CN104017808A - Genetic marker related to growth trait of goat and application thereof - Google Patents
Genetic marker related to growth trait of goat and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of molecular markers and particularly relates to a genetic marker related to growth trait of a goat and application thereof. The genetic marker is a gene fragment which is cloned from an SKIP gene of the goat, shown in a sequence table SEQID NO:1 and related to the growth trait of the goat, wherein the sequence length is 680bp. An allele mutant (base substitution) is arranged at 413bp of the sequence as shown in the sequence table SEQID NO:1, and the mutant leads to Bg1I-RFLP polymorphism. The invention further discloses a preparation method of the genetic marker and application of the genetic marker in detecting polymorphism of the growth trait of the goat. The genetic marker provided by the invention can be applied to marker assisted selection of the goat.
Description
Technical field
The invention belongs to animal molecular marker technical field, be specifically related to a kind of genetic marker relevant to goat growth trait and application.This genetic marker clone is from the gene fragment of a SKIP.
Background technology
Along with the raising of China's expanding economy and living standards of the people, the demand of mutton increases year by year.Than market pig, produce the shortcoming such as Native Breed of Goats ubiquity individuality is little, the speed of growth is slow, dressing percentage is low.The green goat new variety that meet the required fast growth of modern market and have lacol flavor are cultivated in research, and set up efficient breeding technique system, become the new trend of Mutton Sheep Industry.Along with the development of Protocols in Molecular Biology, it is one of main method of taking of current Application of Animal Genetic improvement engineering that molecular marker assisted selection is combined with traditional breeding way.Therefore finding the molecular genetic marker mutually chain with goat growth trait locus, be applied to molecular marker assisted selection and cultivate mutton sheep new variety, is emphasis and the urgent problem of for some time goat biology field research at present and in the future.
First SKIP (5 ' inositol monophosphate enzyme that skeletal muscle is rich in kidney) gene is found by (2000) such as Ijuin and is separated, it is wide expression in each tissue, especially at heart, high expression level (Ijuin in skeletal muscle and kidney, T., et al., Identification and characterization of a novel inositol polyphosphate5-phosphatase.Journal of Biological Chemistry, 2000.275 (15): 10870-10875.).SKIP is accredited as by hydrolysis PI (3,4,5) P3 is PI (3,4) P2 and regulate and control the 5'-lipid Phosphoric acid esterase (Ijuin of insulin signaling, T.and T.Takenawa, SKIP negatively regulates insulin-induced GLUT4translocation and membrane ruffle formation.Mol Cell Biol, 2003.23 (4): 1209-1220.).PI (3, 4, 5) the downstream Akt signal of P3 plays an important role in myogenic differentiation process: the progress (Boonstra of G1 → S phase in the adjustable cell cycle, J., Identification of a restriction point at the M/G1transition during the ongoing cell cycle.Adv Enzyme Regul, 2007.47:208-221.), controlling the expression that myocyte breaks up early stage myogenin, maturation (the Rotwein of myotube, P.and E.M.Wilson, Distinct actions of Akt1and Akt2in skeletal muscle differentiation.J Cell Physiol, 2009.219 (2): 503-511.) etc.In addition, the initial also very important (Hribal of the downstream targets mTOR of Akt to myogenic differentiation, M.L., et al., Regulation of insulin-like growth factor – dependent myoblast differentiation by Foxo forkhead transcription factors.J Cell Biol, 2003.162 (4): 535-541.).Overexpression SKIP gene inhibition in sarcoplast short differentiation factor IGF2 express and the report of PI3K-AKT signal further illustrates the myogenic differentiation (Ijuin that SKIP has participated in Skeletal Muscle Cell, T.and T.Takenawa, Role of phosphatidylinositol3,4,5-trisphosphate (PIP3) 5-phosphatase skeletal muscle-and kidney-enriched inositol polyphosphate phosphatase (SKIP) in myoblast differentiation.J Biol Chem, 2012.287 (37): 31330-31341.).The report of many species all shows that muscle specific gene SKIP and myofiber grow closely related.The weight of SKIP genetic heterozygosis mutant mice soleus muscle and quadriceps muscle of thigh significantly improves (Ijuin, T., et al., Increased insulin action in SKIP heterozygous knockout mice.Molecular and Cellular Biology, 2008.28 (17): 5184-5195.).The also QTL of many distribution influence correlated character of the location of this gene in the economic animal such as pig, ox genome, as pig myofiber number with thigh is heavy and calf birth is heavy.And this intragenic polymorphic multinomial carcass trait (Xiong such as longissimus dorsi muscle height that can remarkably influenced Large white×Meishan pig F2Dai colony, Q., et al., Characterization of porcine SKIP gene in skeletal muscle development:polymorphisms, association analysis, expression and regulation of cell growth in C2C12cells.Meat Sci, 2012.92 (4): 490-497.).It is blank that yet the research of SKIP gene in goat still belongs to, and the polymorphic information of gene has not yet to see report.
