CN101798598B - Maternal genetic marker for identification of domestic cattle of bos and identification method and application thereof - Google Patents
Maternal genetic marker for identification of domestic cattle of bos and identification method and application thereof Download PDFInfo
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Abstract
The invention discloses maternal genetic markers for identification of domestic cattle of bos and an identification method and application thereof, belonging to the technical field of genetic marker-assisted selection. The maternal genetic marker for ordinary cattle comprises specific deletion gap249, the maternal genetic marker for zebu cattle comprises specific deletion gap191, and the maternal genetic marker for gayal and yaks comprises specific deletion gap179-186. In the identification method, the genetic markers of specific deletion of the three types of cattle are used for identification, and the results are completely consistent with cluster results. Therefore, the identification method can completely substitute the complex cluster analysis for convenient and effective identification of zebu cattle, ordinary cattle or gayal and yaks, and can overcome the inaccuracy of cattle appearance identification. The primers provided by the invention for simultaneous expansion of the DNA of the four types of cattle are shown in SEQ ID No. 5 and SEQ ID No. 6.
Description
Technical field
The invention belongs to ox genetic marker assisted Selection technical field, be specifically related to one group and can carry out purebred cattle and differentiate method and application with the matrilinear inheritance mark of cross-bred ox differentiation and primer thereof, discriminating ox kind.
Background technology
See that from phenotype ox (Domestic cattle) can be divided into two main types: zebu (humped zebu) and common ox (humpless taurine).Two types are two of Bos (Bos) independent to plant (Bosindicus and Bos taurus) are by aurochs (aurochs; Twice independent domestication Bos primigenius) (Loftus R.T.et al.Evidence for two independent domesticationsof cattle.Proc.Natl.Acad.Sci.1994,91:2757-2761).Bos also comprises yak (Yak, Bos grunniens) and two tame cowboying kinds of gayal (Gayal, Bos frontalis).
Yak (Bos grunniens) is to be distributed in the geographic rare family of high mountain cowboying kind Qinghai-Tibet and that adjoin.Several local monoids such as existing Kowloon yak, middle pasture yak, Tibet high mountain type yak, Part of Qinghai Plateau type yak, White Yak in Tianzhu, crust state yak, the hollow yak of wheat.The yak Bos mutus that to be the national domestication in plateau distribute with the area and coming is high guanaco fortune, usage, meat, the main poultry kind of giving milk.
Gayal (Bos frontalis) is distributed in the northern mountain area of nation's height above sea level more than 1500 meters of admiring of the Assam of India, eastern Bangladesh, Bhutan and Burma abroad; Only be distributed in western basin, Dulong river, Yunnan and adjacent domain thereof in China, the domestication of approximately having experienced more than 200 year is historical.The Yunnan gayal cries " Dulong ox " again in the locality.The India gayal is very similar with gaur (Bos gaurus) appearance, and many zoologists advocate that the India gayal is the domestic type of gaur.Known that the ox caryogram is 2n=60; Gayal 2n=58; Gaur 2n=58; Gayal and gaur also have same Robertsonian translocation (No. 2 and No. 28 karyomit(e)s) (Gallagher D.S.et al.Chromosome conservation in the Bovidae.JHered.1992,83:287-298; Nie L.et al.The gayal`s genetic diversity andits genetic structure`s enzyme analysis.Acta Genet Sin.1995,22:185-191; Chi J.et al.New insights into the karyotypic relationshipsof Chinese muntjac (Muntiacus reevesi); Forest muskdeer (Moschusberezo vskii) and gayal (Bos frontalis) .Cytogenet.Genome Res.2005; 108:310-316); Both possess identical caryogram, and gayal is tamed by gaur in prompting.Also support gayal and gaur to have nearest sibship (VerkaarE.L.C.et al.Maternal and Paternal Lineages in Cross-Breeding BovineSpecies.Has Wisent a Hybrid Origin from the molecular Evidence of maternal Mitochondrial DNA? Mol.Biol.Evol.2004,21:1165-1170).
Species hybridization between the ox kind is general.Between common ox and the zebu and the filial generation between aurochs (Bisonbonasus) and the bison (Bison bison) all educate and Java wild ox and zebu, yak and the male sterile of zebu filial generation entirely.The local zebu maternal side of Indonesia contains Java wild ox (Bos javanicus) mtDNA haplotype, and the local zebu maternal side of Nepal comprises yak (Bosgrunniens) mtDNA haplotype.Above ox kind chromosome number all identical (caryogram is 2n=60).And gayal (2n=58) is different with common ox and zebu (2n=60) chromosome number, but gayal also can hybridize just male sterile with ox.
