CN107523633A - A kind of method based on pig SINE transposons insertion polymorphism research and development New molecular marker - Google Patents
A kind of method based on pig SINE transposons insertion polymorphism research and development New molecular marker Download PDFInfo
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Abstract
The present invention relates to molecular biology and field of bioinformatics, and in particular to a kind of method based on pig SINE transposons insertion polymorphism research and development New molecular marker.This method is that genome finds insertion point disclosed in pig by the use of SINE nucleotide sequence as search sequence；According to each insertion point searched out, 300 500 nucleotide sequences of trip extension up and down, download every sequence respectively；The sequence of acquisition is removed into redundancy；Detection primer is designed according to the sequence information of acquisition；Different cultivars, or same kind Different Individual genomic samples are expanded using the molecular labeling primer PCR to be verified of gained, the primer that band can clearly be expanded and have polymorphism by choosing combines, and obtains molecular labeling.The inventive method can be the ore grade indexes of pig, and the marker assisted selection in breeding provides useful molecular labeling.
Description
Technical field
The present invention relates to molecular biology and field of bioinformatics, and in particular to one kind is inserted based on pig SINE transposons
The method that polymorphism researches and develops New molecular marker.
Background technology
DNA molecular marker technology is a kind of new Genetic Markers based on explanations of genomic DNA polymorphism, is heredity
Make a variation the direct reflection on DNA level.Marked relative to morphology, biochemical biomarker, cytological marker, DNA molecular mark
The superiority that remembering has has：Most of molecular labelings are codominance, and the selection to the character of recessiveness is very convenient；Genome mutation
Extremely abundant, the quantity of molecular labeling is almost unlimited；It is all available in the different phase of biological development, the DNA of different tissues
In labeled analysis；Molecular labeling discloses the variation from DNA；Neutrality is shown as, does not influence the expression of objective trait, it is and not benign
Shape is without chain；Detection means is simple, rapid.
The development that DNA molecular marker technology is recorded a demerit over the years, mainly there are following several marks, first generation molecular labeling skill
Art：RFLP (RFLP) labelling technique, advantage：The allele of RFLP marks has codominant spy
Point, it is as a result reliable and stable, it is reproducible, it is particularly adapted to build genetic linkage map.Shortcoming：When carrying out rflp analysis, it is necessary to
The DNA fragmentation in the site makees probe, not only dangerous but also be not easy to automate with radio isotope and nucleic acid hybridization technique.In addition,
The sensitivity that RFLP detects to DNA polymorphism is not high, also has very great space region on RFLP linkage maps.Randomly amplified polymorphic
DNA marker technology (RAPD), advantage：Compared with RFLP, RAPD technologies are simple, and DNA dosages are few, and testing equipment is simple, are not required to DNA
Probe, design primer is simple, and does not need isotope, and security is good.Shortcoming：RAPD technologies are influenced by factors, experiment
Stability and poor repeatability, be dominant inheritance first, it is impossible to identify heterozygote site, the genetic analysis of this thing is relative complex,
In the assignment of genes gene mapping, when doing linkage inheritance figure, the accuracy decline of genetic distance between calculating site can be made because of dominant covering effect；
Secondly, RAPD is quite sensitive to reaction condition, including template concentrations, Mg2+Concentration, so the poor repeatability of experiment.Amplified fragments
The polymorphic technology of length (AFLP), advantage：It has the advantages of RAPD and RFLP concurrently, there is higher stability, with a small amount of selectivity
Primer can monitor a large amount of sites within a short period of time, and the number of AFLP marks and chromosome length are in positive on each chromosome
Close, combined by the primer of a small amount of efficiency high, the AFLP marks of covering whole gene group can be obtained.Shortcoming：AFLP is to genome
Purity and reaction condition are higher, easily big by DNA pollution, operating technology difficulty.During for genetic mapping, a small number of mark and figure
Composing tight ness rating, there is any discrepancy.Second generation molecular marking technique, micro-satellite labeling technique SSR, advantage：With very high polymorphism, and
And generally codominance.Shortcoming：Workload is big, and expense is also high, and practical application is few.Third generation molecular labeling：Mononucleotide polymorphic
Property (SNP) advantage：With codominance, obtainable marker number is more, it is easy to accomplish high flux.It is most simple in genome.Most
Common polymorphic forms, there is very high genetic stability.Shortcoming：Chip cost is high, due to the complexity of DNA sample, has
A little SNP can not be picked up.EST (EST) advantage：Cost is low, simple to operate.People are contributed to be managed from molecular level
The diversity of species is solved, and using sequence information wherein interested.Shortcoming：Obtained genomic information is not complete, is such as unable to body
Reveal the information important in gene expression regulation such as introne, regulating and controlling sequence.
Transposons is a kind of moving DNA sequence that can be freely jumped on genome and (replicate or shift), is widely present
In biological genome.Because transposons can replicate in the genome of its host, mobile, accordingly, it is capable to be produced on genome
Transposons insertion polymorphism (Transposon insertion polymorphisims, TIP), to species, kind, subspecies, product
It is that formation has played important function.The New molecular marker technology established using transposons insertion polymorphism (TIP) is entered in heredity
Change the application in research as we have appreciated that the evolution of higher organism genome provides New Century Planned Textbook.
