CN103757004A - Litter size related molecular marker of swine 10# chromosome and primers thereof - Google Patents
Litter size related molecular marker of swine 10# chromosome and primers thereof Download PDFInfo
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Abstract
The invention relates to molecular markers related to swine litter size trait, and particularly relates to a litter size related molecular marker of a swine 10# chromosome and primers thereof, belonging to the field of biotechnologies. The locus of the molecular marker is Chr10: 16018894 of a genome version NCBI Build Sscrofa 10. 2; alleles of the locus are T and C and consist of three genotypes, namely C/C, T/C and T/T, and the locus and a flanking sequence thereof are shown in SEQ ID NO: 1. The primers and extension primers for amplifying the locus of the molecular marker are as follows: Chr10-PCRU: 5'-TGC CAA ACC TGT GTC ACC-3'; Chr10-PCRL: 5'-GGC TGG AGA GGA AAA GGG-3'; Chr10-SNPU: 5'-TGA CTA GAA GCA GCC ATG GGG CCC TGC AGA-3'. The litter size of swine with the genotype of T/T is greater than that of the swine with the genotypes of T/C and C/C. The litter size related molecular marker and the primers thereof have the beneficial effects that trait related molecular markers or candidate genes can be screened rapidly in a batch manner by using a whole-genome re-sequencing method, and the locus Chr10: 16018894 can be applied to the genetic breeding of the swine as a genetic marker, so as to increase the litter size of sows.
Description
Technical field:
The present invention relates to the molecule marker relevant to pig number born character, be specifically related on No. 10 karyomit(e)s of pig a molecule marker relevant to litter size and primer thereof, belong to biological technical field.
Background technology:
The characters of number born of pig is the chief component of pig reproductive trait, improves litter size of pig significant to the overall economic benefit of raising pig industry.But litter size belongs to the quantitative character that heritability is very low, be difficult to carry out genetic improvement with traditional breeding method.The Taihu Lake pig of China is well-known with high yield, nest litter size is than many 4-5 of nest litter size head of other Chinese native pig breed of great majority and external pig kind (< < Chinese pig breeds will > > Zhang Zhongge chief editor, Science and Technology of Shanghai press, 1986).Erhualian is the one of Taihu Lake pig, is the pig kind that reproductivity is the highest in the world.The polymorphic site relevant to high litter size in the pig of Taihu Lake, can be as genetic marker for improving the litter size of sow.
Researchist adopts the method for full genome scanning and candidate gene characters of number born to be carried out to the research of QTL location and candidate gene both at home and abroad.The people such as Li (Li, K., J.Ren, et al.Anim Genet.2009.40 (6): 963-966) utilize and covered 19 chromosomal 183 microsatellite markers pair total litter sizes relevant to litter size of pig of pig, produce the young number of living, produce dead young number and carry out QTL(Quantitative Trait Locus, quantitative trait locus) location.They have studied the F of 299 durocs and the hybridization of Chinese Erhualian
2generation, 6,7, on 8 and No. 15 karyomit(e)s, located and total litter size, produce live young number and the relevant QTL of the dead young number of product.
(the Rothischild such as Rothschild, M., Jacobson, A.1996.93 (1): 201-205 of D.et al.Proc Natl Acad Sci U S) by candidate gene method, two the extreme mixing breeds of utilization including the pig of Prunus mume in China mountain system Taihu Lake, find that a gene (ESR) at estrogen receptor seat on No. 1 karyomit(e) and high litter size major gene are chain.RFLPs by female hormone receptor gene seat analyzes, think the homozygous sow (BB type) with 3.7kb band than there is 4.3kb band homozygous sow (AA type) primiparity time want many 2.3 (P<0.01), average voluminous 1.5 (P<0.01) of every tire.At present reported that the gene relevant to litter size also has OPN (US6410227B1), PRLR (Tomas, A., J.Casellas, et al.J Anim Sci.2006.84 (8): 1991-1998), FSH β (Zhao, Y.Li N, et al.Sci China C Life Sci.1998.41 (6): 664-668) and RBP4 (Spotter, A., S.Muller, et al.Reprod Domest Anim.2009.44 (1): 100-105) etc.
