CN103757009B - A molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig - Google Patents
A molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig Download PDFInfo
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- CN103757009B CN103757009B CN201310729478.3A CN201310729478A CN103757009B CN 103757009 B CN103757009 B CN 103757009B CN 201310729478 A CN201310729478 A CN 201310729478A CN 103757009 B CN103757009 B CN 103757009B
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Abstract
The present invention relates to the molecular marker relevant to pig number born character, be specifically related to a molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig, belong to biological technical field.This molecular marker site is the Chr13:199018050 of genome version NCBI Build Sscrofa10.2；The allele 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 as shown in SEQ ID NO:1.Expand this molecular marker site primer and extend primer be: Chr13 PCRU:5 ' AAG CTG GTG ATG TTG CTA TCA 3 '；Chr13 PCRL:5 ' TAG TGA TGT GGG AGT GGA ACA 3 '；Chr13 SNPU:5 ' GAC TGA CTG ACT GAC TGA CTG TTA CAA TAG ATG TGA AGA CTG ACA 3 '.Litter size of pig with T/T genotype is higher than T/C and C/C genotype.The beneficial effects of the present invention is: use full-length genome weight sequence measurement, can screen the molecular marker relevant to character or candidate gene quickly, in bulk, this site of Chr13:199018050 can be used for the litter size of the genetic breeding raising sow of pig as genetic marker.
Description
Technical field:
The present invention relates to the molecular marker relevant to pig number born character, be specifically related on No. 13 chromosomes of pig one
The individual molecular marker relevant to litter size and primer thereof, belong to biological technical field.
Background technology:
The characters of number born of pig is the key component of pig reproductive trait, improves litter size of pig to improving pig industry
Overall economic benefit significant.But litter size belongs to the quantitative trait that heritability is the lowest, it is difficult to educate by routine
The technology of kind carries out genetic improvement.The Taihu pigs of China is well-known with high yield, and nest litter size is than other China of great majority
Nest litter size many 4-5 head of local pig breed and external pig kind (" Chinese pig breeds will " Zhang Zhongge edits, on
Sea Science Press, 1986).Erhualian is the one of Taihu pigs, is the pig kind that reproductive capacity is the highest in the world.
Polymorphic site relevant to high litter size in Taihu pigs, it is possible to as genetic marker for improving the farrowing of sow
Number.
Research worker uses the method for genome-wide screening and candidate gene to carry out characters of number born both at home and abroad
QTL location and the research of candidate gene.Li et al. (Li, K., J.Ren, et al.Anim Genet.2009.40 (6):
963-966) utilize and cover total relevant to litter size of pig of 183 microsatellite markers pair of 19 chromosomes of pig
Litter size, number born alive, produces dead young number and carries out QTL(Quantitative Trait Locus, Quantitative Trait Genes
Seat) location.They have studied the F of 299 durocs and China's Erhualian hybridization2In generation, 6,7,8
Located and total yield coefficient with on No. 15 chromosomes, the QTL that number born alive is relevant with producing dead young number.
(Rothischild, M., Jacobson, the D.et al.Proc Natl Acad Sci U S such as Rothschild
A.1996.93 (1): 201-205) by candidate gene approach, utilize two including Prunus mume in China mountain system Taihu pigs
Extreme mixing breed, finds a gene (ESR) at estrogen receptor seat on No. 1 chromosome and high litter size
Major gene resistance is chain.Analyzed by the RFLPs at female hormone receptor gene seat, it is believed that there is 3.7kb band
Homozygous sow (BB type) wants many 2.3 than when having homozygous sow (AA type) primiparity of 4.3kb band
(P < 0.01), every tire averagely 1.5 (P < 0.01) of fecund.Report the gene relevant to litter size also at present
Have 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)
With RBP4 (Spotter, A., S.Muller, et al.Reprod Domest Anim.2009.44 (1): 100-105)
Deng.
Application QTL localization method can position region closely linked with character, however it is necessary that research several generations man
A lot of individualities of system, the cycle is long, wastes time and energy, and cannot complete at general laboratory, and some QTL
Position the most wide in range.Utilize candidate gene approach that character is studied, simple direct comparatively speaking, but utilize and wait
Select that gene approach mainly studies is the sudden change of exon region, and the sudden change much playing critical function is likely to
Intron or other functional area；Some complex character is affected by polygenes regulation and control；Can not detect all of
QTL, and some does not find the location of QTL with the gene of candidate gene approach research.