Although the significant challenge that pig genomics research faces at present is the QTL for obtaining a large amount of quantitative variabilities,
Lack the molecular labeling of sufficient density, it is difficult to carry out gene finely positioning, can not effectively carry out molecular marker assisted selection.And
TIP has many advantages, such as preferable molecular labeling：Such as polymorphism height, codominance, genome is widely distributed, detection means is simply fast
It is fast, reproducible low with development cost etc..And SINE has certain length, detection difficulty is less than SNP, microsatellite etc., and
Widely distributed in pig genome, the SINE designed by the method for this patent is marked, can be more with simple and efficient detection insertion
State property, and can be determined that homozygous or heterozygous state.The molecular labeling of the polymorphic research and development of SINE transposons insertion simultaneously will
The research of the process and pig functional gene of molecular breeding is promoted significantly.
The content of the invention
The present invention be directed to the deficiency of existing molecular labeling, has content rich on pig genome based on pig SINE transposons
Richness, it is widely distributed, and provide a kind of method for researching and developing molecular labeling in the presence of the characteristics of insertion polymorphism between different individuals.
The present invention is achieved through the following technical solutions：
A kind of acquisition modes of the New molecular marker based on pig SINE transposons, comprise the following steps：
(1) by the use of SINE nucleotide sequence as search sequence, gene is disclosed to pig in biology common data base
Group is retrieved, and finds insertion point；
(2) for the SINE searched out each insertion point, upstream 300-500 nucleotides is extended with downstream respectively
Sequence, every sequence is then downloaded respectively；
(3) sequence for obtaining step (2) removes redundancy；
(4) sequence information obtained according to step (3) designs detection primer, as molecular labeling primer to be verified, wherein
Side primer is located in 5 ' end genomic flanking sequences of SINE insertion points, opposite side primer be located at SINE 3 ' end groups because
In group flanking sequence；
(5) it is different using the molecular labeling primer PCR to be verified amplification different cultivars obtained by step (4), or same kind
Genes of individuals group sample, the primer that band can clearly be expanded and have polymorphism by choosing combine, and obtain molecular labeling.
Wherein, the members sequences of SINE families are variant, can be by the use of any one as search sequence, and the site of acquisition is all
Belong to the insertion point of SINE transposons.The nucleotide sequence of the SINE includes but is not limited to SEQ ID No.1, SEQ ID
No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、
SEQ ID No.9、SEQ ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14、
SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17.
One example of the above method is：
(1) by the use of SINE nucleotide sequence SEQ ID No.1 as search sequence, in Ensembl data and UCSC numbers
(blat) search is compared to examining genome disclosed in pig according to storehouse, finds insertion points of the SINE on genome；
(2) upstream 300 nucleotides are extended with downstream respectively in each insertion point, the length that then selection compares is more than
95%, similarity is more than 98% site, and downloads every sequence respectively；
(3) sequence of acquisition is merged using CD-HIT programs according to similitude 95%, removes redundancy, it is final to obtain
78 sequences.
(4) interception holds genomic flanking sequence, its own sequence and the 3 ' sequences for holding genomic flanking sequences to form by SINE 5 '
The template as design primer is arranged, according to sequences Design detection primer, wherein making side primer be located at the 5 ' of SINE insertion points
Hold in genomic flanking sequence, opposite side primer is located in SINE 3 ' end genomic flanking sequences, and finally 78 pairs of acquisition is to be tested
Demonstrate,prove molecular labeling primer.
(5) molecular labeling primer PCR to be verified amplification different cultivars, or same kind Different Individual genome sample are utilized
Product, the primer that band can clearly be expanded and have polymorphism by choosing combine, and obtain molecular labeling, obtain 44 marks altogether, corresponding
Primer, primer combination, primer sequence are as shown in table 1.
Wherein, the operation that PCR is expanded in step (5) can be comprised the following steps：
1) artificial synthesized above-mentioned 44 pairs of molecular labeling primers to be verified；
2) the pond DNA of different cultivars and the Different Individual genome of different cultivars are extracted；
3) performing PCR amplification is entered as template using the pond DNA of different cultivars and the Different Individual genome of different cultivars；
4) row agarose gel electrophoresis detection is entered to PCR amplifications.
The present invention mainly by obtaining SINE insertion point sequences, designs each site detection primer, PCR amplifications, agarose
The steps such as gel electrophoresis obtain transposons insertion situation, establish accordingly based on transposons insertion pleiomorphism detecting method.For pig
Ore grade indexes, the marker assisted selection in breeding provide useful molecular labeling.
Brief description of the drawings
Fig. 1 marks amplifications of the SINE-TIP-5 in 11 different cultivars.Marker is the DL2000 of precious biology, from
Left-to-right 1-12 is Lip river gram pig, Landrace, Large White, plum mountain pig, Jiang Shumin, Soviet Union ginger pig, Tibetan pig, wild boar, Ba Maxiang respectively
Pig, Wan Nan flowers pig, the black pig mixutre genome in Yantai and blank control, mixutre genome are 5 individual grade mass mixing bases
Because of group.The signified insertion for representing to have SINE in this mark position of No. 1 arrow in figure, No. 2 arrows are signified to be represented in this mark position
Item chromosome has SINE insertion, and another item chromosome has SINE insertion.No. 3 arrows are signified to represent do not have in this mark position
There is SINE insertion, No. 4 arrows are signified to represent this mark position fragment deletion, and other chart signal justice are unanimously.