Guarantor's kind, pure breeding and the selection cross of common ox, zebu, yak and four ox kinds of gayal all need be known their genetic background, and particularly individual being difficult to of hybridization differentiated its blood lineage source through the appearance build.Prior art mainly adopts appearance to identify and cluster analysis that the former is experimental to the discriminating of each tame ox of Bos, can not prove conclusively, and the latter then needs the Diversity Detection method and complicated clustering method of specialty.Therefore, be applicable to the molecule marker of differentiating each tame ox of Bos from one of heredity screening, undoubtedly will be for purebred and the individual evaluation of hybrid are provided convenience, accurate and effective means.
Still there are not at present the paternal line of differentiating the ox kind, the patent report of maternal molecule marker.Existing so far a large amount of ox mtDNA D-loop sequences report, but this distinguishes and is the hypervariable region, and it is infeasible to seek mark through base pair substitution polymorphism, the special insertion of ox kind/disappearance mark that the present invention provides mtDNA D-loop district to guard through the great amount of samples data.
Summary of the invention
The technical problem that the present invention will solve is to overcome the existing appearance that the discriminating of each tame ox of Bos is adopted to identify with cluster analysis and have inaccurate, inconvenient technological deficiency, its objective is provide a kind of easy, differentiate the matrilinear inheritance mark of Bos (Bos) the common ox of each tame cowboying kind (Bos taurus), zebu (Bos indicus), gayal (Bos frontalis), yak (Bos grunniens) and hybridization individuality thereof accurately and efficiently.This genetic marker is to belong to one section specific DNA fragment that the mtDNA sequence clone obtains from Bos; Each this dna fragmentation of tame ox all possesses special deletion mutantion; Again because mtDNA is a matrilinear inheritance; Therefore this genetic marker is a kind ofly to differentiate relevant matrilinear inheritance mark with the ox kind, and while the present invention also provides the amplimer of said matrilinear inheritance mark and with method and the application of said each tame ox of matrilinear inheritance mark discriminating Bos.
The present invention realizes through following technical proposal:
1, one group of matrilinear inheritance mark of differentiating each tame ox of Bos; Each tame ox of described Bos is common ox, zebu, gayal and yak; The nucleotide sequence of the matrilinear inheritance mark of common ox is shown in SEQ ID No.1; The nucleotide sequence of the matrilinear inheritance mark of zebu is shown in SEQ ID No.2; The nucleotide sequence of the matrilinear inheritance mark of gayal is shown in SEQ ID No.3, and the nucleotide sequence of the matrilinear inheritance mark of yak has a deletion mutantion at the nucleotide sequence 249bp place shown in the SEQ ID No.1 of the matrilinear inheritance mark of described common ox shown in SEQ ID No.4; Be designated as gap249, be the special disappearance genetic marker of common ox; There is a deletion mutantion at nucleotide sequence 191bp place shown in the SEQ ID No.2 of the matrilinear inheritance mark of described zebu, is designated as gap191, is the special disappearance genetic marker of zebu; There is an identical deletion mutantion at nucleotide sequence 179-186bp place shown in the matrilinear inheritance mark SEQ ID No.4 of nucleotide sequence 179-186bp place shown in the SEQ ID No.3 of the matrilinear inheritance mark of described gayal and yak; Be designated as gap179-186, be gayal and the special disappearance genetic marker of yak common.
2, the primer of the matrilinear inheritance mark of a kind of each tame ox of Bos of increasing; Described primer is made up of forward primer and reverse primer; The dna sequence dna of described forward primer is shown in SEQ ID No.5, and the dna sequence dna of described reverse primer is shown in SEQ ID No.6.
3, the described one group of preparation method who differentiates the matrilinear inheritance mark of each tame ox of Bos of above-mentioned the 1st technical scheme, step is following:
(1) identifies that through the individual appearance in sample source the ox of selecting with the breeding registration confirmed is yak, gayal, zebu and the common ox sample of Bos, and extract the poba gene group DNA of these 4 kinds of oxen respectively;
The genomic dna of 4 kinds of oxen of (2) step (1) being extracted with the described primer of above-mentioned the 2nd technical scheme carries out pcr amplification respectively; The purified recovery of PCR product; Order-checking is carried out sequence alignment each other with the sequence of described 4 kinds of oxen of measuring, obtains following matrilinear inheritance mark:
1) the matrilinear inheritance mark of common ox; Its nucleotide sequence shown in SEQ ID No.1, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 249bp place; Be designated as gap249, be the special disappearance genetic marker of common ox;
2) the matrilinear inheritance mark of zebu; Its nucleotide sequence shown in SEQ ID No.2 in the sequence table, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 191bp place; Be designated as gap191, be the special disappearance genetic marker of zebu;
3) the matrilinear inheritance mark of gayal, its nucleotide sequence is shown in SEQ ID No.3; The matrilinear inheritance mark of yak, its nucleotide sequence is shown in SEQ ID No.4; With the nucleotide sequence 179-186bp place shown in the described SEQ ID No.4 an identical deletion mutantion is arranged all at the nucleotide sequence 179-186bp place shown in the described SEQ ID No.3; Be designated as gap179-186, be gayal and the special disappearance genetic marker of yak common.
