CN102643914A - Method for detecting depression sheep fecundity - Google Patents
Method for detecting depression sheep fecundity Download PDFInfo
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Abstract
The invention discloses a method for predicating the depression sheep fecundity by Fec<B> genes. According to the method, the 746th site nucleotide of the Fec<B> genes in the depression sheep genome to be detected is detected, so the genotype of depression sheep is determined, and then, the depression sheep fecundity is determined through the genotype; when the 746th site nucleotide of the Fec<B> genes in the depression sheep genome is A, the homozygote genotype is ++, and when the 746th site nucleotide of the Fec<B> genes in the depression sheep genome is G, the homozygote genotype is BB; the heterozygote genotype is B+; the fecundity of the BB genotype is higher that of the B+ genotype, and the fecundity of the B+ genotype is higher than that of ++ type. The method has the advantages that the depression sheep fecundity is determined through polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restricted fragment length polymorphism (RFLP) genetic marker polymorphism, the depression sheep fecundity can be fast, simply and conveniently detected, and a method for accurately, simply and conveniently detecting the depression sheep fecundity is provided for the depression sheep breeding.
Description
Technical field
A kind of method that detects depression sheep reproductivity.
Background technology
Fec
BGene was found in Australian Booroola Merino sheep variety, and was described to influencing first major gene of sheep ovulation rate and reproductivity in 1985.Booroola sudden change, i.e. the point mutation of coding region, BMPR-IB site gets in other the sheep variety through hybridization, and under state of nature, has also found other the sheep variety of fecund except that Booroola Merino sheep.Fec
BThe assignment of genes gene mapping is in the narrow zone of No. 6 karyomit(e) 6q23～q31 of sheep, between SPP1 and Urogastron.Fec
BThe genotype in site is distributed, and begins to be based on to detect ovulation rate and write down with lambing, is used for getting in touch of genetic marker and Booroola gene afterwards, nowadays can obtain through the molecular gene somatotype at the individuality in BMPR-IB site.A Fec
BCopy increases by 1.3 pieces-1.6 pieces of numbers of eggs ovulated, and two FecB copies increase by 2.7 pieces-3.0 pieces of numbers of eggs ovulated, carry a Fec
BThe ewe reproductivity of copy increases by 0.9-1.2, carries two Fec
BThe ewe reproductivity of copy increase by 1.1-1.7 (foline becomes for storage star, Di Ran, Fang Li, Liu Zhonghui, Peng Zhilan opens with happiness, Jia Lihua, Sun Jie, Feng Tao. sheep polyembryony major gene Fec
BThe foundation of molecular detecting method and application [J]. Journal of Agricultural Biotechnology, 2009,17 (1): 52-58).
Depression sheep (being called for short hollow sheep) is the good local sheep variety that is distributed in the saline and alkaline depression of northern Shandong coastal region.Through textual criticism, depression sheep be from Ming Dynasty period through domestication in more than 400 year, adaptation, seed selection, the good local variety that show unique characteristics that form gradually at coastal region.Depression sheep all can oestrus throughout the year, and spring and autumn is particularly evident.The first-born lambing percentage and the little tail sheep no significant difference of trembling with fear belongs to the polyembryony sheep variety.Depression sheep has crude feed tolerance, the characteristics faster of growing.See that from build depression sheep is acerous, four limbs are short and solid, square tail, and also the personality docility is not bellicose, makes that depression sheep dressing percentage is high, it is fast to grow.Through long-term breeding, depression sheep has adapted to local barren grassland, and continuous grazing has been tempered flock of sheep physique.Especially local strand wetland is saline and alkaline serious, and is very strong to the corrodibility of sheep hoof, and other kind sheep are difficult to adapt to.And depression sheep well adapted local environment, have anti-rotten hoof ability.
