CN108300794A - A kind of and relevant molecular labeling of piglet Splayleg character - Google Patents
A kind of and relevant molecular labeling of piglet Splayleg character Download PDFInfo
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- CN108300794A CN108300794A CN201810356639.1A CN201810356639A CN108300794A CN 108300794 A CN108300794 A CN 108300794A CN 201810356639 A CN201810356639 A CN 201810356639A CN 108300794 A CN108300794 A CN 108300794A
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Abstract
The invention belongs to pig molecule mark screening technique fields, and in particular to the relevant molecular labeling of piglet Splayleg character.The label is screened from pig HOMER1 genes and is obtained, nucleotide sequence such as sequence table SEQ ID NO：Shown in 1, the R at 357 bit base of sequence is A or G, which leads to HOMER1 gene pleiomorphisms.The present invention screening technique include：Genomic DNA is extracted from pig ear tissue, design primer, PCR amplification, PCR product is cloned, is sequenced, sequence comparing analysis, SNP detection, high-flux sequence parting and analyzed with piglet Splayleg correlation using the label of gained.The invention discloses the SNP parting detection techniques of pig HOMER1 gene introns 4, it is found that A357G polymorphic sites are significantly correlated in character with piglet Splayleg.The present invention provides new genetic marker resource for pig marker assisted selection.
Description
Technical field
Invention belongs to the marker assisted selection technical field of pig, and in particular to a kind of and relevant point of piglet Splayleg character
Son label.The molecular labeling detaches 4 region of introne (the i.e. partial nucleotide sequence of the gene from HOMER1 genes
Row).The invention also includes application of the genetic marker in pig marker assisted selection correlation analysis.
Background technology
Heredity piglet Splayleg belongs to disease of multifactorial inheritance, incidence 0.7%[1].Clinical manifestation is newborn piglet
Back leg turns up, and the minority foreleg that is in a bad way can also be affected[2].The disease leads to piglet astasia, seriously affects piglet
The piglet 50% of ability to act and growth and development, morbidity dies of starvation or is pressed, and exerts a certain influence to production[3].It causes
Piglet Splayleg because being known as the factors such as heredity, environment and disease.Utilize molecular marker assisted selection (Marker Assisted
Selection, MAS) technology, by the selection to piglet Splayleg related gene or chain genetic marker, realization is to control
The selection of character, and then applied to the individual for carrying the related gene for leading to Splayleg is eliminated, selection and breeding are without the excellent of genetic defect
Boar has great importance to the genetic improvement of pig variety.
The research of molecular breeding and applying is rapidly developing, and research screens some and the relevant functional gene of pig hereditary disease
And genetic locus.Pig nerve deafness is autosomal inheritance disease, and L.Chen etc. has found the promoter of MITF in Rongchang Pig
Mutation can lead to sensory nerve hearing loss[4].G.447A Stinckens etc. has found on pig MSTN gene promoters>G is prominent
Becoming the binding site of destruction transcription factor MEF3 can cause pig muscle loose[5]。
Before this, the function in relation to pig HOMER1 genes has not been reported.HOMER1 (homer scaffold protein 1,
Homer scaffolding proteins 1) it is mainly distributed on the postsynaptic density of mammalian nervous member, it is mutual with close metabolic pattern glutamate
Effect.HOMER family proteins can pass through Ca2+/ NFAT signal paths adjust the differentiation of myotube and the development of embryonic period, embryonic phase hind leg exists
In musculature, Ca2+Ca can be passed through2+/ NFAT signal paths adjust the expression of gene[6].HOMER1 can be tied with the channels TRPC1
It closes, and TRPC1 is related with the pathological cause of muscular dystrophy；It is logical that the mouse of gene knockout HOMER1 shows as TRPC1
The imbalance in road, Ca2+It flows into myotube to increase, the area of section of muscle fibre is reduced, and skeletal muscle force declines[7]。
Invention content
The purpose of the present invention is screening is a kind of and the relevant genetic marker of piglet Splayleg hereditary disease.Pass through clone pig
4 sequence of introne of HOMER1 genes finds SNP site with direct sequencing, and detects genotype by high-flux sequence.
Analyze the correlation in the mutational site and piglet Splayleg.To establish new label auxiliary for pig of the selection and breeding without genetic disease
Select site.