4, a kind of each tame ox of Bos and individual method of hybridization thereof differentiated, each tame ox of described Bos is common ox, zebu, gayal and yak, the steps include:
Extract cow genome group DNA from the bovine blood of ox kind to be identified, in the cow genome group DNA that is extracted, carry out pcr amplification with the described primer of above-mentioned the 2nd technical scheme, the PCR product purification reclaims and order-checking; The sequence of measuring and the sequence of the described matrilinear inheritance mark of above-mentioned the 1st technical scheme are carried out sequence alignment; Special disappearance genetic marker with the described matrilinear inheritance mark of above-mentioned the 1st technical scheme contains is differentiated the matrilinear inheritance background of individuality to be measured; See from the maternal side source; The individuality to be measured that contains special disappearance gap249 is common ox; The individuality to be measured that contains special disappearance gap191 is a zebu, and the individuality to be measured that contains special disappearance gap179-186 is gayal or yak.
Because hybridization did not also take place at present for gayal and yak, and can distinguish the two from profile, matrilinear inheritance mark provided by the invention does not relate to the evaluation between gayal and the yak.
Extraction cow genome group DNA described in the individual method of each tame ox of above-mentioned discriminating Bos and hybridization thereof, in the cow genome group DNA that is extracted, carry out pcr amplification, the PCR product purification reclaims and order-checking and the sequence measured are the ordinary skill in the art and method through software editing and comparison.
5, the matrilinear inheritance with each tame ox of the described discriminating Bos of above-mentioned the 1st technical scheme is marked at discriminating of each tame ox of Bos and the application in the genetic background detection.Application in discriminating and genetic background that the matrilinear inheritance of each tame ox of the described discriminating Bos of above-mentioned the 1st technical scheme of described usefulness is marked at each tame ox of Bos detect is to differentiate and the application of genetic background in detecting each tame ox of Bos with the special disappearance genetic marker of common ox that is designated as gap249, the gayal and the special disappearance genetic marker of yak common that are designated as the special disappearance genetic marker of zebu of gap191 and are designated as gap179-186 respectively.
Can also carry out pcr amplification to the cow genome group DNA that in application, is extracted with the primer of the matrilinear inheritance mark of each tame ox of the described amplification Bos of above-mentioned the 2nd technical scheme in the application in discriminating and genetic background that the matrilinear inheritance of each tame ox of the described discriminating Bos of above-mentioned the 1st technical scheme of described usefulness is marked at each tame ox of Bos detect.
The invention has the beneficial effects as follows:
Differentiate gap191, gap249 and the special disappearance genetic marker of three ox kinds of gap179-186 that relevant matrilinear inheritance mark contains respectively with provided by the present invention with the ox kind; Only need to detect the special disappearance genetic marker of ox kind; Just can reach the purpose of differentiating the ox kind and need not to carry out complicated cluster analysis; The special disappearance genetic marker of above-mentioned three ox kinds provided by the present invention is to ox kind division result and cluster result strict conformance, and its discrimination method is easy, accurate and effective.
Description of drawings
Fig. 1 is each tame cowboying kind mtDNA haplotype NJ phylogenetic tree of Bos (Bos).Comprising 659 haplotypes altogether, serves as outer crowd with buffalo (Buffallo), and copolymerization is common ox, zebu, yak and four of gayals (the bootstrap value all is more than 70%, 1000 repetitions).Each ox kind haplotype quantity is too many, and haplotype omits in the drawings.Yak prop up with gayal Zhi Suoyou individuality all comprise common disappearance gap179-186, zebu props up and common ox Zhi Suoyou individuality all comprises gap191 and two special disappearances of gap249 respectively.
Fig. 2 is a Yunnan local ox kind mtDNA haplotype NJ phylogenetic tree.Yunnan gayal (Dulong ox) props up gayal (Bos frontalis), common ox (Bos aurus) props up and zebu (Bos indicus) Zhi Douyou distributes, and (the bootstrap value all is more than 70%; Repeat for 1000 times), and the Yunnan ox distributes common ox (Bos taurus) and zebu (Bos indicus) Zhi Douyou.All individualities of every all contain special disappearance mark, and cluster result is divided in full accord with the population of disappearance mark." Hap+ numeral " expression haplotype numbering among the figure.