Montgomery etc. (1993) find Fec first
BGene and microsatellite marker OarAE101 and OarHH55 close linkage, distance is respectively 13cM and 20cM, and a RFLP mark of these two genes secretor type phosphoric acid albumen 1 gene (SSP1) last with being positioned at human chromosome HSA4q11～q12 is chain.This study group adopts PCR RFPL analysis PCR RFPL subsequently; Booroola sheep family half sibs and 17 family full-sibses are studied; Find PDGF receptor alpha gene (platelet-derived growth factor receptor-alpha gene; PDGFRA) with α S1 casein gene (alpha sl-casein gene, CSN1S1) and microsatellite marker BM143 and OarHH55 chain, genetic distance is respectively 12cM, 29cM, 33cM.Little satellite OarAE101 of usefulness such as Lord and BM1329 mark and study Fec
BDuring gene, find Fec
BGene is present on the 162bp allelotrope in 97bp allelotrope and BM1329 site in OarAE101 site, so Fec
BGene further is positioned between No. 6 chromosomal BM1329 and the OarAE101 (Montgomery G W, et al.Nat Genet, 1993,4 (4): 410-414) in the 10cM linkage group.
Montgomory further discovers, Fec
BGene and EGF (Urogastron) gene linkage, its distance is 26cM, and EGF is positioned on the 4q25 of human chromosome, this is the Fec that marches toward
BSudden change position clone's essential step (Montgomery G W, et al.Genomics, 1994,22 (1): 148-153).Wilson etc. (2001) are on the former study basis, with Fec
BGene further is positioned at No. 6 karyomit(e) 6q23～q31 of sheep, with human chromosome 4q21～q25 colinearity.Other has report, and the assignment of genes gene mapping on No. 6 karyomit(e)s of sheep on No. 8 karyomit(e)s of pig, has been found site (Wilson T, et al.Biol Reprod, 2001,64 (4): 1225-1235) of improving ovulation rate at No. 8 karyomit(e)s simultaneously.
Wilson etc., Souza etc., Mulsant etc. (2001) almost find simultaneously, with Fec
BThe last Delicious peptide acceptor IB gene (BMPR-IB) of No. 4 karyomit(e) q22～q23 of people of No. 6 chromosomal region colinearitys of sheep at gene place is associated with the ovulation rate of Booroola Merino sheep.
[0007] Elisha Gootwine etc. (2008) is through to Fec
BThe site ++ with the investigation of B+ genotype reproductivity; The result shows; The B site has the multiplication effect on reproductivity; Because the genotypic reproductivity of B+ is significantly higher than ++ genotypic reproductivity (P＜0.05) (Elisha Gootwine, Shay Reicher, Alexander Rozov.Prolificacy and lamb survival at birth in Awassi and Assaf sheep carrying the FecB (Booroola) mutation. [J] .Animal Reproduction Science; 2008,108 (2): 402-411).
A.K.Mishra etc. (2009) pass through the reproductivity analysis to the hybridization sheep of 51 Garole and Malpura; The result shows; The litter size (CLS) in accumulative total all one's life, the wean number (CWL) of accumulative total lamb with and the reproductive efficiency (CEPE) of accumulative total ewe hybridizing on the ewe ratio that the B+ genotype is relative ++ the high (A.K.Mishra of genotypic ewe; A.L.Arora; S.Kumar, L.L.L.Prince.Studies on effect of Booroola (FecB) genotype on lifetime ewes productivity efficiency, litter size and number of weaned lambs in Garole * Malpura sheep [J] .Animal Reproduction Science; 2009,113 (3): 293-298);
S.Kumar etc. (2008) find Fec through the experimental technique of PCR-RFLP in Kendrapada sheep
BSudden change, in 46 Kendrapada sheep individualities, 26 BB genotype, 15 B+ genotype, 5 ++ genotype, the genotype frequency of B is about 0.73, and the result shows, although Fec
BMutation frequency is very high, but this gene and be unlike in that kind of being reported in the Garole sheep is unfixed (S.Kumar in colony; A.K.Mishra; A.P.Kolte, S.K.Dash, S.A.Karim.Screening for Booroola (FecB) and Galway (FecXG) mutations in Indian sheep [J] .Small Ruminant Research; 2008,80 (4): 57-61).