Along with development and the maturation of technology, genome and gene annotation are more and more perfect.Fast due to sequencing technologies
Speed development, the reduction of order-checking cost, it is possible to utilize genome sequencing or transcript profile to check order to trait related
Cause or mutational site carry out batch and screen.The present invention selects Erhualian (one of Taihu pigs), hides pig, bar
Horse perfume (or spice) pig, Laiwu Pigs, 5 Chinese native pig breeds such as the southern regions of the Yunnan Province microtia pig and wild boar have carried out full-length genome and have resurveyed
Sequence.Wish therefrom to filter out the SNPs(single nucleotide polymorphisms relevant to high litter size, single
Nucleotide polymorphisms) site as genetic marker for the genetic breeding of pig, to improve the litter size of sow.
Summary of the invention:
It is an object of the invention to provide on No. 13 chromosomes of pig a molecular marker relevant to litter size and draw
Thing.
The present invention have selected Erhualian (one of Taihu pigs), hides pig, BaMa miniature pig, Laiwu Pigs, and Yunnan
South 5 Chinese native pig breeds of microtia pig and wild boar have carried out full-length genome and have resurveyed sequence.The sequence of resurveying of 6 pig kinds
Number of individuals is respectively 11,11,9,10,10 and 10.Weight sequencing data is with reference to base
Because group (NCBI Build Sscrofa10.2) is compared, SAMTools software is utilized to carry out SNPs site
Extraction.Inventor compares Erhualian and other 5 pig kinds, finds a SNP on No. 13 chromosomes
Site (Chr13:199018050 of genome version NCBI Build Sscrofa10.2) and the litter size of pig
Relevant.The allele in this site is T and C, has tri-kinds of genotype of C/C, T/C and T/T.This site and
Its flanking sequence (SEQ ID NO:1) is:
After screening this site, inventor comprises the nucleotide in this site with the DNA of pig for template amplification
Sequence fragment also checks order, and verifies this site and correlation analysis.The individuality that wherein inventor selects
It is the F of white Duroc × Erhualian (one of Taihu pigs)2Offspring.Found that farrow with pig in this site
Number has significant correlation.
The beneficial effects of the present invention is: use full-length genome weight sequence measurement, it is possible to quickly, screen in bulk
The molecular marker relevant to character or candidate gene, the Chr13 of genome version NCBI Build Sscrofa10.2:
199018050 these sites can be used for the litter size of the genetic breeding raising sow of pig as genetic marker.
Accompanying drawing illustrates:
Fig. 1 is white Duroc × Erhualian F2Chr13:199018050 site 3 in godmother's pig resource population
The litter size of kind of genotype compare (in figure, * represents when two kinds of genotype litter sizes compare, 0.01≤P≤
0.05；* represents P ＜ 0.01).
Specific embodiments:
(1) the population distribution feature of SNP site
The present invention have selected Erhualian (one of Taihu pigs), hides pig, BaMa miniature pig, Laiwu Pigs, and Yunnan
South 5 Chinese native pig breeds of microtia pig and wild boar have carried out full-length genome and have resurveyed sequence.The sequence of resurveying of 6 pig kinds
Number of individuals is respectively 11,11,9,10,10 and 10.Weight sequencing data is with reference to base
Because group (NCBI Build Sscrofa10.2) is compared, SAMTools software is utilized to carry out SNPs site
Extraction.Inventor compares Erhualian and other 5 pig kinds, finds a SNP on No. 13 chromosomes
Site (Chr13:199018050 of genome version NCBI Build Sscrofa10.2) and the litter size of pig
Relevant.From the point of view of the analysis result of sequence of resurveying, this site equipotential base in other 4 Chinese native pig breeds and wild boar
Because the frequency averaging of C is more than 60%, and it is more than 80% in the frequency of Erhualian allelic T.