Embodiment
Preparation process is following:
(1) according to common ox mtDNA complete sequence (in the GenBank accession number is: V00654) design primer; Described primer is made up of forward primer and reverse primer; The dna sequence dna of described forward primer is shown in SEQ ID No.5, and the dna sequence dna of described reverse primer is shown in SEQ ID No.6.
Above-mentioned primer is to being conservative primer, the mtDNA sequence of the yak of increasing simultaneously, gayal, zebu and these 4 ox kinds of common ox.Above-mentioned primer is synthetic by giving birth to worker's biotechnology (Shanghai) company.
(2) select Bos (Bos) yak (be directed to Chinese yunnan and economize enlightening celebrating state, primary source is economized the Xianggelila country in Chinese yunnan), gayal (be directed to Chinese yunnan province Gong Shan county, primary source is in Chinese yunnan province Gong Shan county), zebu (brahman through sample source individual appearance evaluation and breeding registration confirmed; Be directed to Yunnan Province, Kunming leimocole research institute; Primary source is in Queensland, Australia) and each 10 in common ox (the Qin Chuan ox is directed to Chinese Xian City, Shanxi Province Yangling District Xibei Univ. of Agricultural & Forest Science & Technology animal technical college, and primary source is in Fufeng County, Chinese Shaanxi Province) sample; And extract the poba gene group DNA of these 4 kinds of oxen respectively; Its process for extracting is by phenol/chloroform ordinary method extracting, " genetically engineered ", building intellectual circle; 2002, Science Press.
The genomic dna of 4 kinds of oxen of (3) step (2) being extracted with the described primer of step (1) carries out pcr amplification respectively.PCR reaction system 25 μ L, each component concentrations is in the system: 50ng template DNA, 1 * Taq buffer 2.5mmol/L, 1.5mmol/L MgCl
2, 2.5mmol/L dNTPs, 0.5mmol/LPCR primers, 2U TaqDNA polysaccharase (MBI), the working procedure of PCR is following: 94 ℃ of 4min of sex change in advance; 94 ℃ of 45s, 56 ℃ of 40s, 72 ℃ of 40s, 35 circulations; Last 72 ℃ are continued to extend 10min.The PCR product that obtains reclaims test kit (purchasing China Shun bio-engineering corporation in Shanghai) through a small amount of glue and reclaims, according to the specification sheets recovery and the purifying of this test kit.Directly measure the double chain DNA fragment sequence with the thermally denature method then.Sequencing reagent adopts BigDye Terminator Cycle Sequence ready Reaction Kit (purchasing the PerkinElmer company in the U.S.), and sequencing reaction is undertaken by the method that the said firm is recommended.PCR product order-checking at present can easily send company to accomplish (like the big genome company of China).With the sequences of described 4 kinds of oxen of measuring through DNAstar software (DNASTAR, Madison, WI) editor with carry out sequence alignment each other, obtain as the matrilinear inheritance mark of next each tame ox of group discriminating Bos:
1) the matrilinear inheritance mark of common ox; Its nucleotide sequence shown in SEQ ID No.1, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 249bp place; Be designated as gap249, be the special disappearance genetic marker of common ox;
2) the matrilinear inheritance mark of zebu; Its nucleotide sequence shown in SEQ ID No.2 in the sequence table, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 191bp place; Be designated as gap191, be the special disappearance genetic marker of zebu;
3) the matrilinear inheritance mark of gayal, its nucleotide sequence is shown in SEQ ID No.3; The matrilinear inheritance mark of yak, its nucleotide sequence is shown in SEQ ID No.4; With the nucleotide sequence 179-186bp place shown in the described SEQ ID No.4 an identical deletion mutantion is arranged all at the nucleotide sequence 179-186bp place shown in the described SEQ ID No.3; Be designated as gap179-186, be gayal and the special disappearance genetic marker of yak common.
According to the above-mentioned gap249 of being designated as, gap191 and the special disappearance genetic marker of three ox kinds of gap179-186 to ox kind division result and cluster result strict conformance (seeing embodiment 3).Embodiment 3 all proves with these 3 special disappearance genetic markers with embodiment 4 and differentiates that the ox kind can replace complicated cluster analysis fully, can carry out the discriminating between zebu and common ox, zebu and gayal, zebu and yak, common ox and gayal and common ox and the yak convenient, effectively.Because hybridization did not also take place at present for gayal and yak, only can distinguish the two from profile, genetic marker provided by the invention does not relate to the discriminating between gayal and the yak.