Consider the Main Function that SKIP grows myofiber, the research of carrying out SKIP gene in goat is necessary.The polymorphism of research gene mutation site in colony, and to carry out proterties association analysis be to excavate the relation between molecule marker and proterties, very important means of research gene function.So applicant has carried out polymorphism research and association analysis in goat to this gene, for better using this gene in mutton sheep breed improvement, applicant provides important theory support.
Summary of the invention
The object of the invention is to obtain a genetic marker relevant to goat growth trait, by the specific DNA fragment of cloned goat SKIP gene, and through association analysis, obtain a genetic marker detecting for goat growth trait, this mark belongs to a kind of new mark, can be applied in the association analysis of marker assisted selection of goat.
The present invention realizes by following technical proposal:
Applicant, by screening, obtains one for detection of the genetic marker of goat growth trait, and the nucleotide sequence of this mark is as follows:
GGCTTCCCTTCTCAGCATACATGTCGTGACGTGGAATGTGGCCTCTGCCGCACCCCCTCCAGACCTCAGTGATCTGCTTCAGCTGAACAACCTGAACCTGAACCTGGACATATATGTCATTGGGTAGGTGTCCACAGGACCCGTCACGCATCCCTGTGTCTGAAATCAGGGTCAGGTAAGCCTGGCCAGTCCCAAATTGTCCCCACGGGGAACCACTATAACCTGTTCGCCCTTGCCCTGGAGTGTGAAGGCCAAGAGCTTTCAGCCTGCATCAGAGCCAGGCCACATCTGTTGTCCTGGGTCAGCAGTGGCAAGGGGCTGGCGAGCGACAGCCACAGGGGAGGGAGAACCCAGGGCTAGACAGTCGGCTGAGGGCAGTGCTGGATGTCGGAGCAGAGGCAGGGCCCTCCAGRCCAGGGGGCAGACCTCTGATCTGGGACGGGCAGTTTGCAGGAACTGAACTGTGGGATCATGAGCCTCCTTTCCGACACTGCCTTTGAAGACCCATGGAGCAGTTACTTCATGGACGTGCTTTCCCCTCTGAGCTTCGTCAAGGTAACTTGCAAGTTCATGGGCAATTGGGAAATGTGTATCACCTCTTATTACTAATCCCTAGTCCTTTGTCTCACAGTCCAGCTTTCATGCTATTACCCTTTTATTTAAAGACCCAAACCCCAGTT
At " R " shown in said gene fragment (sequence) (i.e. the allelic sudden change at 413bp place), be G or A, this sudden change causes Bg II-RFLP polymorphism.
Applicant has designed a kind of primer pair (being also the primer pair of amplification genetic marker of the present invention) of increase goat SKIP gene and this gene fragment sudden change of detection, and the DNA sequence dna of this primer pair is as follows:
Forward primer: GGCTTCCCTTCTCAGCATA,
Reverse primer: AACTGGGGTTTGGGTCTTT.
We have set up a kind of preparation method of the genetic marker relevant to goat growth trait, and the method is to comprise the following steps:
With sheep SKIP genomic dna and cDNA, be information probes, do homologous sequence screening, obtain the genome sequence of SKIP on the relevant karyomit(e) of goat.From Goat Blood, extract genomic dna, according to SKIP gene order design primer, the DNA sequence dna of this primer pair is as shown in sequence table SEQ ID NO:2 and SEQ ID NO:3, with the primer pair shown in sequence table SEQ ID NO:2 and SEQ ID NO:3, in goat genomic dna, carry out pcr amplification, PCR product purification, cloning and sequencing, obtain as sequence table SEQ ID NO:1.