Wang Genlin etc. (2003) year is utilized restriction fragment polymorphum round pcr, detects the dna sample of sheep, the cold sheep of little tail and Xinjiang fine-wool sheep, finds to have Fec in the cold sheep of sheep and little tail
BThe polyembryony gene, (Wang Genlin, Mao Xinzhi, George H Davis, Zhao Zongsheng, Zhang Lijun, Ceng Yongqing .DNA analyze and find that there are Booroola (Fec in China sheep and the cold sheep of little tail and the Xinjiang fine-wool sheep is not all carried the Booroola gene
B) polyembryony gene [J]. Agricultural University Of Nanjing's journal, 2003,26 (1): 104-106).
Zhang Liping (2004) is to Fec
BThe discovery of gene, the location, the related grade with application has been comparatively comprehensively analysis revealed, the sudden change of BMPR-IB gene and Fec
BThe behavior of gene is in full accord, proves that the BMPR-IB gene is major gene (the Zhang Liping .Booroola sheep polyembryony gene Fec of control Booroola Merino sheep prolificacy characteristic
BProgress [A] .2003-2004 whole nation sheep raising produce and academic discuss meeting paper collection 2004.).
Liu Nan etc. (2010) have proved in the hybridization colony of the cold sheep of little tail and Suffolk and Du's pool sheep, polyembryony gene Fec
BIt is remarkable to detect the polymorphum effect, can be used as the effective candidate gene (Liu Nan, Liu Xiaoxiao, Liu Meng, Qu Xuxian, Wang Jinwen, the Li Jing .Fec that cultivate the Mutton Sheep new lines
BWith of the research [J] of BMP15 gene as Mutton Sheep breeding candidate gene. HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE, in January, 2010 (on): 39-40).
Cui Xukui etc. (2010) utilize sheep polyembryony gene Fec
BMolecular detection technology, check and analysis Du Han hybridization mutton sheep genotype and influence that reproductivity and lamb are grown.Find Fec
BGene is not only the major gene that influences Du Han hybridization mutton sheep reproductivity, and over a period to come page or leaf influence the growing of lamb (Cui Xukui, Wang Jinwen, Wang Deqin, Zhang Guoping, the king can, Huang Qinghua, Meng Xian cutting edge of a knife or a sword .Fec
BThe influence [J] that gene pairs Du * cold hybridization mutton sheep reproductivity and lamb grow. the Shandong agricultural sciences, 2010,8:98-100).
Wang Jinwen etc. (2010) have proved Fec
BGene possibly be the major gene of control western Shandong mutton sheep how high proterties, this record a demerit for cultivation western Shandong mutton sheep polyembryony strain established theoretical basis (Wang Jinwen, Cui Xukui, Zhang Guoping, Wang Deqin, the king can, Huang Qinghua, Meng Xianfeng. utilize Fec
BGene seed selection western Shandong mutton sheep polyembryony strain research [J]. ecology of domestic animals newspaper, 2010,31 (5): 23-25).
Li Fenge (2010) is to Fec
BAspects such as the proposition of gene, genotype judgement, physiological effect and separating clone done comprehensive summary (Li Fenge, Xiong Yuanzhu. sheep Fec
BGene isolation clone's progress [J] .Animal Science Abroad, 2010,28 (6): 42-44).
Many lambs mechanism of Ren Yanling etc. (2011) sheep from molecular level research depression, the Fec in the sheep of depression that adopted the PCR-RFLP methods analyst
BThe relation of gene pleiomorphism and reproductivity thereof, through statistical study, the reproductivity of BB and B+ genotype depression sheep is significantly higher than ++ and genotype shows Fec
BGene possibly be one of depression many lambs of sheep performance major gene (Ren Yanling, Shen Zhiqiang, Li Min etc. depression sheep Fec
BThe research of gene pleiomorphism and reproductivity relation. Chinese animal and veterinary, 2011,38 (7): 159-162).