Fig. 2 marks amplifications of the SINE-TIP-31 in 11 different cultivars.Marker is the DL2000 of precious biology,
From left to right 1-12 is Lip river gram pig, Landrace, Large White, plum mountain pig, Jiang Shumin, Soviet Union ginger pig, Tibetan pig, wild boar, bar horse respectively
Fragrant pig, Wan Nan flowers pig, the black pig mixutre genome in Yantai and blank control, mixutre genome are 5 individual grade mass mixings
Genome.
Fig. 3 marks testing results of the SINE-TIP-6 in the Different Individual of 9 different cultivars.Marker is precious biology
DL15000, be respectively from left to right wild boar, great Bai, Duroc, Mei Shan, painted face in Beijing opera, hide pig, Rongchang County, BaMa miniature pig, Soviet Union ginger pig,
Pig 3 is wherein hidden, remaining kind is 5.
Fig. 4 marks testing results of the SINE-TIP-7 in the Different Individual of 9 different cultivars.Marker is precious biology
DL15000, be respectively from left to right wild boar, great Bai, Duroc, Mei Shan, painted face in Beijing opera, hide pig, Rongchang County, BaMa miniature pig, Soviet Union ginger pig,
Pig 3 is wherein hidden, remaining kind is 5.
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
First, LINE1 insertion point retrievals, detailed process are as follows in reference gene group：
1st, SINE transposons insertion point in genome is retrieved：By the use of SINE nucleotide sequences SEQ ID No.1 as
Search sequence, in Ensembl databases (http://www.ensembl.org/) and UCSC databases (http://
Genome.ucsc.edu/ the reference gene group (SGCG Sscrofa10.2/susScr3) of pig is carried out using Blat programs in)
Search is compared, operational factor uses respective default setting in database, finds insertion points of the LINE1 on genome.
2nd, each SINE insertion point retrievals in two databases：The retrieval insertion position in Ensembl databases
After point, the length compared into comparison result is more than 95%, 5 ' end upstreams of each insertion point of the similarity more than 98%
Respectively extend 300 nucleotides with 3 ' downstreams, then download every sequence, similarly retrieved again in UCSC databases, rejected
The consistent site with Ensembl databases, then hold upstream and 3 ' end downstreams respectively 300 cores of extension to the 5 ' of each insertion point
Thuja acid, every sequence is downloaded, finally merge the sequence that two databases are downloaded.
3rd, the sequence of acquisition is utilized into CD-HIT-est programs
(http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgiCmd=cd-hit-
Est Sequence identity cut-off parameters) are arranged to 0.95, other specification carries out de-redundancy using default value.
2nd, the design of primer
Using the sequence of acquisition, BatchPrimer3 is used
(http://batchprimer3.bioinformatics.ucdavis.edu/cgi-bin/batchprimer3/
Batchprimer3.cgi) online software carries out design of primers, sense primer is located at the flank at 5 ' ends of SINE insertion points
Sequence, anti-sense primer are located in SINE 3 ' end flanking sequences.Product length is arranged to 500-700bp, and other specification uses soft
Part default setting.Designed each primer is analyzed and evaluated with Olige 7, then by Beijing six directions Hua Da Gene science share
Co., Ltd synthesizes.
3rd, molecular labeling is verified in 11 different cultivars
1st, the preparation of genome：
(1) extracted with TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0
Duroc, Landrace, Large White, plum mountain pig, Jiang Shumin, Soviet Union ginger pig, Tibetan pig, wild boar, BaMa miniature pig, Wan Nan flowers pig, cigarette
Each 5 individual genomes of the black pig of platform, key step are as follows：
A) take 2-25mg ear tissue, shred, add 180 microlitres Buffer GL, 20 microlitres of Proteinase K and
10 microlitres of Rnase A (10mg/ml), 56 DEG C of water-baths to tissue cracking completely.B) 200 microlitres of BufferGB are added into lysate
With 200 microlitre of 100% ethanol, fully mix.C) Spin Column are placed on Collection Tube, solution moves to
In Spin Column, 12000rpm is centrifuged 2 minutes, abandons filtrate.D) 500 microlitres of buffer WA are added into Spin Column,
12000rpm is centrifuged 1 minute, abandons filtrate.E) 700 microlitres of buffer WB are added into Spin Column, 12000rpm from
The heart 1 minute, abandons filtrate.F) step 6 is repeated.G) Spin Column are placed on Collection Tube,
12000rpm is centrifuged 2 minutes.H) Spin Column are placed on new 1.5ml centrifuge tube, in Spin Column films
Centre adds 50-200 microlitres of aqua sterilisa or Elution Buffer, is stored at room temperature 5 minutes.I) 12000rpm centrifuges 2 points
Clock eluted dna (2), which adjusts each genome concentration, turns into 40ng/ μ l, takes each μ l of genes of individuals group 50 to be mixed into kind pond
DNA, totally 11 kinds of mixutre genomes, enter performing PCR amplification in this, as template.
2nd, PCR is expanded
The following PCR reaction systems of design：
(1) 20 μ l reaction systems are prepared in the PCR pipe that sterilizes
(2) PCR pipe is placed in PCR instrument, carries out following response procedures
Note：1：Nanjing Vazyme Biotechnology Co., Ltd.
3rd, Ago-Gel detects
(1) 1.5% Ago-Gel is made：Weigh 1.5g agaroses to be put into conical flask, and added into flask
100ml 1*TAE solution, it is put in heating in micro-wave oven and is allowed to be completely dissolved, when gel is cooled to about 60 DEG C, by agarose gel
Pour into and be inserted with the mould of glue comb, thickness checks for bubble presence typically in 4-5mm.Room temperature places 30min and treats that gel is complete
It is careful to extract glue comb after full solidification, it is immersed in the electrophoresis tank added with 1*TAE electrophoretic buffers.
