CN1749416A - Utilize the method for single nucleotide polymorphism prediction mean lambing number of sheep brood - Google Patents
Utilize the method for single nucleotide polymorphism prediction mean lambing number of sheep brood Download PDFInfo
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Abstract
The invention discloses a kind of method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood.The method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood provided by the present invention, be to use by having SEQ ID № in the sequence table: 1 and SEQ ID №: a pair of primer that 2 nucleotide sequence is formed carries out pcr amplification to the DNA of sheep to be measured, then pcr amplification product being carried out single nucleotide polymorphism detects, determine from SEQ ID №: 5 ' end the 84th bit base of 3 is T or C, and the 174th bit base is T or G.Result of study of the present invention shows that the PRLR gene may be a major gene of control sheep fecundity trait or have genetic linkage closely with it, for the marker assisted selection and the breeding of sheep prolificacy provides the theory and practice basis, can shorten breeding cycle greatly, improve breeding efficiency, and will bring considerable economic to the sheep producer.
Description
Technical field
The present invention relates to a kind of method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood in the bioengineering field.
Background technology
(prolactin PRL) claims prolactin antagonist or prolacrin again to prolactin, is the necessary prepituitary gland peptide hormone of reproductive success.Hprl receptor (prolactin receptor, PRLR) with growth hormone receptor (growth hormonereceptor, GHR), galactagogin acceptor (placental lactogen receptor, PLR) primordial is because of family, this gene family may be by a certain common ancestor's gene evolution (Boutin J M, JolicoeurC, Okamura H, et al.Cloning and expression of the rat prolactin receptor, amember of the growth hormone/prolactin receptor gene family.Cell, 1988,53:69-77).Gene mapping by deer, ox, people compares, and finds that prolactin receptor gene is a member of cytokine receptor superfamily (cytokine receptor superfamily).The hprl receptor family member closely links to each other with the Jak family member.This receptoroid albumen has a kind of common function, promptly by causing various intramicellar reaction with combining of part.
Studies show that the PRL receptor structure has diversity.The diversity of receptor structure is mainly caused by the variation of born of the same parents' internal area.PRL has at least the acceptor of 2 kinds of forms to exist in the known Mammals, i.e. elongated acceptor (long form) and short receptor (short form).The PRL acceptor is made up of film foreign lands, membrane-spanning domain and born of the same parents' internal area 3 parts for only to contain 1 transmembrane protein of striding diaphragm area, has the constitutional features of cytokine receptor superfamily.The difference of Mammals and bird PRL receptor structure is that Mammals has only the outer ligand binding domain of 1 born of the same parents, and bird then has the ligand binding domain of 2 repeating units.
Boutin etc. have cloned short type PRLR cDNA at first from rat, this cDNA is 1635bp, has only an open reading frame, 310 amino acid of codified (Boutin JM, Jolicoeur C, Okamura H, et al.Cloningand expression of the rat prolactin receptor, a member of the growthhormone/prolactin receptor gene family.Cell, 1988,53:69-77).Shirota etc. has cloned elongated PRLR cDNA (the Shirota M that contains 2132bp from the ovary of rat subsequently, Banville D, Ali S.Expression of two forms of PRL receptor in rat ovary and liver.MolEndocrinol, 1990,4:1136-1143).Moore etc. have separated the cDNA of coding elongated PRLR again from the mammary gland of mouse, its length is 1992bp, are respectively 91% and 72% with the homology of rat, rabbit elongated PRLR; Be that 384-524 amino acid is the most conservative by analyzing the middle region of finding mouse elongated PRLR born of the same parents internal area in addition; PRLRcDNA 3 ' non-translational region does not have polyadenylation consensus sequence (AATAAA), this and the rat ovary and consistent (the Moore R C of ox PRLRcDNA sequence that report, Oka T.Cloning and sequencing of the cDNA encodingthe murine mammary gland long-form prolact in receptor.Gene, 1993,134:263-265).