Each tame ox of described Bos is common ox, zebu, gayal and yak, the steps include:
(1) respectively from the bovine blood extraction cow genome group DNA of ox kind to be identified, its process for extracting is by phenol/chloroform ordinary method extracting, " genetically engineered ", building intellectual circle, 2002, Science Press.
The genomic dna of 4 kinds of oxen of (2) respectively step (1) being extracted with 1 designed primer of embodiment carries out pcr amplification.PCR reaction system 25 μ L, each component concentrations is in the system: 50ng template DNA, 1 * Taq buffer 2.5mmol/L, 1.5mmol/L MgCl
2, 2.5mmol/L dNTPs, 0.5mmol/LPCR primers, 2U TaqDNA polysaccharase (MBI), the working procedure of PCR is following: 94 ℃ of 4min of sex change in advance; 94 ℃ of 45s, 56 ℃ of 40s, 72 ℃ of 40s, 35 circulations; Last 72 ℃ are continued to extend 10min.The PCR product that obtains reclaims test kit (purchasing China Shun bio-engineering corporation in Shanghai) through a small amount of glue and reclaims, according to the specification sheets recovery and the purifying of this test kit.Directly measure the double chain DNA fragment sequence with the thermally denature method then.Sequencing reagent adopts BigDye Terminator Cycle Sequence ready Reaction Kit (purchasing the PerkinElmer company in the U.S.), and sequencing reaction is undertaken by the method that the said firm is recommended.PCR product order-checking at present can easily send company to accomplish (like the big genome company of China).
(3) one group of sequence process DNAstar software (DNASTAR that differentiates the matrilinear inheritance mark of each tame ox of Bos that the sequence of measuring and the foregoing description 1 obtain; Madison; WI) editor and sequence alignment, the contained special disappearance genetic marker of matrilinear inheritance mark of 4 ox kinds that obtained by embodiment 1 is respectively differentiated the matrilinear inheritance background of individuality to be identified; See that from the maternal side source individuality to be measured that contains special disappearance gap249 is common ox, the individuality to be measured that contains special disappearance gap191 is a zebu, and containing the individuality to be measured that lacks gap179-186 is gayal or yak.
Because hybridization did not also take place at present for gayal and yak, only can distinguish the two from profile, genetic marker provided by the invention does not relate to the discriminating between gayal and the yak.
The special disappearance genetic marker of ox kind of embodiment 3 the present invention screening and cluster result consistence are relatively
Downloaded yak, gayal, zebu and the common ox sequence that comprises sequence fragment of the present invention from GenBank; Wherein 322 of yaks, 76 of gayals, 391 of zebus, 1511 of common oxes; Totally 2300 samples are downloaded the outer crowd that 7 buffalo sequences are done cluster analysis in addition.These sequences are through DNAstar software (DNASTAR; Madison; WI) editor and comparison, the polymorphic analysis of sequence is by MEGA 2.1 softwares (KumarS.et al.2001 MEGA.Molecular Evolutionary Genetics Analysis Software.Release 2.0.Arizona State University, Tempe A.Z.; USA) accomplish, deletion mutation is not considered in cluster analysis.Haplotype NJ tree and MP tree are by PAUP software (Swofford D.L.2000 PAUP.phylogenetic analysis using parsimony and other methods.Release 4.0.Sinauer Associates; Sunderland; Mass) accomplish; (Ronquist F.et al.MRBAYES 3:Bayesian phylogenetic inferenceunder mixed models.Bioinformatics 2003 19:1572-1574) makes up haplotype Bayes genealogical tree by MrBayes3.1.
Article 2300, sequence totally 659 haplotypes, more than three kinds of haplotype paper mulberry methods obtain consistent results, the NJ tree sees Fig. 1.Yak props up with gayal Zhi Suoyou individuality and all comprises common disappearance gap179-186 in the genealogical tree, and zebu props up with common ox Zhi Suoyou individuality and all comprises gap191 and two special disappearances of gap249 respectively.The result shows according to non-deletion sequence cluster result of ox kind and disappearance mark results strict conformance.
The application of the genetic marker of embodiment 4 the present invention screening in identifying Yunnan man ox
The mtDNA variety of Chinese Cattle shows; South and southwest ox kind maternal side mainly receive influence (Yu Y.et al.Mitochondrial DNA variation in cattle of south China:origin and introgression.Anim.Genet.1999, the 30:245-250 of India's zebu; Lai S.et al.Genetic diversity and origin of Chinese cattle revealed by mtDNA Controlregion sequence variation.Mol.Phylogenet.Evol.2006; 38:146-154), a lot of kinds all have the blood relationship of zebu and common ox concurrently.South Asia region, zebu country of origin is closed in Yunnan, should be the corridor that zebu is introduced China, and the Yunnan ox should have the mixed-blood genetic background of zebu and common ox.