A lot of scientists is also to little satellite gene of sheep and Fec
BGene polymorphic and chainly carried out big quantitative analysis.The result of study of Zhang Baoyun etc. (2009) shows that little satellite seat 300U can only reflect Fec to a certain extent
BSeat information (king is with green grass or young crops, Fang Li, Di Ran for Zhang Baoyun, storage star. little satellite 300U of sheep and Fec
BPolymorphic and the linkage analysis [J] of gene. China Agricultural University's journal, 2009,14 (5): 86-92).The research of Li Yanlu etc. (2009) shows, the linkage disequilibrium analysis shows the little tail sheep Fec that trembles with fear
BThere is certain linkage disequilibrium between the little satellite of gene B allelotrope and the BMS2508 seat 100bp allelotrope; And+all there is certain linkage disequilibrium in allelotrope with the little satellite of BMS2508 seat 110bp and 114bp allelotrope, and (Li Yanlu stores up star, Chen Hongquan; Fang Li; Di Ran, Ma Yuehui, Li Kui. little satellite BMS2508 of sheep and Fec
BPolymorphic and the linkage analysis [J] of gene. heredity, 2009,31 (5): 500-507).The storage star waits the result of study of (2009) to show, the little satellite of OarJL36 seat 182bp allelotrope and the little tail sheep Fec that trembles with fear
BHave stronger linkage relationship between the gene B allelotrope, be with the cold closely linked genetic marker of many lambs of sheep major gene of little tail (storage star, Zhang Baoyun, king be with green grass or young crops, Fang Li, Di Ran, Ma Yuehui, Li Kui. little satellite OarJL36 of sheep and Fec
BPolymorphic and the linkage analysis [J] of gene. Scientia Agricultura Sinica, 2009,42 (6): 2133-2141).The research of Zhang Lin etc. (2009) etc. shows, the little satellite of LSCV043 seat 98bp allelotrope and the little tail sheep Fec that trembles with fear
BThere is certain linkage disequilibrium relation between the gene B allelotrope, is and the cold closely linked genetic marker of many lambs of sheep major gene of little tail (Zhang Lin, Li Chunmiao, storage star; Chen Hongquan, Li Xuewei, Fang Li; Di Ran, Ma Yuehui, Li Kui. the cold little satellite of sheep seat LSCV043 of little tail and Fec
BThe linkage analysis of gene [J]. Journal of Agricultural Biotechnology, 2009,17 (4): 621-628).
According to " China sheep kind will " record (" China sheep kind will " is write group, 1989), it is 2.80 that depression sheep on average produces lamb number alive, is the polyembryony sheep variety of China.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects depression sheep reproductivity.
The method of detection provided by the present invention depression sheep reproductivity is the Fec that detects in the sheep genome of depression to be measured
BThe 746th Nucleotide of gene is that A still sports G, thereby confirms the genotype of depression sheep, confirms depression sheep reproductivity through genotype then; Said Fec
BThe nucleotide sequence GENBANK ACCESSIONNUMBER of gene is 156bp to the 3255bp position Nucleotide of AF357007;
The genotypic method of said definite depression sheep is: if the Fec in the sheep genome of depression
BWhen the 746th Nucleotide of gene was A, its homozygotic genotype did ++; Fec in the sheep genome of depression
BWhen the 746th Nucleotide of gene was G, its homozygotic genotype was BB; Their heterozygote genotype is B+;
The method of confirming depression sheep reproductivity through genotype is: the reproductivity of said BB genotype depression sheep is higher than B+ genotype depression sheep, and the reproductivity of B+ genotype depression sheep is higher than ++ type depression sheep.
