCATTTGA 240
AATTGAGGCCAGAGACCAAGAAAATGCAAATGCCAGCACCAACATTGACTTCAGCAGAGG 300
AGCTAGCACCAGAGCTAACTTTAGCGATGGTGCTAGTATTTGTTTCAGTGGTGCACCCAG 360
CCCCAGTGATGGCTTTGGTGGCAGAGATGGCATTAGTTTTGGTAGCACATGCAGCACCAG 420
TGCCACCTTCAGCAGTACAGCCAGCATTAGCTTTGGTGGTGCAGCCAGCACCAGTTCTAG 480
TTTC
SEQ ID No.3:
<210> 3
<211>108
<212> DNA
<213>nucleotide sequence of fragrant pig SV108 deletion fragment
<400> 3
AGCCAGCATTAGCTTTGGTGGCACACACTACACTAGCGGCAGCTTCAGCAGTGCAGCCAA 60
CATTTGCTTTGGTGGCATGCCCAACACTTGTTCTACTTTCAGTGGTGG
Claims (5)
1. SV108 molecular labeling in a kind of structure variation TRO gene for identifying fragrant pig variety, it is characterised in that: described
SV108 molecular labeling is the chrX:47770108-47770216 of Sscrofa11.1 in pig genome, which becomes
Different 108bp segment, or do not lack.
2. SV108 molecule mark in a kind of structure variation TRO gene for identifying fragrant pig variety according to claim 1
Note, it is characterised in that: the primer pair of the SV108 label are as follows: forward primer SV108F:
GGTACAGAAGAAAGACCCCAAGGA, reverse primer SV108R:GAAACTAGAACTGGTGCTGGCTG.
3. the detection method of SV108 molecular labeling as claimed in claim 1 or 2, it is characterised in that: the following steps are included: (1)
Extract Large White to be measured, the genomic DNA of the black pig of fragrant pig and Guizhou Province north；(2) using the genomic DNA of 3 pig kinds to be measured as template, point
Not Li Yong upstream and downstream primer, carry out PCR detection, analyze the genotype of SV；(3) 0.7% agarose gel electrophoresis detect PCR and produce
Object, gel imaging system record strip band simultaneously judge genotype.
4. the detection method of SV108 molecular labeling according to claim 3, it is characterised in that: carry out PCR in step (2)
Amplification system used, by taking 20 μ L as an example:
；
PCR reaction condition are as follows:
5. SV108 molecule mark in a kind of structure variation TRO gene for identifying fragrant pig variety as claimed in claim 1 or 2
The application method of note, it is characterised in that: the following steps are included: (1) detects the structure variation in different pigs using regular-PCR technology
Distribution situation in varietal population；
(2) frequency for calculating frequency and allele of 3 kinds of genotype in group in each pig variety, analyzes the structure variation and exists
Difference size between fragrant pig and Large White kind；
It (3) whether is deletion Genotype supplementary globe Large White and fragrant pig variety according to structure variation SV320.
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| WO2006076825A1 (en) * | 2005-01-18 | 2006-07-27 | Kui Li | A method for detecting the pork quality traits and carcass traits |
| CN106399523A (en) * | 2016-10-11 | 2017-02-15 | 贵州大学 | Molecular marker SV193 for distinguishing Chinese local pig breeds from Large White pigs and application technology of molecular marker SV193 |
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