(2) PCR primer electrophoresis：6 μ lPCR amplified productions are drawn with pipettor to be added sequentially in gel pore, with the μ of time point 4
LDNAMaker is as reference, 120V constant pressure electrophoresis, to be instructed dose of arrival blob of viscose 2/3 at stop electrophoresis, then gel is put into
In ethidium bromide solution, dye 10 minutes, gel is taken out to be put under uviol lamp and observes and photographs to record.
4th, molecular labeling is verified in 9 different cultivars
1st, the preparation of genome：
(1) extracted with TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0
Wild boar, great Bai, Duroc, Mei Shan, painted face in Beijing opera, Tibetan pig, Rongchang County, BaMa miniature pig, the genes of individuals group of ginger 9 kinds of pig of reviving, it is main
Want step as follows：
A) take 2-25mg ear tissue, shred, add 180 microlitres Buffer GL, 20 microlitres of Proteinase K and
10 microlitres of Rnase A (10mg/ml), 56 DEG C of water-baths to tissue cracking completely.B) 200 microlitres of BufferGB are added into lysate
With 200 microlitre of 100% ethanol, fully mix.C) Spin Column are placed on Collection Tube, solution moves to
In Spin Column, 12000rpm is centrifuged 2 minutes, abandons filtrate.D) 500 microlitres of buffer WA are added into Spin Column,
12000rpm is centrifuged 1 minute, abandons filtrate.E) 700 microlitres of buffer WB are added into Spin Column, 12000rpm from
The heart 1 minute, abandons filtrate.F) step 6 is repeated.G) Spin Column are placed on Collection Tube,
12000rpm is centrifuged 2 minutes.H) Spin Column are placed on new 1.5ml centrifuge tube, in Spin Column films
Centre adds 50-200 microlitres of aqua sterilisa or Elution Buffer, is stored at room temperature 5 minutes.I) 12000rpm centrifuges 2 points
Clock eluted dna (2), which adjusts each genome concentration, turns into 40ng/ μ l, enters performing PCR amplification in this, as template.
2nd, PCR is expanded
The following PCR reaction systems of design：
(1) 20 μ l reaction systems are prepared in the PCR pipe that sterilizes
(2) PCR pipe is placed in PCR instrument, carries out following response procedures
Note：1：Nanjing Vazyme Biotechnology Co., Ltd.
3rd, Ago-Gel detects
(1) 1.5% Ago-Gel is made：Weigh 1.5g agaroses to be put into conical flask, and added into flask
100ml 1*TAE solution, it is put in heating in micro-wave oven and is allowed to be completely dissolved, when gel is cooled to about 60 DEG C, by agarose gel
Pour into and be inserted with the mould of glue comb, thickness checks for bubble presence typically in 4-5mm.Room temperature places 30min and treats that gel is complete
It is careful to extract glue comb after full solidification, it is immersed in the electrophoresis tank added with 1*TAE electrophoretic buffers.
(2) PCR primer electrophoresis：6 μ l pcr amplification products are drawn with pipettor to be added sequentially in gel pore, with the μ of time point 4
L DNA Maker are as reference, 120V constant pressure electrophoresis, to be instructed dose of arrival blob of viscose 2/3 at stop electrophoresis, then gel is put
Into ethidium bromide solution, dye 10 minutes, gel is taken out to be put under uviol lamp and observes and photographs to record.
5th, interpretation of result
Partial detection such as Fig. 1, Fig. 2, Fig. 3, shown in Fig. 4.Molecule based on SINE insertion polymorphisms shown in Fig. 1, Fig. 2
There is good polymorphism in mark, authentication method can be provided for cultivar identification, Fig. 3, is based on shown in Fig. 4 between different cultivars
There is well polymorphic in the molecular labeling of SINE insertion polymorphisms, can be used as Individual identification, molecule auxiliary between Different Individual
The excellent marker of breeding.By two-wheeled experimental verification, a series of molecular labelings based on SINE insertion polymorphisms are obtained.6th, tie
By
By two-wheeled experimental verification, 44 molecular labelings based on SINE insertion polymorphisms shown in table 1, Neng Gou are obtained
Produce clear in kind or between kind and there is polymorphic testing result, authentication method, Ke Yizuo can be provided for cultivar identification
For Individual identification, the excellent marker of marker assisted selection, there is the advantages that testing cost is low, reproducible, as a result clear；And it is
Codominance, the selection to the character of recessiveness are very convenient；In the different phase of biological development, the DNA of different tissues can be used in
Labeled analysis；The expression of objective trait is not influenceed, with bad character without chain；Detection means is simple, rapid.