Genetic marker of the present invention can be applied in goat growth trait detection.Wherein designed primer pair also can be applicable in the detection of goat growth trait.
More detailed technical scheme is as described in < < embodiment > >.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 be clone with the gene order fragment of goat growth trait genes involved SKIP.Sequence length is 680bp, wherein at the 413bp place of this sequence, exists a G/A to replace (allelic mutation, we are by this mutational site writing " R ").Sequence table SEQ ID NO:2 and 3 is nucleotide sequences of the primer pair that designs of the present invention, and it is positioned at 1-19 position and the 662-680 position of SEQ ID NO:1 sequence.
Fig. 1: be the present invention that clone with partial nucleotide sequence goat growth trait genes involved SKIP, in figure, shown 1 pleomorphism site.In figure: underscore is partly primer sequence, the base in bracket is base mutation, and this sudden change is with R representative in claims of the present invention, and the two is same implication.
Fig. 2: the gene mutation site order-checking color atlas that in the present invention, the SKIP portion gene group sequence direct Sequencing of pcr amplification detects.
Fig. 3: be the agarose gel electrophoresis figure of the SKIP portion gene group sequence of pcr amplification acquisition, description of symbols in figure: swimming lane: M is DL2000Marker.
Fig. 4: goat SKIP gene PCR-Bg1I-RFLP detected result.Description of symbols in figure: swimming lane M is DL2000Marker; AA genotype, 680bp; AG genotype, 680bp, 410bp, 270bp; GG genotype, 410bp, 270bp.
Embodiment
Embodiment 1:
(1) clone of .SKIP Gene Partial genome sequence and the foundation of pleiomorphism detecting method
(1) design of primers:
With (the GenBank number of including: NC_019468.1) be information probes of sheep SKIP genomic dna, utilize the BLAST instrument in NCBI in GenBank goat Genomes database, to do homologous sequence screening, obtain and be positioned at the homology SKIP genome sequence that on No. 19 karyomit(e)s of goat, similarity is 97%.According to goat SKIP genome sequence design of amplification primers (forward primer 5'GGCTTCCCTTCTCAGCATA3'; Reverse primer 5'AACTGGGGTTTGGGTCTTT3').Take Macheng black goat and boer goat as test colony, extract the poba gene group DNA of goat, using the DNA mixing constructed dna pond of two Goats Breeds of gained as template amplification goat SKIP portion gene segment.
Through PCR product purification and order-checking, and obtain the nucleotide sequence (seeing Fig. 1) as shown in sequence table SEQ ID NO:1 by sequential analysis; By order-checking, obtain color atlas as shown in Figure 2, find to exist 1 place's single nucleotide polymorphism (SNP).That is: at the 413bp place of SEQ ID N0:1, there is a base mutation of a G/A.
(2) purifying of PCR product, Cloning and sequencing
Pcr amplification: the sequence shown in SEQ ID NO:1.Reaction cumulative volume 25 μ L:10 * Tap buffer2.5 μ L, dNTP final concentration are 200 μ M, and forward and reverse primer is respectively 0.2 μ M, 1U Taq archaeal dna polymerase (purchased from precious biotechnology Dalian company limited).Pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 40s that then circulate 35 times, 59 ℃ of 45s, 72 ℃ of 30s, last 72 ℃ are extended 10min.PCR reaction product detects with 1.5% agarose gel electrophoresis.
The sequence of above-mentioned forward and reverse primer is as follows:
Forward primer: GGCTTCCCTTCTCAGCATA,
Reverse primer: AACTGGGGTTTGGGTCTTT.
