CN105713982A - SNP (single nucleotide polymorphism) marker correlated with characteristic of short stature of Chinese domestic horses and application of SNP marker - Google Patents
SNP (single nucleotide polymorphism) marker correlated with characteristic of short stature of Chinese domestic horses and application of SNP marker Download PDFInfo
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Abstract
The invention relates to an SNP (single nucleotide polymorphism) marker correlated with the characteristic of short stature of Chinese domestic horses. The SNP marker is located in the 18,101,000th base pair of the chromosome 8 of a horse. The SNP marker correlated with the characteristic of short stature of Chinese domestic horses and the application of the SNP marker have the advantages as follows: (1) the provided molecular marker is free from limits of age, sex and the like of the Chinese domestic horses and can be used for early-stage selection of the Chinese domestic horses, even the Chinese domestic horses that are just born can be accurately screened, and the breeding progress of the superior pony variety in Chinese domestic horses can be remarkably promoted; (2) a method for detecting the SNP of TBX3 genes of the Chinese domestic horses is accurate, reliable and simple and convenient to operate; (3) detection of SNP sites of the TBX3 genes of the Chinese domestic horses provides a scientific basis for marker assisted selection of the height of the Chinese domestic horses.
Description
Technical field
The invention belongs to genetic biology field, be specifically related to the SNP marker relevant to Chinese Home horse dwarfism and application thereof.
Background technology
The polymorphism of the DNA fragmentation that SNP mononucleotide polymorphism site is primarily referred to as in genome desoxyribose (DNA) sequence caused by the variation of single Deoxydization nucleotide.The polymorphism of SNP relates only to the variation of single base, and the form of performance has replacement, insertion and disappearance etc..
The detection method of SNP, at present conventional includes, the technology such as sanger order-checking, DNA chip, flight time mass spectrum and the up-to-date secondary order-checking of high flux.SNP has been widely used for the research fields such as gene mapping, clone, genetic breeding and genetic diversity as genetic marker.
Transcription factor TBX3 (T-Boxtranscriptionfactor3) is regulation and control one of growth promoter key genes, and it widely exists in embryonal tissue, embryonic stem cell, conducting system of heart growth in play an important role.Its disappearance or sudden change can cause ulna-mammary gland syndrome.China's Debao pony, Yili horse and Mongolian horse colony carry out whole-genome association and full-length genome selects signal screening, it has been found that the impact of the body measurement trait of Chinese Home horse is reached significant level by the polymorphic variation of TBX3 gene locus.
Pony is individual short and small and flexible, and temper is docile, is particularly suitable as teenager horsemanship and imparts knowledge to students and use horse.Along with the fast development throughout the country of teenager jockey club corporation, Ltd, pony is inestimable in the economic outlook of cultural education industry, but, the whole dependence on import pony of current domestic jockey club corporation, Ltd, every introduces cost about 50,000 yuans.But domestic pony is low due to selection-breeding degree, breeding labelling lacks, and development and utilization level is low, has resulted in China's pony resources advantage and cannot be converted into economic advantages.Abroad navigating to HMGA2 etc. already by genome-wide screening affects the gene loci of pony body measurement trait at present, but early-stage Study also indicates that domestic and international pony has the mechanism that is each independently formed, therefore, it is necessary to develop the molecular marker that domestic pony is special, it is applied to the selection-breeding of domestic pony.
Summary of the invention
First purpose of the present invention is to provide a kind of SNP marker and application thereof assisted and identify Chinese Home horse dwarfism.
It is a further object to provide the primer for detecting the SNP marker relevant to Chinese Home horse dwarfism and the test kit containing this primer.
In order to realize first purpose of the present invention, the invention provides a kind of SNP marker relevant to Chinese Home horse dwarfism, described SNP marker is positioned at No. 8 chromosome the 18101000th base pair (gi194246389:18100500-18101500EquuscaballusisolateTwiligh tbreedthoroughbredchromosome8 of horse, EquCab2.0, wholegenomeshotgunsequence).