And origin of Yunnan gayal and genetic background are also very complicated.Lan Hong etc. have analyzed the mtDNA polymorphum of 1 Yunnan gayal bull; The result shows that the mtDNA type of gayal is identical with zebu; Infer that the Yunnan gayal possibly be descendant (Lan H.et al.Yunnan cattle andgayal ' the s mtDNA research.Acta Genet.Sin.1993 of gayal and the hybridization of female zebu; 20,419-425).Malaysian dragon etc. is analyzed 12 Yunnan gayal Cytb Gene Partial sequences; The result show gayal be in the Bos (Bos) one and independently plant (Ma G.et al.Phylogenetic relationships andstatus quo of colonies for gayal based on analysis of cytochrome B genepartial sequences.J.Genet.Genomics 2008,34:413-419).Lee's generation equality has also been inquired into the taxonomy status of Yunnan gayal from the Cytb gene order; The result show gayal be likely gaur domestic type or the domestication kind (Li S.P.et al.Molecular phylogeny of the gayalinferred from the analysis of cyto-chrome b gene entire sequences.Hereditas 2007,30:65-70).Their result discloses the blood relationship that Yunnan gayal maternal side contains gayal, zebu and common ox.
Therefore, will have very important use value with the genetic marker of the present invention's screening and the complex inheritance background of detection method detection Yunnan gayal and Yunnan ox.
224 Yunnan gayals and local bovine blood sample are gathered in this experiment altogether; Wherein 104 of Yunnan gayals (are directed to Chinese yunnan province Gong Shan county; Primary source is in Chinese yunnan province Gong Shan county), 57 of mountain of papers peak, Yunnan oxen (are directed to Chinese yunnan province Guangnan County; Primary source is economized the Guangnan County in Chinese yunnan), 31 of Nujiang, Yunnan oxes (are directed to Chinese yunnan and economize Nu Jiangzhou; Primary source is in Chinese yunnan province Gong Shan county) and 32 of Deqen in Yunnan Province oxes (be directed to Chinese yunnan and economize enlightening celebrating state, primary source is economized the Xianggelila country in Chinese yunnan).Method according to embodiment 2 detects, and obtains the long mtDNA D-loop region sequence of 457bp.(DNASTAR, Madison WI) edit and compare these sequences, and three deletion mutantions that are designated as gap249, gap191 and gap179-186 respectively of matrilinear inheritance mark are identified individuality according to the present invention through DNAstar software.
Qualification result shows: in 104 Yunnan gayals that detect, wherein 20 belong to common ox type (gap249), and 51 belong to zebu type (gap191), and 33 belong to gayal type (gap179-186); In 57 mountain of papers peak oxen that detect, 48 belong to zebu type (gap191), and 9 belong to common ox type (gap249); In 31 Nujiang oxes that detect, 16 belong to common ox type (gap249), and 15 belong to zebu type (gap191); And in 32 enlightening celebrating oxes that detect, 4 belong to zebu type (gap191), and 28 belong to common ox type (gap249).It is individual that the result shows that Yunnan gayal and Yunnan ox all contain the assorted blood of significant proportion, for the guarantor of these ox kinds plants and selection cross provides clearly matrilinear inheritance background.
In order to verify exactness by disappearance labeled bracketing result; Present embodiment has carried out the haplotype genealogical tree by the molecule clustering method of embodiment 3 to above-mentioned Yunnan gayal and local ox again and has rebuild (Fig. 2), and cluster result and the present invention differentiate that the result of the special disappearance genetic marker discriminating that is designated as gap249, gap191 and gap179-186 respectively of adopting 4 kinds of ox matrilinear inheritance marks in the individual method of each tame ox of Bos and hybridization thereof is in full accord.
