CN105603065B - SNP site relevant to nest litter size and its primer on No. 3 chromosomes of pig - Google Patents
SNP site relevant to nest litter size and its primer on No. 3 chromosomes of pig Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
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- 238000012214 genetic breeding Methods 0.000 abstract description 2
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001237732 Microtia Species 0.000 description 2
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Abstract
The present invention relates to SNP sites relevant to pig number born character, and in particular to a SNP site relevant to nest litter size and its primer on No. 3 chromosomes of pig belong to field of biotechnology.The SNP site is the Chr3:59537592 of genome version NCBI Build Sscrofa 10.2；The allele in the site is T and C, has tri- kinds of genotype of C/C, T/C and T/T, expands the primer and extension primer in the site are as follows: Chr3-1-PCRU:5 '-TTG GTC CTC AGT CAT GTG G-3 '；Chr3-1-PCRL:5 '-AAC CTC TTC CCT TGA CCT GA-3 '；Chr3-1-SNPU:5 '-TGA CTA GTA GGC AGG CAA GAA CAT TCA GAG-3 '.Litter size of pig with T/C genotype is higher than C/C genotype, and T is the beneficial allele and T/C heterozygosis litter size of pig highest for improving litter size of pig.The beneficial effects of the present invention are: full-length genome weight sequencing approach can screen SNP site relevant to character or gene quickly, in bulk, this SNP site of the Chr3:59537592 of genome version NCBI Build Sscrofa 10.2 can improve the litter size of sow as genetic marker for the genetic breeding of pig.
Description
Technical field:
The present invention relates to SNP sites relevant to pig number born character, and in particular to one produces with nest on No. 3 chromosomes of pig
The relevant SNP site of young number and its primer, belong to field of biotechnology.
Background technique:
The characters of number born of pig is the chief component of pig reproductive trait, improves litter size of pig to the total of raising pig breeding industry
Body economic benefit is significant.The Taihu pigs in China are well-known with high yield, and nest litter size is than most of other Chinese native pig breeds
And the nest litter size of external pig kind is 4-5 more (" Chinese pig breeds will " Zhang Zhongge chief editor, Shanghai Scientific & Technical Publishers, 1986).
Polymorphic site relevant to high litter size in Taihu pigs can be used to improve the litter size of sow as genetic marker.
Full-length genome weight sequencing approach is widely used to the correlative study of each species, it quickly can comprehensively be screened
Gene relevant to character, function or mutational site out, compensate for QTL positioning and candidate gene approach is time-consuming and laborious, single
The deficiencies of.
At home and abroad there is not yet SNP site open source literature relevant to Litter Size in Pigs is reported in the present invention.
Summary of the invention:
The object of the present invention is to provide a SNP site relevant to nest litter size and its primers on No. 3 chromosomes of pig.
The present invention has selected Erhualian (one kind of Taihu pigs), hides pig, BaMa miniature pig, Laiwu Pigs and the southern regions of the Yunnan Province microtia pig 5
A Chinese native pig breed and wild boar have carried out full-length genome and have resurveyed sequence.The sequence number of individuals of resurveying of 6 pig kinds is respectively 11, and 11
It is a, 9,10,10 and 10.Weight sequencing data is compared with reference genome (NCBI Build Sscrofa 10.2)
It is right, the extraction in the site SNPs is carried out using SAMTools software.Inventor compares Erhualian and other 5 pig kinds, at No. 3
A SNP site (Chr3:59537592 of genome version NCBI Build Sscrofa 10.2) and pig are found on chromosome
Litter size it is related.The allele in the site is T and C, there is tri- kinds of genotype of C/C, T/C and T/T.
After screening the site, inventor hybridizes resource population F in white Duroc × Erhualian2It is right in godmother's pig individual
It carries out verifying and correlation analysis, as a result, it has been found that there is significant correlation in the site with litter size of pig.
The beneficial effects of the present invention are: full-length genome weight sequencing approach can screen related to character quickly, in bulk
SNP site or gene, this SNP site energy of the Chr3:59537592 of genome version NCBI Build Sscrofa 10.2
The litter size of sow is enough improved for the genetic breeding of pig as genetic marker.
Detailed description of the invention:
Fig. 1 is white Duroc × Erhualian F23 kinds of the site Chr3:59537592 genotype in godmother's pig resource population
Litter size compare (when two kinds of genotype litter sizes compare in figure, * indicate 0.01≤P≤0.05；* indicates P < 0.01).