The purifying of PCR product: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5mL centrifuge tube, with sanprep pillar DNA glue, reclaim test kit (purchased from Shanghai Sheng Gong biotechnology company limited) purifying DNA fragment.Concrete steps are as follows: add 700 μ L sol solutionses, 50-60 ℃ of water-bath is to glue thoroughly melts, and while adding hot melt adhesive, every 2min mixes once, is cooled to room temperature; Centrifugal column is put into collection tube, mixed solution is moved to centrifugal column, room temperature is placed 2min; The centrifugal 1min of 9000r/min, now DNA is adsorbed on post; Outwell waste liquid in collection tube, centrifugal column is put into same collection tube, add 700 μ L elutriants, the centrifugal 1min of 12000r/min; Outwell the waste liquid in collection tube, the centrifugal 1min of 12000r/min; Centrifugal column is put into a preprepared sterilizing 1.5mL centrifuge tube, add 40 μ L elutriants or distilled water (pH>7.0), room temperature or 37 ℃ place 2-3min (improve eluting temperature to 55-80 ℃ of elution efficiency that is conducive to improve DNA, can wash-out twice.); The centrifugal 1min of 12000r/min, the liquid in centrifuge tube is the DNA fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
(3) foundation of PCR-Bg1I-RFLP pleiomorphism detecting method
Above-mentioned PCR product is through order-checking (order-checking entrusts Shanghai Sheng Gong biotechnology company limited to carry out), and the size that obtains fragment is 680bp, and its nucleotide sequence is as shown in sequence table SEQ ID NO:1.Through ClusterW software, carry out sequence alignment, find to be wherein arranged in the G/A of this fragment 413bp place (intron 2) variation and caused that Bg1I restriction enzyme site (GCCNNNN ↓ NGGC) polymorphism changes.
Endonuclease reaction system is 10 μ L, wherein comprises: 10 * buffer1 μ L, restriction enzyme (10U/ μ L) 0.1 μ L, PCR product 3.9 μ L, ddH
2o5 μ L, puts 37 ℃ of isothermal reactions and spends the night, and enzyme cutting type result is observed and recorded to the sepharose gel electrophoresis analysis by 1.8% at gel imaging system.This site of Bg1I nonrecognition when the base at 452bp place is all A, be designated as AA type (680bp), when mutational site base is all G, Bg1I enzyme is identified this site, is designated as GG type (410bp+270bp), is designated as AG type 680bp+410bp+270bp when A and G exist).The enzyme cutting type result of the PCR-Bg1I-RFLP of goat SKIP gene is as Fig. 4.
Embodiment 2:
318 boer goats and Macheng black goat filial generation individuality for the test materials of association analysis with Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences's sheep stud.The proterties of analyzing is mainly growth traits.Growth traits comprises respectively body weight, height, body plagioclase, chest measurement, circumference of cannon bone of at 3,6,12,18,24 monthly ages, recording goat individuality etc.First adopt the PCR-Bg1I-RFLP method that embodiment 1 sets up to carry out individual gene type, adopt (the SAS Institute Inc of SAS8.0 statistical software, Version8Edition) to data analysis, GLM program is carried out the association analysis between mark and proterties, REG program is calculated additive effect and the dominant effect of gene, and carry out test of significance, analyze the correlationship of goat SKIP gene Bg1I-RFLP different genotype and goat growth trait.Set up statistical model, except stochastic effect, other factors are fixed effect, the model that adopts as follows:
Yijklm=μ+Si+Yj+Gk+Fl+Mm+eijklm, wherein Yijklm is proterties observed value, and μ is population average, and Si is seasonal effect, and Yj is time effect, and Gk is genetic effect, and Fl is paternal effect, and Mm is maternal effect, eijklm is random residual.Additive effect represents respectively AA, AG, GG with-1,0,1; Dominant effect is with 0,1, and 0 represents respectively AA, AG, GG.
Chest measurement as can be seen from Table 1 increases day by day SKIP gene polymorphic and the goat 3-6 month, March chest measurement, December circumference of cannon bone, December body weight, the tenth August height be significantly or utmost point significant correlation, wherein GG genotype is preponderant genotype, with respect to AA genotype, GG genotype individuality has higher chest measurement in March, December circumference of cannon bone, December body weight and the tenth height in August (in Table 1).
The relation of table 1 SKIP gene G/A sudden change and goat growth trait
Note: chest measurement increases day by day the DCHC1:3-6 month; CHC1: March chest measurement; CC3: December circumference of cannon bone; BW3: December body weight; BH4: the tenth August height.Significant difference (* P ﹤ 0.05) between the least square average (± standard error) that contains different letters.