Application QTL localization method can be located and the closely linked region of proterties, but need to study a lot of individualities of several generations family, and the cycle is long, wastes time and energy, and cannot complete, and some QTL location is very wide in range in general laboratory.Utilize candidate gene method to study proterties, comparatively speaking simple directly, but what utilize that candidate gene method mainly studies is the sudden change of exon region, and the sudden change of much bringing into play critical function is probably at intron or other functional area; Some complex character is subject to polygene regulation and control; Can not detect all QTL, and some does not find the location of QTL with the gene of candidate gene method research.
Along with development and the maturation of technology, genome and gene annotation are more and more perfect.Due to the fast development of sequencing technologies, the reduction of order-checking cost, can utilize genome sequencing or transcribe group order-checking trait related gene or mutational site are carried out to batch screening.The present invention selects Erhualian (one of Taihu Lake pig), hides pig, the fragrant pig of bar horse, and Laiwu Pigs, 5 Chinese native pig breeds such as the southern regions of the Yunnan Province microtia pig and wild boar have carried out the full genome order of resurveying.Wish therefrom to filter out the SNPs(single nucleotide polymorphisms relevant to high litter size, single nucleotide polymorphism) site as genetic marker the genetic breeding for pig, to improve the litter size of sow.
Summary of the invention:
The object of this invention is to provide on No. 10 karyomit(e)s of pig a molecule marker relevant to litter size and primer thereof.
The present invention has selected Erhualian (one of Taihu Lake pig), hides pig, the fragrant pig of bar horse, and Laiwu Pigs, and 5 Chinese native pig breeds of the southern regions of the Yunnan Province microtia pig and wild boar have carried out the full genome order of resurveying.The order number of individuals of resurveying of 6 pig kinds is respectively 11, and 11,9,10,10 and 10.The order sequenced data of resurveying is followed with reference to genome (NCBI Build Sscrofa 10.2) and is compared, and utilizes SAMTools software to carry out the extraction in SNPs site.Contriver has compared Erhualian and other 5 pig kinds, finds that a SNP site (Chr10:16018894 of genome version NCBI Build Sscrofa 10.2) is relevant to the litter size of pig on No. 10 karyomit(e)s.The allelotrope in this site is T and C, has tri-kinds of genotype of C/C, T/C and T/T.This site and flanking sequence thereof (SEQ ID NO:1) are:
Screen behind this site, contriver, take the DNA of pig as template amplification comprises this site at interior nucleotide sequence fragment and checks order, verifies and correlation analysis this site.The individuality that wherein contriver selects is the F of white Du Luoke × Erhualian (one of Taihu Lake pig)
2offspring.Found that this site and litter size of pig have significant correlation.
Beneficial effect of the present invention is: use the heavy sequence measurement of full genome, can screen fast, in bulk the molecule marker relevant to proterties or candidate gene, the litter size of genetic marker for the genetic breeding raising sow of pig can be served as in this SNP site of Chr10:16018894 of genome version NCBI Build Sscrofa 10.2.
Accompanying drawing explanation:
Fig. 1 is white Du Luoke × Erhualian F
2in godmother pig resource population, the genotypic litter size comparison in 3 kinds, Chr10:16018894 site is (in figure
*while representing that two kinds of genotype litter sizes are relatively, 0.01≤P≤0.05;
*represent P < 0.01).