The 746th Nucleotide of the FecB gene in the sheep genome of said detection depression is that still the suddenly change method of G of A has two kinds; First kind for utilizing the nucleotide sequence GENBANK ACCESSION NUMBER in the sheep genome of PCR method amplification depression to be the fragment of 626bp to 813 Nucleotide of AF357007, and this amplified production is carried out SSCP detect, and all obtains two electrophoretic bands; Two band furthest; Band and thinner do ++ genotype, two bands are nearest, and what band was also thinner is the BB genotype; Two bands are thicker, and apart be between the above two for B+ genotype (like Fig. 2); Second kind for utilizing the nucleotide sequence GENBANK ACCESSION NUMBER in the sheep genome of PCR method amplification depression to be the fragment of 638bp to 778 Nucleotide of AF357007, and amplified production is carried out RFLP detect, if two bands can occur; And lay respectively at 140bp and 110bp, be the B+ genotype, if a band can occur; And be positioned at 140bp, for ++ genotype, if a band can occur; And be positioned at 110bp, be BB genotype (like Fig. 3).
In the said method, the amplimer of said PCR method is to for having sequence 1,2,3,4 described Nucleotide in the sequence table.
In the said method, said reproductivity is every tire litter size.
The inventive method, (single strand conformation polymorphism, SSCP) (restriction fragment length polymorphism, RFLP) method is at Fec with the restriction enzyme segment polymorphism to adopt single strand conformation polymorphism
BGene carries out SNP, and (single nucleotide polymorphism SNP) detects, to compare Fec
BThe polymorphum of gene in the sheep variety of depression; And to the comparative analysis of checking order of the dna fragmentation with SSCP and RFLP polymorphum; Search out and the relevant genetic marker of reproductivity (reproductivity), data statistics is the result show, this genetic marker polymorphum can be confirmed depression sheep reproductivity.Method of the present invention is promptly utilized this genetic marker, sets up the genotype that SSCP and RFLP detect depression sheep, and utilizes this genotype to confirm the effective ways of its reproductivity, experiment showed, the reproductivity of the detection depression sheep that this method can be rapid, easy.Method of the present invention is the method that the molecular breeding of sheep provides accurate easy its reproductivity of detection.
Description of drawings
Fig. 1 is Fec
BThe PCR product of two pairs of primers (P1, P2) of gene.
Fig. 2 for primer P1 to the depression sheep pulsating sscp analysis that increases.
Fig. 3 is the rflp analysis of primer P2 to depression sheep amplified fragments.
Embodiment
The experimental technique of mentioning among the following embodiment is ordinary method if no special instructions.
Used main agents 10PCR Buffer among the following embodiment, dNTP, the Taq archaeal dna polymerase, DNA Maker pBR322/Hae III, AvaII restriction enzyme PCR primers etc. are all available from Shanghai bio-engineering corporation.
The foundation and the compliance test result thereof of the method for embodiment 1, detection depression sheep reproductivity
1, detects the foundation of the method for depression sheep reproductivity
1) mould material is prepared
Gather the ear tissue appearance of depression sheep and write down reproductivity and parity, 98 of the depression sheep of gathering are picked up from the Binzhou Prefecture, Shandong.Adopt the genomic dna of phenol-chloroform method extracting sheep ,-20 ℃ of preservations are subsequent use behind the sterilization TE dissolved dilution.
2) design of primers
Sheep Fec according to the GenBank report
BGene is encoding sequence (AF357007) completely; Locate to design two pairs of primers with Oligo 7.0 softwares at coding region sequence (GENBANK ACCESSION NUMBER is 156bp to the 3255bp position Nucleotide of AF357007), primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.Primer sequence and amplified fragments are big or small as follows:
The genomic gene that the sheep experiment material from the depression that obtains with step 1 is extracted is a template, carries out pcr amplification with above-mentioned primer respectively.