Claims (5)
1. a genetic marker relevant to goat growth trait, its nucleotide sequence is as follows:
GGCTTCCCTTCTCAGCATACATGTCGTGACGTGGAATGTGGCCTCTGCCGCACCCCCTCCAGACCTCAGTGATCTGCTTCAGCTGAACAACCTGAACCTGAACCTGGACATATATGTCATTGGGTAGGTGTCCACAGGACCCGTCACGCATCCCTGTGTCTGAAATCAGGGTCAGGTAAGCCTGGCCAGTCCCAAATTGTCCCCACGGGGAACCACTATAACCTGTTCGCCCTTGCCCTGGAGTGTGAAGGCCAAGAGCTTTCAGCCTGCATCAGAGCCAGGCCACATCTGTTGTCCTGGGTCAGCAGTGGCAAGGGGCTGGCGAGCGACAGCCACAGGGGAGGGAGAACCCAGGGCTAGACAGTCGGCTGAGGGCAGTGCTGGATGTCGGAGCAGAGGCAGGGCCCTCCAGRCCAGGGGGCAGACCTCTGATCTGGGACGGGCAGTTTGCAGGAACTGAACTGTGGGATCATGAGCCTCCTTTCCGACACTGCCTTTGAAGACCCATGGAGCAGTTACTTCATGGACGTGCTTTCCCCTCTGAGCTTCGTCAAGGTAACTTGCAAGTTCATGGGCAATTGGGAAATGTGTATCACCTCTTATTACTAATCCCTAGTCCTTTGTCTCACAGTCCAGCTTTCATGCTATTACCCTTTTATTTAAAGACCCAAACCCCAGTT
In above-mentioned sequence, the R of the 413rd is G or A, and this sudden change causes Bg1I-RFLP polymorphism.
2. detect the primer pair of the base mutation of genetic marker as claimed in claim 1, its DNA sequence dna is as follows:
Forward primer: GGCTTCCCTTCTCAGCATA,
Reverse primer: AACTGGGGTTTGGGTCTTT.
3. a preparation method for the genetic marker relevant to goat natural disposition shape, is characterized in that, comprises the following steps:
With sheep SKIP genomic dna and cDNA, it is information probes, do homologous sequence screening, obtain the genome sequence of SKIP on the relevant karyomit(e) of goat, from Goat Blood, extract genomic dna, Macheng black goat is mixed and is built into DNA pond with the DNA of boer goat, primer according to reference sequences design as shown in claim 2, carry out pcr amplification, obtain goat part SKIP genomic fragment, direct Sequencing after PCR product purification, obtains the nucleotide sequence as shown in claim 1 by sequential analysis.
4. the application of genetic marker claimed in claim 1 in goat growth trait correlation detection.
5. the application of primer pair claimed in claim 2 in goat growth trait correlation detection.
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| CN107022022A (en) * | 2016-03-02 | 2017-08-08 | 湖北省农业科学院畜牧兽医研究所 | Three kinds of goat MC1R defects mutant and its application |
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| WO2022151020A1 (en) * | 2021-01-13 | 2022-07-21 | 深圳华大生命科学研究院 | Nucleic acid molecule related to type of goat horn and use thereof |
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| CN107022022B (en) * | 2016-03-02 | 2020-09-11 | 湖北省农业科学院畜牧兽医研究所 | Three goat MC1R defect mutants and application thereof |
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| CN109680070B (en) * | 2018-11-01 | 2021-11-26 | 天津奥群牧业有限公司 | SNP (Single nucleotide polymorphism) marker and molecular marker remarkably related to Australia white sheep hoof color and application |
| CN109609656A (en) * | 2018-12-07 | 2019-04-12 | 湖北省农业科学院畜牧兽医研究所 | A kind of goat circular rna and its identification method and functional application |
| CN109609656B (en) * | 2018-12-07 | 2022-04-19 | 湖北省农业科学院畜牧兽医研究所 | Goat circular RNA circ _ ZCCHC2 and identification method and application thereof |
| CN112176076A (en) * | 2020-11-05 | 2021-01-05 | 湖北省农业科学院畜牧兽医研究所 | NFAT5 gene molecular marker related to goat growth traits and application thereof |
| WO2022151020A1 (en) * | 2021-01-13 | 2022-07-21 | 深圳华大生命科学研究院 | Nucleic acid molecule related to type of goat horn and use thereof |
| CN117385049A (en) * | 2023-08-02 | 2024-01-12 | 湖北省农业科学院畜牧兽医研究所 | Application of SNP molecular marker rs655589732 related to goat growth traits |
| CN117385049B (en) * | 2023-08-02 | 2024-04-26 | 湖北省农业科学院畜牧兽医研究所 | Application of SNP molecular marker rs655589732 related to goat growth traits |
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