The SINE transposons of table 1 inserts polymorphism mark detection primer list
。
SEQUENCE LISTING
<110>Yangzhou University
<120>A kind of method based on pig SINE transposons insertion polymorphism research and development New molecular marker
<130>
<160> 85
<170> PatentIn version 3.3
<210>1
<211>263
<212>DNA
<213>Pig (Sus scrofa)
<400>1
ggagttcccg tcgtggcgca gtggttaacg aatccgacta ggaaccatga ggttgcgggt 60
tcggtccctg cccttgctca gtgggttaac gatccggcgt tgccgtgagc tgtggtgtag 120
gttgcagacg cggctcggat cccgcgttgc tgtggctctg gcgtaggccg gcggctacag 180
ctccgattcg acccctagcc tgggaacctc catatgccgc gggagcggcc caagaaatag 240
caaaaagaca aaaaaaaaaa aaa 263
<210>2
<211>259
<212>DNA
<213>Pig (Sus scrofa)
<400>2
ggagttcccg tcgtggcgca gcggaaacga atccgactag gaaccatgag gttgcgggtt 60
cgatccctgg cctcgctcag tgggttaagg atccggcgtt gccgtgagct gtggtgtagg 120
tcgcagacgc ggctcggatc csgcgttgct gtggctgtgg cgtaggccgg cagctgcagc 180
tccgattmga cccctagcct gggaacctcc atatgccgcg ggtgcggccc taaaaagaca 240
aaaaacaaaa aaaaaaaaa 259
<210>3
<211>255
<212>DNA
<213>Pig (Sus scrofa)
<400>3
ggagttcccg ctgtggctca gcgggttaag racccgacgt wgtstccgtg aggatgcggg 60
ttcgatccct ggcctcgctc agtgggttaa ggatccggcg ttgccgcaag ctgcggcgta 120
ggtcgcagat gcggctcgga tccggcgttg ccgtggctgt ggcgtaggcc ggcagctgca 180
gctccgattc gacccctagc ccgggaactt ccatatgccg caggtgcggc cntaaaaaga 240
aaaaaaaaaa aaaaa 255
<210>4
<211>254
<212>DNA
<213>Pig (Sus scrofa)
<400>4
ggagttcccg tcgtggcgca gtggttaacg aatccgacta ggaaccacga ggttgcgggt 60
tcgatccctg gcctcgctca gtgggttaag gatccggcgt tgccgtgagc tgtggtgtag 120
gtcgcagacg cggctcggat cccgcgttgc tgtggctctg gcgtaggccg gcggctacag 180
ctccgattng acccctagcc tgggaacctc cntatgccnc aggngcggcc ctagaaaagg 240
caaaaaaaaa aaaa 254
<210>5
<211>216
<212>DNA
<213>Pig (Sus scrofa)
<400>5
ggagttcccg tcgtggcgca gcggaaacga atccgactag gaaccatgag gttgcgggtt 60
cgatccctgg cctcgctcag tgggttaagg atccggcgtt gccgtgagct gtggtgtagg 120
ccggcggctg cagctccgat tngaccccta gcctgggaac ctccatatgc cgcgggtgcg 180
gccctaaaaa aggcaaaaaa aaaaaaaaaa aaaaaa 216
<210>6
<211>216
<212>DNA
<213>Pig (Sus scrofa)
<400>6
ggagttcccg tcgtggctca gcggttaacg aacccgacta gtatccatga ggacgcgggt 60
tcgatccctg gcctcgctca gtgggttaag gatccggcgt tgctgtggct gtggcgtagg 120
ccggcagctg cagctccgat tcgaccccta gcctgggaac ttccatatgc cgcgggtgcg 180
gccctaaaaa aggcaaaaaa anaaaaaaaa aaaaaa 216
<210>7
<211>202
<212>DNA
<213>Pig (Sus scrofa)
<400>7
ggagttcccg ctgtggcgca acgggatcgg cggcgtctcg ggagcgccgg gacgcaggtt 60
cgatccccgg cccggcacag tgggttaagg atccggcgtt gccgcagctg cggcktaggt 120
cgcaactgcg gctcggatct gatccctggc ccgggaactc catatgccgc ggggcggcca 180
aaaaagaaaa aaaaaaaaaa aa 202
<210>8
<211>225
<212>DNA
<213>Pig (Sus scrofa)
<400>8
ggagttcccg ctgtggcgca gtgggttaag gatccgactg cagcggctcg ggtcgctgcg 60
gaggcgcggg ttcgatcccc ggcccggcgc agtgggttaa aggatccggc gttgccgcag 120
ctgtggcgta ggtcgcagct gcggctcgga ttcaatccct ggcccgggaa cttccatatg 180
ccgcgggtgc ggccaaaaaa agaaaaaaaa naaaaaaaaa aaaaa 225
<210>9
<211>221
<212>DNA
<213>Pig (Sus scrofa)
<400>9
gggagttccc gccgtggcgc agtgggttaa gaatccgact gcagcggctc gggtcgctgc 60
ggaggcgcgg gttcgatccc cggcccggcg cagtgggtta aaggatccgg cgttgctgca 120
gctgcggcgt aggtcgcagc tgcggctcgg attcgatccc tggcccggga acttccatat 180
gccgcgggtg cggccgtaaa aagaaaaaaa aaaaaaatcg t 221
<210>10
<211>131
<212>DNA
<213>Pig (Sus scrofa)
<400>10
gggagttctc ttgtggcaca gcaggttaag gatccagcgt tgtcactgca gtggcttggg 60
tcgctgctgt ggcacgggtt caatccctgg cccaggaact tccacatgcc acgggcatgg 120
ccaaaaaaaa a 131
<210>11
<211>119
<212>DNA
<213>Pig (Sus scrofa)
<400>11
ggagttccct ggtggctcag tgggttaagg atccggcatt gtcactgctg tggctcgggt 60
tcgatccctg gcccaggaac ttctgcatgc cgtgggcgcg gccaaaaaaa aaaaaaaaa 119
<210>12
<211>134
<212>DNA
<213>Pig (Sus scrofa)
<400>12
ggagttccct ggtggctcag cgggttaagg atccggcgtt gtcactgctg tggctctggt 60
tacngctgtg gcacgggttc aatccctggc ccgggaactt ccgcatgcca cgggcgcggc 120
caaaaaaaaa aaaa 134
<210>13
<211>127
<212>DNA
<213>Pig (Sus scrofa)
<400>13
ggagttccct tgtggcgcag cgggttaagg atccggcgtt gtcactgcag cggctcgggt 60