Sequence table
< 110>Yunnan Prov Agriculture University
< 120>one groups of matrilinear inheritance mark and discrimination method and application of differentiating each tame ox of Bos
<160>6
<170>PatentIn?version?3.3
<210>1
<211>457
<212>DNA
< 213>common ox (Bos taurus)
<220>
<221>mutation
<222>(249)..(249)
<400>1
gcaaggggta?atgtacataa?cattaatgta?ataaagacat?aatatgtata?tagtacatta 60
aattatatgc?cccatgcata?taagcaagta?catgacctct?a-tagcagta?cataatacat 120
ataattattg?actgtacata?gtacattatg?tcaaattcat?tcttgatagt?atatctatta 180
tatattcctt?accattagat?cacgagctta?attaccatgc?cgcgtgaaac?cagcaacccg 240
ctaggcag-g?gatccctctt?ctcgctccgg?gcccataaac?cgtgggggtc?gctatccaat 300
gaattttacc?aggcatctgg?ttctttcttc?agggccatct?catctaaaac?ggtccattct 360
ttcctcttaa?ataagacatc?tcgatggact?aatggctaat?cagcccatgc?tcacacataa 420
ctgtgctgtc?atacatttgg?tattttttta?ttttggg 457
<210>2
<211>457
<212>DNA
< 213>zebu (Bos indicus)
<220>
<221>mutation
<222>(191)..(191)
<400>2
gcaagaggta?atgtacataa?cattaatgta?ataaagacat?gatatgtata?tagtacatta 60
aattatatac?cccatgcata?taagcaagta?catgatctct?a-taatagta?cataatacat 120
acaattatta?attgtacata?gtacattata?tcaaatccat?cctcaacaac?atatctacta 180
tatacccctt?-ccactagat?cacgagctta?attaccatgc?cgcgtgaaac?cagcaacccg 240
ctaagcagag?gatccctctt?ctcgctccgg?gcccatagac?cgtgggggtc?gctatttaat 300
gaattttacc?aggcatctgg?ttctttcttc?agggccatct?catctaaagt?ggtccattct 360
ttcctcttaa?ataagacatc?tcgatggact?aatgactaat?cagcccatgc?tcacacataa 420
ctgtgctgtc?atacatttgg?tattttttta?ttttggg 457
<210>3
<211>457
<212>DNA
< 213>gayal (Bos frontalis)
<220>
<221>mutation
<222>(179)..(186)
<400>3
acagcgggat?acgtccataa?tattaatgta?acaaagacat?aacatgtata?tagtacatta 60
tattatatgc?cccatgcgta?taagcaagta?cttgaatttc?actaacagta?catagtacat 120
gagcttatta?atcgtacata?gcacataatg?tcaaatctac?ccttgacaac?atgcgtat— 180
------cccc?cccactagat?cacgagctta?atcaccatgc?cgcgtgaaac?cagcaacccg 240
ctaagcagag?gatccctctt?ctcgctccgg?gcccatgaat?tgtgggggtc?gctatttaat 300
gaactttacc?agacatctgg?ttctttcttc?agggccatct?catctaaaac?tgtccattct 360
ttcctcttaa?ataagacatc?tcgatggact?aatggctaat?cagcccatgc?tcacacataa 420
ctgtgctgtc?atacatttgg?tattttttta ttttggg 457
<210>4
<211>457
<212>DNA
< 213>yak (Bos grunniens)
<220>
<221>mutation
<222>(179)..(186)
<400>4
acgggggaat?acgtacataa?tattaatgta?ataaagacat?attatgtata?tagtacatta 60
aattatatgc?cccatgcata?taagcaagta?cataattcct?attgatagta?cataatacat 120
gaaattatta?atcgtacata?acacattatg?tcaaacctac?tcctaacaac?atgtatac— 180
------cccc?tccactagat?cacgagctta?actaccatgc?cgcgtgaaac?cagcaacccg 240
ctaggcggag?gacccctctt?ctcgctccgg?gcccatgaac?tgtgggggtc?gctatttaat 300
gaactttatc?agacatctgg?ttctttcttc?agggccatct?cacctaaaac?cgtccactct 360
ttcctcttaa?ataagacatc?tcgatggact?aatggctaat?cagcccatgc?tcacacataa 420
ctgtgctgtc?atacatttgg?tattttttta?ttttggg 457
<210>5
<211>23
<212>DNA
< 213>yak (Bos grunniens)
<400>5
acacgcccat?acacagacca?cag 23
<210>6
<211>23
<212>DNA
< 213>yak (Bos grunniens)
<400>6
tccaagcatc?ccccaaaata?aaa 23
Claims (5)
1. one group of matrilinear inheritance mark of differentiating each tame ox of Bos; Each tame ox of described Bos is common ox, zebu, gayal and yak; The nucleotide sequence of the matrilinear inheritance mark of common ox is shown in SEQ ID No.1; The nucleotide sequence of the matrilinear inheritance mark of zebu is shown in SEQ ID No.2; The nucleotide sequence of the matrilinear inheritance mark of gayal is shown in SEQ ID No.3, and the nucleotide sequence of the matrilinear inheritance mark of yak is characterized in that shown in SEQ ID No.4: there is a deletion mutantion at the nucleotide sequence 249bp place shown in the SEQID No.1 of the matrilinear inheritance mark of described common ox; Be designated as gap249, be the special disappearance genetic marker of common ox; There is a deletion mutantion at nucleotide sequence 191bp place shown in the SEQ ID No.2 of the matrilinear inheritance mark of described zebu, is designated as gap191, is the special disappearance genetic marker of zebu; There is an identical deletion mutantion at nucleotide sequence 179-186bp place shown in the matrilinear inheritance mark SEQ ID No.4 of nucleotide sequence 179-186bp place shown in the SEQ ID No.3 of the matrilinear inheritance mark of described gayal and yak; Be designated as gap179-186, be gayal and the special disappearance genetic marker of yak common.
