CN105543356B - SNP site relevant to nest litter size on No. 9 chromosomes of pig - Google Patents
SNP site relevant to nest litter size on No. 9 chromosomes of pig Download PDFInfo
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 8
- 108700028369 Alleles Proteins 0.000 claims abstract description 5
- 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
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 238000012163 sequencing technique Methods 0.000 abstract description 6
- 238000013459 approach Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract description 3
- 102100021391 Cationic amino acid transporter 3 Human genes 0.000 abstract description 2
- 108091006230 SLC7A3 Proteins 0.000 abstract description 2
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 241000282898 Sus scrofa Species 0.000 description 39
- 241000282887 Suidae Species 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
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- 238000005119 centrifugation Methods 0.000 description 3
- 238000003205 genotyping method Methods 0.000 description 3
- PMHURSZHKKJGBM-UHFFFAOYSA-N isoxaben Chemical compound O1N=C(C(C)(CC)CC)C=C1NC(=O)C1=C(OC)C=CC=C1OC PMHURSZHKKJGBM-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 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
- 238000010219 correlation analysis Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 244000309715 mini pig Species 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
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- 230000009027 insemination Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000008080 stochastic effect Effects 0.000 description 1
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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. 9 chromosomes of pig belong to field of biotechnology.The SNP site is the Chr9:8179045 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: Chr9-PCRU:5 '-TAT AAA AGA CTG TGT TGC TGC TAG A-3 ';Chr9-PCRL:5 '-AAT GTC AAA CTA GTT TAA GGA GTT GAA-3 ';Chr9-SNPU:5 '-TGA CTT TAG TGG GTG GCT GAG AAC TTA CAT-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 Chr9:8179045 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. 9 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. 9 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. 9
A SNP site (Chr9:8179045 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 Chr9:8179045 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 Chr9:8179045 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. 9
A SNP site (Chr9:8179045 of genome version NCBI Build Sscrofa 10.2) and pig are found on chromosome
Litter size it is related.
(2) identification of SNP site
In design of primers website design SNAPSHOT primer, including a pair of of PCR amplification primer (Chr9-PCRU:5 '-TAT
AAA AGA CTG TGT TGC TGC TAG A-3';Chr9-PCRL:5 '-AAT GTC AAA CTA GTT TAA GGA GTT
GAA-3 ') and extension primer (Chr9-SNPU:5 '-TGA CTT TAG TGG GTG GCT GAG AAC TTA CAT-3 ').It should
Site and totally 24, other 11 sites (12 pairs) primer respectively take 1 μ l to be mixed into new multiple PCR primer pond.12 weights are carried out first
PCR reaction, reaction 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, 24 μ l of Fast Start Taq (5U/ μ l) and
ddH2The 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 weights by 460 μ l of O
Pcr amplification reaction.Amplification 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.Under
One step carries out PCR reaction purification, 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 ddH2O125 μ l is pressed after the above-mentioned purifying mixed liquor centrifugation of 3.5 μ l is added in the PCR product of each sample
Following procedure is reacted: 37 DEG C of 40min, 96 DEG C of 10min.Then single base extension, required reagent are carried out are as follows:
(6.25 μM of each, 12 extensions are drawn for 100 μ l of SNAPSHOT Multiplex ready reaction Mix, extension primer pond
Object respectively takes 1 μ l to mix) 8 μ l, 5 × Sequencing Buffer 200 μ l and ddH2300 μ l of O, by the above 6 μ l of reagent mixed liquor
10 μ l systems, which are mixed into, with the purified product of 4 μ l carries out extension.Extension condition are as follows: 96 DEG C of 10s, 50 DEG C of 5s, 60
DEG C 30s, totally 25 circulations.Then the purifying of extension product, required reagent are carried out are as follows: SAP (1U/ μ l) 120 μ l, 10 ×
SAP Buffer 70 μ l and ddH2Above-mentioned 3.3 μ l purifying mixing is added in 140 μ l of O in the extension product of each sample
Reacted after liquid centrifugation by following procedure: 37 DEG C of 40min, 75 DEG C of 20min, 4 DEG C save backup.Each sample takes 5-4 μ l
High-purity formamide (Hi-Di Formamide) of above-mentioned SNAPSHOT reaction product and 5-6 μ l after purification is mixed into 10 μ l bodies
ABI 3730XL sequenator is splined on after system's centrifugation and carries out Genotyping, 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 179 individuals successfully to divide
The number of individuals of type, 3 kinds of genotype C/C, T/C and T/T is 5,72 and 102 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 10.4509, and standard deviation is
1.489;Average litter size with T/C genotype individuals is 11.6084, standard deviation 0.9154;With T/T genotype individuals
Average litter size be 10.7238, standard deviation 0.887.
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 Chr9:8179045 has significant correlation with litter size.With T/C genotype
F2The litter size of godmother pig be averaged more 1.1575 (P=0.04) than the sow with C/C genotype, as can be seen from Figure 1, with C/
The number born of sow of C genotype is minimum.T is the beneficial allele and T/C heterozygosis litter size of pig highest for improving litter size of pig.
Find out from SNP site and litter size correlation analysis, this SNP site of Chr9:8179045 can be used as molecule mark
Note is applied 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. 9 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: Chr9-PCRU:5 '-TATAAAAGACTGTGTTGCTGCTAGA-3 ';Chr9-PCRL:5 '-
AATGTCAAACTAGTTTAAGGAGTTGAA-3;The nucleotide sequence of extension primer is as follows: Chr9-SNPU5-
TGACTTTAGTGGGTGGCTGAGAACTTACAT-3 ', the pig are that white Duroc × Erhualian hybridizes resource population F2 generation
Sow, the SNP site are the Chr9:8179045 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 C/T be improve litter size of pig desirable genes type.
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CN105969890A (en) * | 2016-07-07 | 2016-09-28 | 贵州大学 | SNP (Single Nucleotide Polymorphism) molecular marker related to litter size of fragrant pig and application of SNP molecular marker |
CN113736890B (en) * | 2021-07-30 | 2023-07-14 | 华南农业大学 | SNP molecular marker related to healthy number and living number rate and application thereof |
CN114085914B (en) * | 2021-10-27 | 2023-07-14 | 华南农业大学 | SNP molecular marker located on chromosome 9 of pig and related to litter size and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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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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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 |
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《中国优秀硕士学位论文全文数据库(电子期刊)》.二花脸猪特异选择位点与猪产仔数的关联性分析.《中国优秀硕士学位论文全文数据库(电子期刊)》.2014, * |
Pekka Uimari et al..Whole-genome SNP association analysis of reproduction traits in the Finnish Landrace pig breed.《Genetics Selection Evolution》.2011,第43卷(第42期), * |
杨怀谷等.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.《广东农业科学》.2014,(第15期), * |
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