Tortonese etc. are reported in and detect PRLR mRNA in the sheep pituitary gland, the PRLR cDNA sequence of cDNA nucleotide sequence that obtains by RT-PCR amplification and ox has homology (the Tortonese D J up to 95.6% simultaneously, BrooksJ, Ingleton P M, et al.Detection of prolactin receptor gene expression inthe sheep pituitary gland and visualization of the specific translation ofthe signal in gonadotrophs.Endocrinology, 1998,139 (12): 5215-5223).In addition, the elongated PRLR separation of also using RT-PCR amplification particular form has obtained 2 cDNA, wherein has born of the same parents' internal area that the insertion fragment of 39bp is arranged.This fragment is identical with the insertion fragment that Anthony etc. finds in sheep fetus liver and adult sheep luteal tissue; The 263rd amino acid that inserts site and ox PRLR is corresponding (Anthony R V, Smith G W, Duong A, et al.Two forms of the prolactin receptormessenger ribonucleic acid are present in ovine fetal liver and adult ovary.Endocrine, 1995,3:291-295).Recently, announced the complete encoding sequence of sheep PRLR cDNA, short type and respectively corresponding 272 and 557 amino acid of two kinds of PRLR of elongated, and confirmed that further short type PRLR cDNA has the insertion fragment of 39bp, genome analysis shows that this segmental existence or disappearance are to cause common transcript to replace the reason of montage simultaneously, is not owing to there is different genes.
The main effect of prolactin is to promote mammogenesis and lactation, by increasing the transformation period of genetic transcription and mRNA, it can stimulate expression (the Bole-Feysot C of milk protein gene, Goffin V, Edery M, Binart N, KellyP A.Prolactin (PRL) and its receptor:actions, signal transduction pathwaysand phenotypes observed in PRL receptor knockout mice.Endocrine Reviews, 1998,19 (3): 225-268).Its function is to cause by combining with the certain height affinity receptor that is arranged in plasma membrane and some tissues.The PRLR gene is expressed in multiple tissues such as mammiferous mammary gland, corpus luteum, ovary, testis, prostate gland, liver, and is also different at the main histoorgan of different animal expressions.Shiu etc. have found PRLR (Shiu R P C in mammary tissue, Kelly P A, Friesen H G.Radioreceptor assay forprolactin and other lactogenic hormones.Science, 1973,180:968-971), discovery PRLR such as Posner also are distributed in (Posner B I in some other tissue in a large number, Kelly PA, Shiu R P C, et al.Studies of insulin, growth hormone and prolactin binding:tissuedistribution, species variation and characterization.Endocrinology, 1974,95:521-531).
Arden etc. are at first hybridized the PRLR assignment of genes gene mapping on people's No. 5 chromosomal galianconism by Southern, by Chromosomal in situ hybridization it further is positioned 5p13-p14 again, database information shows that PRLR sequence of people and rabbit and the PRLR sequence of pig have higher homology simultaneously, and homology is respectively 74% and 67%; In addition Barker etc. also with the PRLR assignment of genes gene mapping of rat in No. 2 karyomit(e), the PRLR assignment of genes gene mapping of mouse is in No. 15 karyomit(e) (Barker C S, Bear S, Keler T, et al.Activation of the prolactin receptor geneby promoter insertion in a moloney murine leukemia virus-induced rat thymoma.Journal of Virology, 1992,66 (11): 6763-6768).
Vincent etc. are by rflp analysis and the somatic hybridization of reference family DNA, No. 16 karyomit(e)s of pig are arrived in the PRLR assignment of genes gene mapping, find itself and 3 mark S0006, GHR1 and S0077 close linkage simultaneously, and with restriction enzyme pcr amplification product is carried out enzyme and cut, found the polymorphism of PRLR gene; Determined the genotype of different varieties source pig simultaneously, find that gene frequency is different (Vincent A L in different varieties, WangL, Tuggle C K, et al.Prolact in receptor maps to pig chromosome 16.MammalianGenome, 1997,8:793-794).The difference of gene frequency in different varieties shows in some colony can carry out an allelic selection, and the research to the effecting allele various trait has very big meaning like this.In addition, Zhang Shujun etc. are by the PCR-RFLP method, to the pig that grows up, restriction fragment length polymorphism analysis (Zhang Shujun has been carried out in a site of the prolactin receptor gene of painted face in Beijing opera pig and Large White, Xiong Yuanzhu, once four gene locuss of all equal .PRLR and ESR polymorphism analysis .Animal BiotechnologyBulletin in the painted face in Beijing opera Large White that grows up, 2000,7 (1): 101-102), the result shows that all there is polymorphism in this site of PRLR gene in two kinds and the sow group that grows up, and its to a certain extent with research (the Vincent AL of Vincent etc., Wang L, Tuggle C K, et al.Prolactin receptor maps to pig chromosome 16.Mammalian Genome, 1997,8:793-794) conform to.