2. the described one group of preparation method who differentiates the matrilinear inheritance mark of each tame ox of Bos of claim 1 is characterized in that:
(1) identify that through the individual appearance in sample source the ox of selecting with the breeding registration confirmed is the yak of Bos, gayal, zebu and common ox sample, and extract the poba gene group DNA of these 4 kinds of oxen respectively;
The genomic dna of 4 kinds of oxen of (2) step (1) being extracted with primer carries out pcr amplification respectively; Described primer is made up of forward primer and reverse primer, and the dna sequence dna of described forward primer is shown in SEQ IDNo:5, and the dna sequence dna of described reverse primer is shown in SEQ ID No:6; The purified recovery of PCR product; Order-checking is carried out sequence alignment each other with the sequence of described 4 kinds of oxen of measuring, and it is following to obtain the described one group of matrilinear inheritance mark of differentiating each tame ox of Bos of claim 1:
1) the matrilinear inheritance mark of common ox; Its nucleotide sequence shown in SEQ ID No.1, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 249bp place; Be designated as gap249, be the special disappearance genetic marker of common ox;
2) the matrilinear inheritance mark of zebu; Its nucleotide sequence shown in SEQ ID No.2 in the sequence table, the long 457bp of described nucleotide fragments, an and deletion mutantion is arranged at described nucleotide fragments 191bp place; Be designated as gap191, be the special disappearance genetic marker of zebu;
3) the matrilinear inheritance mark of gayal, its nucleotide sequence is shown in SEQ ID No.3; The matrilinear inheritance mark of yak, its nucleotide sequence is shown in SEQ ID No.4; With the nucleotide sequence 179-186bp place shown in the described SEQ ID No.4 an identical deletion mutantion is arranged all at the nucleotide sequence 179-186bp place shown in the described SEQ ID No.3; Be designated as gap179-186, be gayal and the special disappearance genetic marker of yak common.
3. differentiate each tame ox of Bos and the individual method of hybridization thereof for one kind; It is characterized in that: extract cow genome group DNA from the bovine blood of ox kind to be identified; In the cow genome group DNA that is extracted, carry out pcr amplification with primer, described primer is made up of forward primer and reverse primer, and the dna sequence dna of described forward primer is shown in SEQ ID No:5; The dna sequence dna of described reverse primer is shown in SEQ ID No:6, and the PCR product purification reclaims and order-checking; The sequence of measuring and the sequence of the described matrilinear inheritance mark of claim 1 are carried out sequence alignment; Special disappearance genetic marker with the described matrilinear inheritance mark of claim 1 contains is differentiated the matrilinear inheritance background of individuality to be measured; See from the maternal side source; The individuality to be measured that contains special disappearance gap249 is common ox, and the individuality to be measured that contains special disappearance gap191 is a zebu, and the individuality to be measured that contains special disappearance gap179-186 is gayal or yak.
4. be marked at discriminating of each tame ox of Bos and the application in the genetic background detection with the described one group of matrilinear inheritance of differentiating each tame ox of Bos of claim 1, each tame ox of described Bos is common ox, zebu, gayal and yak.
5. application according to claim 4; It is characterized in that: the cow genome group DNA that in application, is extracted is carried out pcr amplification with primer; Described primer is made up of forward primer and reverse primer; The dna sequence dna of described forward primer is shown in SEQ ID No:5, and the dna sequence dna of described reverse primer is shown in SEQID No:6.
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| CN2010101346511A CN101798598B (en) | 2010-03-29 | 2010-03-29 | Maternal genetic marker for identification of domestic cattle of bos and identification method and application thereof |
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| CN111455072B (en) * | 2020-05-29 | 2021-09-14 | 华中农业大学 | Genome sequence capable of being used for identifying blood systems of tumor cattle and common cattle and application method thereof |
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Non-Patent Citations (2)
| Title |
|---|
| X. Gou et al.Genetic diversity and origin of Gayal and cattle in Yunnan revealed by mtDNA control region and SRY gene sequence variation.《J. Anim. Breed. Gene》.2010,第127卷第154－160页. * |
| 王威等.多重PCR2RFLP检测XRCC1基因两位点多态性.《中国工业医学杂志》.2008,第21卷(第1期),第3-6卷. * |
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