cgctgctgtg gcgcgggttt gatccctggc ccgggaactt ccacatgctg tgggcgcggc 120
caaaaaa 127
<210>14
<211>131
<212>DNA
<213>Pig (Sus scrofa)
<400>14
ggagttccct ggtggctcag ygggttaagg atctagtgtt gtcactgctg tggctcrggt 60
cactgctgtg gtgcaggttt gatccctggc ctgggaactt ccacatgcyg yrggcatggc 120
caaaaaaaaa a 131
<210>15
<211>123
<212>DNA
<213>Pig (Sus scrofa)
<400>15
ggacttcccg tgtggctcac agcataacta ctcggcgttg cttctgcagt ggctgtggct 60
cgacccctgg cccgggaact tccacatgcc gcgggcgtgg ccaaaaaaaa aaaaaaaaaa 120
aaa 123
<210>16
<211>137
<212>DNA
<213>Pig (Sus scrofa)
<400>16
ggagttccct ggtggtctag tggttaggat ccggcgcttt cactgctgcg gcccgggttc 60
aatcccctgg tctgggaact gggatcccac atcaagccac tgcangcgtg gccaaaaaca 120
aacaaacaaa caaaaaa 137
<210>17
<211>155
<212>DNA
<213>Pig (Sus scrofa)
<400>17
gggagttctc tgatggccta gcgggttgag gctcctgcgt tctcaccgct gtggctctgg 60
ttgctgctgt gcggcgtagg ttcaatccct ggcccaggaa ttcccacata ctgcctgtgt 120
ggcaaaaaag aaaaaaaaaa aaaatacaaa aaaaa 155
<210> 18
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<400> 18
gaaaaatcca ggcaaggaca 20
<210> 19
<211> 24
<212> DNA
<213>Pig (Sus scrofa)
<400> 19
ccacttagga taagaggtcc tcaa 24
<210> 20
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<400> 20
accagctggg attttgattg 20
<210> 21
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<400> 21
gccctgaatg acagacacag 20
<210> 22
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<400> 22
tgtgcatgtc aaatgtggtc 20
<210> 23
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<400> 23
ctacacaacc cccagttggt 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
caagttcccc atgtgctttc 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
gcccaacaaa agatgaatgg 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
cattctaaca accgccaagg 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
aggtgcagcc ttaaaaagca 20
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence
<400> 28
tgagtgtcat ccctgcatgt 20
<210> 29
<211> 22
<212> DNA
<213>Artificial sequence
<400> 29
cagcaacaga gaaatcatag cc 22
<210> 30
<211> 21
<212> DNA
<213>Artificial sequence
<400> 30
gaatgctgtt acacaggcac a 21
<210> 31
<211> 21
<212> DNA
<213>Artificial sequence
<400> 31
ccccacatgg tttactttga g 21
<210> 32
<211> 19
<212> DNA
<213>Artificial sequence
<400> 32
aagcaaggca gagtgagca 19
<210> 33
<211> 20
<212> DNA
<213>Artificial sequence
<400> 33
gggaaaccat cccatttacc 20
<210> 34
<211> 20
<212> DNA
<213>Artificial sequence
<400> 34
ccacgctgtg gctctaaaat 20
<210> 35
<211> 20
<212> DNA
<213>Artificial sequence
<400> 35
ctacctctcc ccaccagtga 20
<210> 36
<211> 20
<212> DNA
<213>Artificial sequence
<400> 36
cagccacacc atggtttcta 20
<210> 37
<211> 20
<212> DNA
<213>Artificial sequence
<400> 37
acccacatca aggaaggtca 20
<210> 38
<211> 20
<212> DNA
<213>Artificial sequence
<400> 38
ggacacccaa ttcccctaat 20
<210> 39
<211> 20
<212> DNA
<213>Artificial sequence
<400> 39
cttttggggg aagggataaa 20
<210> 40
<211> 20
<212> DNA
<213>Artificial sequence
<400> 40
attaccgctg agccactagg 20
<210> 41
<211> 20
<212> DNA
<213>Artificial sequence
<400> 41
tttacagcaa cacggatgga 20
<210> 42
<211> 19
<212> DNA
<213>Artificial sequence
<400> 42
ggagctcctg ggggtaaat 19
<210> 43
<211> 20
<212> DNA
<213>Artificial sequence
<400> 43
tgagggaact caggaaggaa 20
<210> 44
<211> 20
<212> DNA
<213>Artificial sequence
<400> 44
catggtgttt gtcaggagga 20
<210> 45
<211> 24
<212> DNA
<213>Artificial sequence
<400> 45
tggtgttcaa tgagatgact tagg 24
<210> 46
<211> 20
<212> DNA
<213>Artificial sequence
<400> 46
tggtgacgtg tccttcaaga 20
<210> 47
<211> 20
<212> DNA
<213>Artificial sequence
<400> 47
caggaacaca gtcctggtca 20
<210> 48
<211> 20
<212> DNA
<213>Artificial sequence
<400> 48
gggagtcaga aaaaggcaga 20
<210> 49
<211> 20
<212> DNA
<213>Artificial sequence
<400> 49
ccattctcaa agggcattta 20
<210> 50
<211> 20
<212> DNA
<213>Artificial sequence
<400> 50
gggtctatgg atgcacgttt 20
<210> 51
<211> 20
<212> DNA
<213>Artificial sequence
<400> 51
tggccagcat tatcgtaaca 20
<210> 52
<211> 20
<212> DNA
<213>Artificial sequence
<400> 52
ccagggaaca gtttgctctt 20
<210> 53
<211> 20
<212> DNA
<213>Artificial sequence