The cDNA clone that Jenkins etc. utilize ox PRLR with the PRLR assignment of genes gene mapping of sheep in No. 16 karyomit(e), itself and FST (Follistatin) gene and GHR (growth hormone receptor) gene all are positioned at chromosomal the same area (Jenkins Z A No. 16, Henry H M, Sise J A, et al.Follistatin (FST), growth hormone receptor (GHR) and prolact in receptor (PRLR) map onto sheep chromosome 16.Animal Genetics, 1998,29 (Suppl.1): 37); Preliminary linkage analysis shows that the PRLR gene is between two mark McM150 and OarCP99.In this zone, there are 8 microsatellite markers to be determined.Jenkins etc. further find that by colony's multiple spot linkage analysis the PRLR gene is between BMS703 and BM5004 again, the distance of itself and BMS703 is approximately 5cM (Jenkins Z A, HenryH M, Sise J A, et al.Follistatin (FST), growth hormone receptor (GHR) andprolactin receptor (PRLR) genes map to the same region of sheep chromosome 16.Animal Genetics, 2000,31:280).
The PRLR gene is a growth and an important controlling gene that breaks up, and uses digestion with restriction enzyme, finds that by rflp analysis PRLR has polymorphism.The PRLR gene owing to its comprehensive action in several breeding approach be used as reproductive trait a candidate gene (storage star. the progress of pig number born character candidate gene. herding and animal doctor, 2001,33 (1): 36-37).The PRLR number increases at pregnancy duration in the pig lutein cell, and the PRLR gene has navigated to No. 16 karyomit(e) of pig.In view of these characteristics, be well-founded as a candidate gene of pig reproductive trait with the PRLR gene.Vincent etc. with the PRLR gene as by Large White (2 different sourcess), landrace, duroc, a candidate gene of Large White/5 PIC strain reproductive traits that plum mountain pig source is formed has carried out studying (Vincent A L, Evans G, Short T H, et al.The prolactin receptor gene isassociated with increased litter size in pigs.In:Proceedings of the 6th WorldCongress on Genetics Applied to Livestock Production, Armidale, Australia, 1998,27:15-18).To each genotype, all calculated total litter size and the least square mean value of the litter size of living, and carried out the analysis of additivity and dominant effect by strain.The result shows, the total litter size and/or the young digital display work of life birth relevant (P＜0.05) of 3 strains in this gene and 5 strains being measured.Between homozygous genotype, effect-size is that every nest increases by 0.66 to the piglet more than 1, changes with genetic background.Rothschild etc. are reported in an amino-acid residue that is comprised in zone of PRLR gene conservative between several species variation have taken place in pig, this sudden change (Rothschild M F that is associated with the high nest litter size of pig, Vincent A L, Tuggle CK, et al.A mutation in the prolactin receptor gene is associated withincreased litter size in pigs.Animal Genetics, 1998,29 (Suppl.1): 69).When being used in combination with traditional system of selection, the PRLR gene test is potential as a kind of strong instrument to some strains.
Ormandy etc. learn that by the assignment of genes gene mapping of rat embryo stem cell its kind of carrying PRLR is the null mutation gene, and this gene pairs reproductive trait has very big influence.The female mouse of heterozygosis shows as during its primipregnancy and lacks milking capacity, and this is because the growth of mammary gland is suppressed; The female mouse of isozygotying then shows as sterile, but also show multiple abnormal reproduction phenomenon, (the Ormandy C J such as reduction, pre-implantation embryos impaired development that comprises rate of fertilization, CamusA, Barra J.Null mutation of the prolactin receptor gene produces multiplereproductive defects in the mouse.Genes and Development, 1997,11:167-178).