The invention is realized by the following technical scheme：
Seminar's early-stage study where applicant mainly passes through whole-genome association (Genome-wide
Association study, GWAS) filter out piglet Splayleg candidate gene HOMER1.Present invention obtains pig HOMER1
Sequence fragment (i.e. the partial nucleotide sequence of HOMER1 genes) in gene intron 4, the fragment length are 819bp, nucleosides
Acid sequence such as sequence table SEQ ID NO：Described in 1, blast comparisons are carried out to above-mentioned sequence by the websites NCBI, in the amplification piece
Find that site nucleotide polymorphisms (SNP) at one, the site are as shown in Figure 2 in section.Mutational site is specifically in the sequence at this
357th.The site for being related to the mutation in the present specification is named as A357G by applicant.
The present invention selects Splayleg pig and health pig for test material, and genomic DNA is extracted from pig ear tissue, according to
Pig HOMER1 gene orders (gene accession number is shown in described in embodiment 1) design primer pair of Ensemble warehouse publications, this draws
The sequence of object is as follows：
Forward primer：5 '-TGTTCTATAAAGGCACCACC-3 ',
Reverse primer：5’-TCAATCTGGAAGACATGAGC-3’；
Above-mentioned primer pair can be to SEQ ID NO:Genetic marker described in 1 and to the HOMER1 genetic fragments (core of the segment
Nucleotide sequence such as SEQ ID NO:Shown in 1) carry out parting.
PCR amplification, the purifying of PCR product, cloning and sequencing and sequence comparative analysis are carried out using the primer pair.
Screening obtains a kind of and relevant molecular labeling of piglet Splayleg character, and the nucleotide sequence of the genetic marker is as follows
Shown (being mutational site, the i.e. allelic mutation of 357-357bp in bracket)
TGTTCTATAAAGGCACCACCAACACTAAATTAGTGAGTATCGAACCATTTTCCTGGGGGACATACAGGGTAGATTCC
TGTGAGCCTCTGGTCACATTTTCATCAGCCAATCAATAAATAACCTTGTTTGATGTGTGTTTCTATTTAAAGACACC
TTATTTAATATATATTGTTGATTCATTACCATTGACCTCATGGCCAAAAGCACTGTATGTAACTCATGGCTGAAGGA
AGCTTATATAACGTTTTTTCCCCTTGAGGCACATCACCGACTTCTTACACCTGGGAACAGACAGCGCTCAGCACTAT
GCTTGGGGCCCATGTGAAGCAGTGAAAGCACCAACAAAAAGCACAAACR(A/G)
TGCAAAAACCATGGTCTGAATTGATTGCAAACAGGACACTTGTTCATACCAAGAGAGCTGATATAAGAAGGCAGTGT
TGCCTTGATCAATCTCTGGTGGACATGTGCATTTTAGGTCGCTGGAAAAGACCAAGCAAATACCTCAAGTATGGATT
TGGAGGTTACAAATGCATTTTATTGAGTAAGCAAATTCGCAAATATAGCATCTTCTCAAATAATAAGGATTGACAGT
AATTGTTTTAAAGAAGCTTGTGATTGAATACTTAAGAGTAGTCAAGTTTGAAAGTAAGTTTCCCTTACTTTCAAGTA
TGTGGGAGTTTTTGGTTGAGTGGAATCATCCTCATGGTTTTCATGTTCTGTTGTCCAGTGGAATAAAATTGGCTGCT
TTCAGTCTCTCGTTTTGTTTTCTTCCCTCCAGGGAACAACCCATCTTCAGCACTCGAGCTCATGTCTTCCAGATTGA
R in above-mentioned sequence at 357 bit bases is A or G, which leads to HOMER1 gene pleiomorphisms.
Applicant provide a kind of relevant molecule labelling method of screening piglet Splayleg character, the method include with
Lower step：
From pig (such as：Large White, Landrace, Duroc, the white bi-crossbreeding of great Bai-length) genome is extracted in ear tissue
DNA, according to the genome sequence design primer of pig HOMER1 genes, the sequence such as SEQ ID NO of the primer pair：2 and SEQ ID
NO：Shown in 3, PCR amplification is carried out in pig genomic DNA using the primer pair, by direct sequencing, is caused
The nucleotide sequence of HOMER1 gene pleiomorphisms.High-flux sequence is recycled to carry out parting, HOMER1 Gene Partial cores to sample
Nucleotide sequence such as sequence table SEQ ID NO:Shown in 1, in sequence table SEQ ID：A/G is mutated (sequence at the 357bp of base shown in 1
The sequence at 357bp in list is the sequence " G " after mutation).