The pcr amplification system is following: the pcr amplification system of primer 1 and primer 2 is identical.The TV of pcr amplification system is 20 μ L, and wherein 10 * Buffer, 2.5 μ L (contain Mg
2+), 10mmol/LdNTP 2 μ L, each 1.0 μ L of 10 μ mol/L upstream and downstream primers, 100ng/ μ LDNA masterplate 1 μ L, rTaq enzyme 0.3 μ L, deionized water 12.2 μ L.
The pcr amplification condition is following: primer 1 is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, and 55.2 ℃ of annealing 30s, 72 ℃ are extended 30s, 32 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.Primer 2 is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, and 64.6 ℃ of annealing 30s, 72 ℃ are extended 30s, 31 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.
Two pairs of primers that designed are detected with polyacrylamide gel electrophoresis the genome of the depression sheep PCR product that obtains that increases, and the PCR product detects: 5 μ L amplified productions add 2 μ L damping fluids, and (0.05% YLENE is blue; 0.05% bromjophenol blue; 0.02mol/L EDTA adds methane amide to 10ml), DNA Marker consumption is 3 μ L.120V voltage 2.5h under the room temperature, silver dyes colour developing.The result shows amplified fragments and specificity good (Fig. 1) consistent with the purpose clip size, can directly carry out SSCP and rflp analysis.Swimming lane 1-6 is the segmental electrophorogram that the primer 2 amplification obtains among Fig. 1, and 7-11 is the segmental electrophorogram that primer 1 amplification obtains; Swimming lane M is pBR322marker among Fig. 1.
3) sscp analysis and rflp analysis
Sscp analysis
The two pairs of primers amplification PCR products in the sheep of depression that respectively step 1) is obtained is carried out sscp analysis respectively, and concrete grammar is described below: draw 10 μ L pcr amplification products and mix with 10 μ L sex change damping fluids; Place gene-amplificative instrament, 98 ℃ of sex change 10min take out and are placed on-25 ℃ of refrigerator 5min completion of ice chest placement sex change; Cut off the electricity supply, the mixed solution of sex change clicked and entered in each glue hole with the pipettor of 10 μ L, energized, 220V voltage 15min, observe bromjophenol blue and YLENE cyanogen two bands separately after, 120V constant voltage electrophoresis 10h, silver dyes colour developing.Use AlphaImager
TM(CA USA) takes pictures and analyzes 2200 and, 1220 Documentation and Analysis Systems for Alpha Innotech Corporation, San Leandro.
The result finds that primer 1 amplified fragments has 3 kinds of genotype, called after BB, B+, ++ (Fig. 2), promptly amplified production non-denaturing polyacrylamide electrophoresis all obtains two bands.Two band furthest, band and thinner do ++ genotype ( swimming lane 1,2 among Fig. 2,3); Two bands are nearest, and what band was also thinner is BB genotype ( swimming lane 7,8 among Fig. 2,9); Two bands are thicker, and apart be between the above two for B+ genotype ( swimming lane 4,5 among Fig. 2,6).Among Fig. 2, swimming lane 1,2,3 do ++ genotype; Swimming lane 4,5,6 is the B+ genotype; Swimming lane 7,8,9 is the BB genotype.
Rflp analysis
The two pairs of primers amplification PCR products in the sheep of depression that respectively step 1) is obtained is carried out rflp analysis respectively, and concrete grammar is described below:
The PCR-RFLP enzyme is cut system: the pcr amplification product of primer 2 carries out enzyme with the AvaII restriction enzyme and cuts.The TV of endonuclease reaction is 15 μ L.PCR product 5 μ L wherein, AvaII restriction enzyme 1.0 μ L, 10 * Buffer1.5 μ L, deionized water 7.5 μ L.The centrifugal 7500r/min 30s that vibrates, the endonuclease reaction condition is 37 ℃ of water-bath 4h.