Chinese Home horse horses from different regions and kind have been carried out substantial amounts of genotype and height phenotypic correlation Journal of Sex Research by inventor, it has been found that SNP site provided by the present invention is high with family length significantly associates (P < 7.1e-11).Allele A frequency in pony is up to 80.65%, and only has 18.85% at Yili horse and Mongolian horse medium frequency.Frequency of genotypes AA is only present in pony, genotype is that the average height of the horses of AA is significantly short in horses that genotype is GG (X 2 test P value < 2.2e-16), Chinese Home saddlery for being distinguished and filter out dwarfism by genotype has bigger directive significance, can be determined that horses have dwarfism when the genotype of Chinese Home horse is AA, when the genotype of Chinese Home horse is GG be time can be determined that horses have tall and big character, it is possible to improve accuracy rate and the efficiency of the screening of family length height.
In the present invention, to pony height define and the height to pony current in the world defines identical, namely the height of horseback belongs to pony lower than 106 centimetres, belongs to high horse greater than or equal to 106 centimetres.
Present invention also offers the specific primer for detecting SNP marker of the present invention, including:
Forward primer: 5 '-CCAACAACACCTGCCTTTTT-3 '；
Reverse primer: 5 '-GCGACTTCGAAGAGGAGAAA-3 '.
Present invention also offers the application in described SNP marker country's horse dwarfism kind in authentication, comprise the following steps:
1) genomic DNA of horses to be measured is extracted；
2) with the genomic DNA of horses to be measured for template, specific primer is utilized to carry out pcr amplification reaction, it is thus achieved that amplified production fragment；
3) detect the base kind at the 489bp place of pcr amplification product fragment, if base kind is A, then judge that horses to be measured have dwarfism, if base kind is G, then judge that horses to be measured have tall and big character.
Wherein, step 2) described in specific primer include:
Forward primer: 5 '-CCAACAACACCTGCCTTTTT-3 '；
Reverse primer: 5 '-GCGACTTCGAAGAGGAGAAA-3 '.
Wherein, the nucleotide sequence of described amplified production fragment is such as shown in SEQIDNO.1.Described SNP site is positioned at the 489bp place of the nucleotide sequence shown in SEQIDNO.1.In SEQIDNO.1,489bp place letter r is identified, and r represents that base herein is adenine or guanine.
The present invention has no particular limits for the method detecting pcr amplification product fragment, it is possible to use the detection method of this area routine carries out, it is preferred that, it is possible to utilize flight time mass spectrum method to detect the genotype of Chinese Home horse to be measured.
Wherein, step 2) in PCR reaction use amplification system be calculated as with 25 μ l: 50-100ng/ μ l template DNA 1 μ l, 10pmol/ μ l forward primer and each 1 μ l of reverse primer, 10mmol/LdNTPmix2.0 μ l, 5U/ μ lTaqDNA polymerase 0.125 μ l, 10 × PCR reaction buffer 2.5 μ l, surplus is distilled water.
Wherein, step 2) in the condition of PCR reaction be: 94 DEG C of denaturations 5 minutes；94 DEG C of degeneration 30 seconds, anneal 30 seconds for 57 DEG C, and 72 DEG C extend 60 seconds, totally 34 circulations；72 DEG C are incubated 5 minutes.
Present invention also offers the auxiliary containing described specific primer and identify the test kit of Chinese Home horse dwarfism.
Preferably, described test kit also includes dNTPs, Taq DNA polymerase, Mg2+, one or more in PCR reaction buffer.
Preferably, described test kit also includes standard positive template.
Specific primer of the present invention can also combine for the classification of Chinese Home horse and breeding research for the specific primer of Chinese Home horse Phenotypic examination with other.
The SNP marker relevant to Chinese Home horse dwarfism application in Chinese Home horse molecular mark falls within protection scope of the present invention.
The SNP marker relevant to Chinese Home horse dwarfism of the present invention and application thereof have the advantage that
(1) molecular marker provided by the invention is not by restrictions such as the age of Chinese Home horse, sexes, can be used for the early stage selection-breeding of Chinese Home horse, and even just birth can be screened exactly, can remarkably promote the breeding process of Chinese Home horse advantage pony kind.
(2) method of Chinese Home horse TBX3 gene mononucleotide polymorphism is detected accurately and reliably, easy and simple to handle.
(3) detection of the SNP site of Chinese Home horse TBX3 gene, the marker assisted selection for the height size of Chinese Home horse provides scientific basis.
Accompanying drawing explanation
Fig. 1 is three kinds of genotype order-checking peak figure；
Wherein, (a) is AA type；B () is GG type；C () is AG type.