Correlation analysis is carried out to piglet Splayleg as genetic marker using the mutational site.
The present invention provides the genotyping methods for detecting one variation of above-mentioned sequence.
Further, the present invention provides determined between different genotype individual and piglet Splayleg using high-flux sequence method
Association analysis.
More detailed technical solution is shown in《Specific implementation mode》It is described.
Description of the drawings
Sequence table SEQ ID NO：1 is the partial nucleotide sequence of pig HOMER1 genes, i.e., as the heredity mark of the present invention
The nucleotide sequence of note.Generation is sported the allelic mutation of " G " by " A " at 357 bit bases of the sequence.
Sequence table SEQ ID NO：2 and SEQ ID NO：3 be the sequence for the primer pair for expanding HOMER1 genes, the primer pair
It is also the primer pair of detection genetic marker of the present invention.
Fig. 1：The techniqueflow chart of the present invention.
Fig. 2：The partial nucleotide sequence and its mutational site schematic diagram of pig HOMER1 genes.Reference sign：In SEQ
ID NO:R (A/G) at 357 bit bases of nucleotide sequence shown in 1 is allelic mutation, which leads to HOMER1 bases
Because of polymorphism.
Fig. 3：Direct Sequencing testing result of the present invention to HOMER1 genes.Reference sign：According to sequencing result it is found that
There are the mutation of an A/G allele at 357th bit base of the sequence.
Specific implementation mode
Embodiment 1：The acquisition of the DNA fragmentation in 4 region of pig HOMER1 gene introns and the foundation of SNP detection method
The present embodiment selects Splayleg pig (kind is Large White, Landrace, Duroc, the white bi-crossbreeding of great Bai-length)
With health pig (kind be Large White, Landrace, Duroc, the white bi-crossbreeding of great Bai-length), according to pig HOMER1 genome reports
Sequence (the number of the logging in Gene ID in road：10051070) primer pair as described below, is designed：
Forward primer：TGTTCTATAAAGGCACCACC,
Reverse primer：TCAATCTGGAAGACATGAGC；
With above-mentioned primer pair PCR amplification is carried out in " pig " genomic DNA.
PCR reaction systems and PCR reaction conditions are shown in Tables 1 and 2.
PCR reaction systems：
1 PCR reaction systems of table
PCR reaction conditions：
2 PCR response procedures of table
Sequencing is carried out after the PCR product of gained is purified and cloned, Shanghai life work biology is entrusted in examining order
Engineering services Co., Ltd completes.Particular sequence is as described below, and (sequence is exactly the sequence of the genetic marker of the present invention, i.e.,
SEQ ID NO:1 sequence for being)：
TGTTCTATAAAGGCACCACCAACACTAAATTAGTGAGTATCGAACCATTTTCCTGGGGGACATACAGGGTAGATTCC
TGTGAGCCTCTGGTCACATTTTCATCAGCCAATCAATAAATAACCTTGTTTGATGTGTGTTTCTATTTAAAGACACC
TTATTTAATATATATTGTTGATTCATTACCATTGACCTCATGGCCAAAAGCACTGTATGTAACTCATGGCTGAAGGA
AGCTTATATAACGTTTTTTCCCCTTGAGGCACATCACCGACTTCTTACACCTGGGAACAGACAGCGCTCAGCACTAT
GCTTGGGGCCCATGTGAAGCAGTGAAAGCACCAACAAAAAGCACAAACR(A/G)TGCAAAAACCATGGTCTGAATTG
ATTGCAAACAGGACACTTGTTCATACCAAGAGAGCTGATATAAGAAGGCAGTGTTGCCTTGATCAATCTCTGGTGGA
CATGTGCATTTTAGGTCGCTGGAAAAGACCAAGCAAATACCTCAAGTATGGATTTGGAGGTTACAAATGCATTTTAT
TGAGTAAGCAAATTCGCAAATATAGCATCTTCTCAAATAATAAGGATTGACAGTAATTGTTTTAAAGAAGCTTGTGA
TTGAATACTTAAGAGTAGTCAAGTTTGAAAGTAAGTTTCCCTTACTTTCAAGTATGTGGGAGTTTTTGGTTGAGTGG
AATCATCCTCATGGTTTTCATGTTCTGTTGTCCAGTGGAATAAAATTGGCTGCTTTCAGTCTCTCGTTTTGTTTTCT
TCCCTCCAGGGAACAACCCATCTTCAGCACTCGAGCTCATGTCTTCCAGATTGA,
Through blast comparative analyses, it is found that there are an allele, i.e. A/G bases at the 357th bit base in the sequence
The mutation of cause.The mutation causes HOMER1 gene pleiomorphisms.