The PCR-RFLP product detects: 5 μ L enzymes are cut product and are added 2 μ L sample-loading buffers (0.05% YLENE orchid; 0.05% bromjophenol blue; 0.02mol/L EDTA adds methane amide to 10ml), DNA Marker consumption is 3 μ L.120V voltage 3.5h under the room temperature, silver dyes colour developing.Use AlphaImager
TM(CA USA) takes pictures and analyzes 2200 and, 1220 Documentation and Analysis Systems for Alpha Innotech Corporation, San Leandro.
The result finds that the primer 2 amplified fragments has 3 kinds of genotype, called after BB, B+, ++ (Fig. 3), if two bands can occur; And lay respectively at 140bp and 110bp, be B+ genotype ( swimming lane 1,2,5,10,12-16,18-19 among Fig. 3), if a band can occur; And be positioned at 140bp, for ++ genotype ( swimming lane 6,8,9 among Fig. 3), if a band can occur; And be positioned at 110bp, be BB genotype ( swimming lane 3,4,7,17 among Fig. 3).Among Fig. 3, swimming lane 6,8,9 does ++ genotype; Swimming lane 1,2,5,10,12-16,18-19 are the B+ genotype; Swimming lane 3,4,7,17 is the BB genotype.
Experimental result shows that above-mentioned two kinds of methods all can accurately declare type, and reliability is good, and the result who judges is consistent.According to the analytical results of PCR-SSCP and PCR-RFLP, the sample of choosing the different gene type carries out sequencing.The PCR product is transferred to the AB I PRISM of Shanghai biotechnology Services Co., Ltd 377 automatic dna sequencers and is accomplished sequencing after electrophoresis is identified.
2, Fec
BGenotype detection and with the statistical study of reproductivity relation
According to step 2) method carried out Fec with primer 1 and primer 2 to depression sheep
BGenotype detection, and calculated genotype frequency and the gene frequency of depression sheep, statistics is as shown in table 1.
Gene frequency and the genotype frequency of table 1FecB site in the sheep of depression
Annotate: the numeral in the bracket is a sample number.
The least square analysis that association analysis between sheep different genotype and the lambing proterties is adopted makes up following broad sense linear shape model (GLM):
Yi
jk＝μ+G
j+I
k+e
ijk
(Y
IjkBe the character observation value; μ is colony's average; G
jBe the genotype effect; I
kBe the family effect; e
IjkBe the random residual effect), the dependency of employing SPSS bioanalysis software analysis genotype and depression sheep reproductivity.The least-square means and the standard error of the depression sheep reproductivity of different genotype are seen table 2.
The least square MV and the standard error of table 2 different genotype and depression sheep reproductivity
Annotate: same capitalization is represented difference not remarkable (p＞0.05); Different lowercase alphabet differentials different significantly (0.01＜p＜0.05); Different capitalizations are represented difference extremely significantly (p＜0.01).
The result of table 2 shows: the BB genotype compares on reproductivity ++ and genotype is high by 0.90837; This show BB with ++ genotype has certain influence to the lambing proterties of depression sheep, and the different genotype in this site difference on reproductivity reaches utmost point level of signification (P＜0.01); The B+ genotype compares on reproductivity ++ genotype is high by 0.49581, this show B+ with ++ genotype has certain influence to the lambing proterties of depression sheep, and the different genotype in this site difference on reproductivity does not reach level of signification (P＞0.05); The BB genotype is higher by 0.41256 than B+ genotype on reproductivity, and this shows that BB and B+ genotype have certain influence to the lambing proterties of depression sheep, and the different genotype in this site difference on reproductivity reaches utmost point level of signification (P＜0.01).This result shows that this above-mentioned primer 1 capable of using and 2 genotype polymorphisms detect depression sheep polymorphum.