(2) qualification of SNP site
At design of primers website design SNAPSHOT primer, including a pair pcr amplification primer thing
(Chr13-PCRU:5 '-AAG CTG GTG ATG TTG CTA TCA-3 '；Chr13-PCRL:5 '-TAG
TGA TGT GGG AGT GGA ACA-3 ') and extension primer (Chr13-SNPU:5 '-GAC TGA CTG
ACT GAC TGA CTG TTA CAA TAG ATG TGA AGA CTG ACA-3 ').This site and its
Totally 24, its 11 site (12 to) primer respectively takes 1 μ l and is mixed into new multiple PCR primer pond.First enter
Row 12 weight PCR reaction, reaction system agents useful for same is: multiple PCR primer pond (6.25 μMs of each) 8 μ l,
dNTP(10mM each)8μl、10×PCR Buffer II116μl、MgCl2(25mM Stock)234μl、
Fast Start Taq (5U/ μ l) 24 μ l and ddH2O460 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 weight pcr amplification reactions.Amplification reaction condition is: 94 DEG C
4min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, totally 40 circulations.It is pure that next step carries out PCR reaction
Change, required reagent is: Exo I (10U/ μ l) 60 μ l, SAP (1U/ μ l) 130 μ l, 10 × SAP Buffer35 μ l and
ddH2O125 μ l, adds after 3.5 μ l above-mentioned purification mixed liquor is centrifuged by following in the PCR primer of each sample
Program is reacted: 37 DEG C of 40min, 96 DEG C of 10min.Then single base extension, required reagent are carried out
For: SNAPSHOT Multiplex ready reaction Mix100 μ l, extend primer pond (6.25 μMs of each,
Article 12, extend primer and respectively take 1 μ l mixing) 8 μ l, 5 × Sequencing Buffer200 μ l and ddH2O300 μ l, will
The purified product of above reagent mixed liquor 6 μ l and 4 μ l is mixed into 10 μ l systems and carries out extension.Extension
Condition is: 96 DEG C of 10s, 50 DEG C of 5s, 60 DEG C of 30s, totally 25 circulations.Then extension product is carried out
Purification, required reagent is: SAP (1U/ μ l) 120 μ l, 10 × SAP Buffer70 μ l and ddH2O140 μ l,
The extension product of each sample adds after above-mentioned 3.3 μ l purification mixed liquors are centrifuged and carry out by following procedure
Reaction: 37 DEG C of 40min, 75 DEG C of 20min, 4 DEG C save backup.It is above-mentioned after purification that each sample takes 5-4 μ l
SNAPSHOT product and high-purity Methanamide (Hi-Di Formamide) of 5-6 μ l be mixed into 10 μ l systems
It is splined on ABI3730XL sequenator after Li Xin and carries out gene type, use GeneMarker software analysis typing
Result.
(note: the reaction reagent mixed liquor consumption of above 4 steps is amount of reagent needed for 100 samples, from single alkali
Base extension starts, and needs lucifuge to carry out.)
(3) white Duroc × painted face in Beijing opera reproductive performance hybridization resource population builds
Follow-up confirmatory experiment used resource group's individual sample and trait data thereof are given birth to by Agricultural University Of Jiangxi animal
Thing technology National Key Laboratory cultivation base provides.
In December, 1998, will purchase from Erhualian conservation field, 3, Jiangsu Province on Agricultural University Of Jiangxi's scientific research pig farm
The Erhualian sow bought carries out the intersection breeding of blood relationship between field.January calendar year 2001 according to parent's Farrowing Traits, from 3
The pure breeding offspring of head boar and 11 sows have selected 17 Erhualian sows mother for generations as sources group
This.2 high-quality white Duroc boars that Sygen PIC company is given are as the male parent for generations of Resource family.
The F that these 2 white Durocs and 17 Erhualian hybridization are produced1Breed for individuality, it is to avoid Quan Tong
Born of the same parents' copulation, and every boar generally with same head insemination of sows, to obtain the F of large sample2Family half sibs.2003
January in year, March and June, project team is respectively by the 1st batch and the 2nd crowd of F2Sow is sent to Jiangxi Province Yichun City poultry
Herd seed multiplication farm (59), state-run Red Star kind pig farm, Jiangxi Province (52) and quaternionic breeding pig farm, Shangyou County, Jiangxi
(118) are for the mensuration of sow reproductive trait.
(4) SNP site is at hybridization resource population F2The gene type of godmother's swinery body
According to the method described in above-mentioned steps (2), with 192 white Durocs × Erhualian hybridization resource population F2
Godmother pig individuality DNA is the genotype of this SNP site of template detection.192 F2Have in resource population individuality
181 individual success typings, 3 kinds of genotype C/C, the number of individuals of T/C and T/T is 35,88 and 58 respectively.
(5) SNP site regulating effect to characters of number born
At F2For in resource population individuality, the average litter size with C/C genotype individuals is 10.5355, standard deviation
It is 0.9556；Average litter size with T/C genotype individuals is 10.5797, and standard deviation is 0.8858；With
The average litter size of T/T genotype individuals is 11.5227, and standard deviation is 0.9228.
The present invention uses general linear model (GLM) to analyze SNP site to pig total yield coefficient, product son alive
The impact of number.All statistical analysis uses SAS software to carry out.Model is as follows:
Yijklm=u+Ai+Gj+Bk+Pl+eijklm
Wherein YijklmFor pig total yield coefficient or number born alive, u is colony's average, AiFor the additivity effect that i-th is individual
Should, GjFor the fixed effect of jth SNP site genotype, BkFor batch effect (k=1,2,3,4), PlFor with
Machine effect (l=1,2,3).