Embodiment 2：The molecular labeling of the present invention and the correlation analysis of piglet Splayleg and application
In order to determine the SNP in pig HOMER1 gene introns 4 in promoter region and piglet Splayleg character whether phase
It closes, it is test material to select Splayleg pig (sample number is 72) and normal pig (sample number is 110).It is measured using high pass
Sequence method carries out polymorphic detection, and analyzes the phase of pig HOMER1 gene introns region different genotype and piglet Splayleg character
Guan Xing.
The A357G polymorphic sites in 4 region of pig HOMER1 gene introns are detected, are detected in this group
3 kinds of genotype, and analyze this SNP site distribution frequency in Splayleg pig and health pig using Chi-square Test and whether there is
Difference, the results are shown in Table 3：
A357G gene frequency distributional differences in 3 health pig of table and Splayleg pig
3 explanation of table：Ns, P>0.05；*P<0.05；**P<0.01；***P<0.001, N represents normal pig, and A represents Splayleg
Pig.
The mutant allele frequency of this A357G polymorphic site is all higher than in Splayleg pig as can be seen from Table 3
The frequency of wild-type allele.And the distribution frequency in Splayleg pig and health pig in the sites A357G has pole significance difference
Different (P<0.001), this illustrates that the sites A357G are related to piglet Splayleg character, therefore SEQ ID NO:Sequence described in 1 can be made
For the genetic marker of piglet Splayleg.
Leading reference
[1]Thurley D C,Gilbert F R,Done J T.Congenital splayleg of piglets:
myofibrillar hypoplasia[J].Veterinary Record,1967,80(9):302‐304.
[2]Curvers P,Ducatelle R,Vandekerckhove P,De C W,Calus A,Hoorens
J.Morphometricevaluation of myofibrillar hypoplasia in splayleg piglets[J]
.Dtsch Tierarztl Wochenschr,1989,96(4):189‐191.
[3]Dobson K J.Congenital splayleg of piglets[J].Australian Veterinary
Journal,1968,44(1):26.
[4]Chen L,Guo,W.,Wang,J.,Wang,S.,Hu,X.,Li,N.Hereditary sensorineural
hearing loss in Chinese Rongchang pigs result form promoter mutations in Mitf
[J].International Society of Animal Genetics:Abstract P2009,2012,
[5]Stinckens A,Luyten T,Bijttebier J,Van d M K,Dieltiens D,Janssens
S,De S S,Georges M,Buys N.Characterization of the complete porcine MSTN gene
and expression levels in pig breeds
differing in muscularity[J].Animal genetics,2008,39(6):586.
[6]Stiber J A,Tabatabaei N,Hawkins A F,Hawke T,Worley P F,Williams R
S,Rosenberg P.Homer modulates NFAT‐dependent signaling during muscle
differentiation[J].Developmental Biology,2005,287(2):213‐224.