Specific embodiments:
(1) the population distribution feature in SNP site
The present invention has selected Erhualian (one of Taihu Lake pig), hides pig, the fragrant pig of bar horse, and Laiwu Pigs, 5 Chinese native pig breeds such as the southern regions of the Yunnan Province microtia pig and wild boar have carried out the full genome order of resurveying.The order number of individuals of resurveying of 6 pig kinds is respectively 11, and 11,9,10,10 and 10.The order sequenced data of resurveying is followed with reference to genome (NCBI Build Sscrofa 10.2) and is compared, and utilizes SAMTools software to carry out the extraction in SNPs site.Contriver has compared Erhualian and other 5 pig kinds, finds that a SNP site (Chr10:16018894 of genome version NCBI Build Sscrofa 10.2) is relevant to the litter size of pig on No. 10 karyomit(e)s.From the analytical results of the order of resurveying, in other 3 Chinese native pig breeds and wild boar, the frequency averaging of this site allele C is more than 60%, and in the fragrant pig of bar horse, the frequency of this site allele C is 29%, and the frequency of allelotrope T is 100% in Erhualian.
(2) evaluation in SNP site
At design of primers website design SNAPSHOT primer, comprise one couple of PCR amplimer (Chr10-PCRU:5 '-TGC CAA ACC TGT GTC ACC-3 '; Chr10-PCRL:5 '-GGC TGG AGA GGA AAA GGG-3 ') and extension primer (Chr10-SNPU:5 '-TGA CTA GAA GCA GCC ATG GGG CCC TGC AGA-3 ').Totally 24 (12 pairs) primers in this site and other 11 sites are respectively got 1 μ l and are mixed into new multiple PCR primer pond.First carry out 12 heavy PCR reactions, reaction system agents useful for same is: multiple PCR primer pond (6.25 μ M each) 8 μ l, dNTP (10mM each) 8 μ l, 10 × PCR Buffer II, 116 μ l, MgCl
2(5U/ μ is 24 μ l and ddH l) for (25mM Stock) 234 μ l, Fast Start Taq
2o460 μ l, by above reagent mixed liquor 8.5 μ l and 1.5 μ l DNA, (50ng/ μ l) is mixed into 10 μ l systems and carries out 12 heavy pcr amplification reactions.Amplification reaction condition is: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, totally 40 circulations.Next step carries out PCR reaction purification, and required reagent is: Exo I (10U/ μ l) 60 μ l, SAP (1U/ μ is 130 μ l, 10 × SAP Buffer, 35 μ l and ddH l)
2o 125 μ l, react by following program after adding the above-mentioned purifying mixed solution of 3.5 μ l centrifugal: 37 ℃ of 40min, 96 ℃ of 10min in the PCR of each sample product.Then carry out single base extension, required reagent is: SNAPSHOT Multiplex ready reaction Mix 100 μ l, extension primer pond (6.25 μ M each, 12 are extended primers and respectively get 1 μ l mixing) 8 μ l, 5 × Sequencing Buffer, 200 μ l and ddH
2o 300 μ l, are mixed into 10 μ l systems by the purified product of above reagent mixed liquor 6 μ l and 4 μ l and carry out extension.Extension condition is: 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 30s, totally 25 circulations.Then the purifying that carries out extension product, required reagent is: (1U/ μ is 120 μ l, 10 × SAP Buffer, 70 μ l and ddH l) for SAP
2o 140 μ l, by following program react after adding above-mentioned 3.3 μ l purifying mixed solutions centrifugal in the extension product of each sample: 37 ℃ of 40min, 75 ℃ of 20min, 4 ℃ save backup.High-purity methane amide (Hi-Di Formamide) that each sample is got SNAPSHOT reaction product after the above-mentioned purifying of 5-4 μ l and 5-6 μ l is mixed into 10 μ l systems and is splined on ABI 3730XL sequenator after centrifugal and carries out gene type, uses GeneMarker software analysis somatotype result.
(note: the reaction reagent mixed solution consumption of above 4 steps is 100 required amount of reagent of sample, from single base extension, needs lucifuge to carry out.)