Specific embodiment:
(1) the population distribution feature of SNP site
The present invention selects Erhualian (one kind of Taihu pigs), hides pig, BaMa miniature pig, Laiwu Pigs and the southern regions of the Yunnan Province microtia pig 5
Chinese native pig breed and wild boar have carried out full-length genome and have resurveyed sequence.The sequence number of individuals of resurveying of 6 pig kinds is respectively 11, and 11
It is a, 9,10,10 and 10.Weight sequencing data is compared with reference genome (NCBI Build Sscrofa 10.2)
It is right, the extraction in the site SNPs is carried out using SAMTools software.Inventor compares Erhualian and other 5 pig kinds, at No. 3
A SNP site (Chr3:59537592 of genome version NCBI Build Sscrofa 10.2) and pig are found on chromosome
Litter size it is related.From the point of view of resurveying the analysis result of sequence, the site allele in other 4 Chinese native pig breeds and wild boar
The frequency averaging of C is 84.2%, and is 100% in the frequency of Erhualian allelic T.
(2) identification of SNP site
In design of primers website design SNAPSHOT primer, including a pair of of PCR amplification primer (Chr3-1-PCRU:5 '-TTG
GTC CTC AGT CAT GTG G-3'；Chr3-1-PCRL:5 '-AAC CTC TTC CCT TGA CCT GA-3 ') and extend
Primer (Chr3-1-SNPU:5 '-TGA CTA GTA GGC AGG CAA GAA CAT TCA GAG-3 ').The site with it is other
Totally 24,11 sites (12 pairs) primer respectively takes 1 μ l to be mixed into new multiple PCR primer pond.12 weight PCR reactions are carried out first, instead
Answer system agents useful for same are as follows: multiple PCR primer pond (6.25 μM of each) 8 μ l, dNTP (10mM each) 8 μ l, 10 × PCR
Buffer II 116μl、MgCl2(25mM Stock) 234 μ l, Fast Start Taq (5U/ μ l) 24 μ l and ddH2460 μ l of O,
The above 8.5 μ l of reagent mixed liquor and 1.5 μ l DNA (50ng/ μ l) are mixed into 10 μ l systems and carry out 12 heavy pcr amplification reactions.Expand
Increase reaction condition are as follows: 94 DEG C of 4min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, totally 40 recycle.It is anti-that PCR is carried out in next step
Should purify, required reagent are as follows: Exo I (10U/ μ l) 60 μ l, SAP (1U/ μ l) 130 μ l, 10 × SAP Buffer, 35 μ l and
ddH2125 μ l of O is carried out after the above-mentioned purifying mixed liquor centrifugation of 3.5 μ l is added in the PCR product of each sample by following procedure
Reaction: 37 DEG C of 40min, 96 DEG C of 10min.Then single base extension, required reagent are as follows: SNAPSHOT are carried out
(6.25 μM of each, 12 extension primers respectively take 1 μ l for 100 μ l of Multiplex ready reaction Mix, extension primer pond
Mixing) 8 μ l, 5 × Sequencing Buffer 200 μ l and ddH2300 μ l of O, by the pure of the above 6 μ l of reagent mixed liquor and 4 μ l
Change product and is mixed into 10 μ l systems progress extension.Extension condition are as follows: 96 DEG C of 10s, 50 DEG C of 5s, 60 DEG C of 30s, altogether
25 circulations.Then the purifying of extension product, required reagent are as follows: SAP (1U/ μ l) 120 μ l, 10 × SAP Buffer are carried out
70 μ l and ddH2140 μ l of O, be added in the extension product of each sample after above-mentioned 3.3 μ l purifying mixed liquor centrifugation by with
Lower program is reacted: 37 DEG C of 40min, 75 DEG C of 20min, 4 DEG C save backup.Each sample takes 5-4 μ l above-mentioned after purification
High-purity formamide (Hi-Di Formamide) of SNAPSHOT reaction product and 5-6 μ l are mixed into loading after the centrifugation of 10 μ l systems
Genotyping is carried out in ABI3730XL sequenator, analyzes genotyping result using GeneMarker software.
(note: the reaction reagent mixed liquor dosage of above 4 steps is amount of reagent needed for 100 samples, is prolonged from single base
It stretches reaction to start, needs to be carried out in the dark.)
(3) white Duroc × Erhualian reproductive performance hybridization resource population building
Subsequent confirmatory experiment used resource group individual sample and its trait data are by Agricultural University Of Jiangxi's animal organism skill
Art National Key Laboratory cultivation base provides.
In December, 1998, two will bought from the 3 Erhualian conservation fields in Jiangsu Province on Agricultural University Of Jiangxi's scientific research pig farm
Paint face sow carries out the intersection breeding of blood relationship between field.In January, 2001 according to parent's Farrowing Traits, from 3 boars and 11 sows
Pure breeding offspring in selected 17 Erhualian sows as the maternal for generations of sources group.2 that Sygen PIC company is given
For generations male parent of the high-quality white Duroc boars as Resource family.This 2 white Durocs and 17 Erhualians are miscellaneous
Hand over the F generated1Breed for individual, avoid full, and every boar usually with same head insemination of sows, to obtain
The F of large sample2Family half sibs.In January, 2003, March and June, project team is respectively by the 1st batch and the 2nd crowd of F2Sow is sent to Jiangxi
Save Yichun City herding seed multiplication farm (59), Jiangxi Province state-run Red Star kind pig farm (52) and Jiangxi Shangyou County quaternionic breeding pig farm
(118) are used for the measurement of sow reproductive trait.