SEQUENCE?LISTING
< 110>Yangzhou University
< 120>a kind of method that detects depression sheep reproductivity
<130>
<160> 6
<170> PatentIn?version?3.3
<210> 1
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agattggaaa?aggtcgctat?g 21
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accctgaaca?tcgctaatac?a 21
<210> 3
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<212> DNA
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gtcgctatgg?ggaagtttgg?atg 23
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< 213>artificial sequence
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caagatgttt?tcatgcctca?tcaacacggt?c 31
<210> 5
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< 213>Ovis (ovis hircus)
<400> 5
agattggaaa?aggtcgctat?ggggaagttt?ggatgggaaa?gtggcgtggc?gaaaaggtag 60
ctgtgaaagt?gttcttcact?acagaggagg?ccagctggtt?ccgagagaca?gaaatatatc 120
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cggggtc 187
<210> 6
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< 213>Ovis (Ovis hircus)
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gtcgctatgg?ggaagtttgg?atgggaaagt?ggcgtggcga?aaaggtagct?gtgaaagtgt 60
tcttcactac?agaggaggcc?agctggttcc?gagagacaga?aatatatcag?acggtgttga 120
tgaggcatga?aaacatcttg 140
Claims (6)
1. a method that detects depression sheep reproductivity is the Fec that detects in the sheep genome of depression to be measured
BWhether the 746bp place of gene coding region exists the sudden change of an A → G, to confirm the genotype of depression sheep, confirms depression sheep reproductivity through genotype then; The nucleotide sequence GENBANK ACCESSION NUMBER of said FecB gene is 156bp to the 3255bp position Nucleotide of AF357007;
The genotypic method of said definite depression sheep is: if the Fec in the sheep genome of depression
BWhen the 746th Nucleotide of gene was A, its homozygotic genotype did ++; Fec in the sheep genome of depression
BWhen the 746th Nucleotide of gene was G, its homozygotic genotype was BB; Their heterozygote genotype is B+;
The method of confirming depression sheep reproductivity through genotype is: the reproductivity of said BB genotype depression sheep is higher than B+ genotype depression sheep, and the reproductivity of B+ genotype depression sheep is higher than ++ type depression sheep.
2. method according to claim 1; It is characterized in that: the 746th Nucleotide of the FecB gene in the sheep genome of said detection depression is that still the suddenly change method of G of A is to utilize the nucleotide sequence GENBANK ACCESSION NUMBER in the sheep genome of PCR method amplification depression to be the fragment of 626bp to 812 Nucleotide of AF357007, and this amplified production is carried out SSCP detect, and all obtains two electrophoretic bands; Two band furthest; Band and thinner do ++ genotype, two bands are nearest, and what band was also thinner is the BB genotype; Two bands are thicker, and apart be between the above two for the B+ genotype..
3. method according to claim 2 is characterized in that: the amplimer of said PCR method is to for having sequence 1 and 2 described Nucleotide in the sequence table.
4. method according to claim 1 is characterized in that: the 746th Nucleotide of the FecB gene in the sheep genome of said detection depression is that still the suddenly change method of G of A is to utilize the nucleotide sequence GENBANK ACCESSION NUMBER in the sheep genome of PCR method amplification depression to be the fragment of 638bp to 777 Nucleotide of AF357007, and amplified production is carried out RFLP detect; If two bands can occur, and lay respectively at 140bp and 110bp, be the B+ genotype; If a band can occur; And be positioned at 140bp, for ++ genotype, if a band can occur; And be positioned at 110bp, be BB genotype (like Fig. 3).
5. method according to claim 2 is characterized in that: the amplimer of said PCR method is to for having sequence 3 and 4 described Nucleotide in the sequence table.
6. according to any described method among the claim 1-4, it is characterized in that: said reproductivity is every tire litter size.
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| CN109837352A (en) * | 2019-04-17 | 2019-06-04 | 锡林郭勒职业学院 | Identify the primer and method of sheep FecB gene SNP genotype |
| CN111560441A (en) * | 2020-05-28 | 2020-08-21 | 西北农林科技大学 | Method for rapidly identifying FecB gene by using sheep structural variation region |
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