(3) white Du Luoke × painted face in Beijing opera reproductive performance hybridization resource population builds
Follow-up confirmatory experiment resource population individual sample used and proterties data thereof are provided by Animal Biotechnology National Key Laboratory of Agricultural University Of Jiangxi cultivation base.
In December, 1998, in Jiang Cong Jiangsu Province, Agricultural University Of Jiangxi's scientific research pig farm, the Erhualian sow of 3 Erhualian conservation field purchases carries out the intersection breeding of blood relationship between field.January calendar year 2001 is according to parent's Farrowing Traits, selected maternal for generations as resource colony of 17 Erhualian sows from the pure breeding offspring of 3 boars and 11 sows.2 high-quality white Duroc boars that Sygen PIC company is so kind as to give are as the male parent for generations of resource family.By these 2 white Du Luoke and 17 F that Erhualian hybridization produces
1for individuality, breed, avoid full, and every boar conventionally with same insemination of sows, to obtain the F of large sample
2family half sibs.In January, 2003, March and June, project team is respectively by the 1st crowd and the 2nd crowd of F
2sow is sent to Jiangxi Province's Yichun City herding seed multiplication farm (59), state-run Red Star kind pig farm, Jiangxi Province (52) and Shangyou County quaternionic breeding pig farm, Jiangxi (118) mensuration for sow reproductive trait.
(4) SNP site is at hybridization resource population F
2the gene type of godmother swinery body
According to method above-mentioned steps (2) Suo Shu, with 192 white Du Luoke × Erhualian hybridization resource population F
2the individual DNA of godmother pig is the genotype in this SNP site of template detection.F
2for there being 186 successful somatotypes of individuality in resource population individuality, 3 kinds of genotype C/C, the number of individuals of T/C and T/T is respectively 56,92 and 38.
(5) regulating effect of SNP site to characters of number born
At F
2in resource population individuality, with the average litter size of C/C genotype individuality, be 10.5431, standard deviation is 0.9222; With the average litter size of T/C genotype individuality, be 10.6266, standard deviation is 0.9031; With the average litter size of T/T genotype individuality, be 11.9091, standard deviation is 0.9554.
The present invention adopts general linear model (GLM) to analyze the impact of SNP site on the total litter size of pig, product young number alive.All statistical study adopts SAS software to carry out.Model is as follows:
Y
ijklm=u+A
i+G
j+B
k+P
l+e
ijklm
Wherein Y
ijklmfor the total litter size of pig or product young number alive, u is colony's average, A
ibe i individual additive effect, G
jbe the fixed effect of j SNP loci gene type, B
kfor a batch effect (k=1,2,3,4), P
lfor stochastic effect (l=1,2,3).
Analytical results finds that there is significant correlation (as shown in Figure 1) in this SNP site of Chr10:16018894 with litter size.With the genotypic F of T/T
2the litter size of godmother pig is than with the average many 1.36(P=0.0204 of the genotypic sow of C/C), with the genotypic F of T/T
2the litter size of godmother pig is than with the average many 1.28(P=0.0149 of the genotypic sow of T/C), as can be seen from Figure 1, minimum with the genotypic number born of sow of C/C.T is the useful allelotrope that improves litter size of pig.
From SNP site and litter size correlation analysis, find out, this SNP site of Chr10:16018894 can be applied to molecular marker assisted selection (MAS) as molecule marker and improve the litter size of sow.