<400> 53
gccgttcttg catgcttatt 20
<210> 54
<211> 21
<212> DNA
<213>Artificial sequence
<400> 54
caaaggaaag ctgaagccat a 21
<210> 55
<211> 20
<212> DNA
<213>Artificial sequence
<400> 55
tgaacattac gagcggatga 20
<210> 56
<211> 21
<212> DNA
<213>Artificial sequence
<400> 56
ccgtaggagc tcataatgtg g 21
<210> 57
<211> 20
<212> DNA
<213>Artificial sequence
<400> 57
gaaaacggag agcttgatgg 20
<210> 58
<211> 20
<212> DNA
<213>Artificial sequence
<400> 58
tgctggaggg atcaggatag 20
<210> 59
<211> 21
<212> DNA
<213>Artificial sequence
<400> 59
cttccatcat gttccatcac a 21
<210> 60
<211> 20
<212> DNA
<213>Artificial sequence
<400> 60
acggttacgc caatgttttc 20
<210> 61
<211> 20
<212> DNA
<213>Artificial sequence
<400> 61
ccaagtgccc atcaatgaat 20
<210> 62
<211> 20
<212> DNA
<213>Artificial sequence
<400> 62
gcccaatttt ccctctcttc 20
<210> 63
<211> 20
<212> DNA
<213>Artificial sequence
<400> 63
tttaggccct gctcccttat 20
<210> 64
<211> 20
<212> DNA
<213>Artificial sequence
<400> 64
gaggcagcca cagagaagag 20
<210> 65
<211> 25
<212> DNA
<213>Artificial sequence
<400> 65
ctgagggaca tactttaact cattc 25
<210> 66
<211> 20
<212> DNA
<213>Artificial sequence
<400> 66
acaaacagac gctgagcaga 20
<210> 67
<211> 20
<212> DNA
<213>Artificial sequence
<400> 67
cagtcaggga gctgattgct 20
<210> 68
<211> 20
<212> DNA
<213>Artificial sequence
<400> 68
cccctccacc tcagttacaa 20
<210> 69
<211> 20
<212> DNA
<213>Artificial sequence
<400> 69
acacatccac aaggtgacga 20
<210> 70
<211> 20
<212> DNA
<213>Artificial sequence
<400> 70
tcaaacccac atcttcatgg 20
<210> 71
<211> 20
<212> DNA
<213>Artificial sequence
<400> 71
ccatggtggt tagacccaaa 20
<210> 72
<211> 20
<212> DNA
<213>Artificial sequence
<400> 72
tggtggtctt tggggttatc 20
<210> 73
<211> 20
<212> DNA
<213>Artificial sequence
<400> 73
tgtggcaaaa atccagaaac 20
<210> 74
<211> 20
<212> DNA
<213>Artificial sequence
<400> 74
agcacaggga accatatcc 20
<210> 75
<211> 20
<212> DNA
<213>Artificial sequence
<400> 75
gatgccttaa cccactgcac 20
<210> 76
<211> 20
<212> DNA
<213>Artificial sequence
<400> 76
gcttaaatgg cccacaaaga 20
<210> 77
<211> 20
<212> DNA
<213>Artificial sequence
<400> 77
gcacaagttt gcagacaacc 20
<210> 78
<211> 21
<212> DNA
<213>Artificial sequence
<400> 78
ggaaagaaag tgcaatccag a 21
<210> 79
<211> 20
<212> DNA
<213>Artificial sequence
<400> 79
ggagagagag gaggggagaa 20
<210> 80
<211> 20
<212> DNA
<213>Artificial sequence
<400> 80
gagagggaga gggacacaca 20
<210> 81
<211> 20
<212> DNA
<213>Artificial sequence
<400> 81
tttcttgatc tgtgcctcca 20
<210> 82
<211> 20
<212> DNA
<213>Artificial sequence
<400> 82
cctcctctgg ttccttctcc 20
<210> 83
<211> 20
<212> DNA
<213>Artificial sequence
<400> 83
gaggacccat cctagaccaa 20
<210> 84
<211> 20
<212> DNA
<213>Artificial sequence
<400> 84
ccactgaaca tttgccttca 20
<210> 85
<211> 20
<212> DNA
<213>Artificial sequence
<400> 85
tgggaactct gctcttggtt 20
Claims (5)
1. it is a kind of based on pig SINE transposons insertion polymorphism research and development New molecular marker method, it is characterised in that including with
Lower step：
(1) by the use of SINE nucleotide sequence as search sequence, in biology common data base to pig disclosed in genome
Retrieved, find insertion point；
(2) SINE searched out for step (1) each insertion point, upstream 300-500 core is extended with downstream respectively
Nucleotide sequence, every sequence is then downloaded respectively；
(3) sequence for obtaining step (2) removes redundancy；
(4) sequence information obtained according to step (3) designs detection primer, as molecular labeling primer to be verified, wherein making one
Side primer is located in 5 ' end genomic flanking sequences of SINE insertion points, and opposite side primer is located at SINE 3 ' end genome sides
In wing sequence；
(5) the molecular labeling primer PCR to be verified amplification different cultivars obtained by step (4), or same kind Different Individual are utilized
Genomic samples, the primer that band can clearly be expanded and have polymorphism by choosing combine, and obtain molecular labeling.