Linville etc. have studied the influence of PRLR gene pairs pig first tire nest litter size and number of eggs ovulated, the result shows that the PRLR gene frequency is distributed in different (P＜0.005) in the different strain pigs, gene frequency in 524 pigs is A=0.33, B=0.67, low frequency allelotrope A and higher number of eggs ovulated, higher total litter size, higher litter size alive, relevant (the Linville R C of lower mummy, Pomp D, Johnson R K, et al.Candidategene analysis for loci affecting litter size and ovulation rate in swine.J.Anim.Sci., 2001,79:60-67).
The litter size of sheep is a quantitative character that heritability is very low, is difficult to improve the litter size proterties with conventional breeding technique.Marker assisted selection (marker assisted selection, MAS) intensity of the time that can select by influence, selection and accuracy and greatly improve the selection effect of this class low-heritability traits, and find the molecular genetic marker linked with these quantitative trait locuses, then be the prerequisite that realizes marker assisted selection.
The innovation and creation content
The purpose of this invention is to provide a kind of method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood.
The method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood provided by the present invention, be to use by having SEQ ID № in the sequence table: 1 and SEQ ID №: a pair of primer that 2 nucleotide sequence is formed carries out pcr amplification to the DNA of sheep to be measured, then pcr amplification product being carried out single nucleotide polymorphism detects, determine from SEQ ID №: 5 ' end the 84th bit base of 3 is T or C, and the 174th bit base is T or G.
The method that described single nucleotide polymorphism detects can be dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP) or allele specific oligonucleotide oligonucleotide hybridization methods such as (ASO).
For obtaining better detected result, the method that described single nucleotide polymorphism detects is preferably earlier carries out the single strand conformation polymorphism detection to described pcr amplification product, carries out dna sequencing then.
With SEQ ID № in the sequence table: 1 and SEQ ID №: 2 is that the pcr amplification product length that primer obtains is 176bp, has SEQ ID №: 3 nucleotide sequence.If from SEQ ID №: 5 ' end the 84th bit base of 3 is T, and the 174th bit base is T, and its homozygotic genotype is AA; From SEQ ID №: 5 ' end the 84th bit base of 3 is C, and the 174th bit base is G, and its homozygotic genotype is BB; The genotype of heterozygote is AB.
Described sheep comprises the little tail sheep that trembles with fear, many Saites sheep, the assorted generation of the cold sheep ewe of Suffolk and many Saites ram and little tail; Be preferably the cold sheep of little tail.
The present invention is an experiment material with prolificacy sheep variety (as the cold sheep of little tail) and low reproductivity sheep variety (as many Saites sheep, Suffolk etc.), the PRLR gene is carried out single nucleotide polymorphism (single nucleotidepolymorphism, SNP) detect, compare the polymorphism of PRLR gene in different sheep varieties, and to the comparative analysis of checking order of its dna fragmentation with SSCP polymorphism, the result shows from SEQ ID №: there is the base mutation of T → C in 5 ' end the 84th bit base of 3, and there is the base mutation of T → G in the 174th bit base.
Result of study of the present invention shows that the cold average litter size of sheep nest of PRLR locus AB genotype and the little tail of BB genotype Duo 0.52 (P＜0.05) and 0.64 (P＜0.05) than the AA genotype respectively, and PRLR locus B allelotrope and the little tail high litter size of sheep of trembling with fear is remarkable positive correlation.
Result of study of the present invention shows that the PRLR gene may be a major gene of control sheep fecundity trait or have genetic linkage closely with it, for the marker assisted selection and the breeding of sheep prolificacy provides the theory and practice basis, can shorten breeding cycle greatly, improve breeding efficiency, and will bring considerable economic to the sheep producer.