Fig. 2 is three kinds of genotypic flight time mass spectrum SNP typing cluster analysis figure；
Wherein, transverse axis region representation GG genotype individuals；Longitudinal axis region representation AA genotype individuals；Zone line represents GA genotype individuals；Nocall represents that individual one genotype lacks；Other represents other genotype individuals.
Fig. 3 is the analysis result of SNP site.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in detail.
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, the conventional means that in embodiment, technological means used is well known to those skilled in the art, raw materials used it is commercial goods.
The qualification of embodiment 1 Chinese Home horse TBX3 gene polymorphism sites.
1.1 extract the genomic DNA in Chinese Home horse blood to be measured
Gather from 127, In Southwest China pony (include 37 from ground, Debao, 31 from Baise, 31 from Luoping, 11 from Guizhou, 17 from mountain of papers) and the horse of 150 normal heights (include 30 from Tibetan area, 45 from northwest, 32 from the Inner Mongol, 43 are northeast) blood sample, adopt conventional method to extract the genomic DNA in blood.
1.2 amplifications nucleotide fragments containing SNP site: TBX3 gene locus (the gi194246389:18100500-18101500EquuscaballusisolateTwiligh tbreedthoroughbredchromosome8 included according to ncbi database, EquCab2.0, wholegenomeshotgunsequence) primers, including forward primer F:5'-CCAACAACACCTGCCTTTTT-3' and reverse primer R:5'-GCGACTTCGAAGAGGAGAAA-3', with the genomic DNA in 1.1 for template, amplify the nucleotide fragments at SNP place to be measured, as shown in SEQIDNO.1.This SNP site is positioned at the 489bp place of pcr amplified fragment, and base is A or G herein.
The amplification system that wherein PCR reaction uses is calculated as with 25 μ l: 100ng/ μ l template DNA 1 μ l, 10pmol/ μ l primers F and each 1 μ l, 10mmol/LdNTPmix2.0 μ l, the 5U/ μ lTaqDNA polymerase 0.125 μ l of R, 10 × PCR reaction buffer 2.5 μ l, surplus is distilled water.
Wherein the condition of PCR reaction is: 94 DEG C of denaturations 5 minutes；94 DEG C of degeneration 30 seconds, anneal 30 seconds for 57 DEG C, and 72 DEG C extend 60 seconds, totally 34 circulations；72 DEG C are incubated 10 minutes.
1.3 detection pcr amplified fragments, it is thus achieved that SNP marker
Pcr amplification product in 1.2 is carried out order-checking detection, if the base at 489bp place is A in amplified production sequence, then Chinese Home Equus to be measured is in advantage pony kind.Three kinds of genotypic order-checking peak figure are as shown in Figure 1.
1.4 genotype judge: by the analysis method of flight time mass spectrum, the genotype of Chinese Home horse to be measured is detected；Result according to flight time mass spectrum judges SNP site genotype in colony to be measured.Signal distributions position per sample, genotype can be divided into AA type, GG type and AG type.Three kinds of genotypic genotyping result are as shown in Figure 2.
The association analysis of embodiment 2 Chinese Home horse different genotype and height size and detection application.
The genome DNA sample extracting 32 portions of ponies, 32 parts of Yili horse and 32 parts of Mongolian horses according to the method in embodiment 1 carries out DNA chip typing detection, and the genotyping result of TBX3 gene such as sequence 489bp site shown in SEQIDNO.1 is as shown in Figure 3.Use FDR in PLINK software to correct and " adaptive " model carries out whole-genome association, it has been found that above-mentioned SNP site significantly associates (P < 7.1e-11) with height.Allele A frequency in pony is up to 80.65%, and only has 18.85% at Yili horse and Mongolian horse medium frequency.Frequency of genotypes AA is only present in pony, genotype be the average height of the horses of AA significantly short in genotype be the horses of GG.
The pleomorphism site of the TBX3 gene locus of 277 Chinese Home horses is enlarged group analytic by embodiment 3.The X 2 test using R software is analyzed, the frequency (P < 2.2e-16) of the allele A that the frequency of above-mentioned SNP site allele A in pony is significantly higher than in other normal stature horses (non-pony).Demonstrate the dependency (as shown in table 1) of the allele A of this place's SNP site and the dwarfism of Chinese Home horse further.
Table 1SNP site genotypic frequency in Chinese Miniature Horses and non-pony kind and gene frequency
| Phenotype | AA | AG | GG |
| Pony | 79 | 41 | 1 |
| Non-pony | 16 | 70 | 59 |
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (9)
1. the SNP marker relevant to Chinese Home horse dwarfism, it is characterised in that described SNP marker is positioned at No. 8 chromosome the 18101000th base pair of horse.