2. the method according to claim 1 based on pig SINE transposons insertion polymorphism research and development New molecular marker, it is special
Sign is：The step (1) SINE nucleotide sequence includes but is not limited to SEQ ID No.1, SEQ ID No.2, SEQ
ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID
No.9、SEQ ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14、SEQ ID
No.15, SEQ ID No.16 or SEQ ID No.17；As search sequence；In the databases such as NCBI, Ensembl or UCSC
Search is compared to genome disclosed in pig, finds insertion points of the SINE on genome, obtains some sequences.
3. the method according to claim 2 based on pig SINE transposons insertion polymorphism research and development New molecular marker, it is special
Sign is：The step (3) is to merge the sequence of acquisition according to similitude 95% using CD-HIT programs, removes redundancy,
Finally obtain some genomic locations distinguished sequences.
4. the method according to claim 3 based on pig SINE transposons insertion polymorphism research and development New molecular marker, it is special
Sign is：The step (4) makes wherein to make side primer be located at SINE insertion points according to the sequences Design detection primer of download
In 5 ' end genomic flanking sequences, opposite side primer is located in SINE 3 ' end genomic flanking sequences.
5. the method according to claim 4 based on pig SINE transposons insertion polymorphism research and development New molecular marker, it is special
Sign is：The step (5) utilizes molecular labeling primer PCR to be verified amplification different cultivars, or same kind Different Individual gene
Group sample, the primer that band can clearly be expanded and have polymorphism by choosing combine, and obtain molecular labeling.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157811A (en) * | 2019-05-16 | 2019-08-23 | 扬州大学 | SINE transposons multi-state molecular label, detection method and application in a kind of and associated GHR gene of fat thickness at back of pig |
| CN110257481A (en) * | 2019-05-09 | 2019-09-20 | 扬州大学 | A kind of transposons based on comparative genomics is inserted into the method for digging of polymorphic TIP molecular labeling |
| CN110408707A (en) * | 2019-07-23 | 2019-11-05 | 华中农业大学 | Clone the molecular labeling relevant to bristles color character from InDel segment |
| CN111705144A (en) * | 2020-07-17 | 2020-09-25 | 扬州大学 | SINE transposon polymorphic molecular marker in ZNF2 gene associated with pig backfat thickness and detection method thereof |
| CN111876490A (en) * | 2020-07-03 | 2020-11-03 | 扬州大学 | Linkage molecular marker for improving growth speed of pigs, detection method and application thereof |
| CN112094923A (en) * | 2020-09-30 | 2020-12-18 | 扬州大学 | SINE transposon polymorphic molecular marker associated with pig growth rate and detection method and application thereof |
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| CN102174654A (en) * | 2011-03-02 | 2011-09-07 | 西南大学 | High-efficiency functional marker large-scale development and quick QTL (Quantitative Trait Loci) location candidate gene determination method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257481A (en) * | 2019-05-09 | 2019-09-20 | 扬州大学 | A kind of transposons based on comparative genomics is inserted into the method for digging of polymorphic TIP molecular labeling |
| CN110257481B (en) * | 2019-05-09 | 2023-07-28 | 扬州大学 | Transposon insertion polymorphism TIP molecular marker mining method based on comparative genomics |
| CN110157811A (en) * | 2019-05-16 | 2019-08-23 | 扬州大学 | SINE transposons multi-state molecular label, detection method and application in a kind of and associated GHR gene of fat thickness at back of pig |
| CN110157811B (en) * | 2019-05-16 | 2022-06-14 | 扬州大学 | Polymorphic molecular marker of SINE transposon in GHR gene associated with pig backfat thickness, detection method and application |
| CN110408707A (en) * | 2019-07-23 | 2019-11-05 | 华中农业大学 | Clone the molecular labeling relevant to bristles color character from InDel segment |
| CN110408707B (en) * | 2019-07-23 | 2021-02-12 | 华中农业大学 | Molecular marker cloned from InDel fragment and related to pig hair color property |
| CN111876490A (en) * | 2020-07-03 | 2020-11-03 | 扬州大学 | Linkage molecular marker for improving growth speed of pigs, detection method and application thereof |
| CN111876490B (en) * | 2020-07-03 | 2022-07-15 | 扬州大学 | Linkage molecular marker for improving growth speed of pigs, detection method and application thereof |
| CN111705144A (en) * | 2020-07-17 | 2020-09-25 | 扬州大学 | SINE transposon polymorphic molecular marker in ZNF2 gene associated with pig backfat thickness and detection method thereof |
| CN112094923A (en) * | 2020-09-30 | 2020-12-18 | 扬州大学 | SINE transposon polymorphic molecular marker associated with pig growth rate and detection method and application thereof |
| CN112094923B (en) * | 2020-09-30 | 2022-07-12 | 扬州大学 | SINE transposon polymorphic molecular marker associated with pig growth speed as well as detection method and application thereof |
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