Description of drawings
Fig. 1 is the sscp analysis electrophoretogram of the PRLR gene PCR amplified production of the cold sheep of little tail
Fig. 2 is the 84th of the cold sheep PRLR gene of little tail and the 174th AA and the genotypic sequence comparison of BB
Embodiment
The single nucleotide polymorphism of embodiment 1, PRLR gene detects
Experiment material
Have the 1st tire and the 2nd tire litter size record, do not have the cold sheep ewe blood sample of 157 little tails in Shandong (10ml/ only) of sibship to pick up from Jiaxiang, Shandong sheep stud; Assorted 64 ewes of a generation of the cold sheep ewe of 48 ewes of many Saites sheep, 50 ewes of Suffolk, many Saites ram and little tail, blood sample all picks up from Beijing Xing Lvyuan agriculture and animal husbandry Development Co., Ltd sheep stud (the Shunyi District Yang Zhen of Beijing).Institute's blood-sample withdrawal only is 10ml/, uses the ACD anti-freezing, and-20 ℃ frozen.Extract genomic dna with the phenol chloroform extraction method, be dissolved in TE, 4 ℃ of preservations.
Main agents
Taq archaeal dna polymerase, dNTP are available from Beijing ancient cooking vessel state Bioisystech Co., Ltd.
1, design of primers and pcr amplification
According to the sheep PRLR Gene Partial sequence of delivering (GenBank accession number AF042358), at 1 pair of Auele Specific Primer of intron II design, the amplified fragments size is 176bp.Genomic dna with the cold sheep of little tail, many Saites sheep, Suffolk, many Saites ram and the assorted generation ewe of the cold sheep ewe of little tail is a template respectively, carries out pcr amplification.Primer is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd.Primer sequence is:
Upstream primer: 5 '-TGTCAGTAAGCGTCAGAGGGC-3 ' (SEQ ID №: 1);
Downstream primer: 5 '-GGCTGGTGGAAGGTCACTCTT-3 ' (SEQ ID №: 2);
The pcr amplification system is 25 μ L:10 * PCR damping fluid 2.5 μ L, and primer 50ng, dNTP final concentration are 200 μ mol/L, 1U Taq archaeal dna polymerase, Mg
2+Concentration 2.0mmol/L, template 100ng.Reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of renaturation 1min, 72 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.Utilize primer amplification sheep genomic dna, gained PCR product detects with 2% agarose gel electrophoresis, obtains the 176bp specific band, can directly carry out sscp analysis.
2, sscp analysis
The PCR product 2 μ L that get preparation in the step 1 place the PCR pipe, add 5 μ L sex change damping fluids (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene cyanogen, 10mmol/L EDTA (pH 8.0), 10% glycerine), centrifugal mixing, 98 ℃ of sex change 10min, ice bath 10min rapidly.Sample is electrophoresis in 10% non-denaturing polyacrylamide gel.After electrophoresis finishes, carry out silver and dye apparent band.Gel imaging system with U.S. Alpha Innotech company is taken pictures.
Wherein, the SSCP detected result of the cold sheep of little tail as shown in Figure 1.Among Fig. 1, swimming lane 9,10 is the AA genotype; Swimming lane 1-6,8 is the AB genotype; Swimming lane 7 is the BB genotype.
3, the cloning and sequencing of the homozygote individuality of different genotype
In order to determine the position of point mutation in sequence, the PCR product of AA, two kinds of homozygous genotype individualities of BB is carried out cloning and sequencing.The result shows that PCR product length is 176bp, and the BB type is compared with the AA type, only in sequence table 5 of sequence 3 ' there is the base mutation of T → C in end the 84th bit base, there is the base mutation (Fig. 2) of T → G in the 174th bit base.With reference to sheep PRLR gene second intron sequences among the GenBank, the AA type is decided to be wild-type, the BB type is decided to be mutant.This sudden change does not cause amino acid whose change, belongs to silent mutation.
The relation of embodiment 2, the cold average litter size of sheep nest of the little tail of detection and PRLR gene mononucleotide polymorphism
Cooperate following model to carry out the least square variance analysis, the smaller tail difference of the average litter size of sheep nest between the marker gene type of trembling with fear:
y
ijkl＝μ+HYS
i+P
j+G
k+e
ijkl
Wherein: y
IjklRecord value for the average litter size of nest; μ is colony's mean value; HYS
iIt is the fixed effect in i year season; P
jIt is the fixed effect of j parity; G
kIt is the fixed effect of k kind marker gene type; e
IjklBe the random residual effect.GLM (General Linear Model) process with SAS (6.12 version) is finished.