[7]Stiber J A,Zhu‐Shan Z,Jarrett B,Eu J P,Sarah Z,Truskey G A,Malini
S,Naohiro Y,Gerhard M,Ripal S.Mice lacking Homer 1 exhibit a skeletal
myopathy characterized by abnormal transient receptor potential channel
activity[J].Molecular&Cellular Biology,2008,28(8)::2637–2647。
Sequence table
<110>Hua Zhong Agriculture University
<120>A kind of and relevant molecular labeling of piglet Splayleg character
<141> 2018-04-17
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 819
<212> DNA
<213>Pig (Sus scrofa)
<220>
<221> gene
<222> (1)..(819)
<220>
<221> mutation
<222> (357)..(357)
<400> 1
tgttctataa aggcaccacc aacactaaat tagtgagtat cgaaccattt tcctggggga 60
catacagggt agattcctgt gagcctctgg tcacattttc atcagccaat caataaataa 120
ccttgtttga tgtgtgtttc tatttaaaga caccttattt aatatatatt gttgattcat 180
taccattgac ctcatggcca aaagcactgt atgtaactca tggctgaagg aagcttatat 240
aacgtttttt ccccttgagg cacatcaccg acttcttaca cctgggaaca gacagcgctc 300
agcactatgc ttggggccca tgtgaagcag tgaaagcacc aacaaaaagc acaaacgtgc 360
aaaaaccatg gtctgaattg attgcaaaca ggacacttgt tcataccaag agagctgata 420
taagaaggca gtgttgcctt gatcaatctc tggtggacat gtgcatttta ggtcgctgga 480
aaagaccaag caaatacctc aagtatggat ttggaggtta caaatgcatt ttattgagta 540
agcaaattcg caaatatagc atcttctcaa ataataagga ttgacagtaa ttgttttaaa 600
gaagcttgtg attgaatact taagagtagt caagtttgaa agtaagtttc ccttactttc 660
aagtatgtgg gagtttttgg ttgagtggaa tcatcctcat ggttttcatg ttctgttgtc 720
cagtggaata aaattggctg ctttcagtct ctcgttttgt tttcttccct ccagggaaca 780
acccatcttc agcactcgag ctcatgtctt ccagattga 819
<210> 2
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 2
tgttctataa aggcaccacc 20
<210> 3
<211> 20
<212> DNA
<213>Pig (Sus scrofa)
<220>
<221> primer_bind
<222> (1)..(20)
<400> 3
tcaatctgga agacatgagc 20
Claims (3)
1. a kind of and relevant molecular labeling of piglet Splayleg character, it is characterised in that its nucleotide sequence is as follows：
TGTTCTATAAAGGCACCACCAACACTAAATTAGTGAGTATCGAACCATTTTCCTGGGGGACATACAGGGTAGA
TTCCTGTGAGCCTCTGGTCACATTTTCATCAGCCAATCAATAAATAACCTTGTTTGATGTGTGTTTCTATTTAAAGA
CACCTTATTTAATATATATTGTTGATTCATTACCATTGACCTCATGGCCAAAAGCACTGTATGTAACTCATGGCTGA
AGGAAGCTTATATAACGTTTTTTCCCCTTGAGGCACATCACCGACTTCTTACACCTGGGAACAGACAGCGCTCAGCA
CTATGCTTGGGGCCCATGTGAAGCAGTGAAAGCACCAACAAAAAGCACAAACR(A/G)
TGCAAAAACCATGGTCTGAATTGATTGCAAACAGGACACTTGTTCATACCAAGAGAGCTGATATAAGAAGGCAGTGT
TGCCTTGATCAATCTCTGGTGGACATGTGCATTTTAGGTCGCTGGAAAAGACCAAGCAAATACCTCAAGTATGGATT
TGGAGGTTACAAATGCATTTTATTGAGTAAGCAAATTCGCAAATATAGCATCTTCTCAAATAATAAGGATTGACAGT
AATTGTTTTAAAGAAGCTTGTGATTGAATACTTAAGAGTAGTCAAGTTTGAAAGTAAGTTTCCCTTACTTTCAAGTA
TGTGGGAGTTTTTGGTTGAGTGGAATCATCCTCATGGTTTTCATGTTCTGTTGTCCAGTGGAATAAAATTGGCTGCT
TTCAGTCTCTCGTTTTGTTTTCTTCCCTCCAGGGAACAACCCATCTTCAGCACTCGAGCTCATGTCTTCCAGATTGA
R in above-mentioned sequence at 357 bit bases is A or G, which leads to HOMER1 gene pleiomorphisms.
2. a kind of primer pair expanding genetic marker as described in claim 1 and parting is carried out to HOMER1 genetic fragments,
Nucleotide sequence is as described below：
Forward primer：TGTTCTATAAAGGCACCACC,
Reverse primer：TCAATCTGGAAGACATGAGC.
3. application of the genetic marker described in claim 1 in piglet marker assisted selection correlation analysis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961731A (en) * | 2020-07-08 | 2020-11-20 | 四川农业大学 | Positioning method of CpG-based pork color character whole genome methylation sites |
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2018
- 2018-04-19 CN CN201810356639.1A patent/CN108300794B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| M.SALANOVA等: "Homer proteins and InsP3 receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres", 《CELL CALCIUM》 * |
| XINGJIE HAO等: "Genome-wide association study identifies candidate genes for piglet splay leg syndrome in different populations", 《BMC GENETICS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961731A (en) * | 2020-07-08 | 2020-11-20 | 四川农业大学 | Positioning method of CpG-based pork color character whole genome methylation sites |
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