2. require the specific primer of SNP marker described in 1 for test right, it is characterised in that including:
Forward primer: 5 '-CCAACAACACCTGCCTTTTT-3 '；
Reverse primer: 5 '-GCGACTTCGAAGAGGAGAAA-3 '.
3. the application in the country's horse dwarfism kind in authentication of the SNP marker described in claim 1, comprises the following steps:
1) genomic DNA of horses to be measured is extracted；
2) with the genomic DNA of horses to be measured for template, specific primer is utilized to carry out pcr amplification reaction, it is thus achieved that amplified production fragment；
3) detect the base kind at the 489bp place of pcr amplification product fragment, if base kind is A, then judge that horses to be measured have dwarfism, if base kind is G, then judge that horses to be measured have tall and big character；
Wherein, described specific primer includes:
Forward primer: 5 '-CCAACAACACCTGCCTTTTT-3 '；
Reverse primer: 5 '-GCGACTTCGAAGAGGAGAAA-3 '.
4. application according to claim 3, it is characterized in that, step 2) in PCR reaction use amplification system be calculated as with 25 μ l: 50-100ng/ μ l template DNA 1 μ l, 10pmol/ μ l forward primer and each 1 μ l of reverse primer, 10mmol/LdNTPmix2.0 μ l, 5U/ μ lTaqDNA polymerase 0.125 μ l, 10 × PCR reaction buffer 2.5 μ l, surplus is distilled water.
5. application according to claim 4, it is characterised in that step 2) in the condition of PCR reaction be: 94 DEG C of denaturations 5 minutes；94 DEG C of degeneration 30 seconds, anneal 30 seconds for 57 DEG C, and 72 DEG C extend 60 seconds, totally 34 circulations；72 DEG C are incubated 5 minutes.
6. the auxiliary containing specific primer described in claim 2 identifies the test kit of Chinese Home horse dwarfism.
7. test kit according to claim 6, it is characterised in that described test kit also includes dNTPs, Taq DNA polymerase, Mg2+, one or more in PCR reaction buffer.
8. the test kit according to claim 6 or 7, it is characterised in that described test kit also includes standard positive template.
9. the SNP marker relevant to Chinese Home horse dwarfism described in claim 1 application in Chinese Home horse molecular mark.
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Cited By (3)
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| CN108410866A (en) * | 2018-02-05 | 2018-08-17 | 中国农业科学院北京畜牧兽医研究所 | With the relevant SNP marker of middle country's horse adaptation to high altitude character and its application |
| CN108950014A (en) * | 2018-08-02 | 2018-12-07 | 中国农业科学院北京畜牧兽医研究所 | SNP marker relevant to middle country's horse adaptation to high altitude and application |
| CN110541041A (en) * | 2019-09-06 | 2019-12-06 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to Chinese domestic horse dwarf trait and application thereof |
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| CN103160589A (en) * | 2013-04-03 | 2013-06-19 | 哈尔滨体育学院 | Method for predicting height of excellent ice-snow athlete by using polymorphism of COL11A1 gene promoter |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410866A (en) * | 2018-02-05 | 2018-08-17 | 中国农业科学院北京畜牧兽医研究所 | With the relevant SNP marker of middle country's horse adaptation to high altitude character and its application |
| CN108410866B (en) * | 2018-02-05 | 2021-08-06 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to high-altitude adaptability of Chinese domestic horses and application thereof |
| CN108950014A (en) * | 2018-08-02 | 2018-12-07 | 中国农业科学院北京畜牧兽医研究所 | SNP marker relevant to middle country's horse adaptation to high altitude and application |
| CN108950014B (en) * | 2018-08-02 | 2021-02-02 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to high altitude adaptability of Chinese domestic horses and application |
| CN110541041A (en) * | 2019-09-06 | 2019-12-06 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to Chinese domestic horse dwarf trait and application thereof |
| CN110541041B (en) * | 2019-09-06 | 2021-02-02 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to Chinese domestic horse dwarf trait and application thereof |
| US11542562B2 (en) * | 2019-09-06 | 2023-01-03 | Institute Of Animal Sciences Of Chinese Academy Of Agricultural Sciences | Single nucleotide polymorphism marker related to Chinese horse short stature trait and use thereof |
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