Analysis result finds that this SNP site of Chr13:199018050 has significant correlation (such as figure with litter size
Shown in 1).F although with T/T genotype2Between litter size and the sow with C/C genotype of godmother pig
There is no significant difference (P=0.0835), but with the F of T/T genotype2The litter size of godmother pig than with
The sow of C/C genotype averagely many 0.98, with the F of T/T genotype2The litter size ratio of godmother pig is with T/C
The average many 0.94(P=0.0393 of sow of genotype), as can be seen from Figure 1, the sow with C/C genotype produces
Young number is minimum.T is the useful allele improving litter size of pig.
Find out with litter size correlation analysis from SNP site, this SNP site energy of Chr13:199018050
Enough being applied to molecular marker assisted selection (MAS) as molecular marker improves the litter size of sow.
SEQUENCE LISTING
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
Agricultural University Of Jiangxi
<120>molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig
<130> 7
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 601
<212> DNA
<213> Sus scrofa
<400> 1
tagttcttcc tctgatttat gtaaaatcat caaaacggta tcctgattca ttacttacgc 60
atggcacaac tataaggagt tgaaatgttt ttattcatca cgaagacagt gccagggtca 120
gttttcccaa cgtgtttaac ttcaatcttc tcagtccggg gagttaagga tagctgcctg 180
tttttctctt tgttaaagag atcattctgc atctctatca gttcggtcgg tgacagctga 240
gaagctggtg atgttgctat caatcctggg cagaagttac aatagatgtg aagactgaca 300
taacagtaac ttaaaaatga atatgacaaa ataaataacc tactaaccta ccctgttcca 360
ctcccacatc actaccctcc tacaacaaga gcaagaggca agggaatttt aaacgccctt 420
tatcaaaatg agctcatcgg taaacttaca ccctttccaa tactaatcat taaaacgccg 480
acagaagaaa actgcagcct gcccagatcc gcaccttcat ttcaactgtg ctctctcacc 540
atcgagctcc acttcccagt ttcctcggcc gtatcccgat ctgtgcttta cagtaagggc 600
a
601
SEQUENCE LISTING
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
Agricultural University Of Jiangxi
<120>molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig
<130> 7
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Sus scrofa
<400> 1
aagctggtga tgttgctatc
a
21
<210> 2
<211> 21
<212> DNA
<213> Sus scrofa
<400> 2
tagtgatgtg ggagtggaac
a
21
<210> 3
<211> 45
<212> DNA
<213> Sus scrofa
<400> 3
gactgactga ctgactgact gttacaatag atgtgaagac tgaca
45
Claims (1)
1. one kind is detected on No. 13 chromosomes of pig SNP site in a molecular marker relevant to litter size
Primer detection litter size of pig in application, it is characterised in that: described primer include pcr amplification primer thing and
Extending primer, wherein the nucleotide sequence of pcr amplification primer thing is as follows: Chr13-PCRU:5 '-AAG
CTG GTG ATG TTG CTA TCA-3’；Chr13-PCRL:5 '-TAG TGA TGT GGG AGT
GGA ACA-3’；The nucleotide sequence extending primer is as follows: Chr13-SNPU:5 '-GAC TGA CTG
ACT GAC TGA CTG TTA CAA TAG ATG TGA AGA CTG ACA-3 ', described pig is white
Duroc × Erhualian hybridization resource population F2 godmother pig, described SNP site is genome version NCBI
The Chr13:199018050 of Sscrofal10.2, the allele in this site is T and C, have C/C, T/C and
Tri-kinds of genotype of T/T, the litter size of pig with T/T genotype is higher than the pig with T/C and C/C genotype,
Its allelic T is the useful allele improving litter size of pig.
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| CN201310729478.3A CN103757009B (en) | 2013-12-26 | 2013-12-26 | A molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig |
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| CN201310729478.3A CN103757009B (en) | 2013-12-26 | 2013-12-26 | A molecular marker relevant to litter size and primer thereof on No. 13 chromosomes of pig |
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| CN103757009B true CN103757009B (en) | 2016-08-17 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109554487B (en) * | 2019-01-19 | 2021-04-16 | 华中农业大学 | Molecular marker associated with boar semen volume character and application |
| CN111518916B (en) * | 2020-02-28 | 2022-07-15 | 南京农业大学 | SNP marker significantly related to pig chromosome 13 and number of live piglets of Erhualian pigs as well as detection method and application of SNP marker |
Citations (1)
| 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 |
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Patent Citations (1)
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| Quantitative trait loci for litter size and prenatal loss in a White Duroc Chinese Erhualian resource population;K. Li et al.;《Animal Genetics》;20090420;第40卷;全文 * |
| ss726817199;NCBI;《NCBI dbSNP数据库》;20130315;第1页 * |
| 猪产仔性状的分子标记研究与QTLs定位;雷雪芹等;《洛阳农业高等专科学校学报》;20011227;263-266 * |
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