1, the different sheep PRLR of colony genotype frequencies and gene frequency
Method according to embodiment 1 has been carried out the PRLR genotype detection to the cold sheep of little tail, many Saites sheep, Suffolk, many Saites ram and the assorted generation ewe of the cold sheep ewe of little tail, calculated the genotype frequency and the gene frequency of different sheep colony, statistics is as shown in table 1.
Gene frequency and the genotype frequency of PCR-SSCP in 4 sheep colonies of table 1.PRLR gene
The population sample base is because of the frequency genotype frequency
A B AA AB BB
The little tail sheep 157 0.7898 0.2102 0.6178 (97) 0.3440 (54) 0.0382 (6) that trembles with fear
Many Saites sheep 48 0.6146 0.3854 0.3542 (17) 0.5208 (25) 0.1250 (6)
Suffolk 50 0.92 0.08 0.84 (42) 0.16 (8) 0 (0)
Assorted generation sheep 64 0.6484 0.3516 0.4844 (31) 0.3281 (21) 0.1875 (12)
Annotate: the number of individuals of the numeral different genotype in the bracket.
Table 1 shows in the PRLR of 4 sheep colonies gene intron 2 and has all detected base mutation.In the cold sheep of little tail, many Saites sheep, many Saites ram and the assorted generation ewe of the cold sheep ewe of little tail, all detect wild homozygous genotype (AA), sudden change heterozygous genes type (AB) and sudden change homozygous genotype (BB), and in Suffolk, do not detect sudden change homozygous (BB), only detect 2 kinds of genotype (AA, AB).
2, the different sheep PRLR of colony genotype distributional difference checks
The different sheep PRLR of colony genotype distributional difference assays are as shown in table 2, show PRLR genotype distributional difference remarkable (P＜0.05) between cold sheep of little tail and the Suffolk, PRLR genotype distributional difference extremely significantly (P＜0.01) between the cold sheep of little tail and the many Saites sheep, PRLR genotype distributional difference extremely significantly (P＜0.001) between cold sheep of little tail and the assorted generation sheep; PRLR genotype distributional difference extremely significantly (P＜0.001) between many Saites sheep and the Suffolk, PRLR genotype distributional difference not significantly (P＞0.05) between many Saites sheep and the assorted generation sheep; PRLR genotype distributional difference extremely significantly (P＜0.001) between Suffolk and the assorted generation sheep.
The check of table 2. PRLR of 4 sheep colonies genotype distributional difference
Many Saites sheep Suffolk generation sheep of mixing
The little tail sheep 12.31** 8.98* 13.87*** that trembles with fear
Many Saites sheep 25.32*** 4.23
Suffolk 18.04***
Annotate: df=(2-1) (3-1)=2, x
2(df=2,0.05)=5.99, x
2(df=2,0.01)=9.21, x
2(df=2,0.001)=13.82,
*P＜0.05，**P＜0.01，***P＜0.001
3, the least square mean value and the standard error of the cold average litter size of sheep nest of the little tail of PRLR gene different genotype
The least square mean value and the standard error of the cold average litter size of sheep nest of the genotypic little tail of different PRLR are as shown in table 3, show that the cold average litter size of sheep nest of the little tail of BB genotype Duos 0.64 (P＜0.05) than the AA genotype, the cold average litter size of sheep nest of the little tail of AB genotype is Duoed 0.52 (P＜0.05) than the AA genotype, and B allelotrope is 0.625 to the allelic degree of dominance of A.Illustrate that the cold high litter size of sheep of B allelotrope and little tail is remarkable positive correlation.Infer that the PRLR gene may be a major gene of the cold sheep fecundity trait of the little tail of control or have genetic linkage closely with it.
The least square mean value and the standard error of the cold average litter size of sheep nest of the genotypic little tail of the different PRLR of table 3.