(4) SNP site is in hybridization resource population F2The Genotyping of godmother's pig groups
According to method described in above-mentioned steps (2), hybridize resource population F with 192 white Duroc × Erhualians2Godmother
Pig individual DNA is the genotype of the template detection SNP site.192 F2For resource population individual in there are 176 individuals successfully to divide
The number of individuals of type, 3 kinds of genotype C/C, T/C and T/T is 60,84 and 32 respectively.
(5) regulating effect of the SNP site to characters of number born
In F2For in resource population individual, the average litter size with C/C genotype individuals is 10.5507, and standard deviation is
0.7672；Average litter size with T/C genotype individuals is 11.595, standard deviation 0.7568；With T/T genotype individuals
Average litter size be 10.9155, standard deviation 0.8394.
The present invention analyzes influence of the SNP site to pig total yield coefficient, number born alive using general linear model (GLM).
All statistical analysis is carried out using SAS software.Model is as follows:
Yijklm=u+Ai+Gj+Bk+Pl+eijklm
Wherein YijklmFor pig total yield coefficient or number born alive, u is group's mean value, AiFor the additive effect of i-th of individual, Gj
For the fixed effect of j-th of SNP site genotype, BkFor batch effect (k=1,2,3,4), PlFor stochastic effects (l=1,2,
3)。
Analysis is as a result, it has been found that this SNP site of Chr3:59537592 has significant correlation with litter size.With T/C gene
The F of type2The litter size of godmother pig as can be seen from Figure 1 has than sow more 1.04 (P=0.0181) that are averaged with C/C genotype
The number born of sow of C/C genotype is minimum.T be improve litter size of pig beneficial allele and T/C heterozygosis litter size of pig most
It is high.
Find out from SNP site and litter size correlation analysis, this SNP site of Chr3:59537592 can be used as molecule
Apply the tag to the litter size of molecular marker assisted selection (MAS) Lai Tigao sow.
Claims (1)
1. primer the answering in detection litter size of pig of SNP site relevant to nest litter size on a kind of detection No. 3 chromosomes of pig
It include PCR amplification primer and extension primer with, it is characterised in that: the primer, the wherein nucleotide sequence of PCR amplification primer
It is as follows: hr3-1-PCRU:5 '-TTGGTCCTCAGTCATGTGG-3 '；Chr3-1-PCRL:5 '-
AACCTCTTCCCTTGACCTGA-3'；The nucleotide sequence of extension primer is as follows: Chr3-1-SNPU:5 '-
TGACTAGTAGGCAGGCAAGAACATTCAGAG-3 ', the pig are that white Duroc × Erhualian hybridizes resource population F2 generation
Sow, the SNP site are the Chr3:59537592 of genome version NCBI Sscrofal10.2, the allele in the site
For T and C, there are tri- kinds of genotype of C/C, T/C and T/T, the litter size of pig with T/C genotype, which is higher than, has C/C and T/T genotype
Pig, wherein genotype T/C be improve litter size of pig desirable genes type.
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| CN201610003243.XA CN105603065B (en) | 2016-01-04 | 2016-01-04 | SNP site relevant to nest litter size and its primer on No. 3 chromosomes of pig |
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| CN106399524B (en) * | 2016-10-11 | 2019-11-19 | 贵州大学 | One kind PAV3 molecular labeling relevant to fragrant litter size of pig and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007133075A2 (en) * | 2006-05-12 | 2007-11-22 | Institute For Pig Genetics B.V. | Igf2 marker assisted selection for porcine reproductive traits |
| CN103757003A (en) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | Litter size related SNP (Single Nucleotide Polymorphism) locus of swine 13# chromosome and primers thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007133075A2 (en) * | 2006-05-12 | 2007-11-22 | Institute For Pig Genetics B.V. | Igf2 marker assisted selection for porcine reproductive traits |
| CN103757003A (en) * | 2013-12-26 | 2014-04-30 | 中国科学院昆明动物研究所 | Litter size related SNP (Single Nucleotide Polymorphism) locus of swine 13# chromosome and primers thereof |
Non-Patent Citations (3)
| Title |
|---|
| Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed;Pekka Uimari et al.;《Genetics Selection Evolution》;20111201;第43卷(第42期);1-8 * |
| 二花脸猪特异选择位点与猪产仔数的关联性分析;林国珊;《中国优秀硕士学位论文全文数据库（电子期刊）》;20140131;D050-189 * |
| 利用选择性清除方法鉴别影响太湖猪（二花脸、梅山猪）及野猪产仔数差异的SNP位点;杨怀谷等;《广东农业科学》;20141231(第15期);100-106 * |
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