SEQUENCE LISTING
<110> Kunming Institute of Zoology, Chinese Academy of Sciences
Agricultural University Of Jiangxi
Molecule marker relevant to litter size and primer thereof on No. 10 karyomit(e)s of <120> pig
<130> 7
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 601
<212> DNA
<213> Sus scrofa
<400> 1
ttttcttttc agggaggcac ctgtggcatt tggagctaca gctgctggcc acagctgcag 60
ccacggccat ccaggatcct agccacgtct gcgacctaca ccacagctga cggttatgcc 120
agctccttaa tccactgagt gaggccaggg atagaacctg cgtcctcatg gatgctagct 180
agggttctta acccactgag ccacagtggg aactcctggg gcatttttat ttaccagctg 240
gccagcgtgc ctcctgccaa acctgtgtca ccccaagaag cagccatggg gccctgcaga 300
cacccctttc cttcagcaca ggacacccct tttcctctcc agcctgggaa ctgctgaaca 360
ggtgaacccc ctcccctgac tccagactgc ggggcccaca ctccaggagg aaatatgcat 420
ttgggaccta atgctggagc ctgaggcctg tggactccgg aaagccccac atcctcctgg 480
ccctgccagc cccttgggcc cttgtctcct ggcactgggt tccctcggcc tggcctgtgt 540
cccggggccc tccatgtccg cccactgggt gtgcttccca ctgctccccc actcacctgc 600
c 601
SEQUENCE LISTING
<110> Kunming Institute of Zoology, Chinese Academy of Sciences
Agricultural University Of Jiangxi
Molecule marker relevant to litter size and primer thereof on No. 10 karyomit(e)s of <120> pig
<130> 7
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Sus scrofa
<400> 1
tgccaaacct gtgtcacc 18
<210> 2
<211> 18
<212> DNA
<213> Sus scrofa
<400> 2
ggctggagag gaaaaggg 18
<210> 3
<211> 30
<212> DNA
<213> Sus scrofa
<400> 3
tgactagaag cagccatggg gccctgcaga 30
Claims (8)
1. a molecule marker relevant to litter size on No. 10 karyomit(e)s of pig, it is characterized in that: this molecule marker site is the Chr10:16018894 of genome version NCBI Build Sscrofa 10.2, the allelotrope in this site is T and C, has tri-kinds of genotype of C/C, T/C and T/T.
2. the molecule marker of claim 1, wherein said three kinds of yielding characteristicses are: with the genotypic litter size of pig of T/T higher than with T/C and the genotypic pig of C/C.
3. the molecule marker of claim 1, wherein said allelotrope T is the useful allelotrope that improves litter size of pig.
4. the molecule marker of claim 1, is characterized in that: this molecule marker site and flanking sequence thereof are as shown in SEQ ID NO:1.
5. the molecule marker of claim 1-4, wherein said pig is the offspring of Taihu Lake pig.
6. take the DNA of pig as molecule marker described in template amplification comprises claim 1-5 is at interior nucleotide sequence fragment.
7. take the DNA of pig as template, to comprising molecule marker described in claim 1-5, at interior nucleotide sequence fragment, check order.
8. described in amplification claim 1, the primer in molecule marker site and extension primer are:
Chr10-PCRU：5’-TGC CAA ACC TGT GTC ACC-3’；
Chr10-PCRL：5’-GGC TGG AGA GGA AAA GGG-3’；
Chr10-SNPU：5’-TGA CTA GAA GCA GCC ATG GGG CCC TGC AGA-3’。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046442A (en) * | 2019-12-26 | 2021-06-29 | 广西扬翔股份有限公司 | SNP molecular marker related to pig litter size trait and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "CM000821.4", 《GENBANK》, 7 September 2011 (2011-09-07) * |
| K. LI ET AL.: "Quantitative trait loci for litter size and prenatal loss in a White Duroc · Chinese Erhualian resource population", 《ANIMAL GENETICS》, vol. 40, 20 April 2009 (2009-04-20) * |
| NCBI，DBSNP数据: "登录号：rs336014538", 《NCBI，DBSNP数据》, 15 March 2013 (2013-03-15) * |
| PEKKA UIMARI ET AL.: "Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed", 《GENETICS SELECTION EVOLUTION》, vol. 43, 1 December 2011 (2011-12-01) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046442A (en) * | 2019-12-26 | 2021-06-29 | 广西扬翔股份有限公司 | SNP molecular marker related to pig litter size trait and application thereof |
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