Genotype sample number least square mean value and standard error
AA 97 2.01
b±0.18
AB 54 2.53
a±0.21
a 0.32
d 0.20
D 0.625
Annotate: same proterties has different letter shoulder target mean value differences significantly (P＜0.05).
D＝d/a；a＝(BB-AA)/2；d＝AB-(BB+AA)/2
Sequence table
<160>3
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tgtcagtaag?cgtcagagggc 21
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
ggctggtgga?aggtcactct?t 21
<210>3
<211>176
<212>DNA
＜213〉sheep (0vis aries)
<220>
<221>misc-feature
<222>(84)
＜223〉n=t or c
<220>
<221>misc-feature
<222>(174)
＜223〉n=t or g
<400>3
tgtcagtaag?cgtcagaggg?caatgaaaga?aagccaagaa?aacaccagca?cacaggaatg 60
cttgttaggt?ttatcagatc?tgtncataag?ccctgtcata?aacattttat?acaaataatc 120
agttgagaaa?gtgggtgaat?atttgatgaa?tacaaaagag?tgaccttcca?ccancc 176
Claims (5)
1, a kind of method of utilizing single nucleotide polymorphism prediction mean lambing number of sheep brood, be to use by having SEQ ID № in the sequence table: 1 and SEQ ID №: a pair of primer that 2 nucleotide sequence is formed carries out pcr amplification to the DNA of sheep to be measured, then pcr amplification product being carried out single nucleotide polymorphism detects, determine from SEQ ID №: 5 ' end the 84th bit base of 3 is T or C, and the 174th bit base is T or G.
2, method according to claim 1 is characterized in that: the method that described single nucleotide polymorphism detects is dna sequencing, restriction fragment length polymorphism, single strand conformation polymorphism or the hybridization of allele specific oligonucleotide oligonucleotide.
3, method according to claim 2 is characterized in that: the method that described single nucleotide polymorphism detects detects for earlier described pcr amplification product being carried out single strand conformation polymorphism, carries out dna sequencing then.
4, according to claim 1 or 2 or 3 described methods, it is characterized in that: described sheep comprises the little tail sheep that trembles with fear, many Saites sheep, the assorted generation of the cold sheep ewe of Suffolk and many Saites ram and little tail.
5, method according to claim 4 is characterized in that: described sheep is the cold sheep of little tail.
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| CN106498080A (en) * | 2016-12-14 | 2017-03-15 | 兰州大学 | Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms |
| CN111118178A (en) * | 2020-02-18 | 2020-05-08 | 天津奥群牧业有限公司 | Detection primer pair, kit, method and application of sheep GHR gene insertion/deletion polymorphism |
| CN111154895A (en) * | 2020-03-02 | 2020-05-15 | 天津奥群牧业有限公司 | Detection primer pair, kit, method and application of sheep GHR gene insertion/deletion polymorphism |
Family Cites Families (1)
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| CN1274842C (en) * | 2003-02-17 | 2006-09-13 | 中国农业科学院畜牧研究所 | Method for predicting the born lamb number and its application |
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Cited By (6)
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| CN102643914A (en) * | 2012-04-17 | 2012-08-22 | 扬州大学 | Method for detecting depression sheep fecundity |
| CN106498080A (en) * | 2016-12-14 | 2017-03-15 | 兰州大学 | Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms |
| CN106498080B (en) * | 2016-12-14 | 2019-08-06 | 兰州大学 | The method and its application of sheep NELF gene mononucleotide polymorphism are quickly detected using PCR-SSCP |
| CN111118178A (en) * | 2020-02-18 | 2020-05-08 | 天津奥群牧业有限公司 | Detection primer pair, kit, method and application of sheep GHR gene insertion/deletion polymorphism |
| CN111154895A (en) * | 2020-03-02 | 2020-05-15 | 天津奥群牧业有限公司 | Detection primer pair, kit, method and application of sheep GHR gene insertion/deletion polymorphism |
| CN111154895B (en) * | 2020-03-02 | 2023-04-28 | 天津奥群牧业有限公司 | Sheep GHR gene insertion/deletion polymorphism detection primer pair, kit, method and application |
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